2018 Biotech Primer WEEKLY Compendium

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B I O T E C H P R I M E R ’ S

W E E K LY
C O M P E N D I U M
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2018 BIOTECH PRIMER WEEKLY COMPENDIUM

Contents
1 Swallowing A Biologic Drug? 61 Vaccines: Schooling The Herd
3 Taking A Swing At Allergies 63 Eye Of The Cytokine Storm
5 New Hope For Spinal Muscular Atrophy 65 The Multiple Myeloma Landscape
7 GMO Apples Hit Marketplace 67 From Fantasy To Reality: Xenotransplantation
9 Stem Cell Snapshot 69 Picturing Disease
11 Therapeutic Antibody Primer 71 Circadian Rhythm & Disease
13 The Mechanics Of Melanoma 73 Vaccines: Powerful Simplicity
15 The Intrigue Of HIF 75 DNA Vaccines Explained
17 The PARP Race Is On 77 Natural Born Cancer Killers
19 The Science Of CRISPR/CAS9 79 Off-Color: The Science

Behind Color Vision Deficiency
21 Pills, Proteins & Peptides
81 The Science Behind Opioid Addiction
23 Putting The NA in DNA
83 Bye-Bye Opioids? Introducing Electroceuticals
25 RNA Therapeutics March Onward
85 Zinc Finger Nucleases
27 Decoding Your Genes
86 Plants That Heal
30 Phage — More Than Just A Phase
88 Drug Discovery 101
32 Viruses Blasting Cancer
34 Nanobodies: These Are Not Your Mother’s mAbs
92 Gene Therapy Cures
36 Epigenome: Writing, Reading & Erasing
38 A Skin Cell With Stem Cell Diversity?
96 From Drug Development to Approval: Phase I/II
40 The Biotech Primer BIO 2017 Preview
98 From Drug Development To Approval: Phase III
42 Cell Signaling Explained
100 From Drug Development To Approval: Phase IV
45 Breast Cancer Subtypes
102 Market Access
47 Stopping A Big Problem: Blood Clots
104 From Drug Development to Approval: A Recap
49 Unpacking Digital Medicine
106 Who’s Your Daddy? The Science of 23andMe
51 Chaperoning The Rare Disease Dance
108 Meatless Meat: Biotech Burger Ain’t No Bean Patty
53 Putting The CAR-T Before The Horse
110 Alzheimer’s Disease: A Tough Nut To Crack
55 The Science Of CRISPR/Cas9
112 Exploring Different Strategies to Fight Alzheimer’s
57 Harnessing Your Immune System For Good
114 Cancer Diagnostic In A Drop
59 The Microbiome Magnified
116 Turning On Cellular Garbage Disposals
2018 BIOTECH PRIMER WEEKLY COMPENDIUM

Swallowing A Biologic Drug?
SURVIVING THE STOMACH IS KEY • When it enters the intestine, its outer coating
dissolves at the slightly higher pH of the intestine.
Over the past two decades, biologic drugs — drugs
composed of proteins produced by living cells — have • This pH change also triggers a chemical reaction to
become the safest, most effective top sellers within the occur, which releases carbon dioxide which blows up
pharmaceutical industry. a tiny “balloon” found within the pill.
Approved to treat a variety of diseases including • This supplies the force needed to drive the
rheumatoid arthritis, diabetes, multiple sclerosis, drug-containing needle into the intestinal wall —
Crohn’s disease, and a whole range of cancers, these delivering the biologic to the bloodstream.
drugs include monoclonal antibody therapeutics, • The needle itself then dissolves, and the balloon
hormones, and immune system signaling molecules. is excreted.
Their safety and efficacy depend largely on the complex,
This may sound like the stuff of science fiction — and
three-dimensional structure of the protein product
indeed, the device is still in preclinical trials — but the
itself — which is incredibly delicate and time-consuming
robotic pill has attracted big name pharma partners,
to develop on a large scale. The majority of these are
including AstraZeneca (London, UK) and Novartis
administered via injection directly into the bloodstream
(Basel, Switzerland), as well as investment from
for maximum potency.
Google Ventures (Mountain View, CA) among other
So, why can’t we just swallow a biologic pill? The simple venture funds. If the device ultimately succeeds, it will
answer: biologics would not survive the acidic pH and revolutionize the delivery of biologic drugs.
digestive enzymes of the gut. Even if survival were
possible, the next issue to contend with is absorption;
ENGENE’S GUT CELL FACTORIES
if the protein is not broken down properly, it will not
be absorbed into the bloodstream. Companies seeking enGene (Vancouver, Canada) is bypassing the delivery
to develop oral delivery of biologics must overcome obstacle by attempting to turn the cells of the gut into
both hurdles. drug-producing factories. The trick is to deliver a gene —
the instructions for a specific therapeutic protein — to
Injectable delivery is cumbersome at best. It has a
those cells. enGene is using tiny carbohydrate-based
significant impact on a patient’s quality of life, which
nanoparticles to encase the gene. The carbohydrate
in turn affects compliance to drug therapy regimes.
coating protects the gene as it passes through the
In many cases, patients must make regular trips to an
stomach, yet allows it to be absorbed into the intestines.
infusion center to receive required treatments. Thus
The protein is then made in the intestines.
oral administration is the holy grail in terms of delivering
biologics. In this WEEKLY, we’ll take a look at the different This approach is especially appealing for diseases
paths being pursued to make this ambition a reality. affecting the colon and small intestine, such as
inflammatory bowel syndrome or Crohn’s disease.
enGene is currently using this platform in the preclinical
THE ROBOTS OF RANI THERAPEUTICS
development of the anti-inflammatory protein IL-10, for
One of the hottest companies in the oral delivery of the treatment of inflammatory bowel disease.
biologics space is Rani Therapeutics (San Jose, CA).
Their product is a “robotic pill” — a small device capable
APPLIED MOLECULAR TRANSPORT
of traversing the intestinal tract and injecting a biologic
drug directly into the body. It works in the following way: Applied Molecular Transport South San Francisco,
CA) is using a protein scaffold adapted from pathogenic
• After the pill is swallowed, it makes its way through
microbes such as salmonella, which colonize the gut by
the digestive process, keeping its fragile biologic
secreting immune-crippling proteins into our body.
drug cargo tucked safely inside. Its capsule is
pH-sensitive and stays intact at the low pH of Proteins from these “gut bugs” work by tricking the
the stomach. intestines into absorbing toxic proteins in the same way
2018 BIOTECH PRIMER WEEKLY COMPENDIUM 1

that they absorb nutrients from food. Applied Molecular on the biopharma industry, innovative companies are
Transport scientists have tweaked these microbial approaching the drug delivery problem with a range of
proteins to carry a therapeutic payload, rather than strategies. It is likely that no single strategy will work
the toxins. Development efforts are currently aimed at for all types of biologics, but any success with a handful
delivering anti-inflammatory proteins to the intestines. of products would represent a major breakthrough for
Swallowing biologic drugs is no longer a pipe dream. the industry.
Sensing the opportunity to make a significant impact

Taking A Swing At Allergies
HOW DO ALLERGIES DEVELOP? TERM OF THE WEEK: HISTAMINES
Watching a game at the ballpark and digging into a Histamines are small molecules produced by mast cells.
bag of peanuts is a source of entertainment for many Once released, histamines bind to receptors on the
Americans. For the 15 million who suffer from peanut surface of blood vessels, increasing their permeability —
allergies, the idea of being taken out to the ballgame the ease with which fluid moves out of the blood vessels.
elicits concern — or even anxiety. Histamines also bind to receptors on certain types of
Food allergies — think tree nuts, milk, eggs, wheat, nerve cells, resulting in muscle contraction. A host of
soy, fish, and shellfish — are on the rise. The mere dust symptoms can be triggered, ranging from annoying to
particle of a freshly cracked peanut can be responsible deadly. Typical signs of histamine release include:
for an unpredictable cascade of reactions, including • Increased blood vessel permeability resulting in a
death brought about by anaphylaxis. runny nose and watery eyes.
This WEEKLY takes a swing at explaining how allergies • Increased muscle contraction leading to throat
develop, the current treatments, and what new products constriction and difficulty breathing.
might change the way allergen desensitization therapy • Extreme fluid release from tissues causing a sudden
is delivered. drop in blood pressure, potentially bringing on a
heart attack.
SOMETHING TO SNEEZE AT
• Difficulty breathing and swallowing, swelling, heart
The host of symptoms dubbed “allergies” are the palpitations, and unconsciousness — sometimes
end result of the immune system’s response to a causing death.
normally harmless substance, as if that harmless
Mild allergy symptoms such as runny nose and watery
substance were a threat. An initial allergen exposure
eyes can often be successfully controlled by the use of
results in the production of a class of antibodies called
an over-the-counter antihistamine, a drug that works by
Immunoglobulin E (IgE). A second exposure to the
blocking the interaction of histamines with receptors on
allergen results in an “allergen-IgE antibody complex”
nerve and muscle cells. However, once anaphylaxis — a
These newly produced complexes bind to and activate
severe allergic reaction that includes difficulty breathing
mast cells — a type of immune cell. As the image below
and heart palpitations — has occurred, it is too late for
shows, activated mast cells send out chemical alarms in
antihistamines to be effective. These symptoms can only
the form of histamine.
be treated with an injection of the hormone epinephrine
— and the sooner, the better in cases where a life is on
the line.
Epinephrine helps to reverse histamine’s effects by
decreasing blood vessel permeability, relaxing muscle
cells, and stimulating the heart. People at risk for
anaphylaxis need access to an epinephrine auto-injector
— a spring-loaded syringe that makes the lifesaving shot
readily available. This type of product is referred to as
a “combination product” because it combines a device
(the auto-injector) with a medicine (epinephrine). Mylan
(Canonsburg, PA) EpiPen is an epinephrine auto-injector,
as is Amedra’s (Horsham, PA) Adrenaclick.

THE HYGIENE HYPOTHESIS allergy was assessed – and an 81 percent reduction
in allergy was found in the children who regularly
Epidemiologists have noticed an interesting trend as
consumed peanuts when compared to those who
countries rise from developing to developed status:
avoided them
an improvement in sanitation and access to antibiotics
means less pathogenic exposure and lower infection
rate. As the infection rate drops, the incidence of ALLERGENS BY THE DOSE
allergies shoots up. The current allergen immunotherapy market includes
Many scientists think early exposure to infection helps allergy shots (which require monitoring by a physician)
shift the immune response towards fighting pathogens and drops or tablets dissolved under the tongue
while minimizing the production of IgE antibodies. (which can sometimes be taken at home). Aimmune
Exposure to potential allergens (while the immune Therapeutics’ (Brisbane, CA) AR 101 is a dissolvable
system is still developing) helps to desensitize the tablet made of pharmaceutical grade peanut protein that
allergic response. can be mixed with food as a means of delivery.
AR 101 Phase II studies report patients becoming
THE STRATEGY BEHIND desensitized to doses at least twenty times greater
DESENSITIZATION THERAPY than the original allergy-inducing dose — and in some
cases, more than 100-fold greater. The goal is to reach a
The idea of allergy desensitization through controlled tolerance level that offers protection against accidental
exposure has been around for decades. Desensitization eating of peanuts. The product was awarded Fast Track
is the principle behind allergy shots shown to be status by the FDA and is now in Phase III. Aimmune has
effective against pet dander, dust mites, and pollen. plans for clinical studies on immune system training for
Desensitization therapy was once considered to be too egg and milk allergies to begin later this year.
risky for food allergies, but a number of new studies
support the idea that gradual exposure to food allergens DBV Technologies (Bagneux, France) is also in the
may be beneficial. allergy fight. They are currently conducting clinical trials
on their Viaskin skin patch which delivers low doses of
In January 2017, the National Institutes of Health either peanut, milk, or dust mite allergens. By delivering
(NIH) released new guidelines recommending the early an allergen through the skin instead of the blood,
introduction of peanuts into children’s diets, including the body will react less severely, reducing the risk of
those considered to be at high risk for developing peanut anaphylaxis as a side effect. The Viaskin peanut patch,
allergies because they already have severe eczema or currently in Phase III clinical testing, has been awarded
egg allergies. These new guidelines are based on the Fast Track designation by the FDA.
results of the NIH-funded Learning Early About Peanut
Allergy (LEAP), a study which randomly assigned 600 As allergen desensitization treatments continue to make
high-risk infants to either peanut-avoidance or the their way through the drug pipeline, allergy sufferers
regular inclusion of small amounts of peanut products in remain hopeful for better and easier treatment options,
their diet for the first five years of life. At age five, peanut and maybe even one day — a cure.

New Hope For Spinal
Muscular Atrophy
FIRST THERAPY APPROVED FOR SMA is non-functional and degrades shortly after being
produced. Patients with less severe forms of the disease
Squeaking by on December 23rd as the last new drug
usually have extra SMN2 copies because ultimately,
approval of 2016, Biogen’s (Cambridge, MA) Spinraza
even tiny amounts of SMN protein provides some motor
now provides hope for the thousands of families
nerve function.
affected by a debilitating neuromuscular disorder known
as spinal muscular atrophy (SMA). SMA robs people of The four generally accepted classifications of SMA are:
their ability to walk, eat, and ultimately, breathe. • Type 1: The most severe and the most common.
In addition to Spinraza, there are 13 other new therapies Babies do not move, but lay perfectly still in their
making their way through the clinic, according to the cribs. As the disease progresses, toddlers have
patient advocacy group Cure SMA. The increase is trouble with swallowing and respiratory function.
largely due to a better understanding of the disease SMA Type 1 is usually fatal by age two.
and a surge in funding for basic and clinical research. • Type 2: Symptoms manifest between six and
SMA affects about 1 in 10,000 babies born in the eighteen months. These children can typically sit
United States. but not stand or walk. Respiratory function is often
In this weekly, we’ll decipher the science behind SMA, compromised, however with the help of machines
explain the novel mechanism of action used by Biogen’s many of these patients live into adulthood.
new drug, and find out how other drugs in development • Type 3: Symptoms occur after age one. These kids
are zeroing in on this genetic disease. are usually able to walk, but may lose that ability as
the disease progresses. Respiratory function is less
SMA PRIMER impaired, and life expectancy is often near normal.
Our nervous system consists of the brain, spinal cord, • Type 4: This is the adult-onset form, typically
and a vast network of nerves that feed into every tissue developing at age 30 or later. Muscles gradually
of the body. Motor neurons are a type of nerve cell weaken, and the patient often needs to use
that sends messages from the spinal cord to muscles, a wheelchair later in life. Life expectancy is
enabling movement. not affected.
In order for the motor neurons to do their job, a SMA Type 1 is the most common and most severe,
functional protein called the survival motor neural (SMN) making up 60% of cases. As a result, many companies
protein is necessary. The survival motor neuron 1 (SMN1) are looking to tackle this segment of the disease
gene is responsible for producing most of the SMN population. Below find a few treatment approaches for
protein used by the body. A second, closely related SMA Type 1.
gene is the survival motor neuron 2 (SMN2) gene, which
produces a much smaller amount of SMN protein and is ON THE MARKET: ANTISENSE THERAPY
seen as a sort of “back-up” version to SMN1.
Developed by Biogen in partnership with Ionis
SMA is caused by a variety of mutations in the SMN1 Pharmaceuticals (Carlsbad, CA), Spinraza is one of a
gene. Without functional SMN protein, the neurons do small but growing class of drugs: antisense therapeutics.
not work correctly and eventually die. How soon they It is a synthetic mRNA molecule that binds to the
die depends on the extent of the SMN deficiency, which naturally occurring SMN2 mRNA in such a way that more
correlates with the severity of the disease: the less SMN of the mRNA is used to make the protein. The result is
produced, the more severe the disease. greater amounts of full-length, functional SMN protein.
The back-up gene, SMN2, produces a small amount of Recall from high school biology that mRNA provides the
functional SMN protein. However, differences in the way instructions to make proteins. If the mRNA is working
SMN2 functions means most (but not all) of the protein properly, the correct, functional protein is made.

IN DEVELOPMENT: SMALL that has been stripped of its disease-causing ability,
MOLECULE ENHANCERS and modified to carry a correct version of the mutated
gene. The viral vector can also be engineered to drop its
PTC Therapeutics (South Plainfield, NJ) — in partnership genetic cargo into specific cells. In the case of SMA Type
with Roche (Basel, Switzerland) — has begun Phase 1, the AAV9 vector crosses the blood-brain barrier and
II clinical studies on its proprietary small molecule delivers corrected copies of the SMN1 gene into motor
drug, RG67800. RG67800 is similar to Spinraza in that it neuron cells in the brain.
changes the way nerve cells process the SMN2 mRNA,
resulting in increased production of functional SMN AveXis (Bannockburn, IL) has a gene therapy candidate,
protein. However, a notable difference is the delivery AVXS-101, in Phase I/II clinical studies. The company
mechanism—Spinraza is an injectable while RG67800 reported promising results in mid-2016, noting that
is a pill. Novartis (Basel, Switzerland) is preparing for the drug appears to be safe and effective. Babies
Phase I clinical testing of a small molecule modulator of who received this gene therapy showed marked
SMN2 mRNA. increases in SMN production and in movement. Also
in the gene therapy mix is Voyager Therapeutics
(Cambridge, MA) with their treatment currently in
IN DEVELOPMENT: A GENE THERAPY CURE?
preclinical development.
As a single gene disorder, SMA is an ideal candidate for a
With additional approvals hopefully to follow in the wake
gene therapy approach. Scientists deliver the corrected
of Spinraza, SMA patients and their family members are
version of the mutated gene by a viral vector — a virus
hoping to kiss this devastating illness goodbye.

GMO Apples Hit Marketplace
COMING SOON TO A Recall that RNA serves as the intermediary between DNA
SUPERMARKET NEAR YOU and proteins. The information in DNA is converted to
RNA, which is then translated into a protein by organelles
Coming soon to a supermarket near you: apples that called ribosomes. One way to stop PPO production is to
have been genetically modified to resist browning. get rid of the PPO RNA before it enters the ribosome.
Dubbed Arctic Apples, the plants were approved two Enter RNAi.
years ago by the USDA and will begin appearing on select
retailers’ shelves in Midwestern U.S. states this month. RNAi is a short, complementary RNA sequence that
attaches to a target RNA, in this case the target is PPO
Developed by Okanagan Specialty Fruits (Summerland, RNA. This creates a double-stranded RNA, which is
B.C., Canada), the Arctic Apple also represents a new universally recognized as viral RNA and is destroyed
type of genetically altered food — one that has been by another enzyme known as RNA-induced silencing
engineered to directly appeal to and benefit the complex (RISC). In the case of the Arctic Apple, RNAi
consumer rather than the farmer. Their debut may also attaches to the PPO RNA and is destroyed by RISC. No
help ease lingering safety concerns voiced by GMO critics PPO RNA ever makes it to the ribosome, so no PPO is
due to the innovative technology used to create Arctic ever produced by the apple. No enzyme, no chemical
Apples. Let’s take a look at what causes apple browning, reaction, no browning.
how science is able to produce a remedy, and preview
what’s on the horizon for consumer-oriented genetically
WHY LESS BROWN?
modified food.
Why go to all this trouble to make apples less brown?
OXIDATION CAUSATION This modification is expected to benefit growers,
packers, shippers, and retailers by limiting the amount
The browning observed in conventional apples is the end of bruising and other visual imperfections in their
result of an oxidation reaction — a chemical reaction apples — meaning less waste and more product to sell.
between oxygen and another substance. Typically, Food processors, likewise, will be able to produce more
oxidation reactions result in a color change (think rusting consistent juices, sauces, and sliced apple products,
car — the oxidation of metal). In apples, browning is the without relying on antioxidant treatments currently
oxidation of “phenolic compound” by an enzyme called in use.
polyphenol oxidase (PPO).
And since Arctic Apples contain no foreign DNA, they
In order for oxidation to occur, PPO has to come into are likely to be more palatable to GMO-wary consumers.
contact with the phenolic compounds. Within the apple The apples have undergone 10 years of field testing,
cells, phenolic compounds are typically sequestered and do not differ in any significant respect from non-
inside of membrane-bound compartments. If the cell modified apples, apart from the lack of PPO enzyme.
is disrupted, by slicing or dropping the apple, the seal Based on field studies, blocking the production of PPO
of these compartments is broken and the phenolic does not make apple trees more susceptible to pests,
compounds exposed. PPO is then able to act, oxidizing and so their adoption should not result in any increased
the compounds to create browning. pesticide use.
SILENCING THE GENE ON THE MARKET

Scientists at Okanagan Specialty Fruits chose to tackle The first two varieties of modified Arctic Apples
apple browning by blocking the production of the PPO appearing on the market are Golden Delicious and
enzyme. No enzyme, no chemical reaction, no browning. Granny Smith. If successful, they could pave the way
They used a gene-silencing technique known as RNA for other consumer-oriented products such as non-
interference (RNAi) to block the production of PPO. browning cherries and pears, also in development at
Okanagan Specialty Fruits.

A similar product — the Innate Potato — was approved in When potatoes are cooked at high temperatures,
November 2014 by the USDA. Developed by J.R. Simplot asparagine synthetase-1 reacts with the potato sugars
(Boise, Idaho), Innate also uses RNAi technology to to produce a chemical called acrylamide — which has
decrease production of the PPO protein, again with the been linked to cancer in rodents. The Innate potatoes are
goal of reduced browning. Innate potatoes also use RNAi already on the market in the U.S. at select retailers.
to knock out a second protein, asparagine synthetase-1.

Stem Cell Snapshot
INDUCING STEM CELLS TO HEAL Let’s take a look at companies trying to boost the
regenerative capacity of these tissues less adept at self-
Headlines touting stem cells often claim the therapies
repair.
heal everything from hair loss to hearing loss. While
many of these treatments are not FDA approved, there
are some promising innovations winding through PUSHING THE PROGENITOR
preclinical and clinical development. Here at WEEKLY Progenitor Therapeutics’ (Stevenage, UK) name hints to
headquarters, we like to tease out the science behind the company’s main technology. A progenitor cell is one
the scene, so let’s review regenerative medicine basics that has taken the first steps to becoming a specialized
and survey the companies attempting to repair damaged cell, such as a cardiac cell. Once these steps are taken,
tissue with these high potential cells. the stem cell is “committed” to becoming that specialized
cell type.
STEM CELL PRIMER Progenitor Therapeutics is focused on discovering small
Stem cells are unspecialized cells that have the ability molecule drugs that will nudge adult stem cells down
to develop (differentiate) into 1 of 200 cell types in the the differentiation pathway. They do this by testing
body. There are two general classifications: hundreds of thousands of drugs on stem cells to see
which bring out progenitor cell types. Although still in
• Embryonic stem cells, found only in developing
preclinical development, their work offers a tantalizing
embryos, can become any cell type within the
future of popping a pill to regenerate damaged tissue.
adult body.
• Adult stem cells, found within organs of a fully
IGNITING NEURAL STEM CELLS
developed body, can become only certain cell types.
Typically these cell types come from the organ in Neuronascent (Clarksville, MD) and Neuralstem
which they are found. (Germantown, MD) are leading the charge in developing
small-molecule activators of neurogenesis, or the
Embryonic stem cells have the most therapeutic
generation of neurons (brain cells) from stem cells.
potential. A main focus of research is sussing out which
By screening large chemical libraries, scientists at
combo of hormones cause a stem cell to commit to
these companies have identified compounds that
becoming a specific tissue; for example, spinal cord
activate neurogenesis from adult neural stem cells
tissue vs. brain tissue. The billion dollar market: grow
both in the lab and in mouse models of various
and transplant stem cells into patients who have lost
neurodegenerative disorders.
tissue due to acute trauma.
Neuralstem’s lead neurogenesis candidate, NSI-189,
Since embryonic stem cells may come from unrelated
increased the size of the hippocampal region of mice
donors, there is the likelihood of tissue rejection. Thus
brains by 20%. Neuralstem is beginning Phase II of NSI-
there is considerable interest in developing therapies
189 for major depressive disorder. The drug is already in
using a patient’s own adult stem cells.
preclinical development for Alzheimer’s disease.
In certain tissues such as bone marrow, muscle, liver,
Neuronascent’s first focus is the development of small
and skin, damaged or worn out areas are naturally
molecule compounds that promote neurogenesis for
and regularly replaced by activated adult stem cells. In
the treatment of Alzheimer’s disease. In an Alzheimer’s
other tissues such as heart and brain/spinal cord, adult
mouse model, the lead compound NNI-362 promoted
stem cells exist in such small numbers, our body cannot
the growth of new hippocampal neurons that not only
readily activate them to replace tissue. Enter stem
migrated to the correct functional location, but also
cell therapy.
differentiated and survived long enough to reverse
previously observed cognitive declines. Neuronascent is
preparing for Phase I of NNI-362.

ON THE FAST TRACK WITH ANGIONETICS
Last month, Angionetics (San Diego, CA) won fast track
designation for its gene therapy candidate, Generx,
which is currently in Phase III clinical studies.
Generx is a viral vector — a virus that has been modified
to deliver a specific gene to a target cell type — that
delivers a gene for fibroblast growth factor 4 (FGF4) to
cardiac tissue damaged due to lack of oxygen during
a heart attack. The damaged tissue then produces
the FGF4 protein, which promotes the formation of
new blood vessels in damaged regions, increasing the
heart’s ability to deliver oxygen-rich blood. The FGF4
gene delivered by Generx does not incorporate into the HEAR, HEAR
cardiac cell’s genome. This means the growth factor is
Good hearing depends on thousands of tiny hair cells
only produced for a few weeks — just long enough to
within the inner ear that mechanically amplify low-
jump start the growth of new blood vessels.
level sound. As these hair cells are damaged or lost
through exposure to excessive noise, toxic drugs, and
normal aging, hearing loss occurs and we are not able to
generate new hair cells.
That may change. The leaders behind Cambridge-based
startup Frequency Therapeutics reported the discovery
of a small-molecule activator of hair cell precursors —
LGR5+ cells — which are a type of adult stem cell. Once
activated, LGR5+ cells differentiate into hair cells. The
work was done on animal models in the lab using cochlea
– the portion of the inner ear which contains the hair
cells. The findings suggest Frequency may be on the path
to developing a drug to restore hearing loss.
NAKED DNA
WANTING THE WNT PATHWAY
Juventas (Cleveland, OH) is also developing a gene
Just last month, a new regenerative medicine company,
therapy for heart disease. However, instead of using
Surrozen, was launched in South San Francisco.
modified virus, Juventas relies on plasmids — small,
The company founders include scientists who have
circular pieces of DNA engineered to contain the
spent decades deciphering a cell signaling pathway
therapeutic gene. In this case, the plasmids are injected
known as the Wnt pathway, which plays a central role
directly into damaged cardiac tissue. Advantages to this
in stem cell maintenance and tissue regeneration.
“naked DNA” approach include reduced patient immune
Surrozen’s focus is on developing drugs that activate
response and lower manufacturing cost.
this pathway, potentially leading to treatments for
Dubbed JVS-100, the Juventas plasmid contains a degenerative diseases.
gene for stromal-cell derived factor-1 (SDF-1), which
If any of these companies are successful a shot of stem
recruits cardiac stem cells and promotes new blood
cells — not major surgery — may repair previously
vessel growth. Like Generx, JVS-100 does not integrate
unfixable tissue damage. We’ll keep a close eye on these
into the recipient’s own DNA, making the production
exciting developments and be sure to report them back
of SDF-1 temporary. JVS-100 has completed Phase I
to you.
clinical studies.

Therapeutic Antibody Primer
BASICS & INNOVATIONS cancer cell. When injected into a patient’s body, this
binding triggers the patient’s immune system to attack
Monoclonal antibody (mAb) therapeutics burst onto the
the cancer.
healthcare scene 20 years ago, and today they remain
one of the most versatile and effective therapeutics
available. The tried and true mAbs are still in high EASILY CONFUSED: MONOCLONAL
demand, and we suspect this first wave of derivative VS. POLYCLONAL ANTIBODIES
products clamoring their way through the pipeline will Commercially available antibodies are divided into two
be equally as successful. In this WEEKLY, we’ll review the categories: Monoclonal and polyclonal. Monoclonal
basics of monoclonal antibodies and highlight some of antibodies are antibodies that are all produced by the
the recent innovations within this therapeutic modality. same B-cell, or by the clones (descendants) of that B-cell.
This means they all recognize the same epitope on a
MAB PRIMER target immunogen. Therapeutic antibodies are always
Antibodies are proteins produced by B-cells, a type of monoclonal — this is required to ensure a consistent
white blood cell, in the immune system. Each antibody therapeutic effect.
has a distinct shape that recognizes a unique target, Polyclonal antibodies are produced by a collection of
often that unique target is a protein on the surface different B-cells, and recognize multiple epitopes on
of a pathogen. These foreign proteins are known as the same immunogen. Polyclonal antibodies are used in
immunogens. When an antibody recognizes its specific diagnostic and research applications, where detection of
immunogen, it physically binds to it. This binding action a specific immunogen is the only requirement, and the
alerts the immune system to attack and eliminate specific epitope recognized doesn’t matter.
the pathogen.
The specific protein that triggers antibody recognition A BISPECIFIC CONNECTION
is called an immunogen, and the exact part of the Antibodies are Y-shaped proteins. In antibodies
immunogen that is recognized by the antibody is called produced by our own immune system, as well as most
an epitope. therapeutic antibodies, the two “arms” of the Y are
identical, meaning that each antibody recognizes only
one target. Bispecific antibodies, in contrast, have been
genetically engineered by splitting and fusing the genes
for two different monoclonal antibodies together to
make a new Y. This way, the bispecific antibody is able
to recognize two different targets and bring them in
contact with one another.
For example, one arm might recognize a cancer cell,
while the other arm might recognize and bind to a killer
T-cell — a type of white blood cell that has the ability
to inject toxins directly into the cancer cell. By bringing
a cancer cell into direct contact with a killer T-cell,
This ability of antibodies to recognize and bind the T-cell is activated to kill that cancer target. This is
to a specific target is what makes them effective the mechanism of action used by Blincyto (Amgen;
therapeutics. And researchers have expanded on this Thousand Oaks, CA), the first and so far only bispecific
concept — for example, scientists may identify an antibody approved by the FDA.
antibody that binds to a protein on the surface of a

Currently, there are two ADCs on the market: Seattle
THE ABCS OF ADCS Genetics’ (Seattle, WA) Adcetris for the treatment of
Antibody-drug conjugates (ADCs) are highly potent, Hodgkin’s lymphoma, and Roche’s (Basel, Switzerland)
targeted therapeutics that work by combining the Kadcyla for the treatment of HER2-positive breast
targeting capabilities of monoclonal antibodies with the cancer. There are dozens more in clinical development
cancer-killing ability of toxic drugs, enabling the killing of by companies including AstraZeneca (Cambridge, UK),
cancer cells with less impact on healthy cells. ADCs have Roche, Immunogen (Waltham, MA), Novartis (Basel,
three key components: Switzerland), and Fortis Therapeutics (San Diego, CA).
• A monoclonal antibody that is highly specific for a

tumor-associated immunogen that has little to no PHOTOIMMUNOTHERAPY
expression on healthy cells. DELIVERS A PUNCH
• A small molecule drug that is highly toxic and Photoimmunotherapy delivers the latest twist on
designed to kill the cancer cell once internalized. antibodies. Developed by the National Institutes of
Health, this technology uses light to deliver a punch.
• A chemical linker that connects the small molecule
drug to the antibody. The linker is stable in blood • A light sensitive, non-toxic drug is attached to the
circulation but releases the toxin once inside end of an antibody. This drug becomes toxic when
the tumor. exposed to infrared light.
How does it work? • Infrared light is shone, activating the drug and
damaging the cancer cell membrane. This damaged
• The antibody binds to its target immunogen on the
membrane allows water to enter, causing the cancer
surface of the cancer cell.
cell to burst and die.
• The antibody-drug conjugate is then taken up or
• Upon bursting, the cancer cell releases tumor
internalized by the cancer cell.
immunogens that serve to activate the immune system
• Once inside the cancer cell, the linker is degraded to further recognize and target more tumor cells.
and the active drug released.
One caveat to using photoimmunotherapy is the
The ability to target only cancer cells allows drug tumor must be accessible to an infrared laser in
designers to use drugs that are much more toxic order to activate the photosensitive drug. Aspyrian
than traditional chemotherapy. The ADC has a higher Therapeutics (San Diego, CA) is in Phase II clinical
specificity and only attacks cancer cells, avoiding studies of photoimmunotherapy antibodies that target
nearby healthy tissue which is often destroyed head and neck cancer.
by chemotherapy.
Over the years, monoclonal antibodies have proven
to be safe and effective therapeutics in a number of
different indications, most notably various cancers
and autoimmune disorders. Innovations in antibody
technology ensure monoclonal antibodies will
remain one of the most powerful tools in the drug
development arsenal.

The Mechanics Of Melanoma
SPECTRUM OF THERAPIES GENETIC FACTORS: P53 & BRAF
Melanoma accounts for less than 1% of skin cancer Although most cases of skin cancer are traceable to
cases yet causes the vast majority of skin cancer deaths. excessive UV radiation from sun exposure, about
If detected early enough, melanoma is almost always 10% are likely due to genetic factors. The gene most
curable. If not, its ability to metastasize makes it difficult commonly mutated in familial melanoma is p53. p53 is
to treat. a “tumor suppressor” — it detects DNA damage in cells
Melanoma is more common in young adults than many and triggers either DNA repair pathways or activates
other types of cancer, with 25% of new cases occurring cell death if the damage cannot be repaired. Another
in people under age 45. Its prevalence is growing — gene, known as the BRAF gene, regulates cell growth
the number of new cases per year relative to the total and is mutated in inherited forms of melanoma. About
population has doubled since 1973. In 2017, there will half of all genetically-based melanomas have the
be an estimated 87,110 new cases of melanoma in the BRAF mutation.
U.S. and 9,730 melanoma-related deaths, according to Let’s take a closer look at BRAF. BRAF codes for a protein
the Aim at Melanoma Foundation. Let’s review the required for the transmission of a growth signal from a
basics and find out the latest treatments in the battle cell surface receptor to the cell nucleus (growth signal
against melanoma. transduction). Growth signaling is initiated by a growth
factor binding to its receptor. This binding transmits
MELANOMA’S METHOD a signal through the membrane, causing the internal
portion of the receptor to interact with and activate
Melanoma is the uncontrolled growth of melanocytes,
a protein inside of the cell. This activation is then
the pigment-producing cells located in the bottom layer
transferred to the next protein in the pathway, and so on
of the skin’s outermost layer (the epidermis). In skin
until the signal reaches the last protein in the pathway.
cancer, a tumor initially grows and spreads within the
When this protein is activated, it enters the nucleus,
epidermis due to DNA damage, which is usually caused
where it turns on specific genes that make proteins
by ultraviolet (UV) radiation.
which initiate cell division. BRAF is one of the proteins
If melanoma is detected during the epidermal level in this pathway. In BRAF-associated melanoma, the
growth stage, it can often be surgically removed. mutated BRAF is always turned on even when no growth
Penetrating the deeper layers of the skin as it grows, factor is present.
it will eventually come into contact with lymph and
blood vessels — which act as a cancer highway. When
melanoma spreads to other parts of the body it is known
as metastatic melanoma.
Lighter-skinned people are at higher risk for melanoma
because the increased skin pigmentation found in darker
skin tones helps to block UV rays from penetrating and
damaging skin cell DNA. However, darker-skinned people
can and do get skin cancer. Another general risk factor
for melanoma is atypical moles, or moles with irregular
shape, color, or size. Moles are clusters of melanocytes
and so sudden changes in them may be an early warning
sign of melanoma.
The small molecule drugs Zelboraf (Genentech; South
San Francisco, CA) and Tafinlar (Novartis; Basel,
Switzerland) inhibit overactive BRAF and are approved
for the treatment of late-stage melanoma.

IMMUNOTHERAPIES IN THE FIGHT INCYTE’S INSIGHT
A few different checkpoint inhibitor therapies are on Incyte (Alapocas, Delaware) has a new type of small
the market for metastatic melanoma. These therapies molecule immunotherapy in Phase III called Epacadostat
enable killer T-cells – immune system cells that recognize – it inhibits the enzyme IDO1. IDO1 helps regulatory
and kill threats such as cancer cells – to become fully T-cells to develop and become activated. Regulatory
activated and able to kill tumor cells. Keytruda (Merck, T-cells suppress the immune response, and therefore
Kenilworth, NJ) and Opdivo (Bristol-Myers Squibb, New help cancer cells to escape immune surveillance.
York, NY) both target PD-1, an inhibitory protein on the Inhibiting IDO1 should suppress the development of
surface of T-cells; Yervoy (Bristol-Myers Squibb) targets regulatory T-cells, bolstering the immune response
a second inhibitory protein, CTLA-4. Both PD-1 and against melanoma.
CTLA4 essentially act as “off switches” for killer T-cells.
By inhibiting these off switches, the killer T-cells become THE IMPLICATIONS OF MICRORNA
fully activated, and able to kill melanoma cells.
Researchers at Tel Aviv University examined how
A second type of immunotherapy approved for melanoma metastasizes in an article last summer:
melanoma is Amgen’s (Thousand Oaks, CA) oncolytic the melanoma cells release tiny vesicles containing
virus therapy, talimogene laherparepvec (T-VEC). An microRNA, a type of regulatory RNA produced by all cells.
oncolytic virus is a virus that infects and kills cancer cells.
The microRNA-filled vesicles induce changes in the
The cancer cells are killed through cell lysis: as the virus
layer of skin just below the epidermis, where melanoma
multiplies inside of the cells, it causes them to burst
originates. That lower layer of skin, known as the dermis,
open. This in turn releases new infectious particles that
contains blood vessels which cancer cells are able to
can target remaining tumor cells. In addition to killing
access. The Tel Aviv team is identifying drug candidates
cancer via lysis, the presence of an actively replicating
that may interfere with this process, preventing the
virus triggers the immune response to target the tumor.
metastasis that makes melanoma so deadly.
T-VEC was designed to infect and kill melanoma cells, and
is so far the only FDA-approved oncolytic virus. Increased understanding of the molecular pathways
that contribute to melanoma’s development and
spread will provide additional tools to fight those cases
of metastatic melanoma that inevitably will continue
to arise.

The Intrigue Of HIF
THE VERSATILITY OF HIFS Erythropoietin production can be enhanced by
increasing the amount of HIF present.
Quite a few headlines touting a term called “HIF
compound” or “hypoxia-inducible factor compound” Under normal oxygen conditions, small amounts of
have intrigued us here at WEEKLY headquarters. HIF are produced, but are quickly degraded though the
GlaxoSmithKline (London, England), Akebia action of an enzyme called HIF prolyl-hydroxylase (HIF
Therapeutics (Cambridge, MA) and more have HIF- PHD). HIF PHD is inhibited in low-oxygen conditions,
inducing drugs in the pipeline which may prove to be enabling HIF levels to increase. Several companies are
attractive alternatives to Amgen’s (Thousand Oaks, CA) developing small molecule, orally available drugs to
injectable Epogen currently on the market. In earlier inhibit HIF PHD, facilitating the activation of HIF under
stages of research, HIFs are being studied for their normal oxygen levels. HIF PHD inhibitors in clinical
connection to tumor growth. How can one compound development include:
be versatile enough to affect both anemia and cancer? • Akebia’s vadadustat, Phase III clinical development
Without further ado, let’s take a look at the story behind for CKD anemia
hypoxia-inducible factors.
• FibroGen’s (San Francisco, CA) roxadustat, Phase III
clinical development for CKD anemia
TERM OF THE WEEK: HYPOXIA-
• GlaxoSmithKline’s daprodustat, Phase III clinical
INDUCIBLE FACTOR
development for CKD anemia
Hypoxia occurs when the amount of oxygen reaching a
person’s cells and tissues is inadequate. Hypoxia may DISRUPTING ANGIOGENESIS IN CANCER
be triggered by lower oxygen concentrations at higher
altitudes or by disease processes seen in pulmonary HIF is also thought to contribute to the process of
disorders, anemia, or circulatory deficiencies. angiogenesis — the growth of blood vessels into tumors.
Most solid tumors have a hypoxic environment, due to
The low-oxygen environment in hypoxia causes cells to their high cell density and lack of supporting vascular
make a protein called hypoxia-inducible factor (HIF). HIF networks. This hypoxic environment causes cancer cells
is a transcription factor — a protein that binds to cellular to produce HIF, which in turn activates the secretion
DNA in a defined location and “turns on” specific genes of vascular endothelial cell growth factor (VEGF). VEGF
(which then make their intended proteins). triggers angiogenesis, which provides a way for tumors
HIF activates genes involved in the production of oxygen- to get oxygen and nutrients, enabling the tumor to
carrying red blood cells and the formation of blood continue growing. Angiogenesis also provides a possible
vessels (angiogenesis). Both of these processes assist in route for individual tumor cells to exit the tumor and
increasing oxygen delivery to hypoxic (oxygen-deprived) spread to other parts of the body.
tissues. Because of this relationship between HIF, angiogenesis,
and cancer, there is considerable interest in developing
ACTIVATING HIF FOR ANEMIA HIF inhibitors in an attempt to block angiogenesis.
Anemia is the decrease in the total amount of red blood The drugs Torisel (Pfizer; New York, NY) and
cells in the body, resulting in a lowered ability of the Zortress (Novartis; Basel, Switzerland) stop mTOR, a
blood to transport oxygen. In healthy people, when protein that activates HIF. It is thought that the anti-
the number of red blood cells in circulation drops, angiogenesis effects of these mTOR inhibitors are a
the kidney releases a hormone called erythropoietin, result of suppressing HIF. Torisel is FDA-approved for the
which stimulates the bone marrow to produce more red treatment of renal cell cancer; Zortress is FDA-approved
blood cells. In chronic kidney disease (CKD), the kidneys for advanced kidney cancer, metastatic pancreatic
don’t produce adequate amounts of erythropoietin in neuroendocrine tumors, hormone-positive, and HER2-
response to reduced circulating red blood cells, leading negative breast cancer.
to anemia.

COCKTAIL FODDER: THE HIF ADVANTAGE lower elevations, creating a slightly hypoxic environment
inside the athlete’s cells. This hypoxia increases the
You may have heard of “altitude training” — the
levels of HIF, leading to more erythropoietin and
practice of training at high altitudes in order to increase
subsequent red blood cell production. The enhanced
performance, especially in endurance events such as
oxygen-carrying capacity lasts for about ten to twenty
long distance running or cycling. Altitude training works
days after returning to lower elevations, so an athlete
because at elevations higher than about 5,000 feet there
who trains at a higher altitude and then competes at sea
are fewer oxygen molecules per volume of air due to
level will have an advantage over those who complete all
reduced atmospheric pressure. Every breath taken at
of their training at sea level.
higher elevations delivers less oxygen than it would at

The PARP Race Is On
PARP1 INHIBITOR LINEUP prone to sustaining DNA damage at a much higher rate
than normal. This higher rate of DNA damage increases
PARP1 inhibitors are making a strong statement!
the chances of cancer developing in those cells. BRCA1/
Tesaro’s (Waltham, MA) just-approved Zejula
BRCA2 positive cancer is cancer that is associated with
has garnered predictions of blockbuster status.
mutations in the BRCA1/BRCA2 genes. The mutations are
AstraZeneca’s (Cambridge, UK) Lynparza was the first
most strongly associated with breast and ovarian cancer,
PARP1 inhibitor to make it to market back in 2014, and
but are also associated with increased risk of developing
their recent clinical trial results showed significant
stomach, pancreatic, prostate, melanoma, leukemia,
survival benefit in ovarian cancer. Clovis Oncology
lymphoma, and colon cancer.
(Boulder, CO) achieved the second FDA approval of a
PARP1 inhibitor with Rubraca in December 2016. AbbVie
(North Chicago, IL) and Medivation (San Francisco, CA) THE POINT OF PARP
both have PARP1 inhibitors in late-stage development. Poly ADP ribose polymerase 1 (PARP1) is a DNA repair
The race is in full swing, so let’s pick up the science of protein. By stopping the PARP1 repair pathway in cells
PARP1 inhibition. already deficient in BRCA1/BRCA2-mediated repair,
cancer cells become extremely vulnerable to DNA
damage. Because of this, DNA damage accumulates
DNA DAMAGE RUNS DEEP
and triggers apoptosis. A PARP1 inhibitor is usually
Simply put, PARP1 inhibitors work by exploiting the administered in combination with chemotherapy or
cellular pathways found in DNA damage repair. So, how radiation therapy, which increases the incidence of
exactly does DNA get damaged? apoptosis-triggering DNA damage. Healthy cells, which
DNA incurs approximately 10,000 to 1,000,000 still have BRCA repair pathways intact, are less sensitive
“molecular lesions” per day from breaks or “nicks” to the to additional DNA damage.
double helix, or chemical modification to the A, C, G, or
T bases. This may sound high — but remember, our DNA
contains 6 billion bases (3 billion base pairs), so this is
equivalent to .001% to .1% of the total DNA in each cell.
This damage occurs as a result of normal DNA replication
errors and environmental exposures, such as ultraviolet
radiation, X-rays, and chemicals.
The good news is our cells have mechanisms to fight
against this damage before it causes harm. DNA repair
proteins find and fix different types of DNA damage. If
DNA damage exceeds a threshold amount (beyond which
repair is possible) a protein called p53 triggers cell death
— also known as apoptosis. DNA repair proteins prevent What cancers are PARP1 inhibitors aiming to fight?
errant cells from turning into cancerous cells, a likely AstraZeneca’s Lynparza and Clovis Oncology’s Rubraca
outcome if the damage accumulates in genes important are both approved for ovarian cancer. Zejula (Tesaro)
for regulating cell growth and division. targets ovarian, fallopian tube, and primary peritoneal
cancer. The Phase III clinical pipeline includes:
BEHIND BRCA • Lynparza: Prostate, gastric, breast, and pancreatic
Arguably the most famous DNA repair proteins, BRCA1 • Rubraca: Prostate
and BRCA2, are found in breast and ovarian cells. If • Talazoparib (Medivation): Breast
these repair proteins themselves are non-functional, • Veliparib (AbbVie): Breast, lung, and ovarian
the cells in which they would normally do their job are • Zejula: Breast

BEYOND CANCER A DNA mutation is a change to the actual base sequence
(A, C, G, or T). Mutations can arise if DNA damage
Preclinical research suggests that PARP1 inhibitors
is not corrected. Recall that in undamaged DNA, an
may also be relevant to other disease areas, such as
“A” base always pairs with a “T” base, and a “C” base
autoimmune and inflammatory disorders. PARP1 has
always pairs with a “G” base. These base-pairing rules
been shown to play a role in activating proteins that
are what enable DNA to replicate faithfully from one
drive inflammation. Preclinical models demonstrate
generation of cells to the next. However, uncorrected
that in cases without the PARP1 gene, subjects were
DNA damage may cause that “A” base to mistakenly pair
less vulnerable to rheumatoid arthritis than with the
with a “G” during replication; or a “C” to pair with a “T.”
gene. Inhibiting PARP1 resulted in reduced signs of
This results in a sequence change – a mutation - in the
inflammation in models of multiple sclerosis, irritable
replicated DNA. The gene now provides incorrect genetic
bowel disease, and allergic airway inflammation.
information to the cell.
EASILY CONFUSED: DNA DAMAGE

VS. DNA MUTATION
BRCA1, BRCA2, PARP1, and other DNA-repair proteins
correct DNA damage, but they don’t fix mutations.
What’s the difference?
DNA damage refers to alterations in the chemical
structure of DNA. This may mean a break in the
DNA strand, a substitution to one of the bases that
make up DNA (A, C, G, or T), or even a missing base.
These changes are detected and corrected by DNA
repair enzymes.

The Science Of CRISPR/CAS9
CRISPR/CAS9 UPDATE sequences referred to as “spacers” between the repeats.
They dubbed these regions a tongue twister of a name,
CRISPR/Cas9 can’t seem to stay out of the news — from
“clustered regularly interspaced short palindromic
first in human to patent disputes, we here at the WEEKLY
repeats,” or CRISPR. Scientists also noticed CRISPR
want to update you on this hot technology.
sequences were always located near a gene that coded
A group of scientists from the State Key Laboratory of for an enzyme that cut DNA. This enzyme became known
Proteomics (Beijing, China) and the National Center as Cas, short for “CRISPR-associated”.
for Protein Sciences (Beijing, China) recently reported
In the mid-2000’s, scientists realized the “spacer
the first ever edit using CRISPR/Cas9 in healthy human
sequences” matched DNA sequences of invading viruses
embryos — just two years after researchers from the
— the bacteria were storing away bits of invading viral
University of Guangzhou (Guangzhou, China) used the
DNA between its own bacterial CRISPR sequences! These
genome-editing technique on abnormal embryos. This
bits of viral DNA create a “genetic memory” of the virus,
news comes just weeks after a report released by an
enabling the bacteria to fight back if reinfected.
international committee convened by the U.S. National
Academy of Sciences weighed in on the topic, which Reinfection triggers the following steps:
stated the technique may be permissible in embryos if • Viral DNA present in the spacer sequences is copied
the goal is to cure or prevent serious disease. into viral RNA.
CRISPR technology and its applications were discovered • The DNA-cutting enzyme Cas is made, and
by two different research teams, one at University attaches itself to the viral RNA produced from the
of California, Berkeley, and another at the Broad spacer sequence.
Institute (Cambridge, MA). Both have filed patents on
• This newly minted viral RNA/Cas complex finds its
various aspects of the CRISPR/Cas9 system. The Broad
“match” on the invading viral DNA.
Institute had granted an exclusive license to Editas
Medicine (Cambridge, MA), while Berkeley had granted • The Cas enzyme is now positioned to cut up viral
licenses to Caribou Biosciences (Berkeley, CA), CRISPR DNA, destroying the invading virus.
Therapeutics (Basel, Switzerland and Cambridge,
MA), Intellia Therapeutics (Cambridge, MA), and ERS USE IN HUMANS
Genomics (Dublin, Ireland). In February, the U.S. Patent
In 2013, researchers adapted this bacterial defense for
Office ruled in favor of the Broad Institute and its
use in human cells. Human cells were engineered to
licensee, while in March the European Patent Office
contain both specially-designed RNA and Cas genes.
ruled in favor of U.C. Berkeley patents. The legal battle is
When these human cells produce the RNA/Cas complex,
certainly far from over.
the dynamic duo is ferried to its complementary DNA
CRISPR/Cas9 entered its first human clinical trial at target. Once in position, Cas goes to work cutting the
Sichuan University (Chengdu, China) last fall, and is DNA. The particular Cas protein chosen for this work was
widely expected to do so in the U.S. by the end of 2017. one discovered in Streptococcus bacteria, Cas9 — hence
With all of these new developments making waves in the the moniker CRISPR/Cas9.
industry let’s review the basics.
The ability to cut human DNA in precise locations is an
exciting innovation because of what the cell does next.
CAS TO THE RESCUE
CRISPR was originally discovered as a key component of BREAKING & FIXING
the bacterial immune response. Bacteria, like people,
Cas9 creates double-stranded breaks (DSB) in the
are plagued by viral infections, and have evolved
specified DNA sequence. Double-stranded breaks cut
clever ways to attack invading viruses. In the 1980s,
both strands of the double-stranded DNA helix. Think
scientists observed an interesting pattern in bacterial
of DNA as a two-lane bridge that, after experiencing
genomes: repeating, palindromic sequences, with unique

an earthquake, has a section break off and fall into the CRISPR IN THE CLINIC
water below.
A clinical trial initiated last fall by Chinese researchers
DSBs activate two repair pathways to fix the break in at Sichuan University uses CRISPR/Cas9 to disable the
the DNA: PD-1 gene in T-cells.
• Non-Homologous End-Joining (NHEJ) closes the gap The PD-1 gene produces the PD-1 protein, which is
between the break by joining the two sections located on the T-cell’s surface. When the PD-1 protein
back together—visualize pushing the two sides of is activated, the T-cell doesn’t function. When the PD-1
the bridge together, leaving the fallen section in protein is deactivated, the T-cell functions. Aggressive
the water. An unintended byproduct of NHEJ is the cancers take advantage of this on/off switch turning PD-1
possibility of sequence error, much like the sections on, effectively shutting down the T-cell. By turning PD-1
of the bridge not lining up properly. If the repair off, the T-cells can’t be suppressed—freeing them up to
occurs in the middle of a gene, the minor error can attack cancer cells.
be enough to disrupt gene function and halt the
production of the corresponding protein. CRISPR COMING SOON
• Homology Directed Repair (HDR) relies on a highly A U.S. clinical trial of CRISPR to disrupt PD-1 in T-cells is
similar (homologous) DNA segment to repair the expected to begin before the end of 2017. This two-year
break—visualize the missing bridge section built study is funded by the Parker Institute (San Francisco,
elsewhere and helicoptered in to fill the break. CA).
By engineering double-stranded breaks to occur at A number of private companies also have plans for
specific locations, scientists activate the NHEJ and HDR CRISPR/Cas9 clinical trials that include both gene
cell repair pathways. By activating the NHEJ pathway, disruption and gene correction. The table below
scientists can disrupt a disease-associated gene, summarizes some key players in the genome-editing
preventing the production of a protein that causes arena and their approaches to applying CRISPR. In vivo
the disease. By activating the HDR pathway, a short means the therapy will take place inside the human
sequence of DNA is delivered with CRISPR/Cas9 to and Ex vivo means the treatment will be performed in
correct the mutated sequence. cells taken from the body and then injected back into
the patient.
As these and other potential treatments move through

clinical trials, the world will be watching to see if this
revolutionary technology will live up to the hype and
change the way we prevent and treat disease.

Pills, Proteins & Peptides
PROMISING PEPTIDE THERAPIES which include sensitivity to digestive enzymes, delivery
by injection, and high specificity for their target.
The front runners in the game of drug delivery include
small molecule and large molecule drugs, but there is
another class that lands right in between: peptides.
Several companies, including Rhythm Pharmaceuticals
(Boston, MA), Kalos Therapeutics (San Diego, CA),
Aileron Therapeutics (Cambridge, MA), and Bicycle
Therapeutics (Cambridge, MA) have emerged as
prominent players in the peptide arena.
Let’s review the differences between the drug classes
and explain where peptides fit into the picture. Then
we’ll take a spectator’s interest in the companies and
products leading the charge in peptides therapeutics.
Examples of peptide drugs on the market today include
EASILY CONFUSED: SMALL MOLECULE glucagon-like peptide-1 (GLP-1) receptor activators, such
VS. LARGE MOLECULE VS. PEPTIDE as Byetta (AstraZeneca; Cambridge, England), Victoza
Small molecule drugs are chemically synthesized — made (Novo Nordisk; Bagsvaerd, Denmark), and Trulicity
by a series of chemical reactions in the lab. They are (Eli Lilly; Indianapolis, Indiana). These peptide drugs
typically taken as a pill or capsule. The pill or capsule work by interacting with a receptor on the surface of
dissolves in the gastrointestinal tract and the active pancreatic beta cells and stimulate the release of insulin
ingredient is easily absorbed into the bloodstream for diabetes.
via the intestinal wall. The molecules that make up
these drugs are so tiny they are able to penetrate cell IN THE RHYTHM
membranes and get inside of cells. Rhythm Pharmaceuticals is prepping to enter Phase
In contrast, large molecule drugs — protein-based III clinical studies of their anti-obesity peptide drug
therapeutics known as biologics — are made by living setmalnotide. Designated as a breakthrough therapy
cells. They must be administered via injection because by the FDA, early clinical trial results in rare genetic
they will be destroyed by digestive enzymes in the forms of obesity were promising, helping to attract $41
gastrointestinal tract if given orally. Their large size, million from key investors including Pfizer Venture
anywhere from 50 to 1,000 times larger than a typical Investments and Third Rock Ventures to fund the
small molecule drug, makes it impossible for them to upcoming Phase III.
penetrate cells. On the flip side, large molecules are Setmalnotide works by activating the melanocortin-4
highly specific for their target — typically a cell-surface receptor (MC4R), a receptor present on the surface of
receptor on the outside of the cell. cells in the hypothalamus of the brain, a region involved
The FDA defines a peptide therapeutic as a chain of in regulating both appetite and satiety. Mutations in
amino acids (the building blocks of proteins) containing MC4R that result in reduced activation are the most
40 amino acids or less, and regulates them as small common genetic cause of obesity, and account for
molecules. Peptide therapeutics are similar to small approximately 6-8% of obesity cases.
molecule drugs in that they can be synthesized in the
lab using a peptide synthesis machine — a machine that KALOS FIGHTS CANCER
links amino acids together in a specified order. Peptides
Kalos Therapeutics has a peptide drug in development
also share key characteristics with large molecule drugs
based on a straightforward observation: despite
constant activity, heart muscles don’t get bigger, and

cancers of the heart are extremely rare. At least part of cancer cells but is inactivated in a range of malignancies.
the reason for this is a peptide known as atrial natriuretic ALRN-6924 is in Phase II clinical studies for lymphoma.
peptide (ANP), which is produced in the heart. It helps The company is pursuing the development of stapled
to control cell growth and division, making sure that peptides in a range of therapeutic areas, including
the heart doesn’t get too big for the chest. Since cancer inflammation and endocrine and metabolic diseases.
is caused by out-of-control cell growth and division, a Bicycle Therapeutics also uses chemically linked
connection was made: perhaps these peptides could play peptides to increase stability, target interaction, and
a role in controlling tumor cell growth. penetrate cells. Their peptides are formed — using a
Kalos Therapeutics has identified a portion of ANP and chemical linker — into the shape of a bicycle.
is synthesizing and testing it as a potential anti-cancer Bicycle’s lead candidate, BT1718, is a “bicycle drug
agent. Dubbed KTH-22, the agent is cytostatic, meaning conjugate” — a bicyclic peptide with a toxic drug
it halts the growth and division of cancer cells, but attached. The peptide targets a protein called
does not directly kill them as a cytotoxic (toxic to cells) “membrane type 1 matrix metalloproteinase” (MT1-
agent would. KTH-22 is in preclinical research, with data MMP) which is overexpressed in many tumors. BT1718
supporting its use in the treatment of pancreatic and delivers its toxic payload to tumors overexpressing MT1-
ovarian cancers. MMP. Preclinical trials have shown high efficacy against
these tumors, and clinical trials are expected to start by
STAPLES & BICYCLES the end of 2017.
Most peptide therapeutics do not penetrate cell
membranes. Designing peptides that could enter cells
would truly endow them with the best characteristics
of both large and small molecule therapies. Aileron
Therapeutics and Bicycle Therapeutics are aiming to
do just that.
Aileron Therapeutics is developing “stapled peptides.”
These peptides are synthesized according to an
optimized amino acid sequence. Next, a chemical linker
is used to connect two amino acids within the chain,
creating a folded or “stapled” version of the peptide.
These stapled peptides still recognize their target
protein, are more stable, and better able to penetrate Already capable of affecting a range of therapeutic
cell membranes than the linear versions. targets with high specificity, continued innovations in
Aileron’s leading stapled peptide candidate, ALRN- peptide design and delivery should make this class of
6924, activates p53, a protein that triggers cell death in drug an important player.

Putting The NA in DNA
NUCLEIC ACID THERAPEUTICS complementary base pairs — “A” complements “T”
and “C” complements “G.”
Small molecule, peptide, and biologic drugs aren’t the
only players in the game of drug development. A fourth • Bases include Adenine (A), Cytosine (C), Guanine (G),
class of therapeutics differs from all three of these: Thymidine (T).
nucleic acid-based drugs. These drugs are rising in RNA
prominence due to their potential to specifically target a
• The ribose sugar group is a less chemically stable
wide range of diseases, including various types of cancer,
sugar group because “ribose” has a highly reactive
autoimmune, and infectious diseases. Companies like
oxygen atom.
Moderna (Cambridge, MA) are garnering unprecedented
investor interest, while improvements in delivery • RNA is typically single stranded.
methods have increased the efficacy of nucleic acid- • Bases include Adenine (A), Cytosine (C), Guanine (G),
based therapies. Uracil (U).
In this WEEKLY — the first of a two part series — we’ll
dig up the different types of nucleic acids and unearth
mRNA-based therapeutics in development.
TERM OF THE WEEK: NUCLEIC ACID

Nucleic acids are long chains of repeating units of
nucleotides. Nucleotides are made up of a phosphate
group, a sugar group, and a base.
WHY MRNA?
You probably recognize DNA as the molecule of heredity,
and may recall that it provides cells with the instructions
for making proteins. Enter messenger RNA (mRNA) — the
literal messenger that relays the DNA instruction to the
ribosome where the protein-making process takes place.
There are two types of nucleic acids: DNA and RNA.

The nucleotides (or building blocks) of these two
varieties of nucleic acids are quite similar, but there are
marked differences.
DNA
• The deoxyribose is a more chemically stable sugar
group because “deoxyribose” lacks the highly
reactive oxygen atom.
• Two individual strands of linked nucleotides join
together to make the double helix by forming

So, why all this talk about mRNA? Well, protein IN THE PIPELINE
therapeutics — the injectable protein-based drugs
Moderna Therapeutics (Cambridge, MA) has
discussed last week — have revolutionized the
received nearly $2 billion to fund ongoing mRNA drug
treatment of a range of diseases, from diabetes to
development. The company now has five different
cancer to autoimmune disorders. However, they are
products in Phase I clinical studies. Four of these are
time consuming and expensive to produce. Cells must be
vaccine candidates: two against different strains of
engineered to develop the desired protein, then grown in
influenza virus, one against Zika virus, and one against
large (thousands of liters) tanks. Finally, the therapeutic
an undisclosed target in partnership with Merck
protein must be painstakingly purified away from other
(Kenilworth, New Jersey). A mRNA-based vaccine uses
proteins and cellular debris in the cell.
lipid nanoparticles to deliver the instructions for making
What if we could eliminate the huge biomanufacturing a particular viral protein to a cell. The cell then makes the
tanks and just have the patient make the therapeutic viral protein and displays segments of it on its surface,
protein using their own cells? That is the idea behind activating an immune response to fight infection.
mRNA therapeutics — figure out a way to provide the
The fifth drug for which Moderna has initiated clinical
information contained in mRNA directly to the patient’s
trials — in partnership with AstraZeneca (Cambridge,
ribosomes and let the patient’s cells do the work. Not
UK) — is a mRNA that codes for the protein known as
only would this be more efficient, it would also enable
vascular endothelial growth factor, or VEGF. This protein
therapeutic proteins to be introduced directly inside cells
promotes the growth of blood vessels, and may help
or embed into the cell membrane. Recall that protein
to improve blood supply in cardiac tissue after a heart
therapeutics injected into the bloodstream are too large
attack, or in diabetic wound healing.
to enter cells and are limited to interacting with proteins
on the surface of cells or in the blood. CureVac (Tubingen, Germany) is focused on mRNA
vaccines as well, with a prostate cancer therapeutic
vaccine in Phase II clinical studies. Therapeutic vaccines
THEORY VS. REALITY
train the patient’s immune system to recognize a specific
Like much in biotech, the concept of mRNA-based cancer associated protein, priming immune cells to
therapeutics is elegant in theory, yet rough in reality. attack the tumor that bears those proteins. CureVac
• Reason 1: The relative instability of the mRNA also has a mRNA-based rabies vaccine in Phase I clinical
molecule itself; mRNA traveling through the studies, with several more infectious disease and
bloodstream would typically be degraded by therapeutic cancer vaccines in preclinical development.
nucleases — enzymes that break down nucleic acids. Other companies to watch in this space include:
• Reason 2: “Foreign mRNA” coming from outside of the • BioNTech (Mainz, Germany): Phase I studies
cell could trigger an immune response; our immune completed on a mRNA-based therapeutic vaccine
systems have evolved to recognize foreign mRNA for melanoma; preparing to enter clinical studies
as bad. on therapeutic cancer vaccines for head and neck
• Reason 3: Delivery of mRNA therapy is difficult. cancer and personalized vaccines.
Right now the approach that appears to be having • Arcturus (San Diego, CA): Preclinical development of
the most success is encasing the mRNA in a lipid mRNA drugs to treat protein deficiency disorders.
nanoparticle for delivery to cells.
• RaNA (Cambridge, MA): Preclinical development of
Bringing mRNA drugs to market involves designing mRNA drugs to treat protein deficiency disorders.
chemically modified mRNA that is more stable (resistant
mRNA drugs show much promise and we will continue
to nucleases) and less visible to immune cells than
to closely follow this area for new developments. Next
unmodified mRNA. These modified mRNA molecules are
week, we’ll continue our discussion of nucleic acid-based
called “nucleotide analogs” because they are similar but
therapeutics as we look at additional types of RNA and
different from naturally occurring nucleotides.
DNA based drugs.

RNA Therapeutics March Onward
TAKING STEPS WITH ANTISENSE renewed itself in 2013 thanks to the FDA approval of
Ionis Pharmaceuticals’ (Carlsbad, CA) Kynamro for the
With their high specificity and relative low manufacturing
treatment of familial hypercholesterolemia.
cost, RNA therapeutics may be tomorrow’s biotech
sweetheart. In fact, chances are good that previously Kynamro targets apolipoprotein B, a key component of
“undruggable” targets that cannot be accessed by small LDL cholesterol, to lower cholesterol levels:
or large molecule drugs, are now within reach. However,
the main roadblock continues to be delivery—getting
the RNA drug where it needs to be, in high enough
concentrations, to be effective.
Last week we looked at the emerging class of
nucleic acid-based drugs known as messenger RNA
therapeutics. This week, we’ll continue our discussion
by examining the RNA therapeutic known as antisense.
Let’s march!
RNA RELAY
Messenger RNA (mRNA) is a molecule that carries a
copy of the protein-making instructions from DNA in the
MEET INTRONS & EXONS
nucleus of a cell to the ribosomes located just outside
of the nucleus. The information contained in mRNA is Exondys 51 (Sarepta Therapeutics; Cambridge, MA)
used by the ribosomes to assemble a protein. Without and Spinraza (Ionis and Biogen; Cambridge, MA) are
mRNA relaying the instructions, there would be no recently-approved antisense drugs that work by altering
protein produced. how the cell processes pre-mRNA — an immature
template of mRNA. Pre-mRNA is a long strand of RNA
from which specific pieces are cut out or added in before
ANTISENSE EXPLAINED
becoming the mature mRNA that is able to relay the
Antisense drugs are short (between 10 and 30 protein-making instructions from DNA to the ribosomes.
nucleotides), synthetic pieces of nucleic acid whose
Think of pre-RNA as a master grocery list that includes
sequence is complementary to the mRNA that codes for
all of the most common foods you buy over the year.
a disease-associated protein. When the antisense drug
Envision mRNA as your adjusted grocery list that
enters a patient’s cells, it binds to the disease-causing
includes only what you need to buy for this week.
mRNA. This binding triggers an enzyme called RNAse H
to destroy the double-stranded antisense-mRNA hybrid. When pre-mRNA is first produced, it contains two types
Our bodies recognize that double-stranded RNA as a of sections that are either cut out or left in depending on
mistake and destroys it. Without the mRNA, the disease- the instructions:
associated protein simply is not made — stopping • Introns: regions cut out of the final mRNA; grocery
disease in its tracks. items you delete from your master list because you
The history of antisense in drug development is quite don’t need them this week.
fickle. Over the years the inability to get antisense • Exons: regions included in the final mRNA; grocery
drugs to accumulate at therapeutically effective items you keep from your master list because you
concentrations in the target tissue was the undoing of need them this week.
many clinical trials. But the love affair with antisense

the right item was left off during the list-making process.
Spinraza fixes this error, in other words, the antisense
drug glues itself to the exon which needs to stay on so it
can remain on the list. This results in the production of a
full-length mRNA and a more functional SMN protein.
Duchenne’s muscular dystrophy (DMD) and spinal

muscular atrophy (SMA) are diseases in which errors
occur in the processing of pre-mRNA, resulting in
proteins that are not fully functional. Harking back to our
grocery list analogy, this means your weekly grocery list
is incorrect. Some wrong items were left on and some right
were left off, resulting in a failed shopping trip.
Antisense drugs alter the way pre-RNA is processed THE FUTURE OF ANTISENSE
by either including or excluding exons to make a more
The future of antisense looks bright. By targeting the
functional protein. Antisense technology fixes the weekly
right tissues (the most promising targets are in the liver)
grocery list.
and developing more stable formulations, we can expect
In DMD, the wrong items were left on during the list- to see more success stories. Check out some of the
making process. Exondys 51 is an antisense drug that antisense drugs currently in clinical development:
binds to the “wrong item” to ensure it stays off. In
reality, it cuts a mutated exon out of the dystrophin
gene. By cutting out this exon, DMD patients produce
a more functional dystrophin protein than the disease-
associated version. The dystrophin protein maintains
muscle integrity.
The complex yet fascinating world of antisense marches

forward. They promise several key advantages, including
high specificity, low manufacturing costs, and the
potential to target previously “undruggable” targets. We
here at the WEEKLY look forward to reporting on new
approvals in this arena.
SMA patients don’t produce enough of a protein called
spinal motor neuron (SMN) because it is missing an exon;

Decoding Your Genes
THE SKINNY ON DNA TESTING Many SNPs have no significant impact on an individual’s
health, but other SNPs are associated with disease
23andMe (Mountain View, CA) recently found itself back
susceptibility; having one variant instead of another
in the limelight after the disease risk section of its mail-in
makes someone more likely to get a particular disease.
DNA kit received an OK from the FDA.
SNP genotyping is characterizing the SNP profile of
The Silicon Valley biotech had to halt sales of its
an individual — finding out which A, C, G, or Ts are
direct-to-consumer genetics testing back in 2013 after
in positions differing from the norm. In the case of
regulatory officials grew concerned over marketing
23andMe, that information is used to assess disease
claims and the possibility of consumers misinterpreting
risk, carrier status for certain genetic diseases, wellness
the test results. 23andMe rebooted a limited part of their
information, and ancestry.
genetics section in 2015 after a preliminary go ahead,
and last month marked a full return with the approval of
their Genetic Health Risk (GHR) reports. A CHIP OFF THE OLD GENE
In this issue, we’ll get the skinny on the science 23andMe’s core technology involves the use of DNA
driving 23andMe. microarrays, also known as DNA “gene chips.” The
technology relies on the very specific base pairing rules
followed by double-stranded DNA: A’s always pair with
TERM OF THE WEEK: SNP GENOTYPING
T’s, and C’s always pair with G’s.
23andMe’s moniker is a nod to the 23 pairs of
A DNA microarray is simply a tiny piece of glass or silica
chromosomes that make up the human genome. Recall
that has had a microscopic checkerboard etched into it
the human genome is all the DNA found in a human
— each square is about 11 micrometers (10-6 meters) by
cell. The tests, which are available to anyone with a
11 micrometers big, just big enough to hold one single-
mailbox and a credit card, rely on a technique called
stranded piece of DNA.
SNP genotyping.
“SNP” (pronounced snip) stands for “single nucleotide
polymorphism” — a change in a single DNA nucleotide
(A, C, G, or T) that occurs at a specific position in the
genome. An example of a SNP is demonstrated in the
image below: within a specific gene sequence the most
common variant for individuals is a “T” at a particular
location, but in some individuals the “T” has been
replaced by a “C” SNP.
Each square represents one gene, or gene variant, within

the human genome. The entire gene sequence need not
be present — typically, between 25 and 60 bases are
used (though we are only showing 6 in our image above).
Computer software keeps track of the location of gene
sequences within the array. In this manner, the specific
variants within each DNA sample can be identified.

EASILY CONFUSED: GENOME Mutations in susceptibility genes, in contrast, do not
SEQUENCING VS. SNP GENOTYPING necessarily mean the patient has or will develop the
disease, it only means the patient is at risk for developing
The term DNA sequencing is often mistakenly used when the disease. One well-characterized susceptibility gene
the correct term is SNP genotyping. is apolipoprotein E (ApoE). The ApoE4 variant of the
• DNA sequencing means to determine the order ApoE gene is associated with a higher risk of Alzheimer’s
of every single base pair in a given gene (gene disease (AD). Those with two copies of ApoE4 may have
sequencing) or in an entire genome (whole as much as 20 times the risk of developing AD; however,
genome sequencing). some individuals with two copies never develop the
• SNP genotyping means to identify single base disease according to statistical studies of different ApoE
changes between a given gene sequence and a variants. On the other hand, another variant — ApoE2 —
reference sequence. may reduce the risk of developing AD.
By identifying which ApoE gene variant an individual
MUTATIONS & SUSCEPTIBILITY GENES has, researchers can say whether or not that person has
an average risk, a higher than average risk, or a lower
To qualify as a diagnostic test in the eyes of the FDA,
than average risk of developing AD — but they cannot
genetic testing companies must show that a specific DNA
definitively diagnose or predict AD onset.
sequence can confirm whether one has (or doesn’t have)
a particular disease.
For example, Huntington’s disease is caused by a known
mutation in the huntingtin gene. If a specific mutation
is detected, a doctor can say with certainty whether the
patient has or will develop Huntington’s disease. It is a
dominant genetic disorder, meaning that one copy of the
disease-associated gene variant is enough to cause the
disease (recall that we have two copies of each gene, one
from each parent).
Most genetic disorders are recessive — meaning two
copies (one from each parent) of the disease-associated
gene must be present to actually develop the disease.
23andMe now offers 10 Genetic Health Risk tests that
If someone has only one copy of the gene variant that
look at susceptibility genes, including ApoE4. Part of the
causes cystic fibrosis, they are deemed a “carrier” — they
FDA’s earlier concerns about consumer misinterpretation
themselves do not have cystic fibrosis, but they have a
involved the distinction between susceptibility and
25% chance of passing the disease on to their child if the
diagnosis — making sure consumers understood the
other parent is a cystic fibrosis carrier.
test results did not, for example, diagnose them with AD,
but merely reported their susceptibility in the form of a
statistical risk factor.
23andMe also offers carrier status tests for more than
40 different conditions. They also provide information on
gene variants associated with “wellness” — such things
as sensitivity to caffeine and lactose intolerance, as well
as non-health-related traits such as dry or wet earwax
and skin pigmentation.

MORE THAN A SNP (in partnership with Genentech; South San Francisco,
CA), wherein they recruited patients with those diseases
Direct-to-consumer genetic testing is only a part of
to participate in identify SNPs more common in those
23andMe’s business. Since its founding in 2006, the
populations. In 2015, the company announced it
company has been collecting genetic data on patients
would begin to do its own drug discovery research and
along with their self-reports of symptoms and health
launched 23andMe Therapeutics.
status — about 85% of users from the 23andMe
database have opted in. From new options for consumer genomics to
breakthroughs in drug discovery, we expect 23andMe to
The company has also initiated research efforts around
stay in the news for some time to come.
specific diseases such as inflammatory bowel disease (in
partnership with Pfizer; New York, NY) and Parkinson’s

Phage — More Than Just A Phase
OLD WAY OF FIGHTING BACTERIA RENEWED coexisted with bacteriophage for a long time, suggesting
that there should be few safety issues with their use
One of the greatest public health challenges of the 21st
as therapeutics.
century is antibiotic resistance, which occurs when a
few bacteria in a given population develop a genetic Scientists have known about the bacteria-devouring
mutation that enables them to survive — even in the ability of phage for about 100 years, but with the advent
presence of antibiotics. of antibiotics in the late 1920s, medicine’s focus shifted
to these new wonder drugs because they were easier to
How do bacteria become drug resistant? Suppose a
manufacture and test in controlled settings. Now that
particular antibiotic inhibits an enzyme required for
antibiotic resistance is emerging, so too is a renewed
bacterial replication. If one bacterium mutates so the
interest in bacteriophage, which are now starting to
enzyme has a slightly different shape, the antibiotic is
be manufactured and tested in a standardized way for
no longer effective. The mutated bacterium lives on and
the first time. Let’s take a look at some of the biotech
continues to replicate, even as all the others die off. Over
companies delving into the world of bacteriophage-
time, this resistant strain becomes dominant, spreading
based therapeutics.
from person to person, remaining unchecked and
thriving. It is not uncommon for a strain of bacteria to
become resistant to several different antibiotics, giving THE COCKTAIL APPROACH
rise to the term multi-drug resistant bacteria. The first multicenter clinical trial examining the use
In this issue of the WEEKLY, we’ll take a look at an of bacteriophage as antibacterial treatments was
entirely novel approach to fighting bacterial infections — initiated in 2015 by French biotech Pherecydes (Paris,
bacteriophage. France). Preparative work for the trial began in 2013 as
researchers established protocols for producing phage
that met good manufacturing practice guidelines. The
TERM OF THE WEEK: BACTERIOPHAGE
researchers are studying two different “cocktails” of
A bacteriophage — also referred to as a phage — is a bacteriophage — mixtures of different bacteriophage
virus that infects bacteria. By attaching to a bacterium’s that have shown activity against different substrains
surface, a phage punches holes in the membrane and of a particular bacteria in the lab. The first contains 13
injects its own genetic material inside. The phage then different phages targeting P. aeruginosa; the second, 12
replicates inside of the bacterium, creating so many new phages that target E. coli. Both are being evaluated for
viruses that the bacterium breaks open, releasing newly the treatment of burn wound-associated infections.
produced viruses, which can then go on to infect other
Other companies testing phage cocktails in human
bacteria, continuing the cycle.
patients include:
The word “bacteriophage” is derived from the Greek
• AmpliPhi Biosciences (Richmond, VA): Phage
word phagein — “to devour.” So we can think of
cocktail AB-SA01 is in Phase I clinical testing for
bacteriophage as, literally, devouring bacteria — a
antibiotic resistant S. aureus in two different clinical
potentially very useful trait! Typically each phage is
settings: chronic rhinosinusitis as well as acute and
specific for a type of bacteria, meaning that if adapted
chronic wound and skin infections.
for therapeutic use, researchers can select viruses
that will only attack harmful bacteria, and leave the • TechnoPhage (Lisbon, Portugal): Phage cocktail TP-
many strains of “friendly” bacteria that make up our 102 targets bacteria associated with chronic ulcers,
gut microbiome alone. And since each type of phage respiratory and skin infections.
has coevolved for millennia with its chosen strain of • Intralytix (Baltimore, MD): Completed Phase I
bacteria, each adapting and changing in response to the studies using a bacteriophage for the treatment of
other, resistance is much less likely to evolve as has been infected wounds.
the case for antibiotics. Likewise, humans have safely

• EpiBiome (San Francisco, CA): Phage cocktails THE COMPONENT APPROACH
targeting E. coli and S. dysenteriae diarrheal infections
A third approach to tapping into the therapeutic power
in preclinical development.
of bacteriophage lies in isolating the component or
components that make them toxic to bacteria. For
THE ENGINEERED APPROACH example, in order to inject their genome into bacteria,
Creating a phage cocktail can be a challenging phage must essentially punch a hole in the membrane,
biomanufacturing problem. An alternative approach to which is itself very damaging to the bacteria. The viral
combining beneficial characteristics of different viruses protein that creates these tears in the membrane
is to genetically engineer a synthetic virus that combines are called “lysins” — enzymes that essentially chew
the properties of multiple phages into a single genome. holes in the bacterial cell wall. ContraFect (Yonkers,
NY) has completed Phase I clinical studies of its drug
For example, scientists could insert genes into a phage
CF-301 — a lysin — for the treatment of S. aureus
genome to increase the range of bacteria subtypes
bloodstream infections.
an individual phage can attack, yet still maintain the
specificity that prevents the phage from raiding friendly Although still in the early days of clinical testing,
bacteria. Researchers could also add in genes to make bacteriophage offer the possibility of being a safe and
the bacteriophage’s antibacterial response even effective solution to the current antibiotic resistance
stronger. Companies with engineered bacteriophage in public health crisis.
preclinical development include Synthetic Genomics
(San Diego, CA) and EnBiotix (Cambridge, MA).

Viruses Blasting Cancer
ENGINEERING VIRUSES TO ATTACK against cancer. Let’s take a look at the unique features
of a selection of oncolytic viruses on the market and
Getting bacteria-eating viruses to combat antibiotic
in development.
resistance isn’t the only way viruses are being hacked
to defend team homo sapien. This week, we’ll turn our
attention to another benevolent use of viruses: cancer- INSIDE OF IMLYGIC
fighters known as oncolytic viruses. Viruses can be thought of as very simple packages of
genetic material — DNA or RNA — encapsulated in a
ONCOLYTIC VIRUS PRIMER protein package. Like the human genome, viral genomes
code for proteins required by the virus. Some of these
Oncolytic viruses are an immunotherapy — a type of
proteins enable the virus to make copies of itself
therapy that harnesses the power of a patient’s immune
(replicate), or to evade the human immune response. It
system to combat a disease. Getting a virus to trigger
is often necessary to modify the viral genome in order to
the immune response to fight cancer is no easy task,
safely use a virus as a therapeutic, but how?
the process involves engineering the virus to selectively
infect and kill cancer cells. Oncolytic viruses are created Amgen’s (Thousand Oaks, CA) Imlygic is the only FDA
in the lab by genetically modifying existing viruses in at approved oncolytic virus, aiming to attack melanoma.
least two ways: The virus used in Imlygic is a modified herpes simplex 1
virus. The modifications made to Imlygic to ensure safety
• Making the virus safe by removing genes that cause
and efficacy include:
the virus to make people sick
• Deletion of viral gene ICP34.5. This gene codes for a
• Engineering viral surface proteins so the virus
protein that enables the virus to replicate in human
recognizes and binds to the cell receptors of
cells by blocking a human protein known as PKR.
cancerous cells, disregarding the healthy, non-
PKR prevents viral replication. It is less active in
cancerous cells
most tumor cells, so this makes the virus able to
The oncolytic virus follows the same life cycle as any selectively replicate in tumor cells.
virus—once inside the human body it hunts down,
• Deletion of viral gene ICP47. This gene codes for a
attaches to, and enters its host cell. In this case, the host
viral protein that thwarts the immune response by
happens to be cancer cells! The virally infected cancer
turning off a process called antigen presentation.
cells are destroyed via the process of cell lysis—as the
Normally, one of the key ways the immune system
oncolytic virus multiplies inside of the cells, it causes
“knows” to attack a virally-infected cell is by
the cancer cells to burst open which kills them. Spewing
recognizing antigens (or fragments of viral proteins)
from the burst cells are new infectious viruses that
displayed on the infected cell’s surface. Turning
further target remaining tumor cells. The presence of a
this process off helps the virus evade the immune
replicating virus also activates the immune response, so
system. Turning it back on prompts the immune
the cancerous area is further attacked.
system to attack virus-infected tumor cells.
Additional modifications may also be made to the virus,
• Activating the earlier expression of the viral gene
depending on the characteristics of the targeted cancer.
US11, resulting in increased viral replication in
For example, an oncolytic virus might be modified to
tumor cells.
produce proteins that stimulate the immune system or
directly attack the tumor. • Insertion of a gene for the human protein GM-
CSF, which activates the immune system, aiding in
Most oncolytic viruses are tested both as “stand-
the overall immune response toward the tumor
alone treatments” and “in-combination with other
triggered by viral infection.
immunotherapies” — such as checkpoint inhibitor
therapies — to help fully activate the immune response Taken together, these modifications create a virus that
selectively replicates in tumor cells, resulting in their

direct destruction as well as activation of a host immune for “therapeutic payloads” — proteins that will boost
response targeting the virus-infected tumor cells. the patient’s immune response to the cancer, or even
therapeutic antibodies that will then be produced inside
ONCORUS ON THE OFFENSE of the cancer cell. This approach is a clever response
to the fact that due to their relatively large size, most
Oncorus (Cambridge, MA) is also developing a modified
therapeutic antibodies are not able to completely
herpes virus, ONCR-001, for the treatment of cancers,
penetrate solid tumors. Using an oncolytic virus to
including the notoriously difficult-to-treat brain cancer,
penetrate the tumor and deliver genes instructing the
glioblastoma. Like Imlygic, ONCR-001 has been modified
tumor itself to make the antibody could be a game-
to selectively target tumor cells. Unlike Imlygic, ONCR-
changing work around.
001 retains all viral genes needed for viral replication.
Finally, Genelux is also creating engineered virions that
So, how is safety maintained in the presence of an
incorporate “imaging” proteins. For example, GL-ONC1
oncolytic virus that is actively replicating? Oncorus
delivers a gene for a fluorescent protein directly to tumor
scientists have figured out a clever way to take
cells. As the virus replicates inside of the tumor, the
advantage of a key difference: the types of microRNAs
fluorescent signal increases. In preclinical animal testing,
produced by healthy cells vs. cancer cells.
this has allowed non-invasive detection and imaging of
MicroRNAs are a type of “regulatory RNA” that promotes tumor progression and regression in real time, and may
the degradation of a target messenger RNA (mRNA — one day be a powerful guide to physicians monitoring
the RNA that gets turned into proteins). This means that cancer patients. GL-ONC1 has successfully completed
different target sequences will be recognized in healthy Phase I and is preparing to enter Phase II for a variety of
cells vs. cancer cells. By engineering sequences that solid tumors including peritoneal carcinomatosis.
microRNAs from healthy cells will recognize, Oncorus
scientists can ensure the viral mRNA will be destroyed ONCOLYTIC PIPELINE
in any healthy cells it infects. No viral mRNA, no viral
proteins, no virus. Because these microRNAs are not With the approval of Imlygic in 2015, biotech companies,
present in tumor cells, ONCR-001 is able to produce viral investors, and regulatory officials have recognized the
proteins and new viral particles freely when it infects strong potential of oncolytic viruses to treat cancer.
those target cells. ONCR-001 has shown strong ability in The race is on to get the next oncolytic virus through
fighting glioblastoma in preclinical models. the clinic:
THE GENESIS OF GENELUX

San Diego-based Genelux is adapting the vaccinia virus
as an oncolytic virus for the treatment of a variety of
solid tumors. Vaccinia is the scientific name for the
cowpox virus — the virus that is used as a vaccine for
smallpox. Because of its decades-long use as a vaccine,
researchers have confidence the virus is safe to use
in humans, although the modified version must still
undergo safety testing.
Their lead product, GL-ONC1, selectively replicates in
tumor cells and tumor-associated blood vessels, directly
We expect to see this new therapeutic class become
killing tumors while cutting off their blood supply. The
increasingly common, opening up novel approaches for a
company is also developing oncolytic viruses with genes
whole range of cancers.

Nanobodies: These Are Not
Your Mother’s mAbs
THE DRUG KINGPINS also selected for their ability to inhibit (stop the activity
of) a particular protein. For example, the breast cancer
Monoclonal antibodies (mAbs) are the undisputed drug
drug Herceptin (Genentech, South San Francisco)
kingpins. In 2013, the mAb market raked in $75 billion in
inhibits the activity of the growth factor receptor HER2
combined sales, covering a whole range of indications
by preventing it from interacting with the growth
from cancer and infectious disease, to autoimmune
factor HER2:
disorders, and even high cholesterol.
Despite the success, mAbs have one chink in their armor:
they cannot enter cells due to their large size, hampering
their range as therapeutics. To date, mAbs can only
target proteins on the surface of cells, such as receptor
proteins, or proteins circulating in the bloodstream, such
as inflammatory cytokines. The development of cell-
penetrating mAbs would open up a world of therapeutic
targets and patient benefits.
Let’s review the fundamentals of therapeutic antibodies
and explore a new type of therapeutic antibody that may
be able to go where no antibody has gone before.
By developing antibodies that can enter cells, this
MAB RECAP inhibitory power can be used against targets inside of the
Antibodies are proteins naturally produced by our cell. Let’s take a look at the newest contender.
immune system to help defend against foreign invaders
such as viruses and bacteria. Each antibody produced NANOBODIES
has a unique shape that enables it to recognize unique
Scientists at Ablynx (Ghent, Belgium) are developing
targets (antigens) which are typically proteins on the
a new type of therapeutic antibody from an unlikely
surface of pathogens. By binding to these pathogens,
source — camels and llamas, members of the biological
antibodies act as a flag to alert the rest of the immune
family Camelidae. These antibodies are structurally
system to attack.
and functionally very similar to human antibodies,
with a few important differences that could add up to
something big!
Like all antibodies, Camelidae antibodies work because
they have a specific shape that enables them to
recognize and bind to a specific target. However, they are
a tenth the size of other mammalian antibodies — giving
rise to the moniker “Nanobodies.” Nanobodies have the
ability to recognize targets hidden inside of cells. Their
small size may potentially also enable them to cross
the challenging blood brain barrier, or penetrate the
interior cells of tumors – two activities that conventional
antibody therapies lack.
Antibody therapeutics also rely on the ability of
In addition to their small size, Nanobodies also exhibit
antibodies to interact with a specific target. Scientists
a less complex structure overall. Because of this, they
have developed antibodies that recognize and bind
have been successfully produced in bacterial cells. If
proteins on the surface of tumor cells, thereby alerting
Nanobodies can be scaled-up, it would significantly
the immune system to attack the tumor. Antibodies are

reduce production costs as compared to standard immune system may prove a useful treatment for
antibody production in mammalian cells. Preliminary autoimmune disorders. Vobarilizumab is being tested
studies in mice also suggest that Nanobodies can be for the treatment of rheumatoid arthritis and lupus, in
maintained in the stomach and intestine — opening up partnership with AbbVie (North Chicago IL). Next up
the possibility of oral delivery for some indications such is ALX-0171, which binds the fusion (F) protein on the
as Crohn’s disease. surface of the respiratory syncytial virus (RSV). The F
protein enables RSV to lock onto lung cells. ALX-0171 is
IN THE CLINIC expected to interfere with the interaction of F protein
and lung cells, thereby preventing RSV infection.
We can expect to see the first Nanobodies on the
market this year. In February of 2017, Ablynx submitted In partnership with Boehringer Ingelheim (Ingelheim,
an application to the European Medicines Agency for Germany), Ablynx is also entering the oncology space
the first of its Nanobody therapeutics, caplacizumab. with Phase I testing of a Nanobody that inhibits the
Caplacizumab is being tested for the treatment vascular endothelial growth factor (VEGF) protein. VEGF
for a rare disease known as acquired thrombotic is a growth factor secreted by tumor cells to encourage
thrombocytopenic purpura (aTTP), a blood-coagulation the growth of blood vessels into the tumor, a process
disorder that results in extensive microscopic clots called angiogenesis. By inhibiting VEGF and angiogenesis,
forming in small blood vessels throughout the body. the flow of blood and nutrients into the tumor is
The disease is triggered by excess von Willebrand stopped, essentially starving the tumor. It is hoped that
factor (vWF), a protein that initiates blood clotting. Nanobodies may be even better angiogenesis inhibitors
Caplacizumab inhibits vWF, thereby preventing than monoclonal antibodies have proven to be, due to
clot formation. their enhanced tumor-penetrating abilities.
The company has two more Nanobody products in As Nanobodies continue to be tested for safety and
Phase II clinical testing: Vobarilizumab, which reduces efficacy, a whole new kingdom of potential antibody
the activity of interleukin-6 (IL-6). IL-6 is a protein targets may begin to emerge.
that stimulates the immune response; inhibiting the

Epigenome: Writing,
Reading & Erasing
FOUNDATIONS OF EPIGENETICS A second type of modification is called acetylation — the
addition of an acetyl group (CH3 CO) to the histones.
Genetic mutations — changes in the order of the A, C,
Acetylation loosens the association of the DNA with
G, and T nucleotide bases that make up a gene — have
the histones, making the DNA more accessible to the
been the primary focus of cancer researchers over
enzymes used in gene expression, ultimately increasing
the last several decades. By sussing out mutations
protein production.
involved in regulating cell growth and division, scientists
better understand the molecular range of different
cancers and consequently develop more targeted and
effective therapeutics.
In recent years, another type of genetic variation has
captured the attention of researchers: epigenetic
modifications. Best characterized in cancer, epigenetic
changes are also thought to play a role in a range
of other diseases, including autoimmune disease,
cardiovascular disorders, diabetes, neurodegenerative
disorders such as Alzheimer’s disease, and potentially
even male infertility.
In this WEEKLY, we’ll tell the epigenetics story and discuss
Deacetylation — the removal of an acetyl group —
how it’s being used to develop new treatments.
increases the association or “grip” of the DNA around the
histones. Deacetylation makes the DNA less accessible
TERM OF THE WEEK: EPIGENETICS to enzymes used in gene expression, thereby decreasing
Epigenetic modifications are changes to DNA that do the production of proteins.
not alter the actual gene sequence; they are chemical
modifications to the DNA itself. These changes typically ADDING IT ALL UP
affect gene expression, or how often the gene is read by the
Epigenetic modification is a normal part of development.
cell. Epigenetic modification can occur either directly to the
This is in part why different genes are expressed in the
nucleotide bases themselves (A, C, G, or T) or to the histones,
heart than, say, the liver — the two different tissue types
which are small proteins that package and order DNA.
contain the same genome, but tissue-specific differences
in epigenetic modification lead to differences in gene
expression in the two tissues.
Problems may arise, however, if variations in epigenetic
modifications result in changes to gene expression.
If a cell or tissue type begins to make too much of a
protein that activates cell growth, for example, the cell
could begin to divide too often — potentially leading to
cancer. Alternatively, a cell could begin to make less of
a protective protein, for example, a “tumor suppressor”
protein (a protein that deactivates cell division), and
again cancer could ensue.
Epigenetic medicine seeks to identify disease-associated
One of the most common types of epigenetic
differences in epigenetic modifications, and to develop
modification is methylation — the addition of a methyl
drugs to restore the epigenome to that of healthy cells.
(CH3) group to cytosine (C) nucleotides. The end result:
methylation reduces or even blocks gene expression.

BREAKING IT DOWN • Histone-methyltransferase (EZH2) inhibitors: EZH2’s
are also writers — these enzymes transfer methyl
Epigenetic drugs are small molecule drugs that target
groups to histone proteins. One EZH2 is associated
epigenetic regulators, or proteins that write, read, or
with over activity in a number of different cancers.
erase epigenetic modifications.
There are no EZH2 inhibitors currently approved, but
• Writers are the enzymes that make the chemical several are in development, including Constellation
modifications — methylation or acetylation Pharmaceuticals’ (Cambridge, MA) CPI-1205
as described above — to DNA molecules or in Phase I for advanced B-cell lymphomas, and
histone proteins. Epizyme’s (Cambridge, MA) tazemat, currently
• Erasers are enzymes that remove these in Phase II for non-Hodgkin lymphoma, certain
chemical groups. genetically-defined solid tumors, and mesothelioma.
• Readers are the proteins that detect and respond to

these modifications, causing the DNA to be more or THE NEW CLASS: READERS
less tightly wrapped around the histone protein. Any A class of proteins called “Bromodomain and Extra
one of these proteins could be inhibited or activated Terminal motif” (BET) proteins are reader proteins. They
to affect changes in epigenetic modifications. recognize and bind to specific patterns of acetylation on
histone proteins. Upon binding, they recruit additional
OLD SCHOOL: WRITING & ERASING proteins that regulate gene activity. Irregularities in
histone acetylation, then, may send the wrong message
The disease that has been best classified in terms of
to a BET protein. By inhibiting the interaction between
epigenetic variations is cancer. Currently, there are five
BET protein and histone proteins, researchers have
epigenetic drugs on the market to treat cancer, and more in
found that they can prevent incorrect messages from
development. Those on the market fall into two categories:
being received by the BET proteins. Currently, there
• Histone deacetylase (HDAC) inhibitors: HDACs are are no BET inhibitors (BBI) approved, but several are in
erasers — they remove acetyl groups from histone clinical development. The farthest along is Resverlogix’s
proteins, resulting in increased expression of (Calgary, Canada) apabetalone, which is in Phase III
associated genes. Three HDAC inhibitors have been testing for atherosclerosis and associated cardiovascular
approved by the FDA: Zolinza (Merck; Kenilworth, disease. Additional BBIs in clinical development are
NJ) and Istodax (Celgene; Summit, NJ) both treat shown in the table below:
cutaneous T-cell lymphoma, and Farydak (Novartis;
Basel, Switzerland) for the treatment of multiple
myeloma. HDAC inhibitors in development include:
Epigenetics promises to change the way we look at the

human genome. Scientists have made great strides in
understanding how epigenetic modifications contribute
• DNA-methyltransferase (DNMT) inhibitors: DNMT’s are to both health and disease; however, a complete
writers — they add methyl groups to DNA, resulting understanding of these modifications is still very much a
in decreased expression of associated genes. Two work in progress. As that work develops, researchers will
DNMT inhibitors have been approved by the FDA: undoubtedly uncover new drug targets and approaches
Vidaza (Celgene) and Dacogen (Otsuka; Tokyo, to disease management. Stay tuned!
Japan). Both drugs are used to treat myelodysplastic
syndrome and acute myeloid leukemia.

A Skin Cell With Stem
Cell Diversity?
INDUCED PLURIPOTENT STEM TERM OF THE WEEK: INDUCED
CELLS SHOW PROMISE PLURIPOTENT STEM CELL
Imagine being able to reprogram one of your own skin An induced pluripotent stem cell (IPSC) is a type of
cells to produce a functioning nerve cell or section of pluripotent stem cell that can be generated directly from
cardiac tissue. This may sound like science fiction — but adult cells. In theory, this means that a scientist could
the groundwork for this to become a reality is already in create a stem cell treatment using cells from a patient’s
the works as researchers expand their ability to create own body by following these steps:
and manipulate induced pluripotent stem cells. • Remove a skin or other easily accessible cell type
In this WEEKLY, we’ll get the details on what these from the patient.
multifaceted cells are all about and discover their • Manipulate the cell in the lab to produce an induced
therapeutic potential. pluripotent stem cell.
• Add the required growth and differentiation
STEM CELL PRIMER
factors to get the induced pluripotent stem cell to
Stem cells are unspecialized cells that have the ability differentiate into the desired tissue type.
to develop (differentiate) into 1 of 200 cell types in the
This newly differentiated tissue could then be
body. There are two general classifications:
transplanted back into the patient’s body without fear of
• Embryonic stem cells (found only in developing immune rejection since it is derived from their own cells!
embryos) can become any cell type within the adult
body. These are pluripotent stem cells. AN EASIER RECIPE
• Adult stem cells (found in the organs of an adult) The development of IPSCs was so significant that the
can only become certain cell types. Typically, these scientists at Cambridge University (Cambridge, UK) and
cell types come from the organ in which they are Kyoto University (Kyoto, Japan) who figured out how to
derived from. create them received the 2012 Nobel Prize in Medicine.
Due to their ability to differentiate (change into) into any Their discovery was based the observation that when
cell type in the adult body, pluripotent stem cells show pluripotent stem cells differentiate into specialized cell
the most promise as a therapeutic. The idea is they can types, certain genes are deactivated or switched off.
be induced in the lab to form a specific type of adult They wondered if the reverse might also be true — if by
tissue and then transplanted into a patient who needs reactivating or turning on those same genes, they could
that tissue. arrive at pluripotency. Through a series of experiments,
For example, someone who damages their spinal cord they hit upon the correct combination of genes to
may potentially benefit from a transplant of replacement reactivate, and succeeded in inducing pluripotency.
spinal cord tissue. In fact, these types of clinical trials
are currently ongoing and show promise. However, RESEARCH APPLICATIONS
transplanting tissue derived from an embryo carries the The best way to decipher a disease is to examine the
same risks as an organ transplant — rejection by the affected cells from a patient — for example, a blood
immune system. Thus, patients undergoing this type of sample from a leukemia patient or a tumor biopsy from
therapy must receive immunosuppressive drugs, which a breast cancer patient. What if the affected tissue is
carries its own set of risks. impossible or dangerous to access — such as brain or
What could be a possible alternative that solves the cardiac tissue? IPSCs to the rescue! To better understand
rejection issue? Alzheimer’s, researchers are creating IPSCs from
Alzheimer’s patients’ skin cells, which are then induced
to become brain cells. In this manner, the disease models
reflect the genetics of an Alzheimer’s patient without the

need to directly access their brain cells. These patient- develop IPSC-derived heart muscle cells and neurons
based disease models are being used as both a way to for drug discovery applications.
better understand the disease process as well as for
drug discovery research. CLINICAL APPLICATIONS
Companies developing IPSC-based drug discovery Ultimately, scientists and physicians want to use patient-
platforms include: derived IPSCs for individualized treatments as described
• Evotek’s (Hamburg, Germany) collaboration with above. Currently, there is only one ongoing clinical
Celgene (Summit, NJ) to use their IPSC platform trial using IPSCs, taking place at the RIKEN Institute
to discover and develop new drugs for a range of (Wako, Japan) for the treatment of wet age-related
neurodegenerative disorders, including Alzheimer’s, macular degeneration. The scientific community is
Parkinson’s, and ALS. closely watching this trial — if IPSC treatments prove to
be safe and effective, they will revolutionize an already
• Axiogenesis’ (Cologne, Germany) collaborations
revolutionary field.
with Metrion Biosciences (Cambridge, UK) to

The Biotech Primer
BIO 2017 Preview
BIOTECH PRIMER AT BIO 2017 deliver this type of course in various locations and
can customize it and bring it in-house to your team.
Biotech Primer will be headlining a few events at the
For a list of upcoming classes click here. To learn more
BIO 2017 Annual International Convention in San
about our in-house classes, contact Stacey at Franklin@
Diego, CA next week. Will you be there? If so, please
BiotechPrimer.com.
join us!
• Learn the fundamentals of biotechnology in our all-
READER’S CHOICE: TOP TOPICS
day class BioBriefing: Biotech for the Non-Scientist
on Monday, June 19th. SOLD OUT
FROM BIOTECH PRIMER WEEKLY
• What are the most popular topics in the WEEKLY?
Attend Readers’ Choice: Biotech Primer WEEKLY Top
Topics on Tuesday, June 20th at 10:45am to learn
about genome editing, immunotherapy and much
more. Event open to BIO 2017 registrants.
• Be sure to visit us at booth #5225 to pick up your
free copy of our book, The Biotech Primer: An insider’s
guide to the biotech and pharma industry!
Dr. Emily Burke
• Come by our booth for a pair of tickets to the
PABNAB party on Wednesday night. One pair per On Tuesday, June 20th, I will be giving a talk, Readers’
person, until all tickets are gone. Choice: Biotech Primer WEEKLY Top Topics, that
highlights the most popular Biotech Primer WEEKLY
topics over the past year. Two of the topics chosen by our
BIOTECH PRIMER’S BIOTECHNOLOGY
readers are summarized below:
FOR NON-SCIENTISTS COURSES
• Genome editing: we often refer to a person’s
What can you expect to learn when you take a class with genome—the complete set of genes—as a blueprint
us? Starting with the basics, we build upon this base for life. Sometimes, there are errors in this blueprint.
to deliver more complex, industry-relevant knowledge Wouldn’t it be life changing if we could edit and
about disease, diagnostics and drugs. correct these errors? We might soon be able to!
Some of my favorite topics to teach include the genetic Researchers have developed tools that make it
variation, genomics, and immunology sections of the possible to cut an individual’s DNA at a specific site,
course. Starting with the concept of the gene as the and either disrupt or correct the gene sequence at
basic unit of human inheritance, I love to surprise my that location. The first of these therapies are now
students with information on just how highly similar we entering clinical trials. Come learn more about how
all are at the level of DNA—but how in some cases, very this technology works!
small differences can lead to a fatal disease state. We • Immunotherapy: the term “immunotherapy” covers
then discuss how advances in next generation whole a wide range of topics, including vaccinations,
genome sequencing technologies are enabling us to monoclonal antibody therapeutics, checkpoint
better identify disease specific mutations, and how this inhibitor therapies, CAR-T, and antibodies that
information is already being used to develop better deliver toxic compounds directly to cancer
diagnostics and drugs. In the immunology section, I cells. During Tuesday’s talk, I’ll give a detailed
give an overview of how our immune system works and overview of how each of these different types of
explain how biotech companies have leveraged this immunotherapies work.
knowledge to develop cutting-edge therapies.
Please join me on June 20th, from 10:45am-12:15pm at
This is a BIO2017 affiliate event and registration is the San Diego Convention Center in room 10. This talk is
now closed because we have sold out. However, we

only open to BIO 2017 full registrants. FYI: Last year the and whether targeting it is likely to be both safe and
room was filled to capacity and many were turned away effective. Target validation is a very important step in the
at the door. Get there early and get your seat! drug discovery process, since research and development
gets progressively more expensive—if a drug is unlikely
NO SCIENCE SINCE HIGH to be successful, millions of dollars can be saved if this is
realized early on.
SCHOOL? WE CAN HELP!
Target validation will most often include cell-based
assays (in vitro testing) and animal models (in vivo
testing). Since the goal of many therapeutic interventions
is to inhibit the activity of the selected target, many
validation assays attempt to measure the effects of
inhibition. In some cases, a selected target may play a
role in disease progression – but if it is inhibited, another
cellular protein will simply take its place, nullifying
the potential therapeutic effect of an inhibitor. In
other cases, inhibiting a selected target may have the
desired therapeutic effect—halting cancer cell growth,
for example—but may also result in unexpected side
effects, such as the death of healthy cells.
One of the most popular ways of testing the effects of
inhibition in cell-based assays is through the use of RNAi,
described in detail in the chapter “From Gene to Protein.”
RNAi is an effective way to quickly determine the results
of blocking the production of a particular protein, thus
Biotech Primer has published a book titled The Biotech
mimicking the effects of a strong inhibitor.
Primer: An insider’s guide to the biotech and pharma
industry—though some refer to it as “Biotech for If the cell models show promise, the researchers will
Dummies.” The 200-page book explains the science move on to animal models, most likely designing
behind the biotech industry, and includes a glossary of experiments using so-called “knockout” mice—
commonly used terms. Similar to our classes, The Biotech mice in which a particular gene has been disrupted.
Primer starts out with the fundamentals of biology used Researchers can ask similar questions to those asked
by researchers and progresses to how those basics are in the cell model, but on the scale of the whole animal:
employed to create therapeutics. The text is written to do the experimental mice still get cancer, Parkinson’s
be understood by all — even those who have not taken disease, diabetes, or heart disease when the target
a science class since high school. The illustrations and gene is silenced or absent? The animal model also
cocktail fodder (so you can impress your friends at your provides valuable information about targeting safety
next party) keep things interesting. Stop by booth #5225 that might not be addressed in cell models because it is
to pick up your free copy! possible to examine the effects of gene targeting on the
whole organism.
Excerpted below are a few paragraphs from Chapter 8:
The Science of Discovery:
COCKTAIL FODDER
VALIDATING THE TARGET Proteins are easily broken down by the body’s digestive
system, therefore patients receiving biologics (large
Once a potential drug target has been identified,
molecule drugs) do not take them orally, but rather
researchers will try to validate the target by determining
as injections.
whether the target plays a key role in the disease process

Cell Signaling Explained
UNDERSTANDING THE DISEASE PROCESS Another class of membrane proteins that aid in cellular
communication is channel proteins. These proteins
Greetings from BIO 2017! It’s been a busy week here at
act as molecular gates that allow the passage of small
the BIO International Convention here in San Diego,
molecules and ions, for example, glucose and sodium,
CA. At our convention booth, this year’s giveaway
across the membrane in response to a stimulus, such
was our book written especially for non-scientists:
as an electrical current in the case of ions or insulin
The Biotech Primer: An insider’s guide to the biotech and
signaling in the case of glucose. In neurons, ion transport
pharma industry. If you weren’t at the convention to
between cells serves as a principle means of signal
stop by and get a copy, please enjoy this excerpt below
transduction. The influx of calcium ions (Ca++) into a
on cellular communication – a topic fundamental to
neuron results in the release of neurotransmitters –
understanding both normal and diseased cellular
chemical messengers specific to the nervous system.
processes, and a process that is modulated by many
Different types of neurotransmitters regulate a variety
drugs on the market today.
of brain functions, including muscular activity, memory
and learning, and mood regulation.
CELLULAR COMMUNICATION
In multicellular organisms, cells must communicate
with each other. Since cells don’t have mouths, ears, or
access to email, they must rely on chemical messengers.
A chemical message – for example, a hormone – is
released by one cell, and received by a second cell – the
target cell. The target cell receives the message through
proteins inserted into its membrane known as receptors
– proteins that control the passage of molecules and the
flow of information across the membrane. When the
signaling protein binds its receptor, the receptor changes
shape and transduces (converts from one form to
another) the chemical message across the membrane to
the cell interior. This process of cellular communication
is known as signal transduction. The most common
end result of signal transduction, and a key step in cell
decision making, is the switching on, or off, of protein
production – more commonly called gene expression.
The regulation of blood sugar levels by the

protein hormone insulin is an example of cellular

communication. After you eat, beta cells in your receptors for epidermal growth factor (EGF), while nerve
pancreas sense increased blood glucose and respond by cells express receptors for nerve growth factor (NGF).
releasing insulin into the bloodstream. Insulin molecules
attach to specific insulin receptors on muscle cells and,
in doing so, deliver a signal to the inside of the muscle
cell to send glucose channels to the membrane, resulting
in glucose uptake. In this way blood glucose levels are
kept constant.
CELL SIGNALING: A CLOSER LOOK

Some cells send signals while others receive signals, but
most cells do both. The signals are chemical hormones,
such as adrenaline, or proteins, such as insulin. They
are produced within specialized cells (the signaling cell)
and released to find their target cells. The signal is often
called a ligand. In some cases, the signaling cell and After receiving the initial growth factor signal, the
target cell may be the same cell. The target cell may be enzymatic activity of the internal portion of the growth
in direct contact with the signaling cell, or it may be in a factor receptor is activated. The particular type of
different part of the body and receive a signal that has activity switched on is protein kinase activity – or the
been transported through the bloodstream. ability to transfer a phosphate group from one molecule
to another. These types of receptors are sometimes
referred to as receptor tyrosine kinases (RTKs), because
they selectively transfer phosphate groups to the amino
acid tyrosine on the recipient protein. This transfer, in
turn, causes a slight shape change in the protein which
received the phosphate group, typically leading to the
activation of that protein’s own kinase activity. This
newly activated protein kinase then goes on to activate
yet another kinase protein, and so on, in what is referred
to as a signal transduction cascade. The last element in
this cascade to be phosphorylated is typically a protein
called a transcription factor. Once phosphorylated, the
transcription factor enters the nucleus, where it binds to
the DNA at a particular location, activating expression of
Alternatively, the signal and target may be in close
a specific gene.
proximity and the signal can be transported by diffusion
through the intracellular space. After receiving a signal,
the target cell responds in a manner that is determined
by the nature of the signal received.
GROWTH FACTOR SIGNALING

Growth factors are proteins that signal a cell to multiply.
For instance, epidermal growth factor (EGF) stimulates
the proliferation of skin cells during wound repair.
Cells are constantly exposed to many different growth
factors, and the particular ones they respond to depends
on their cell surface receptors. Skin cells, as well cells
covering the gut, lung and breast, have or express

Defects in the growth factor signaling process are
associated with different types of cancer. A major
challenge in oncology lies in understanding the
complex signaling pathways that trigger cell division
and determining what has gone wrong in each type of
cancer. Once these signaling pathways are understood,
it is possible to develop targeted therapies for the
particular cancer.

Breast Cancer Subtypes
FOUR MOLECULAR VARIANTS EXPLAINED Kadcyla, also made by Genentech, is an antibody-drug
conjugate — a monoclonal antibody that delivers a
Hearing your doctor utter the words HER2-positive,
highly toxic drug directly to HER2+ breast cancer cells.
HR-positive, triple-negative, or BRCA mutation can be
Kadcyla binds the HER2 receptor like Herceptin, but
devastating — even for the most resilient person. Simply
also delivers a toxic payload (which is actually attached
put, breast cancer is a complex disease. A diagnosis can
to the monoclonal antibody). As a normal part of the
be derived from any combination of the factors listed
cell’s lifecycle, cell-surface receptors get internalized or
above — or, none at all.
“taken up” by the cell on a regular basis. When Kadcyla
The National Cancer Institute (Bethesda, MD) has is attached to a receptor that gets internalized, the
outlined four molecular subtypes of the disease. toxic payload is released from the antibody and kills the
Each subtype is categorized by the cancer’s hormone cancer cell internally.
receptor (HR) status and the level of expression from
the HER2 gene. These cellular distinctions lead patients
HR-POSITIVE
on different treatment journeys because the cancer
subtype determines the drugs used in a treatment plan. About 70% of breast cancer diagnoses involve a
significant number of receptors for either estrogen or
In this WEEKLY, we present a quick primer on the science
progesterone, making them hormone receptor positive
behind HER2-positive, HR-positive, triple-negative, and
(HR+). HR+ cancers may respond positively to treatments
the BRCA gene.
that block either the action or the production of
estrogen. In some cases, these treatments may continue
HER2-POSITIVE to be used for up to five years after initial treatment in
HER2-positive (HER2+) breast cancer patients — about order to prevent recurrence.
20% of all breast cancer cases — have the most highly Two common types of medication for HR-positive breast
effective therapies available on the market. HER2+ cancers are Tamoxifen and aromatase inhibitors. Both
cancer cells produce, and therefore present, larger than types of drugs may also be prescribed as a preventive
normal numbers of the HER2 receptors on their cell treatment in women who are at high risk for breast
surface. These HER2 receptors capture growth factors, cancer. In fact, Tamoxifen is named on the World
which trigger the cell to grow and reproduce more Health Organization’s List of Essential Medicines, a list
rapidly than normal. Mutations are more likely with rapid of the most important medications needed in a basic
reproduction and thus, a tumor is born. healthcare system.
The overexpression of the HER2 receptor is a result Tamoxifen works by inhibiting the estrogen receptor
of having extra copies of the HER2 gene — known in and was originally discovered by AstraZeneca (London,
the world of genomics as gene amplification. Gene U.K.). On the other hand, aromatase inhibitors block
amplification events are thought to be caused by the production of estrogen by inhibiting an enzyme
mutations that occur after a person is born — it is not an whose activity is required for estrogen production. The
inherited form of cancer. different aromatase inhibitors on the market include
Genentech’s (South San Francisco, CA) Herceptin is Arimidex (AstraZeneca), Femara (Novartis; Basel,
a monoclonal antibody that binds to and blocks the Switzerland, and Aromasin (Pfizer; New York, NY).
activity of the HER2 receptor on cancer cells. When the Selective estrogen receptor degraders (SERDs) are drugs
HER2 receptor is blocked, the HER2 growth factor can no that bind to estrogen receptors and cause them to be
longer bind and send a growth signal to the cell, so the degraded. Fewer estrogen receptors mean that the cells
cancer cells stop dividing. The presence of an antibody receive growth signals from estrogen. Currently, there is
on the surface of HER2+ breast cancer cells also signals only one selective estrogen receptor degrader approved
the patient’s immune system to attack that cell. — Faslodex, marketed by AstraZeneca. A second SERD,

Elacestrant, is in Phase I clinical testing by Radius Health repair. Testing positive for BRCA1/2 mutations may
(Waltham, MA). indicate there is an accumulation of DNA damage, which
Another new class of therapies for estrogen-receptor may eventually lead to cancer. BRCA is normally active in
positive breast cancer are small molecule inhibitors of breast and ovarian cells, which is why certain mutations
cellular enzymes known as cyclin-dependent kinases in BRCA1/2 are associated with a significantly increased
(CDKs). CDKs promote the development and division risk of developing breast or ovarian cancer. It must be
of cancer cells and inhibiting CDKs help to arrest stressed that BRCA1/2 mutations in and of themselves
cancer growth. do not cause cancer; they simply make it more likely
to occur.
The first CDK inhibitor, Ibrance (Pfizer) was approved in
2015. Kisqali (Novartis) was approved in March of 2017, A new class of drugs known as PARP1 inhibitors gives
and Eli Lilly’s (Indianapolis, Indiana) Abemaciclib is in hope to women whose breast cancer is associated
Phase III clinical development. with non-functioning BRCA genes. PARP1 is a second
type of DNA repair protein. By inhibiting this pathway,
DNA damage becomes so extensive that the cancer
TRIPLE-NEGATIVE
cells commit “cell suicide” (or apoptosis.) When the
Triple-negative breast cancers lack receptors — they cell in question is a cancerous cell, apoptosis is a very
are estrogen-receptor negative, progesterone-receptor good outcome.
negative, and HER2-negative. Since there are no receptor
The first FDA approved PARP1 inhibitor drug, Lynparza
drug targets, this subtype is challenging to treat and
(AstraZeneca) was approved for BRCA associated ovarian
there are no targeted therapeutics to date. If detected
cancer in December 2014. Clovis Oncology (Boulder, CO)
early enough, triple-negative breast cancer may respond
achieved the second FDA-approval of a PARP1 inhibitor,
well to chemotherapy.
Rubraca, in December 2016, and Tesaro (Waltham, MA)
garnered the most recent approval in March 2017.
THE BRCA GENE
Not all triple-negative breast cancers are BRCA
BRCA stands for “BReast CAncer susceptibility gene” associated, but many BRCA associated cancers are triple-
and everyone has the BRCA 1 and BRCA 2 genes. The job negative. For this reason, triple-negative breast cancer
of BRCA is to scan cellular DNA for damage and trigger patients may find hope in PARP1 inhibitor drugs.
DNA repair processes when mutations are found. BRCA
Breast cancer is a complex disease, and a better
genes are passed down from one generation to the next
understanding of its molecular causes has enabled
— a good thing, unless the version passed down is a
researchers to develop more effective therapies. As our
mutated variation.
understanding of the disease continues, we can expect
Mutated BRCA1/2 genes are non-functioning, so they to see additional novel therapeutics.
cannot locate DNA damage, nor can they enlist DNA

Stopping A Big Problem:
Blood Clots
MEDS FOR THINNER BLOOD CAN another part of the body, where it may interfere with
EQUAL FEWER CLOTS, BUT HOW? blood circulation and impair organ function. If this
occurs in a major organ such as the lungs, brain, or
The FDA’s recent approval of Portola Pharmaceuticals’ heart, critical injury or death may result. The clots may
(South San Francisco, CA) new blood thinner drug also grow to a size large enough to block the flow of
Bevyxxa paved the way for the prevention of blood clots blood in the blood vessel in which it originally developed.
in patients hospitalized for conditions such as heart Risk factors for VTE may be acquired (including older age,
failure, stroke, and pulmonary disease. The medical term major surgery, prolonged immobilization, certain type
for blood clot is venous thromboembolism (VTE), but if we of cancers, pregnancy and hormonal contraceptives)
take it apart: or inherited.
• ”venous” means relating to a vein or the veins.
• ”thrombo” is a blood clot. MECHANISM OF ACTION: BEVYXXA
• ”embolism” involves the lodging of an embolus, Bevyxxa and other drugs that prevent the formation
a blockage-causing piece of material, inside of a of blood clots belong to a class of drugs called
blood vessel. anticoagulants which thin the blood. Bevyxxa works
by directly inhibiting one of the key clotting factors,
Hospitalized patients are at high risk for VTE because
Factor Xa. This differs from older anticoagulants such
of their restricted mobility — not being able to move
as warfarin that works by inhibiting Vitamin K, which is
causes blood to pool and collect in the body. An
required for complete activation of clotting factors.
especially dangerous type of VTE is deep vein thrombosis
— blockage of a vein that is deep within the body, as Some key benefits of direct Factor Xa inhibition include
opposed to near the surface of the body. If a portion of faster onset, less interaction with other medicines or
a deep vein thrombosis breaks off, it may travel to the certain foods, and fewer bleeding events observed
lungs, causing a potentially fatal pulmonary embolism. during clinical trials, leading to a better safety profile.
Bevyxxa is the first oral Factor Xa inhibitor to be
An estimated 24 million people are hospitalized annually
approved, and has been approved for use for up to 42
due to VTE, so let’s find out how blood clotting is
days. These attributes mean Bevyxxa can be prescribed
activated and learn the science of Bevyxxa.
to a patient to continue taking the anticoagulant after
release from the hospital.
A CLOT IN THE DARK
Patients were selected for treatment with Bevyxxa
In healthy people, blood clotting is activated when based on increased levels of “D-dimers” in their blood.
tissue or a blood vessel is damaged, and involves D-dimers are degradation products of fibrin, the key
specialized blood cells known as platelets — also known protein component of blood clots. When our body
as thrombocytes. Either type of aforementioned damage breaks down blood clots, D-dimers are produced. Thus,
results in activating platelets, which then form an initial having higher than normal blood levels of D-dimers is a
“plug” at the site of injury. At the same time, proteins sign that higher levels of blood clots are present.
known as clotting factors are also activated. Clotting
factors work together to produce a protein called fibrin,
AN ANTICOAGULANT U-TURN
which is a fiber-like protein that forms a network of
strands that, together with the platelets, form a clot Anticoagulant drugs can be life-saving; however
at the site of injury. Clot formation in response to their inhibitory effects may need to be reversed due
injury prevents excessive bleeding and enables healing to major bleeding, or in the case of an emergency
to begin. surgery. Portola’s andexanet alfa, currently in late stage
development, reverses Factor Xa inhibition. Andexanet
VTE occurs when blood clots form in the absence of
alfa works by irreversibly binding Bevyxxa, preventing
an injury. These clots may break free and migrate to
it from binding clotting Factor Xa. If Bevyxxa can’t

interact with and inhibit Factor Xa, it no longer prevents Foods that we eat may also impact blood clotting ability.
blood clotting. For example, foods high in vitamin E such as almonds
and hazelnuts, as well as spices such as cayenne pepper,
COCKTAIL FODDER: BLOOD garlic, ginger, and onion have some natural anticoagulant
effects, while foods high in vitamin K such as leafy
THINNERS IN THE WILD
green vegetables, egg yolk, and soybeans may promote
Ever wonder how mosquitos and ticks are able to keep coagulation. For most healthy people, the relatively
the blood flowing from their point of attack until they’ve small amounts of these foods consumed in a normal diet
had their fill? It turns out that their saliva contains a would not have a significant impact on blood clotting;
natural anticoagulant which prevents platelets from however, those on anticoagulant medicines may want to
being activated. Fortunately, the effect is only temporary consult their physician about any possible dietary impact
and localized to the site of the insect bite. on their medicine’s efficacy.

Unpacking Digital Medicine
CAN APPS PROGRAM BETTER HEALTH? diagnosed with the disease has quadrupled over the
past 35 years, from 5 million to 20 million (Centers for
Digital medicine is defined by the field’s pioneer Dr. Eric
Disease Control).
Tool of the Scripps Translational Science Institute
(La Jolla, CA) as “the ability to digitize human beings, WellDoc’s (Columbia, MD) BlueStar smartphone-based
by a variety of means (sequencing, sensors, imaging, app is the first FDA-cleared mobile prescription therapy.
etc.), fully exploiting our digital infrastructure of ever- The app allows users to enter data including levels of
increasing bandwidth, connectivity, social networking, blood glucose, carbohydrates consumed, medications
the Internet of all things, and health information taken, exercise amount, hours of sleep, and perceived
systems.” This new field is changing the way diseases stress levels. BlueStar then makes personalized
from diabetes to substance abuse are prevented recommendations regarding diet, exercise, and
and treated. medication, and even pinpoints the best times of day for
the patient to test blood glucose levels. This information
“Digitizing human beings” may sound impersonal — but
is easily shared with a physician.
in fact the opposite is true. By enabling better access
to individual health data, patients and physicians can In clinical testing, patients assigned the BlueStar
create truly personalized health management plans. In app showed an average 1.9% drop in glycosylated
this WEEKLY, we’ll take a look at this emerging biotech hemoglobin (HbA1C) levels when compared to patients
sector and the companies leading the way into the land treated according to the current standard of care and
of digital medicine. no continuous intervention app. HbA1C is a reflection of
average blood glucose levels over the past three months.
THERE’S AN APP FOR THAT Patients must receive a doctor’s prescription for the
BlueStar app.
There is a wealth of potential to delay the onset of
diseases confronting our population, and a number Another leader in digital therapeutics diabetes
of companies are developing digital medicine apps — management is Omada Health (San Francisco, CA),
similar to the ones you have already downloaded onto whose interactive behavioral intervention program
your phone such as your favorite music streaming app. reduces the development of Type 2 diabetes in
Most of today’s digital medicine falls under the heading prediabetics through personal coaching via the
of “medication augmentation” — interventions meant to integration of web, mobile, and smart devices. The goal
supplement rather than replace medication. is to help patients lose weight and increase physical
activity. Although not FDA-approved, the platform is
Chronic diseases such as diabetes can be managed much recognized by the Centers for Disease Control as an
more effectively with “continuous intervention” — a effective diabetes prevention tool.
day-to-day monitoring of the patient’s lifestyle choices
and medication compliance. In many cases, these apps
CHRONIC RESPIRATORY DISEASE
are highly sophisticated, clinically validated, and FDA-
approved. The disease areas most commonly being In some cases, digital health companies are partnering
tackled include Type 2 diabetes, chronic respiratory with pharma companies to ensure better use of a
disease, chronic cardiovascular conditions, and mental therapeutic drug. Propeller Health (Madison, WI) has
health conditions. joined forces with GlaxoSmithKline for a “digitally
guided therapy” platform for use with inhalers to treat
Let’s unpack some of the digital medicine coming to an
asthma and chronic obstructive pulmonary disease
app store near you.
(COPD).
DIABETES The platform consists of sensors, provided by Propeller,

that are attached to patients’ inhalers; a smartphone
Diabetes management is a prime target for digital app for patient use; a website for physician use and
medicine intervention, as the number of people data from a network of air-quality sensors. Propeller

monitors inhaler use, tracking patients over time and with a clinician-facing web interface. It is designed to
providing data on disease management as it relates to deliver behavioral therapy through a series of learning
environmental factors (air quality). The Propeller system modules. The goal is to keep patients interested and
has four FDA clearances which allow the company to engaged in their treatment between therapist visits. The
claim the system can be used to increase medication apps have been clinically tested in five different trials.
adherence, predict exacerbations, and reduce the Patients who received reSET treatment in addition to
frequency of symptoms and exacerbations in asthma standard addiction therapy showed better rates of drug
and COPD. abstinence. The app has been submitted to the FDA
for approval.
HEART DISEASE Big Health (San Francisco, CA) has developed a digital
No discussion of digital medicine would be complete therapy, Sleepio, to help with insomnia. The app consists
without a nod to the legendary story of Dr. Eric of cognitive behavior therapy (CBT)-based exercises
Topol using AliveCor’s (Mountain View, CA) mobile delivered by an online, animated therapist dubbed
electrocardiography (ECG) device and app to diagnose The Prof. When tested against an online version that
a heart attack mid-flight. Dr. Topol was actually using a included interaction with the Prof but lacked CBT-based
prototype model during his in-flight diagnosis; today, the activities, Sleepio was more effective at helping 75% of
Kardia Mobile device — essentially two sensor pads — is the participants to fall asleep.
FDA-cleared and available for $99. After downloading the
accompanying smartphone app, users can get an ECG THE FUTURE
reading in 30 seconds by opening the app on a phone The companies and apps described here are really
placed nearby and placing their fingertips on the sensor just the tip of the iceberg of this new therapeutic
pads. An irregular reading indicates possible atrial world. Additional disorders for which digital therapies
fibrillation, potentially indicating a heart attack. are being developed include obesity, hypertension,
hyperlipidemia, smoking cessation, chronic pain,
ADDICTION & SLEEP coronary artery disease, and even serious mental
Pear Therapeutics (Boston, MA) is tackling substance illnesses such as schizophrenia. The field is still in its
abuse disorders with its digital therapy. Their lead infancy, but the dramatic benefit already seen by many
product, reSET, is a smartphone app for patients adopters suggests a bright future.

Chaperoning The Rare
Disease Dance
PROPERLY FOLDING MISFOLDED cause the enzyme to be misfolded and therefore non-
DISEASE PROTEINS functional.
Amicus Therapeutics (Cranbury, NJ) found itself in The only Fabry’s disease treatment on the market in
the news earlier this month when the FDA agreed to the U.S. is Fabrazyme, which is made by Genzyme
review the company’s new drug application for their (Cambridge, MA). Fabrazyme is an enzyme-replacement
investigational therapy to treat Fabry’s disease. The therapy: since the patients don’t make enough functional
drug under consideration, migalastat, has already galactosidase enzyme, scientists produce it in the lab
been approved by the European Medicines Agency. using cells that have been genetically engineered to
It belongs to a small, but growing class of therapeutics produce the enzyme, which is then purified and injected
known as pharmacological chaperones that properly fold into patients.
improperly folded proteins that cause disease.
MECHANISM OF ACTION: MIGALASTAT
Let’s take a look at which chaperones are on the dance
floor and find out the steps they are taking to treat Amicus’ drug migalastat, if approved, would be the first
disease caused by proteins. small molecule treatment for Fabry’s. The potential
availability of swallowing a drug (vs. injecting) would give
TERM OF THE WEEK: CHAPERONE PROTEIN those with Fabry’s another drug delivery option.
Chaperone proteins are proteins that assist in the correct In the lab, migalastat binds to and inhibits galactosidase.
folding and assembly of other proteins. Many of the In the body, this high affinity is taken advantage of by
proteins produced by our cells require chaperone migalastat binding to mutated galactosidase during
proteins to ensure their correct molecular structure. the process of folding, where it then shifts the folding
towards the correct conformation. The now correctly
A pharmacological chaperone is a small molecule drug folded protein makes its way to a cellular compartment
that targets specific misfolded proteins and encourages known as the lysosome, where it carries out its job of
them to fold correctly. digesting lipids. The inside of the lysosome has an acidic
Protein misfolding plays a role in many different rare pH, which causes migalastat to disassociate, leaving
diseases, including enzyme deficiencies like Fabry’s behind a functional galactosidase enzyme for the body
and the related Niemann-Pick disease, as well as to pick up and use.
Huntington’s disease, and some cases of amyotrophic Fabry’s is caused by a variety of different mutations
lateral sclerosis (ALS). Some of the mutations in the within the galactosidase gene; not all of them are
genetic disease cystic fibrosis (CF) involve misfolded amenable to treatment with migalastat. Amicus
proteins. Diseases caused by misfolded proteins scientists estimate that between 35% to 50% of patients
that disrupt cellular function are sometimes called will be responsive to migalastat.
proteopathies, where proteo = protein, pathy = disease.
MECHANISM OF ACTION: LUMACAFTOR

MECHANISM OF ACTION: FABRAZYME
Another disease that can be traced to protein misfolding
A type of lysosomal storage disorder, Fabry’s disease is cystic fibrosis. CF is a genetic disease caused by one
involves the inability to process certain types of lipids of several possible mutations in the gene encoding the
(fats), because they lack functional versions of critical “cystic fibrosis transmembrane conductance regulator”
enzymes, resulting in a range of symptoms, including (CTFR) protein. The CTFR protein is critical for the
kidney, heart, and skin disorders. The enzyme in production of sweat, digestive fluids, and mucus.
question here, galactosidase, helps to break down
glycolipids — lipids with a carbohydrate attached. The most common mutation, responsible for about
Production of functional galactosidase enzyme is limited two-thirds of CF cases, results in a protein that is so
because of mutations in the galactosidase gene that misfolded, it never makes it to the cell surface where it is
required to do its job. Vertex Pharmaceuticals’ (Boston,

MA) drug lumacaftor serves as a pharmacological disease; and Niemann-Pick disease, a lysosomal storage
chaperone for these proteins, assisting them with disorder similar to Fabry’s disease.
correct folding so that they can make it to the cell Chaperone Therapeutics has a drug in preclinical
surface. Lumacaftor is one piece of the CF puzzle; it is development for Huntington’s disease, which
often used in combination with other therapies to fight is associated with disordered folding of the
various aspects of the disease. huntingtin protein.
MORE DANCE CHAPERONES COCKTAIL FODDER: SHOCKING

Instead of creating pharmacological chaperones, THE CHAPERONE
another approach to getting mutated proteins to fold
The largest family of naturally-occurring chaperone
correctly is to stimulate diseased cells to produce greater
proteins are called “heat shock proteins” because they
amounts of natural chaperone proteins. This can be
were first discovered as part of a cellular response
done by identifying small molecules that induce cells to
to heat shock — exposure to a higher than normal
express heat shock proteins, a common class of cellular
temperature. These proteins were later discovered to
chaperones (described below). Two companies following
be induced in response to other types of cellular stress
this approach are Orphazyme (Copenhagen, Denmark)
such as ultraviolet light exposure or wound healing.
and Chaperone Therapeutics (Research Triangle Park,
It’s thought that these cellular stressors can disrupt
NC).
protein folding, and the production of heat shock protein
Orphazyme’s lead product, arimoclomal, has completed chaperones can help to counteract the disruption.
Phase II clinical testing for amyotrophic lateral
Pharmacological chaperones that activate or mimic
sclerosis (ALS) associated with mutations in the gene
these protective proteins may prove to be the fresh new
for superoxide dismutase 1 (SOD1) enzyme; sporadic
approach that can make a difference in a whole range of
inclusion body myositis (sIBM), a rare muscular atrophy
different diseases.

Putting The CAR-T
Before The Horse
THE STORY BEHIND CAR-T WHY CAR-T?
The hottest cancer therapy in the pipeline — chimeric In theory our immune system should recognize the
antigen receptor therapy (CAR-T) — got a big boost unique proteins presented on all diseased/cancerous
last month when an FDA advisory panel unanimously cells; however there are two main reasons this doesn’t
recommended approval of the treatment for children always happen:
and young adults with a severe form of leukemia who • Early on in the tumor development, the cell
have run out of other options. Developed by Novartis composition is similar enough to healthy tissue that
(Basel, Switzerland), this elegant hack of the immune it can be overlooked by the immune system.
system is one of many horses in the race for a FDA
approval, with Kite Pharmaceuticals (Los Angeles, CA) • Later as a tumor progresses, it releases chemical
and Juno Therapeutics (Seattle, WA) rounding out the signals that suppress the immune response, helping
pack. Let’s take a moment to review these revolutionary it to evade detection. This trickery is known as
therapeutics and understand how they attack cancer. the tumor microenvironment and once again the
dangerous cancer cells can pass by undetected.
TERM OF THE WEEK: KILLER T-CELLS So what’s a scientist to do?! Figure out a way to train
killer T-cells to ALWAYS recognize and destroy cancer
CAR-T therapy is modeled after a cell in the immune
cells… enter CAR-T.
system known as the killer T-cell. The job of a killer T-cell
is exactly what the name implies — to kill dangerous
cells. They target diseased cells in the body via their HOW TO TRAIN AN IMMUNE SYSTEM
receptors: each one has a uniquely shaped receptor, Killer T-cells are “trained” to go after early and late stage
and will recognize its intended target because the shape cancer by having their physical structure altered. This
of its receptor “matches” or fits into a uniquely shaped alteration is accomplished by fusing an antibody with the
surface protein found only on diseased cells. Once the receptor of a killer T-cell to create a chimeric molecule —
Killer T-cell “docks” onto its target, it injects an enzyme or the “C” in CAR-T.
which triggers death. The result: no more bad cells. Training day begins by having killer T-cells drawn out of
a patient’s body and isolated in the lab. Next, scientists
deliver a gene to the T-cells that encodes the chimeric
receptor. This receptor consists of two parts:
• A targeting domain: This is the part of the chimeric
receptor that will be outside of the T-cell. It is
composed of an antibody that will recognize
and dock onto a unique surface protein of the
patient’s cancer.
• An activation domain: This part of the receptor will be
triggered once the targeting domain is engaged. It
will signal to the killer T-cell to:
1. Stay alive.
2. Make copies of itself.
3. Release signaling molecules called cytokines.
Cytokines are chemical signals that activate
other white blood cells to join the fight against
the tumor.
4. Kill the target cell.

The T-cell/antibody hybrid is now a CAR-T therapeutic.
It is then multiplied in the lab and infused back into the WHAT’S IN A NAME?
patient’s body. Once inside, the CAR-T locks onto its Chimeric antigen receptor therapy broken down:
cancer target, replicates, sends out cytokines, and kills
• Chimeric: Composed of components from two
the designated cancer cells. The CAR-T will continue
distinct parts, such as an antibody and a killer T-cell.
to replicate and kill any and all cancer cells recognized
by the initial antibody component, with the goal of • Antigen: A protein that is recognized by an antibody,
eliminating the disease. such as a protein on the surface of a tumor cell.
• Receptor: A protein that is embedded in a cell
membrane and transmits signals to itself in response
to being activated, for example a T-cell receptor
transmits signals to the T-cell when it docks onto
its target.
• Therapy: A treatment meant to manage or cure
a disease.
As these therapies begin to move from clinical trials
into clinical practice, the treatment of cancer will truly
be revolutionized, offering new hope to patients and
their families.

The Science Of CRISPR/Cas9
CRISPR/CAS9 UPDATE In the mid-2000’s, scientists realized the “spacers”
matched DNA sequences of invading viruses — the
As CRISPR/Cas9 adds new indications to its resume,
bacteria were storing away bits of invading viral DNA
legal battles over its IP continue to be waged in the US
between its own bacterial CRISPR sequences! These
and Europe.
bits of viral DNA create a “genetic memory” of the virus,
On the clinical front, CRISPR/Cas9 entered its first enabling the bacteria to fight back if reinfected.
human trial at Sichuan University (Chengdu, China) last
Reinfection triggers the following steps:
fall for metastatic lung cancer, and is widely expected to
do so in the U.S. by the end of the year. This past March, • Viral DNA present in the spacer sequences is copied
a team of scientists at Oregon Health and Science into viral RNA.
University (Portland, OR) announced that they had • The DNA-cutting enzyme Cas is made, and
successfully edited a gene linked to severe heart defects attaches itself to the viral RNA produced from the
in human embryos. spacer sequence.
On the patent front, CRISPR technology and its • This newly minted viral RNA/Cas complex finds its
applications were discovered by two different research “match” on the invading viral DNA.
teams, one at University of California, Berkeley, and
• The Cas enzyme is now positioned to cut up viral
another at the Broad Institute (Cambridge, MA). Both
DNA, destroying the invading virus.
have filed patents on various aspects of the CRISPR/Cas9
system. The Broad Institute had granted an exclusive
license to Editas Medicine (Cambridge, MA), while USE IN HUMANS
Berkeley had granted licenses to Caribou Biosciences In 2013, researchers adapted this bacterial defense for
(Berkeley, CA), CRISPR Therapeutics (Basel, Switzerland use in human cells. Human cells were engineered to
and Cambridge, MA), Intellia Therapeutics(Cambridge, contain both specially-designed RNA and Cas genes.
MA), and ERS Genomics (Dublin, Ireland). In February, When these human cells produce the RNA/Cas complex,
the U.S. Patent Office ruled in favor of the Broad the dynamic duo is ferried to its complementary DNA
Institute and its licensee, while in March the European target. Once in position, Cas goes to work cutting the
Patent Office ruled in favor of U.C. Berkeley patents. DNA. The particular Cas protein chosen for this work was
The legal battle is certainly far from over. one discovered in Streptococcus bacteria, Cas9 — hence
With all of these new developments making waves in the the moniker CRISPR/Cas9.
industry, let’s review the basics. The ability to cut human DNA in precise locations is an
exciting innovation because of what the cell does next.
CAS TO THE RESCUE
CRISPR was originally discovered as a key component of BREAKING & FIXING
the bacterial immune response. Bacteria, like people, are Cas9 creates double-stranded breaks (DSB) in the
plagued by viral infections, and bacteria have evolved specified DNA sequence. DSBs cut both strands of the
clever ways to attack invading viruses. In the 1980’s, double-stranded DNA helix. Think of DNA as a two-lane
scientists observed an interesting pattern in bacterial bridge that, after experiencing an earthquake, has a
genomes: repeating, palindromic sequences, with unique section break off and fall into the water below.
sequences referred to as “spacers” between the repeats.
DSBs activate two repair pathways to fix the break in
They dubbed these regions a tongue twister of a name,
the DNA:
“clustered regularly interspaced short palindromic
repeats,” or CRISPR. Scientists also noticed CRISPR • Non-Homologous End-Joining (NHEJ) closes the gap
sequences were always located near a gene that coded between the break by joining the two sections
for an enzyme that cut DNA. This enzyme became known back together—visualize pushing the two sides of
as Cas, short for “CRISPR-associated”. the bridge together, leaving the fallen section in

the water. An unintended byproduct of NHEJ is the The PD-1 gene produces the PD-1 protein, which is
possibility of sequence error, much like the sections located on the T-cell’s surface. When the PD-1 protein
of the bridge not lining up properly. If the repair is activated, the T-cell doesn’t function. When the PD-1
occurs in the middle of a gene, the minor error can protein is deactivated, the T-cell functions. Aggressive
be enough to disrupt gene function and halt the cancers take advantage of this on/off switch turning PD-1
production of the corresponding protein. on, effectively shutting down the T-cell. By turning PD-1
• Homology Directed Repair (HDR) relies on a highly off, the T-cells can’t be suppressed—freeing them up to
similar (homologous) DNA segment to repair the attack cancer cells.
break—visualize the missing bridge section built
elsewhere and helicoptered in to fill the break. COMING SOON
By engineering double-stranded breaks to occur at A U.S. clinical trial of CRISPR to disrupt PD-1 in T-cells is
specific locations, scientists activate the NHEJ or HDR expected to begin before the end of 2017. This two-year
cell repair pathways. By activating the NHEJ pathway, study is funded by the Parker Institute (San Francisco,
scientists can disrupt a disease-associated gene, CA).
preventing the production of a protein that causes A number of private companies also have plans for
the disease. By activating the HDR pathway, a short CRISPR/Cas9 clinical trials that include both gene
sequence of DNA is delivered with CRISPR/Cas9 to disruption and gene correction. The table below
correct the mutated sequence, perhaps allowing a summarizes some key players in the genome-editing
missing protein that causes disease to be made. In both arena and their approaches to applying CRISPR. In vivo
scenarios cures for many different types of diseases may means the therapy will take place inside the human
be realized. and Ex vivo means the treatment will be performed in
cells taken from the body and then injected back into
the patient.
CRISPR IN THE CLINIC

A clinical trial for metastatic lung cancer, initiated last As these and other potential treatments move through
fall by Chinese researchers at Sichuan University use clinical trials, the world will be watching to see if this
CRISPR/Cas9 to disable the PD-1 gene in T-cells. revolutionary technology will live up to the hype and
change the way we prevent and treat disease.

Harnessing Your Immune
System For Good
YOUR INNER IMMUNE WORKINGS bacteria and virus based on a special receptor called a
PAMP (Pathogen-Associated Molecular Pattern) found
What do monoclonal antibodies, CAR-T therapy, and
only on the bad ones. Other non-specific defenders
checkpoint inhibitor treatments all have in common?
include neutrophils (which also recognize the PAMP and
They are immunotherapies, or therapies that activate
engulf invaders) and natural killer cells, which inject the
the immune system to fight or prevent a disease. While
protein granzyme B into invaders, triggering cell death.
an activated immune system can help save a life, an
overactive immune system can attack the body it is Once activated, these non-specific defenders release
charged with protecting. This over-activity is the basis for “inflammatory cytokines,” or signaling molecules
autoimmune disorders. that switch on other immune cells. The inflammatory
response is kicked into gear, ensuring a rapid and
The biotech industry has elegantly hacked the immune
comprehensive retaliation.
system — a highly complex network of signaling
molecules, cells, and tissues — to make some of the
leading immunotherapies such as Abbvie’s (North SPECIFIC IMMUNITY: T-CELLS AND B-CELLS
Chicago, IL) Humira that battles psoriasis or Merck’s When non-specific defenses are unable to rid the body
(Kenilworth, NJ) Keytruda that fights different cancers. of pathogens, it’s time for back up. Waiting for the call
Let’s discover how the immune system operates and find are T-cells and B-cells, which make up your specific (or
out how our best and brightest are applying the immune adaptive) immunity. These cells are highly specialized
approach to disease treatment. to recognize unique targets, called epitopes, thanks
to their distinctly shaped receptors. Once the B-cell or
IMMUNE SYSTEM PRIMER T-cell receptor binds to the pathogen’s epitope, they
are activated. Each T- or B-cell recognizes only one
The immune system is devoted to protecting us from
unique epitope.
foreign invaders. These include viruses, bacteria,
parasites, fungi, and even cancer cells. The immune
system consists of many different players, all working
together as a team. The first defense consists of physical
barriers, such as the skin and mucus membranes, which
attempt to thwart these pathogens from entering our
bodies in the first place. If these barriers are breached,
then our cellular defense mechanisms kick in – first in
the form of non-specific immunity, and then in the form
of specific immunity.
NON-SPECIFIC IMMUNITY:
MACROPHAGES AND NEUTROPHILS
Non-specific (or innate) immunity fends off pathogens at
the cellular level. The troops are specialized white blood
cells (WBC). Most WBC in the body are non-specific,
meaning these foot soldiers attack in the same fashion Activated T-cells divide rapidly and produce three types
without stopping to consider the specific characteristics of descendants: killer T-cells, helper T-cells, and memory
of the enemy. T-cells. All recognize the same target as the originally
Macrophages are one type of non-specific defender that activated T-cell.
freely circulate in the bloodstream. When they encounter • Killer T-cells roam the body in search of their pre-
a bug, they engulf it – essentially eating it. Macrophages programmed epitope, and seal the deal by injecting
have the ability to differentiate between good and bad granzyme B, triggering cell death.

• Don’t let the label “helper” fool you – helper T-cells cancer patient’s tumors. This compels white blood cells,
are critical, they release inflammatory cytokines that such as killer T-cells and macrophages, to attack the
activate antibody-producing B-cells, killer T-cells, tumor. Herceptin acts just like a naturally-occurring
and macrophages to respond en masse. The human antibody, only it is produced in the lab. Chimeric antigen
immunodeficiency virus (HIV) only infects helper receptor T-cell (CAR-T) therapies, which to date are still
T-cells, and in so doing completely cripples the in clinical testing, are T-cells whose receptors have been
immune response. engineered to recognize and destroy cancer.
• Memory T-cells don’t defend against an initial
infection, but if these cells encounter the same CHECKPOINTS ON THE CASE
epitope a second time, they are very quickly The body has natural checkpoints to prevent
converted into killer T-cells and helper T-cells, inflammatory disorders; this screening process stops
ensuring a rapid response. T-cells and B-cells from mistakenly killing its own tissues.
These checkpoint proteins send a “stop attacking”
signal to the T- and B-cell when they encounter their
body’s healthy cells. Many types of cancer cells have
evolved to express these checkpoint proteins, tricking
the T and B-cell into thinking the cancer is a healthy
cell. Checkpoint inhibitor therapies prevent the cancer
from activating their checkpoint proteins, enabling the
immune system to more fully go after the tumor.
THE OTHER SIDE OF THE COIN:

AUTOIMMUNE DISEASE
The immune system prevents us from falling deathly ill
as it responds to constant microbe exposure. However,
an overactive immune system can cause serious
problems, potentially leading to autoimmune disease.
Activated B-cells also reproduce rapidly and produce Chronic inflammatory disorders such as Crohn’s disease,
two types of descendants: plasma cells and memory rheumatoid arthritis, and psoriasis wreak havoc by
B-cells. activating white blood cells to target innocent cells in
the body and release inflammatory cytokines to sustain
• Plasma cells secrete antibodies – proteins that
the barrage.
recognize and bind to any bacterium or virally-
infected cell that bears a matching epitope. The Biologic drugs that treat these disorders—like Humira
binding action triggers other immune cells, such (AbbVie, North Chicago, IL), Enbrel (Amgen, Thousand
as killer T-cells or macrophages, to sweep in and Oaks, CA), and Rituxan (Genentech, South San Francisco,
destroy the invader attached to the antibody. CA)—work by shutting down key parts of the response.
Humira and Enbrel inhibit a specific inflammatory
• Like memory T-cells, memory B-cells lie in wait,
cytokine known as TNF-alpha. Both of these drugs are
preparing for future attacks instigated by the same
approved for a range of inflammatory diseases. Rituxan,
foreign invader.
approved for rheumatoid arthritis, works by reducing the
number of B-cells that target the synovial tissue of joints.
APPLIED IMMUNOLOGY
From one side of the coin to the other, the immune
Immunotherapies use strategies from specific immunity system continues to both challenge and reward the
to their advantage. Monoclonal antibody therapies are industry as new pathways and targets are discovered.
developed by selecting antibodies that recognize and A delicate balance of the body’s toughest fighters,
bind to a disease-specific epitope. The classic example, understanding and optimizing the immune system is
Genentech’s (South San Francisco, CA) therapeutic central to the immunotherapy paradigm.
antibody Herceptin, binds to the HER2 epitope which is
present at high levels on the surface of 25% of breast

The Microbiome Magnified
DECODING THE GUT-BRAIN AXIS TRAIL BLAZERS: AXIAL &
There is no shortage of microbiome-focused startups THE MAZMANIAN LAB
in biotech right now. The link between the gut Axial Biotherapeutics (Cambridge, MA) is focusing
microbiome — the entire collection of microbes living much of its initial attention on work originally done at
in the gut — and diseases such as inflammatory bowel microbiologist Sarkis Mazmanian’s lab in the California
disease are well-established. New research has made Institute of Technology (Pasadena, CA) — mostly
it clear, however, that the gut microbiome also impacts relating to the gut-brain axis’ role in Parkinson’s disease
neurological health, leading to the phrase “the gut-brain (PD) and autism.
axis.” Let’s explore this connection and examine the
Parkinson’s Disease
early efforts by a few innovative biotechs to translate
these new discoveries into the clinic. Parkinson’s disease (PD) is a chronic and progressive
movement disorder, according to the Parkinson’s
GUT MICROBIOME PRIMER Disease Foundation. Symptoms include tremor of
the hands, arms, legs, jaw, and face; slowness of
The human microbiome is the complex collection of movement; rigidity of the limbs and trunk; and impaired
microbes (mostly bacteria, but also includes small balance and coordination. These symptoms are
numbers of fungi and viruses) that reside on and caused by the malfunction and death of neurons that
inside the human body, including our skin, mouth, produce the neurotransmitter dopamine. PD affects
nose, respiratory tract, and digestive tract (gut). The nearly one million people in the U.S., and the cause is
microbiome is huge — microbial cells outnumber human unknown. About 75% of PD cases are accompanied by
cells by a ten to one ratio! Human cells are much larger gastrointestinal disorders such as constipation, which
than bacteria cells, however, so don’t worry — you’re still provided an initial impetus to examine a possible
mostly human. For every 100 pounds that you weigh, it connection between gut health and the disease.
is estimated that about two pounds of that weight come
from bacteria. A key molecular characteristic of PD is the aggregation
of a protein called alpha-synuclein (αSyn) within cells of
Most of us think of bacteria as harmful and certainly the brain and gut. Researchers in the Mazmanian lab
some types are; however, those that have evolved with used a strain of mice that overproduce αSyn to study
humans to become part of the human microbiome the disease. One group of the αSyn mice were bred in
are either neutral — causing no harm — or beneficial. a completely sterile environment to create “germ-free”
Scientists are busy trying to better understand and (GF) αSyn mice. The other αSyn mice had the normal
characterize these beneficial bacteria and the role that collection of gut bacteria. On a series of tests designed
they play in human health. to assess motor skills, the GF αSyn mice performed
The gut contains the largest number of bacteria, significantly better — suggesting that even in mice that
as well as the greatest diversity of bacteria, when overproduced the αSyn protein, the presence of certain
compared to other parts of the body. Thus much of microbes are required for the disease to progress.
the attention directed towards the human microbiome Further work suggested that a molecule called butyrate,
has been focused on the gut microbiome in particular, produced by certain gut bacteria, can enter the brain
which continues to surprise us with its influence on and activate an immune response, leading neurons to
diseases such as obesity, diabetes, and, increasingly, malfunction or die.
brain disorders. There is reason to believe that this connection is also
at work in humans with PD. In collaboration with Rush
University (Chicago, IL) gastroenterologists, Mazmanian
lab researchers transferred fecal samples from PD
patients into the GF αSyn mice. Fecal transplants are
an established way to “reset” the gut microbiome of

the recipient to make it match that of the donor. After lab demonstrated that feeding ASD mice a specific
transplantation, the mice began exhibiting symptoms of strain of bacteria called B. fragilis — a part of the
PD. Transfer of fecal matter from healthy people did not human microbiome — altered the mouse microbiome
trigger these symptoms. These experiments suggest that and reduced some of the ASD-like behaviors such as
the gut microbiome is a major contributor to the disease anxiety and repetitive behavior, and increased levels
process in PD patients. of communication with other mice. These experiments
Of course, these promising early stage findings still need suggest a possible probiotic therapy for autism.
to be translated into human therapeutics. This may be
easier than traditional neurological approaches because CARB LOADING WITH SYMBIOTIX
it is much easier to deliver drugs to the gut than to get Another early-stage company making headlines in
them to cross the blood-brain barrier. Following up with the gut-brain axis space is Symbiotix (Boston, MA).
a targeted approach to modulate the production of Focusing on multiple sclerosis (MS), their lead candidate
butyrate and other inflammatory compounds produced is a carbohydrate molecule produced by B. fragilis. This
in the gut may bring the first truly effective PD therapy therapy increases the production of regulatory T-cells,
into the clinics. which are a class of T-cells that “turn down” an overactive
Autism Spectrum Disorder immune response by releasing anti-inflammatory
signaling molecules. Symbiotix is preparing to enter
Autism spectrum disorder (ASD) is a developmental brain
clinical trials with an orally-administered product for the
disorder characterized by impaired social interaction,
treatment of MS and inflammatory bowel disease.
communication, and restrictive and repetitive behavior.
These symptoms impact a child’s educational, social, The emerging work described here gives credence to the
emotional, and physical development. More than 3.5 old expression “think with your gut.” In addition to the
million Americans live with an autism spectrum disorder diseases discussed above, scientists are discovering links
according to the Autism Society. The cause is unknown, between the gut microbiome and other brain disorders
although genetics is thought to play a role. such as anxiety and depression. As the story continues
to unfold, we are likely to see new therapeutics based on
Similar to PD, a significant portion of ASD patients
restoring the balance that millions of years of human-
exhibit gastrointestinal problems. The Mazmanian
microbe co-evolution has fine-tuned.

Vaccines: Schooling The Herd
VACCINATION NATION accumulates mutations that make it better able to
survive in a new environment, but when injected
Back to school means shopping for new school supplies,
into a human, it is no longer virulent. Attenuation
adjusting to a new schedule, and making sure all
may also be more direct and occurs when genes
required vaccinations are up to date.
associated with pathogenesis or replication are
Every state requires school-age children to be vaccinated removed in the lab. In general, attenuated vaccines
against certain infectious diseases including tetanus, induce a strong and long-lasting immune response.
hepatitis B, measles, mumps, rubella, polio, pertussis Examples of live, attenuated vaccines include
(whooping cough), and chicken pox. Vaccination policies measles, mumps, rubella, chicken pox, and polio.
are highly effective at eliminating many types of sickness
• Subunit vaccines: In some cases, just one protein
from the most perfect incubator — the classroom.
from the virus can be enough to induce an immune
In this WEEKLY, we’ll go to the chalkboard to learn the response. These are called subunit vaccines and are
basic science of vaccines. typically made using recombinant DNA techniques to
produce the desired protein. Advantages of subunit
BASIC SCIENCE vaccines include easier production and a better
The idea behind vaccination is simple: by exposing safety profile for patients. Examples of subunit
someone’s immune system to a harmless version of a vaccines include pertussis and hepatitis B.
pathogen, we can train it to recognize and respond to the • DNA vaccines: DNA vaccines are the next frontier in
bug in the wild. After an initial exposure to a virus, our vaccine development. Rather than delivering a whole
immune system creates memory cells which are then pathogen or pathogen subunit, DNA vaccines deliver
ever-ready to spring into action and attack the same just a gene. Once inside the body, the patient’s own
disease later on down the line. cells reproduce the pathogenic protein. If successful,
Vaccination is required because creating these pathogen- this technology would mimic a natural infection
specific memory cells takes a few weeks — a length of and elicit a strong immune response. The technical
time that is long enough for a virus to do serious damage challenge that remains to be solved is the delivery of
to the body. the pathogenic gene. Ichor Medical Systems (San
Diego, CA) and Inovio Pharmaceuticals (San Diego,
CA) are both developing electroporation-mediated
ABCS OF VACCINES
DNA delivery systems to solve this problem.
There are a few ways to create a vaccine and below we
list the most common methodologies. TERM OF THE WEEK: HERD IMMUNITY
• Inactivated vaccines: The most obvious type is the Herd immunity means a significant portion of a
inactivated vaccines — the use of heat or chemicals population has immunity to a particular pathogen. There
to kill the pathogen. Inactivated vaccines produce a is little opportunity for an outbreak, so even those who
dampened immune response in comparison to other cannot be vaccinated such as immunocompromised
vaccine methods and often require “booster” shots. individuals, pregnant women, or newborn babies
Inactivated vaccines include polio, influenza, and are unlikely to become infected despite their
pertussis vaccines. unprotected state.
• Live, attenuated vaccines: A live, attenuated
vaccine simply means that the weakened (not A UNIVERSAL FLU VACCINE?
killed) pathogens are unable to cause disease.
Unlike most other vaccines, you must get the flu shot
Attenuation occurs by a process called “passage,”
every year in order to be afforded protection. Current
or growing viruses at temperatures slightly lower
flu vaccines work by mounting an antibody response
than the human body or in cells different from the
against two large proteins on the surface of the virus —
human host cells. Under these conditions, the virus

hemagglutinin (H) and neuraminidase (N). The catch? The will be protective over several flu seasons. M-001 is in
structure of those two proteins changes every season Phase II clinical testing.
due to a high mutation rate. Once the structure changes, Researchers at Crucell Vaccine Institute of Janssen
the immune system no longer recognizes it, and the Pharmaceuticals (Leiden, Netherlands) have discovered
body must be retrained. This is also why the vaccine is an antibody that recognizes and binds a portion of the
only 60-70% effective — when formulating each year’s HA protein that doesn’t mutate very rapidly. Studies
vaccine, scientists attempt to predict the influenza in animals suggest treatment with this antibody
strains that will be circulating in winter, and they are significantly reduces the amount of active virus present.
seldom 100% correct. Human clinical trials are in the works.
For many years, scientists have talked of producing A clinically tested and FDA-approved universal flu
a universal flu vaccine. Recent advances in vaccine vaccine is still years away, but these early results are
technology have led to some promising developments. promising. The new vaccine would most likely need
BiondVax (Ness Ziona, Israel) has identified nine to be administered every five or 10 years, rather than
epitopes — short sequences of proteins that elicit an annually—but the real advantage will be its ability to
immune response — that do not vary much between protect against a range of different influenza strains,
different strains of the virus. These sequences were inching closer to 100% efficacy. And since most of us
combined to make one recombinant protein referred to have suffered through a bad case of the flu, we can
as Multimeric-001 (M-001). The hope is this combination all agree that a universal flu vaccine cannot come
of epitopes will invoke a strong immune response which soon enough!

Eye Of The Cytokine Storm
THE FLASH OF THE FIRST CAR-T A cytokine storm is the adverse event most associated
with CAR-T treatments and next generation CAR-T
Last week’s much anticipated FDA approval of the first
treatments are being developed to have built-in controls
chimeric antigen receptor T-cell (CAR-T) therapy for acute
to regulate cytokines so the storms can be stopped.
lymphocytic leukemia hails as the first gene therapy on
the US market.
AN ON/OFF SWITCH
Classified as a “cell-based gene therapy,” Novartis’
(Basel, Switzerland) Kymriah works by removing patients’ In first generation CAR-T, maximum activation occurs.
T-cells, using a viral vector to introduce a gene that will With a full cytokine barrage, there is no way to tamp
allow the T-cells to recognize and kill cancer, and then down the cytokine response.
infusing these modified T-cells back into the patient. In next generation CAR-T, a small molecule drug may be
Recall T-cells are found in the blood and fight disease. co-administrated with the therapy. The drug’s function
Along with its significant potential, Kymriah also carries is to activate CAR-T to fight cancer, or turn it “on.” If a
serious risks. Its approval came with a boxed warning cytokine storm ensues, the small molecule drug can be
because of the potential for “cytokine release syndrome immediately withdrawn — the “off” switch — essentially
(CRS),” also referred to as a cytokine storm, which has deactivating CAR-T and stopping cytokine release.
caused fatalities in clinical trials of other CAR-T products. This second iteration of CAR-T is made possible by a
Making CAR-T safer while maintaining efficacy are goals handful of companies who are designing drugs that
of next generation CAR-T. Let’s explore cytokine storms will act as an “on/off switch” to control CAR-T. Bellicum
and find out how scientists aim to circumvent this Pharmaceuticals (Houston, TX) is developing a CAR-T
roadblock to fighting cancer. product, BPX-601, that uses small molecule-activation.
BPX-601 entered Phase I clinical trials in February 2017.
Intrexon (Germantown, MD) has a similar product in
TERMS OF WEEK: CYTOKINE
Phase I clinical development.
& CYTOKINE STORM
Cytokines are small proteins which play an important GETTING BISPECIFIC
role in relaying messages from one cell to surrounding
Juno Therapeutics (Seattle, WA) is developing a CAR-T
cells and tissue. Cytokines serve two main functions
product that uses a sort of two-step verification process.
involving white blood cells:
It turns out that tumor cells have many proteins on their
• Activate additional white blood cells to fight surface — so it is a challenge to find a distinct protein
off pathogens that is also unique to any given cancer cell. Instead of
• Stimulate white blood cells to move towards sites relying upon finding that one special protein, why not
of inflammation target a more common one and use another protein to
double check the work?
Cytokine signaling makes for a very quick and strong
immune response. Usually, the response is kept in check, Juno’s approach: bispecific chimeric antigen receptors.
and dissipates when the bad cells have been eliminated. This means each engineered killer T-cell has not one, but
two chimeric antigen receptors (CARs).
However, in some cases, this positive feedback loop —
activated cells releasing still more activating cytokines • One CAR is activated in the presence of a protein
— spins out of control, resulting in a cytokine storm. found on the surface of cancer cells. Once activated
Acute inflammation with accompanying symptoms such this CAR-T cell would produce more copies of itself,
as high fever, swelling, and nausea can occur. In severe release cytokines, and attack the tumor.
cases, serious tissue damage and death can result — for • A second CAR called an inhibitory CAR (iCAR), is
example, lung failure induced by excessive amounts of activated in the presence of a different protein found
fluids and cells moving into the lungs. only on healthy cells — NOT on cancer cells. If an

iCAR is activated, an inhibitory signal is sent to the the cancer cell, where it interacts with other proteins in a
first CAR, preventing the CAR-T from working. cytokine signaling pathway.
Simply put, one CAR finds the target protein while the Scientists at the Blood Research Institute of Blood
other iCAR verifies the cell is “unhealthy” via another Center of Wisconsin (Milwaukee, WI) have developed
protein. This bispecific CAR-T, now in preclinical decoy molecules that interfere with the protein-protein
development, aims to eliminate the off-target effects interactions in the pathway. These decoy molecules are
and decrease the amount of cytokines released. short protein fragments called peptides. By binding to
proteins in the cytokine pathway, the signal to produce
more cytokines is blocked.
OXYGEN DEPRIVED
Earlier this year, Cellectis (Paris, France) published a The decoy peptides reduce cytokine production by
paper describing work they’ve done to engineer CAR-Ts ~70%, which is likely enough to prevent the immune
with an oxygen-sensitive domain. response from spinning out of control. In the words of
Laura Savatski of the Blood Research Institute, “By
Under normal cellular conditions, this domain signals the
reducing cytokine production by CAR-T cells, you prevent
CAR-T to remain inactive. Under low oxygen, or hypoxic
a cytokine storm from happening. So instead of dealing
conditions, this domain sends an activation signal to
with a problem at the back end of the therapy, you solve
the CAR-T. Since most solid tumors have a hypoxic
it at the front end through intelligent design.” Decoy
environment, an oxygen-sensitive CAR-T should be
molecules are currently in preclinical development.
activated within the tumor but not outside of it.
With some of the best minds in the biopharma industry
working on CAR-T design, this landmark FDA approval is
INTELLIGENT DESIGN THROUGH DECOYS
likely to be just the first shot in a treatment revolution.
It turns out that CAR-T activation results in cytokine
production because part of the CAR-T is actually inside

The Multiple Myeloma Landscape
BLOOD CANCER: MULTIPLE MYELOMA reabsorbing cells, osteoclasts, is increased. The resultant
damage to the bone structure creates soft spots or
Plasma cells are the antibody-producing cells of our
lesions which may extend from the inner bone marrow
immune system which happen to play a critical role in
to the outside surface of the bone. Bone lesions result
our defense against infections. In multiple myeloma,
in significant pain and increase the risk of fracture. Bone
plasma cells begin to grow and divide in an uncontrolled
destruction also releases excessive calcium into the
manner, forming a cancerous mass known as a
bloodstream, leading to a range of symptoms including
plasmacytoma. Marrow — which produces plasma — no
confusion, nausea, and loss of appetite. Excess blood
longer functions in our defense, it simply takes up space
calcium, combined with high levels of M protein, also
inside the bone.
contributes to impaired kidney function seen in multiple
What does biotech have in store to fight multiple myeloma patients.
myeloma? Let’s find out the treatments on the market
and the up-and-comers in development.
UNMASKING MULTIPLE MYELOMA
There is no one diagnostic test for multiple myeloma.
EASILY CONFUSED: PLASMA
Blood and urine tests to detect some of the symptoms
CELLS VS BLOOD PLASMA listed above such as low blood cell counts, elevated
Plasma cells are specialized white blood cells that blood calcium levels, and impaired kidney function may
produce infection-fighting antibody proteins. Most suggest multiple myeloma. These tests are followed by a
plasma cells are found in the bone marrow. bone marrow biopsy for confirmation.
Blood plasma is the straw-colored liquid component Most cases of multiple myeloma have no known cause,
of blood that holds blood cells in suspension, made although some research suggests that regular exposure
up of water (95%), proteins, glucose, clotting factors, to herbicides, insecticides, petroleum products, heavy
electrolytes, hormones, carbon dioxide, and oxygen. metals, and asbestos increases the risk of developing
the disease. And although there is not a specific gene
PICKING APART PLASMACYTOMA yet associated with multiple myeloma, abnormalities in
chromosome structure or number are associated with
Plasmacytoma formation can lead to a host of
the disease.
problems with recognizable clinical symptoms. Instead
of producing normal disease-fighting antibodies,
plasmacytoma cells produce abnormal antibodies called ON THE MARKET
M proteins, which don’t provide any benefit to the body Once considered incurable, there are now a number of
and crowd out normally functioning antibodies. And effective treatments for multiple myeloma, and several
because all blood cells are formed in the bone marrow, more are in the pipeline.
overproduction of plasma cells can also crowd out Darzalex ( Johnson & Johnson; New Brunswick, NJ)
normal blood-forming cells. This can lead to anemia, and Empliciti (Bristol Myers Squibb; Princeton, NJ) are
caused by a shortage of oxygen-carrying red blood both monoclonal antibody therapeutics approved to
cells; increased bruising and bleeding due to a reduction treat multiple myeloma. They work by recognizing and
in clot-promoting platelets; and an increased risk of binding to proteins on the surface of multiple myeloma
infection due to lower levels of healthy infection-fighting cells, activating the patient’s immune system to destroy
white blood cells. those cells.
Although multiple myeloma is classified as a blood Ninlaro (Takeda; Osaka, Japan) is a small molecule
cancer, it has a significant impact on bone health. proteasome inhibitor therapy. A proteasome is a
As the plasmacytoma grows, bone-forming cells specialized compartment within the cell that gets rid of
called osteoblasts are suppressed. At the same damaged proteins by digesting them. If the proteasome
time, production of a substance that activates bone- is inhibited, damaged proteins build up within the cell.

This triggers a process called apoptosis — essentially, Selinexor helps to increase the number of tumor
cell suicide. In other words, the cancer cell kills itself. suppressor proteins present in the nucleus of cancer
Farydak (Novartis; Basel, Switzerland) is a small cells. These proteins help to protect against cancer by
molecule “histone deacetylase (HDAC) inhibitor.” HDACs detecting DNA damage and promoting apoptosis in
are enzymes that modify chromosomes (strands of those cells that have high levels of DNA damage. In many
DNA that contain our genes) and influence how often types of cancer cells, tumor suppressor proteins are
specific genes are activated. Some cases of multiple transported out of the nucleus, where they can no longer
myeloma are associated with changes in gene activation. do their job of detecting DNA damage. By blocking this
By inhibiting HDACs, Farydak can correct this changed transport, Selinexor enables tumor suppressor proteins
gene expression. to do their job of triggering apoptosis is cancer cells.
Selinexor began Phase III clinical testing for myeloma in
June 2017.
IN THE PIPELINE
CAR-T therapies are also in development for multiple
Two novel drugs in the multiple myeloma pipeline are
myeloma. Bluebird Bio (Cambridge, MA), in partnership
Mivebresib (AbbVie; North Chicago, IL) and Selinexor
with Celgene (Summit, NJ), and Nanjing Legend Biotech
(Karyopharm Therapeutics; Newton, MA).
(Nanjing, China) have announced promising results in
Similar to Farydak, Mivebresib influences the activation early phase CAR-T trials for multiple myeloma.
of specific genes by inhibiting a group of proteins
Multiple myeloma is a complex cancer. In recent years,
called Bromodomain and Extra Terminal motif (BET)
a better understanding of the disease has led to the
proteins. In some types of cancer, genes are activated
approval of several new therapeutics. In the coming
or deactivated inappropriately due to BET activity. By
years, we can look forward to additional approvals as
inhibiting BET, Mivebresib may restore normal gene
novel therapeutics move through the pipeline.
activity to these cells. Mivebresib is currently in Phase I
clinical testing for multiple myeloma.

From Fantasy To Reality:
Xenotransplantation
TRANSPLANTING ORGANS FROM • Preventing immune rejection: Immunosuppressive
ANIMALS INTO HUMANS drugs need to be given at a higher dose for
xenotransplant recipients than for those
Every ten minutes, a new person is added to the national receiving organs from human donors. High
transplant waiting list. A little more than 75,000 people doses of immunosuppressive drugs can lead to
are active waiting list candidates — meaning they are serious infections.
medically eligible for transplantation according to the
Organ Procurement and Transplantation Network. • Lingering concerns that viral genes present within
Over the past decade, the gap between organ supply and animal genomes, which do not harm the animal,
demand has continued to grow; an estimated 20 people could be harmful to humans.
a day die as they wait for an organ transplant. Today, pigs are considered to be the best potential
The idea of xenotransplantation — transplanting organs animal organ donor for humans, due to their availability
from animal donors to humans — has been the subject and the size of their organs closely matches human
of discussion for decades in science circles. Now, with organ size. Let’s see what biotech is doing to address the
the tools provided by modern biotech, we may soon two remaining challenges.
see this seemingly science-fiction idea become a life-
saving reality. Let’s explore the past, present, and future THE FUTURE
of xenotransplantation. Cambridge, MA-based eGenesis is using CRISPR/Cas9
genome editing to tackle the problems of immune
THE PAST rejection and potential interspecies virus transfer —
The dream of xenotransplantation has been around referred to as “porcine endogenous retroviruses (PERVs)”
for over a century. In 1905, a French scientist inserted in pigs. CRISPR can be used to “knockout” (remove)
portions of a rabbit’s kidney into a child suffering from undesirable genes, or to “knock-in” (add) desirable
kidney failure. The patient’s kidney function improved; gene sequences.
however long-term follow-up was not possible as the Last month, eGenesis scientists published a paper in
child died of pneumonia within a few weeks. In the which they described their successful use of CRISPR
early 1960s, a small group of critically ill patients at genome editing combined with a cloning technique
Tulane University received kidney transplants from called “somatic cell nuclear transfer” to produce dozens
chimpanzees. None of these patients survived long- of healthy pigs whose genomes no longer contain PERVs.
term, and the establishment of working cadaver organ The pigs were produced using the following steps:
procurement programs put xenotransplantation on the • Use CRISPR to inactivate PERV sequences present in
back burner until 1984 when “Baby Fae” captured the an adult pig’s DNA.
public’s imagination as the first infant to receive not
only a heart transplant, but a xenotransplant, when she • Transfer the modified DNA into a pig egg whose own
received a baboon heart. The transplant operation itself nucleus has been removed. This technique is known
was successful; however, the baboon heart was rejected as somatic cell nuclear transfer.
by Baby Fae’s immune system 21 days after surgery. • The egg, now containing a PERV-free pig genome,
is stimulated with an electric shock to trigger
THE PRESENT cell division.
A few key challenges have prevented xenografts from • After several rounds of cell division, an early-stage
solving the problem of organ shortages for patients in embryo is formed, which can then be transferred
need. They include: into a surrogate mother to complete development.
• Determining the correct animal donor, taking into This was the first time that as many as 25 genes had
account both anatomical and ethical considerations. been simultaneously and precisely deactivated with

CRISPR — a significant accomplishment and a major step Genomics (La Jolla, CA), in collaboration with United
towards making xenotransplants safe. Therapeutics (Silver Spring, MD), is also working on
More work still needs to be done, however, before pig- modifying pig genomes to make their organs safe for
to-human heart transplants are routine. In addition to human transplant.
deleting PERV genes, scientists will need to deactivate We are still several years away from making
pig genes that help to trigger immune rejection and add xenotransplants a routine clinical reality. The next
in gene sequences to help the human immune system step will be testing genetically modified pig hearts in
recognize the transplant as safe. baboons. But with two of the leading genomics pioneers
As for the cloned pigs, the next step will be to see if the behind eGenesis and Synthetic Genomic — George
changes made to their genome will be transmitted to Church and Craig Ventor, respectively, this is certainly
subsequent generations because breeding pigs are much an area to keep close tabs on.
easier than cloning them. And speaking of pig breeding,
Smithfield Foods (Smithfield, VA) — better known for COCKTAIL FODDER: ORGANS FOR SALE?
producing pork products for consumption — has thrown The only place one may buy an organ legally is in the
its hat in the ring by establishing a bioscience unit to be country of Iran; however, citizenship is required in order
used for supplying pig parts for medical use, ultimately to lessen transplant tourism. Australia and Singapore
including organs for xenotransplantation. have legalized monetary compensation for living organ
eGenesis is not the only company using genome donors, in order to cover associated medical expenses
editing in the pursuit of xenotransplants. Synthetic and compensate for time of work.

Picturing Disease
USING MEDICAL IMAGING TO gamma rays, can be created, manipulated, and
INVESTIGATE DISEASE detected for various applications, including medical
imaging technology.
Medical imaging — using various modalities to take a
snapshot of the body’s interior structure — has been
CT VS. PET
around since 1895, with the discovery of X-rays by
Wilhelm Roentgen. Medical X-rays, as described above, work by beaming
electromagnetic radiation at the body from an outside
X-rays are a type of electromagnetic radiation (more on
source (the X-ray machine). “Positron emission
that later!) that are able to pass through soft tissues such
tomography” — a PET scan — in contrast, assesses
as skin, fat, and muscle — but not bone. When an X-ray
bodily function from the inside out. A radioactive tracer
beam is aimed at the body, it passes through the soft
is injected and seeps through the body and attaches
tissue but is blocked by the bone, casting a shadow to
itself to certain tissues, then the gamma rays emitted by
create an X-ray image on a piece of film. The introduction
the tracer are detected by the PET scan. The radioactive
of X-rays revolutionized medicine, making it possible
tracer (also called a radiopharmaceutical) consists of a
to accurately diagnose broken bones and to identify
“carrier molecule” that is bonded tightly to a radioactive
the location of harmful objects such as bullets inside of
atom. The carrier molecule used has an affinity for the
patients’ bodies.
part of the body to be imaged.
Newer technologies such as PET and CT scans
CT scans — “computerized tomography” — are
supplement X-rays, aiding in the diagnosis and
essentially 3D X-rays. X-rays are taken from many
management of a disease. Let’s find out how
different angles, creating cross-sectional images or
these pictures are worth much, much more than a
“slices.” These cross-sections are then processed by a
thousand words.
computer to produce a 3D anatomical image. A contrast
agent — which is used to enhance the body’s structure
TERM OF THE WEEK: or fluids — is typically given to patients prior to a CT
ELECTROMAGNETIC SPECTRUM scan, making the soft tissues denser, therefore enabling
Electromagnetic radiation is a combination of electric the agent to block X-rays and thus become visible in
and magnetic fields from various wavelengths that move the CT.
through space and carry energy. The electromagnetic This combination of both types of tomography — the
spectrum encompasses the following: use of waves to penetrate the body to create images — is
very useful when information about both metabolism
(PET) and anatomy (CT) are required to study disease.
PARKINSON’S & ALZHEIMER’S

Radioactively labeled glucose — fluorodeoxyglucose
(FDG) — is widely used for metabolic imaging studies in
PET scans because they detect deficits in brain activity
that may show signs of neurodegenerative diseases such
as Alzheimer’s or Parkinson’s.
Since all types of tissue use glucose for energy, increased
uptake of glucose-loving FDG indicates more metabolic
activity. In Alzheimer’s or Parkinson’s disease, a brain
We can only see a small portion of the electromagnetic
PET scan that uses FDG as the tracer may detect
spectrum — visible light. The remaining types of energy,
lower than normal FDG consumption in certain brain
from low frequency radio waves to high frequency
areas, meaning there is a reduction of metabolic

activity in those regions — a possible indication of the (Basel, Switzerland; Phase I), and Janssen (Raritan, NJ;
early neurodegeneration. preclinical). Challenges that must be overcome when
Although FDG-based PET scans have emerged as a developing new radiopharmaceuticals for targets inside
useful tool in neurodegenerative disease, they are of the brain include developing an agent that will cross
not capable of clearly differentiating one type of the blood-brain barrier and be extremely target-specific.
neurodegeneration from another. To do that, disease-
specific radiopharmaceutical detection agents have DETECTING CANCER
been developed. Tumor cells consume glucose at a much higher rate
DaTSCAN (GE Healthcare; Chicago, IL) is a (approximately 200 times higher) than healthy cells.
radiopharmaceutical that binds to cells in the brain that This difference makes FDG-based PET scans a reliable
release dopamine, a chemical messenger. Loss of this method for detecting and monitoring cancer as follows:
type of brain cell is associated with Parkinson’s disease • After the body is injected, tumor cells consume the
(PD). PET imaging with DaTSCAN enables physicians to radiolabeled glucose (FDG) much more rapidly than
visualize the loss of dopamine-associated neurons, and non-tumor cells.
to diagnose PD.
• The PET machine detects the gamma rays emitted
Preclinical efforts are now being directed at the by FDG.
development of radiopharmaceuticals for the detection
• Areas of high activity are referred to as “hotspots”
of alpha-synuclein, a protein that is elevated in the brains
and may indicate the presence of a tumor.
of PD patients. Alpha-synuclein elevation is thought to
occur earlier in PD progression than dopamine signaling PET scans may be used as part of an initial diagnosis,
irregularities; thus, the ability to detect it could enable and as a way to monitor treatment. Tumors shrink in
earlier diagnosis and intervention. This is considered response to therapy while the spreading of cancer to
such an important goal in the field that in 2016, the other parts of the body can point to metastases.
Michael J. Fox Foundation announced a $2 million
dollar award to the first team that successfully develops NOT SO RADIOACTIVE
a PET tracer for the visualization of α-synuclein. Leaders
The prospect of being injected with a radioactively-
in this race include AC Immune (Lausanne, Switzerland)
labeled imaging agent may give some patients pause.
and ICBI International (La Jolla, CA).
However, the total radiation dose received by that
Three radiopharmaceuticals — Vizamyl (GE Healthcare), patient is equivalent to the exposure caused by two
NeuraCeq (Piramal Imaging; Berlin, Germany) and routine chest X-rays according to the National Institute
Amyvid (Eli Lilly; Indianapolis, IN) — have been approved of Biomedical Imaging and Bioengineering. This is due
for the detection of beta-amyloid protein plaques in largely to the rapid decay of the radioactive label used
the brain, which could indicate the presence of the for PET tracers. Within a few hours of administration,
Alzheimer’s disease (AD). They are approved for clinical there is no detectable radioactivity in the patient’s
use in PET scans, however they are not considered to be body. Patients are encouraged to drink plenty of
stand-alone diagnostics. In other words, a negative scan fluids after a PET scan to aid in flushing any remaining
may be used to rule out AD, but a positive result is not radiopharmaceutical out of the body.
enough on its own to diagnose.
The ability to see inside of a patient’s body, directly
The second type of abnormal protein cluster found in measuring metabolic activity or anatomical structure, is
AD patients’ brains are tau proteins. PET tracers for critical to detecting, monitoring, and treating diseases.
the detection of tau are in development by several With new discoveries in the field of imaging we can hope
companies, including AC Immune (Phase I), Piramal to catch signs of disease sooner rather than later.
Imaging (Phase I), Merck (Kenilworth, NJ; Phase I), Roche

Circadian Rhythm & Disease
AND THE BEAT GOES ON traveling through several time zones, even if they get an
adequate amount of sleep. These tiny molecular clocks
Earlier this week, the 2017 Nobel Prize in Physiology
are signaling that it is later (or earlier) than the local
or Medicine was awarded to three American scientists
time—and a whole range of physiological functions from
( Jeffrey Hall and Michael Rosbash, of Brandeis
body temperature to heart rate, blood pressure, and
University, and Michael Young, of Rockefeller
alertness levels respond accordingly.
University) for their work in deciphering the molecular
basis of circadian rhythm – the 24-hour cycle that Clock proteins are regulated by a “master clock” in the
governs the inner workings of all life on Earth. Although brain. Technically referred to as the suprachiasmatic
the work that garnered the Prize was done over twenty nucleus, this master clock consists of about 20,000 nerve
years ago, its implications to human disease and new cells just above the region where the optic nerve crosses
therapies are still being worked out today. In this issue of into the brain. This location, called the optic chiasm,
the Weekly, we’ll take a look at this Nobel science. explains why many of these neurons are sensitive
to light.
CIRCADIAN RHYTHM GOVERNS
MORE THAN 24 HOURS ON THE MARKET: HETLIOZ
Overnight shift workers, students pulling an all-nighter People who are totally blind cannot receive the proper
to cram for a final exam, and business people rushing light input to control their master clock. As a result,
between time zones all share one thing in common: many suffer from non-24-hour sleep-wake disorder. It
significant disruption to their circadian rhythm. This becomes virtually impossible to fall asleep at standard
roughly 24-hour activity cycle responds primarily to light times, which severely impacts their ability to function
and darkness and is found in most living organisms— professionally and socially.
people, plants, animals, and even some microbes. In July 2014, the FDA approved Vanda
Disruption in the cycle can cause more serious Pharmaceuticals’(Washington, DC) Hetlioz for patients
consequences on top of a few days of disorientation. with non-24-hour sleep-wake disorder. Hetlioz is an
Abnormal circadian rhythms are correlated with agonist; it binds directly to and activates the melatonin
insomnia, diabetes, increased cardiovascular events, receptor. Melatonin is a sleep-inducing hormone with
some types of cancer, Parkinson’s disease, and more. levels increasing at the onset of darkness, linking it to
the sleep-wake cycle. Since melatonin production is
In this issue, we’ll take a closer look at how circadian dysregulated in totally blind people, Hetlioz aims to
rhythms are regulated, examine some links with various normalize sleep patterns. Although melatonin itself is
disease states, and find out how drug discovery efforts available in pill or liquid form over the counter, synthetic
are taking this important phenomenon into account. melatonin receptor activators such as Hetlioz are
designed to be more stable.
PROTEINS ON THE CLOCK
The circadian rhythm is regulated by a combination of IN DEVELOPMENT: RESET YOUR PRESET
environmental and internal cues. Environmental cues are Aptly-named Reset Therapeutics (South San Francisco,
based on light and dark, while internal cues are simply CA) is focusing on circadian-rhythm disruptions as they
“biological clocks.” These biological clocks are groups of relate to a whole range of diseases including type 2
interacting proteins in cells throughout the body. diabetes, Cushing’s syndrome (elevated cortisol levels),
The most extensively characterized biological clocks are high blood pressure, obesity, sleep apnea, cancer,
appropriately dubbed Clock proteins. The Clock proteins and inflammation. Scientists at Reset have identified
interact with each other and regulate levels of expression potential circadian-modulating compounds and are
of other proteins throughout an approximately 24-hour testing them in animal models of Cushing’s syndrome
period. This is why most people feel disoriented after and type 2 diabetes.

PRECLINICAL: SHUTTING DOWN AM and 3:00 AM. In people with high blood pressure,
THE CANCER CLOCK however, this drop often doesn’t occur naturally. By
taking blood pressure medication at night, patients can
A possible link between circadian rhythm dysfunction restore this normal decrease in pressure.
and cancer has caught the eye of drug development. One
of the functions of the Clock proteins is to set restrictions Dosage timing may also be important for certain types
on when cells can divide, so circadian disruptions may of cancer drugs. For example, PARP1 inhibitors work
affect cancer-related cell division. Researchers at the by shutting down a DNA repair enzyme in cancer
University of California, Santa Cruz are zeroing in on cells. The shutdown results in so much DNA damage
a protein known as PASD1, which is expressed in many to the cells that they initiate a cell-suicide program
different cancer cell lines. PASD1 interacts with the Clock known as apoptosis—the cancer cells pretty much end
proteins, essentially interfering with their function and up killing themselves! Researchers at University of
shutting them down. Early preclinical work suggests North Carolina (Chapel Hill, NC) have discovered that
that the inhibition of PASD1 causes the Clock proteins to DNA repair enzymes are more active later in the day,
reactivate—making PASD1 a possible drug target. translating into PARP1 inhibitors possibly being more
effective if given in the morning; inhibiting the enzymes
should be easier when they are not at their peak
TIMING IS EVERYTHING
activity level.
Taking medicine at the same time every day is a good
Circadian rhythms govern more than the 24 hours of
way to make sure a dose isn’t forgotten. For certain
your day, and uncovering various mechanisms at the
medications, however, the time of day the pill is
cellular level of the sleep-wake cycle might just open up
swallowed may influence drug efficacy.
new avenues to treat a whole array of diseases.
In most healthy people, blood pressure is tied to
circadian rhythm, naturally dropping between 12:00

Vaccines: Powerful Simplicity
VACCINES: ELEGANT, COCKTAIL FODDER: 40,000 SAVED!
POWERFUL SIMPLICITY According to the CDC, flu season runs from October
Anyone who’s suffered the aches and fever of influenza through May in the US. The organization’s latest study
has good reason to value the simple flu shot. In fact, estimates that the flu vaccine saved 40,000 lives in the
millions roll up their sleeves and literally take their US during a nine-year period.
medicine. The US Centers for Disease Control (CDC)
(Atlanta, GA) estimates that about 146 million doses TYPES OF VACCINES
of influenza vaccine went to doctors’ offices, health Vaccines come in different varieties including: inactivated
departments, and the corner drugstore among whole, live attenuated, and subunit vaccines. Each
other places, to help keep us flu-free during 2016-17. necessitates different manufacturing requirements.
Drug companies keep up with this high demand by
manufacturing these vaccines well in advance—six to Inactivated whole vaccine. Made with dead
nine months before the start of flu season in October. microorganisms (viruses or bacteria), these stimulate an
immune response. Among the most famous is Dr. Jonas
The influenza vaccine is one of many life-saving Salk’s polio vaccine, developed in 1955.
vaccines that keep people healthy. This week, we look
at different types of vaccines and how drug companies Live, attenuated (weakened) vaccine. These are
manufacture them. created by reducing a pathogen’s strength so that they
become harmless. Live vaccines tend to produce the
strongest immune reaction.
JENNER’S NEEDLE IN THE HAYSTACK
Subunit vaccine. These vaccines use only one part of a
Vaccines have been around a long time—dating from the
pathogen, an antigen. The antigen provokes an immune
late 19th century during the smallpox pandemic. This
response. One method of making subunit vaccines
deadly disease killed or disabled hundreds of thousands
involves isolating a specific protein from a virus and
of people just in England! In 1796, an English doctor,
administering only this protein.
Edward Jenner, noticed that local milkmaids were
immune to smallpox. Jenner observed that these women Scientists are also currently developing DNA-based
had all suffered from cowpox, a related but harmless vaccines. These consist of a gene encoding a pathogenic
virus. Jenner hypothesized that the cowpox imparted protein as opposed to the protein itself.
some type of immunity, so to test his theory Jenner took The Whole Story
pus from a cowpox blister and inoculated his gardener’s
Most whole pathogen vaccines protect against viruses,
small son, James, with the virus through shallow
such as rabies, not bacteria. But making any vaccine
scratches. James developed a slight fever afterward, but
means first growing lots of virus. This “virus-farming”
when intentionally exposed to smallpox later, the little
involves selecting and obtaining a strain of a particular
boy never became ill.
virus, the seed strain, and choosing what to grow it in
Jenner’s methods were a little rough (not to mention (the medium).
unethical by today’s standards), but his thinking was
Where do manufacturers buy a pathogen in the first
spot on. The idea behind vaccines is simple. First expose
place? They come from one of two sources. Viruses
someone to a harmless version of a disease-causing
(and other microorganisms and biological materials)
microorganism, or pathogen. Amazingly, this “trains”
are produced and housed in well established “culture
their immune system to recognize and fight the germ.
collections,” such as the American Type Culture
Exposure to the disease forces the body to create special
Collection (Manassas, VA.) Some companies or
white blood cells, known as memory cells, which combat
academic institutions also develop strains of particular
any further exposure to the disease.
viruses “in-house.” For the flu vaccine, new strains
are selected annually based on the World Health

Organization’s assessment of the virus’s evolution over more of themselves.The last step in making vaccines
the previous season. is formulation. The inactivated virus gets mixed into
Choosing the seed strain is only the first step of vaccine a sterile water or salt solution along with stabilizers
manufacture. Although dangerous and often deadly, and preservatives. Some vaccines also contain
viruses are powerless without a host—someone or adjuvants at this point. An adjuvant is a substance
something to grow on. Two of the most common “host- that boosts the immune response to a vaccine. Vials
cell platforms” are chicken eggs and animal cell culture. are then filled, inspected, labeled, and shipped. Most
vaccines require refrigerated storage and shipping.
Chicken eggs? Yes, the incredible edible egg provides a
fantastic growth medium for influenza and other viruses.
ALIVE, NOT KICKING
(Side note: The CDC recommends that most people with
egg allergies be vaccinated. However, it also suggests Producing a live, attenuated vaccine follows similar
that those with severe, life-threatening allergies receive steps, without inactivation. In addition, it starts with a
only certain vaccines.) seed strain that has been rendered harmless. The new
organism continues to grow but produces immunity
Some viruses thrive in certain types of animal cells.
without causing illness. Attenuated vaccines produce
Two of the most commonly used in vaccine production
stronger, usually longer-lasting, immune responses than
include one from the kidney of the African green
inactivated vaccines because they more closely mimic
monkey—known as Vero cells, and one derived from the
actual infection. Attenuated vaccines should not be given
kidney cells of a cocker spaniel (MDCK cells). Though it is
to people with weakened immune systems, such as
easier and quicker to scale up animal cell culture vaccine
cancer patients or the elderly.
production, it is much more expensive than egg-based
vaccine growth. Safe, attenuated vaccine strains are produced in a few
different ways. Sometimes, it may simply be a related,
Regardless of medium, once there’s enough virus, the
but harmless virus that kicks in the immune response.
manufacturer needs to separate or isolate it from the
Jenner’s vaccine, which was essentially the cowpox
host material. Isolation involves centrifuging and filtering
virus, is a classic example. Today’s smallpox vaccine
to divide virus particles from the host cells.
uses a related virus, vaccinia. Another common method
Production of whole pathogen or inactivated vaccines to produce vaccines is raising several generations of
involves the critical step of inactivation. This means a clinical isolate, or a laboratory-pure version of a
disabling a virus’s ability to infect without eliminating pathogen. Growing in non-human cells, it adapts to its
the parts of the virus that trigger an immune response. new host, becoming less infectious over time. Examples
Inactivation involves a variety of strategies, including include the measles, yellow fever, and poliovirus vaccine
detergent treatment, heat treatment, or exposure to strains. Scientists can also use recombinant DNA
UV light. technology to delete the portions of a virus’s genome
• Detergent-treated: Specific for envelope viruses, that cause infection.
detergents break the chemical bonds that hold the Vaccines have a simple premise, but the science and
virus’s envelop (outside surface) together disabling manufacturing that make them possible is complex.
its ability to invade a host. Different microorganisms require individual approaches.
• Heat, chemical, and pH-treated: Viruses use Through trial and error, microbiologists, virologists, and
proteins on their surface to infect host cells. Altering other scientists determine the best formulation.
their shape destroys their ability to recognize and In part two of this series, we’ll discuss other kinds
infect cells. of vaccines, including virus-like particle (VLP),
• Ultraviolet light-treated: A virus’s basic building polysaccharide, and further discuss subunit vaccines.
blocks—their DNA or RNA—are destroyed by UV We’ll also give an overview of how vaccines are tested
light. With no genetic code, viruses cannot make and approved.

DNA Vaccines Explained
MORE ON THE POWERFUL, ELEGANT Sometimes a viral subunit or subunits form what’s called
SIMPLICITY OF VACCINES a virus-like particle (VLP)–a protein structure whose
shape closely mimics a virus with none of its genetic
Last week, we overviewed vaccine development material. In these cases, the body’s immune system
and manufacture, focusing on those that use whole responds very robustly.
pathogens to protect us from a disease. This week, we
examine subunit and polysaccharide vaccines, which use A subunit vaccine can also derive from toxins produced
different strategies to fight infection. We also take a brief by dangerous bacteria. For example, Clostridium tetani
look at the US Food and Drug Administration (FDA)’s (tetanus) secretes tetanospasmin, a neurotoxin that
vaccine approval process. causes severe muscle spasms, potentially leading to
death. The vaccine contains inactivated toxin, which
helps develop antibodies that prevent future illness.
A PART IS SOMETIMES GREATER
Because subunit vaccines contain none of a pathogen’s
THAN THE WHOLE
genetic code, they are generally very safe.
The structure of viral cells is much simpler than that of
our own cells. Despite the damage they can do, viruses
CARBS+PROTEIN=IMMUNITY (SOMETIMES)
consist only of one or more strands of DNA or RNA,
encased in a protein shell such as in the viruses you In general, bacterial infections tend to be more difficult
see below. to protect against by vaccine than viral infections.
That’s because the surface of some bacteria is covered
in long chains of carbohydrate molecules. Called
polysaccharides, they mask the bacteria’s proteins. This
“cloaking device” means the body doesn’t recognize the
threat and mount an immune response. But molecular
biologists and other scientists have discovered that it’s
possible to link polysaccharides to a harmless protein,
thereby coaxing an immune response. These vaccines
are known as conjugated polysaccharide vaccines because
the carbohydrate is conjugated (connected) to a protein.
The Haemophilus influenzae type B (or Hib) and some
types of pneumococcal and meningococcal vaccines are
made this way.
This simple structure means that sometimes only part,
or subunit, of a virus is enough to stave off infection.
GETTING DOWN TO THE ESSENTIALS
Different subunit vaccines use different bits of a
pathogen. Often, they consist of nothing more than The basics of immunization have been around over a
one of a virus’s surface protein. Subunit vaccines work century—use a disease-causing microbe, or just a part,
because our immune systems recognize and respond against itself. However, the latest step in the evolution of
readily to these surface proteins. vaccines takes a different tack, delving more deeply into
the building blocks of life—DNA.
For subunit vaccines, drug manufacturers alter yeast,
bacteria, or Chinese hamster ovary (CHO) cells to Instead of immunizing someone with a whole pathogen
produce a protein by transferring the gene encoding the or fragment, a DNA vaccine injects only a small bit of a
virus into them. These host cells make the viral protein, virus’s genetic code. Drug companies nestle the code
which the manufacturer then isolates and formulates in plasmids— small, circular DNA molecules within a
into the vaccine. Subunit vaccines include those for pathogen. As you can see below, the “visiting” DNA
hepatitis B and human papillomavirus (HPV). prompts the host to produce the target viral protein and

consequent immune response within their own cells, but TESTING, TESTING
without an infection.
How does a new vaccine come to market? The FDA
requires companies test vaccines for safety and
effectiveness on human subjects. The process differs
somewhat from clinical drug trials. Researchers test a
new drug on sick volunteers to see if it makes them well;
researchers administer an investigational vaccine to
healthy volunteers to see if it prevents illness.
Clinical vaccine trials involve three distinct steps.
• Phase 1 is typically a small study in which healthy
volunteers get the vaccine. Doctors monitor them for
side effects. If no unacceptable reactions occur, the
vaccine advances to the next stage.
The key challenge for DNA vaccines is getting patients’ • In Phase 2, more subjects, typically hundreds, get the
cells to accept the introduced DNA. So far, the most vaccine. The same number of subjects participate in
effective technique seems to be electroporation– the study without receiving the vaccine. Scientists
delivering short pulses of electrical current to the patient refer to this second group as the control. Both
with the vaccine. The electricity creates temporary pores groups are at the same level of risk for contracting
in a patient’s cell membranes, enabling the DNA to enter. the target disease. Researchers observe the
“vaccinatees” for two years or more to see if they
The FDA has yet to approve any DNA vaccines for human
contract the disease at lower rates than those in the
use. The prospect of DNA vaccines, however, presents
control group. Researchers also monitor immune
some important advantages, which includes producing
response by measuring levels of anti-pathogen
a strong immune response and somewhat easier
antibodies in the participants’ blood.
manufacturing. Producing large volumes of viral gene-
containing plasmids still means growing lots of bacteria • For Phase 3, even more subjects—often thousands-
in which to reproduce the plasmids, but purifying and -at high risk for the disease receive the vaccine and
formulating these vaccines is more straightforward due are monitored as described in Phase 2, from three to
to the relative simplicity of DNA’s structure. In addition, five years. This trial also includes a control group.
DNA vaccines don’t require refrigeration, extending their
shelf life and transportation time. COCKTAIL FODDER:
Inovio Pharmaceuticals (San Diego, CA) currently has VACCINATION ACTIVATION
DNA vaccines for hepatitis B and C, and the Ebola, HIV, Getting a shot won’t make you sick. Sometimes, people
and Zika viruses in the early stages of clinical testing. feel mild symptoms such as fatigue, headache, and low-
grade fever after receiving a shot. These are signs that
an immune response is being activated.

Natural Born Cancer Killers
FURTHER DOWN THE CANCER
TREATMENT ROAD WITH CARS
This past August, to much fanfare, the FDA approved the
first chimeric antigen receptor (CAR) T-cell therapy for
blood cancer. Called Kymriah (Novartis), it promises to
revolutionize treatment by genetically altering a patient’s
own cells to fight cancer. Less than eight weeks later,
Kite Pharma, now a part of Gilead Sciences (Foster City,
CA), had its new CAR T-cell therapy, Yescarta approved
as well.
Meanwhile, biotech companies continue to push the
boundaries of immunotherapy by creating engineered Need reminding how CAR-T cells work? Check out our
immune cells. WEEKLY on the topic.
A CAR THAT DOES WHAT? NATURAL BORN KILLERS

But first, what exactly is a chimeric antigen receptor? Our immune system inherently includes NK (natural
CARs are manufactured proteins that molecular killer) cells. They are the body’s first responders. At the
biologists and others engineer to appear on the surface first sign of illness, NK cells attack the infection for two
of a white blood cell such as a killer T-cell. This new, reasons. First, pathogens lack surface proteins called
revved-up receptor then targets the white blood cell to MHC1 that the body identifies as normal. Second, the
attack cancer cells. presence of abnormal proteins tells the body that the
A CAR consists of: invader poses a threat.
• Targeting domain: This part of the CAR exists outside Many immunologists believe that prompt action by NK
the white blood cell. It is composed of an antibody cells helps eliminate cancer cells early on– before they
that recognizes and docks onto a specific cancer grow into a serious problem. However, in the early stages
surface protein. of tumor development, there are often not enough red
flags – abnormal proteins on the cancer cell surface – to
• Activation domain: This component kicks into gear tag them as dangerous. Engineering NK cells to display
once the targeting domain locks onto the intended a CAR “trains” them to recognize and respond to tumor
cancer surface protein. In CAR-T cells, the activation cells. Once activated, the CAR-NKs behave much like
domain signals T-cells to do three things: 1) make killer T-cells, releasing cytokines that bolster the immune
copies of themselves; 2) release signaling molecules response to the cancer cells--killing the nasty cells by
called cytokines (proteins that prompt other white injecting even nastier toxins.
blood cells to attack the tumor); and 3) finally—the
really good bit-- kill cancer cells.
HOMEGROWN ISN’T ALWAYS BEST
CAR-NK cells have two important advantages over
CAR-T cells: safety and accessibility. CAR-T cells must
come from the patient’s own T-cells to avoid triggering
graft-versus-host-disease (GVHD). This potentially
deadly illness occurs when the patient’s immune system
responds badly to foreign tissue. Donor NK cells, in
contrast, don’t appear to cause GVHD.

Besides avoiding potentially life-threatening reactions fragments of the alien cell’s proteins or antigens are
in already very ill patients, medical professionals can displayed on the macrophage’s surface. These leftovers
obtain donor NK cells relatively easily—for example, help activate some of the immune system’s other
from umbilical cord blood. Labs modify these donor cells defenses, such as killer T-cells.
to express a CAR, which can then be given to the patient.
Removing and engineering a patient’s own T-cells, then MMM…CANCER?
transfusing them back into the patient is much more
Researchers are now exploring the potential power of
time-consuming. The ability to more easily use donor NK
CAR-macrophages to destroy specific cancer cells. The
cells means that biotech companies can create “off-the-
enhanced macrophages will simultaneously activate
shelf” products for this type of CAR therapy more readily.
other immune cells to also recognize and attack those
In addition, the resulting lower production costs mean
same antigen-bearing cells. Like other macrophages,
more available treatments.
CAR-macrophages can penetrate solid tumors much
more effectively than “plain old” T-cells.
TEST-DRIVING CAR-NKS
If a typical T-cell does make it into a solid tumor, the
This past June, scientists at the MD Anderson Cancer cancer’s own defenses makes short work of it. In
Center (Houston, TX) started a Phase I/II trial of CAR- contrast, by modifying macrophages to treat solid
NK cells. The research focuses on patients with chronic tumors, doctors may be able to effectively get at cells
lymphocytic leukemia (CLL), acute lymphocytic leukemia inside the tumor. At the same time, the super-powered
(ALL), or non-Hodgkin lymphoma. The trial cells macrophages will “wake up” the patient’s suppressed
contain a “suicide” gene that is triggered by excessive T-cells to fight the cancer as well.
inflammation. Researchers hope this built-in safety
feature will reduce problems caused by overactive Preclinical data from CARMA Therapeutics
immune responses in patients from earlier trials of (Philadelphia, PA) shows that its scientists can modify
CAR-T cells. CAR-macrophages to recognize and engulf different
types of solid tumor cells. They’ve also demonstrated
Researchers elsewhere are also looking into the that infusing cancerous mice with tumor-specific CAR-
development of CAR-NK cells that treat other cancers— macrophages leads to long-term tumor control and
specifically targeting the HER2 protein in breast and longer survival. CARMA plans to begin clinical testing the
ovarian cancers. effect of CAR-macrophages on specific ovarian cancers
in 2019.
CHOWING DOWN ON DISEASE
As biotech companies continue to translate these new
Of course, NKs, modified or otherwise, aren’t the only applications of CAR into treatments, both patients and
white blood cells going toe-to-toe with cancer. Another doctors can look forward to seeing an increase in the
approach involves the immune system’s scavengers number of different types of cancer that respond to
or macrophages. “Macrophage” comes from Greek, these cutting-edge immunotherapies.
meaning “big eater.” These cells kill invading or diseased
cells by surrounding and digesting them. Leftover

Off-Color: The Science Behind
Color Vision Deficiency
You’re at the supermarket, puzzling over whether those two copies of each opsin gene, it’s unlikely both would
peaches for the pie are ripe. Maybe you’re watching your contain mutations interfering with color perception.
child’s soccer team, and struggling to separate the Green By far, most cases of genetic color blindness come
Hornets from the Scarlet Knights. As if determining from genetic mutations that code for red or green-
offsides isn’t hard enough! Or more seriously, you’re detecting opsins. Consequently, those affected have
approaching a stoplight on a busy street and can’t tell trouble distinguishing shades of red or green. Rather
if the signal is red or green. People with color vision than responding to only clearly distinct wavelengths
deficiency (CVD), more commonly known as color of light, the mutated opsin detects a broader range,
blindness, face challenges like these all the time. That’s essentially bleeding over into the neighboring detection
not to mention the inability to fully experience some of wavelength. The brain detects color by comparing
the world’s wonders—the blazing of sugar maples in the signals from two different types of cone cells. Too much
fall or the first flush of April’s flowers. overlap between signals interferes with a person’s
perception of color.
TERM OF THE WEEK: PHOTORECEPTOR CELL
While most people with color vision deficiency have
Photoreceptors are cells in the eye’s retina that respond trouble distinguishing colors in the red and green
to light and convert its signals into information the brain parts of the spectrum, other conditions exist, such as
uses to create visual representations of images. We have tritanomaly—reduced sensitivity to blue light.
two kinds of photoreceptors: rods and cones. Rods work
Much more rarely, two different color-detecting opsins
in low levels of light and detect only black and white.
are affected, which results in significantly reduced
Cones enable us to see colors.
color vision. Even though people with this condition
Photoreceptors contain light-sensitive proteins called retain one-color-detecting opsin, the brain needs to
opsins. Opsins change chemically in response to light. compare signals from different types of cones to see
Photoreceptors detect those changes, enabling them color. The most severe and rare form of CVD is called
to convey visual information. Rods contain only one of achromatopsia—true color blindness. People with this
these proteins, called rhodopsin. Cones, in contrast, condition perceive no color at all, and suffer from other
contain three different opsins, one of which responds to vision problems as well.
red, green, or blue wavelengths of light. For most people,
our opsins bring us the world in technicolor. IMPROVING THE OPTICS
There is no cure for color blindness at present.
SEEING RED—OR NOT
However, two remarkable adaptive technologies exist.
The most common types of color blindness are inherited. A company called Enchroma (Berkeley, CA) produces
In these cases, the genes for one or more cone opsin glasses that help people with red-green color blindness
are defective and make abnormal proteins. Physical or perceive colors normally. They combine the latest in
chemical damage to the eye, optic nerve, or brain can lens development with color perception neuroscience.
also cause color blindness. Enchroma’s proprietary technology filters specific
wavelengths to create sharp distinctions between red
YES HONEY, THE DRESS IS PURPLE and green, significantly improving color vision. Available
off-the-shelf online or at vision centers nationwide, the
Color blindness affects far more men than women.
glasses can incorporate prescription lenses.
The National Eye Institute (Bethesda, MD) estimates
that nearly one in 12 men are color blind, versus one in Colormax (Timonium, MD) also markets color vision
200 women. This difference arises because genes for correction contact lenses and glasses that filter specific
the different opsin proteins are on the X chromosome. wavelengths of light. The lenses are custom-made
Women have two X chromosomes; men only one. With and so require a thorough in-person exam. Colormax
guarantees that the lenses will enable wearers to pass

the color vision tests required for certain occupations, PHILOSOPHICAL QUESTION: RED OR RED?
such as firefighter, pilot, and railroad engineer.
How does a person know if the red they see is the same
red someone else sees? Even among the majority of
TINKERING WITH THE FAULTY GENES people, who see color “normally,” there are probably
Scientists at Adverum Biotechnologies (Menlo Park, CA) variations in color perception. That’s because the eye
are looking at biotech approaches to treating inherited and brain work in concert to translate light into color.
forms of color blindness. Possible gene therapies could Add to that the physical differences in saturation,
entail delivering a correct version of the mutated gene. hue, and tone and the panoply of color names in
Adverum’s work stems from gene therapy treatment of the English language alone—crimson, blood, garnet,
CVD squirrel monkeys at the University of Washington in vermilion, maroon…and we’ll all be seeing red for a
Seattle, WA. . loooonnng time.

The Science Behind
Opioid Addiction
THE SCIENCE BEHIND OPIOIDS (Malvern, PA) Percocet, work by binding to mu receptor
proteins on the surface of cells in the central nervous
Concerns over the opioid epidemic continue to grow,
system (CNS) —think brain and spinal cord. While the
with deaths from narcotic overdoses the leading cause
CNS is tasked with relaying pain signals, opioids decrease
of death in people under 50 last year. Nearly half of
the excitability of nerve cells delivering the message,
those deaths were attributable to prescription opioids.
resulting in pain relief—along with a feeling of euphoria
The directors of both the Center for Disease Control
in some users.
(Atlanta, GA) and the Food and Drug Administration
(Silver Spring, MD) have called for urgent action on the
crisis, and President Trump declared the opioid epidemic LESSENING THE PAIN
a public health emergency. Short term medical used of opioid painkillers rarely
This stark reality highlights the dark side of a class of leads to addiction when properly managed. Due to
treatments serving a vital need. Opioid pain medications the euphoria-inducing effects of the drugs, long term
manage the severe short-term or chronic pain of millions regular use, or use in the absence of pain, may lead
of Americans. While these medications mitigate needless to physical dependence and addiction. And because
suffering, the US government, healthcare industry, and regular use increases drug tolerance, higher doses are
medical community are joining forces to battle against required to achieve the same effect, leading abusers to
opioid abuse and addiction. consume pain pills in unsafe ways such as crushing and
snorting or injecting the pills. According to the Centers
Here at the WEEKLY, we always wonder: What is the
for Disease Control, 91 Americans die every day due to
science behind the headlines? So, let’s talk about how
opioid overdose, which includes prescription opioids and
pain medications work, the different types on the
heroin. At the same time, chronic pain is also a serious
market, and the approaches to developing less addictive
problem, affecting approximately 100 million U.S. adults,
versions of opioid drugs.
while millions of others suffer acute pain due to injury
or surgery. The medical need for these drugs is very real
OPIOIDS VS. NSAIDS despite the dark side.
There are two main categories of pain medications, The answer to developing less addictive drugs may
opioids and non-steroidal anti-inflammatory drugs be found in a drug that blocks pain without inducing
(NSAIDs). Although these two categories of drugs work euphoria. These new drugs will need a different
differently, they do share one thing in common: both mechanism of action than traditional opioid drugs, which
are derivatives of natural products. NSAID commonly bind to the mu receptors of cells inside the CNS. Cara
known as Aspirin was developed by Bayer (Leverkusen, Therapeutics (Shelton, CT) is developing drugs that
Germany). It is a synthetic version of an extract from bind to a different type of opioid receptor, the kappa
willow tree bark. Opioids are synthetic versions of opium opioid receptor. These receptors are present on sensory
and morphine, which come from poppy flowers. nerves outside of the CNS. Preclinical studies suggest
Aspirin works by stopping an enzyme called that targeting these receptors could be effective at
cyclooxyrgenase 1 (COX-1). Once stopped, COX-1 can reducing pain without driving addictive behaviors. Their
no longer produce prostaglandins and thromboxanes. lead candidate, CR845, is currently in Phase III clinical
Prostaglandins and thromboxanes are produced in testing for post-operative pain and pruritus (severe
response to injury or infection and cause inflammation, itching), and in Phase II clinical testing for chronic pain.
which is associated with symptoms of fever, swelling, and Cara Therapeutics is also developing compounds that
pain. Other NSAIDs, such as ibuprofen and naproxen, selectively activate cannabinoid (CB) receptors outside
also work by inhibiting COX-1 or its sister enzyme COX-2. of the CNS. It’s interesting to note that CB receptors
inside the CNS are linked to marijuana’s psychoactive
Opioid pain medications, such as Purdue Pharma’s
qualities. Cara’s lead CB receptor activator, CR701, is in
(Stamford, CT) Oxycontin and Endo Pharmaceuticals’
preclinical development.

Hydra Biosciences (Cambridge, MA) is developing small relief and mild euphoria without the detrimental side
molecule drugs that inhibit ion channels, proteins on effects associated with opioid drugs such depressed
the surface of nerve cells that help transmit pain signals. respiration, motor impairment, and addiction. Scientist
This plays a critical role in sending the pain signal to have tried before to develop safer pain medications
the brain, yet because it works on nerves outside of based on endogenous opioids, but have not been
the brain, it has less of a potential for addiction. Hydra successful due to the molecule’s instability. The Tulane
is currently in Phase II clinical studies of HX-100 for the team created a stable derivative of endomorphin that
treatment of painful diabetic neuropathy. binds to the mu receptor in such a way that pain relief
Centrexion Therapeutics (Baltimore, MD) lead occurs, but not the negative side effects listed above.
candidate is a derivative of capsaicin, a naturally- Clinical testing is expected to begin within a few years.
occurring compound found in chili peppers. Capsaicin
has pain relieving properties and is used as a natural ANTIDOTE TO AN OVERDOSE
remedy. Centrexion’s lead candidate, CNTX-4975, is a Overdosing can be fatal since respiratory failure
highly potent, synthetic form of capsaicin designed to be occurs at high opioid concentrations. If an overdose is
administered via injection into the site of pain. CNTX- suspected, the individual should be treated as quickly
4975 targets the capsaicin receptor, an ion channel as possible with naloxone—a “competitive antagonist”
protein on the surface of nerve cells. When CNTX-4975 of the mu-opioid receptor. Simply put, a competitive
binds the capsaicin receptor, the influx of calcium ions antagonist binds the receptor without activating it.
results in desensitization of the nerves, making them Since naloxone doesn’t activate the receptor, it doesn’t
unresponsive to other pain signals. This effect can have any pain relieving or euphoria inducing qualities;
last for months, and only affects nerves near the site rather, it prevents the opioid drugs from binding. It may
of injection. Centrexion recently announced positive also displace opioids that have already bound the mu
Phase IIB results for CNTX-4975 in a study testing its receptor, aiding in the stoppage of an overdose.
effectiveness against pain in knee osteoarthritis. The
drug is also in Phase II clinical studies for Morton’s
COCKTAIL FODDER: RUNNER’S HIGH
neuroma, a sharp pain in the foot and toe caused by a
thickening of the tissue around one of the nerves leading Some folks love to run; others avoid it at all costs.
to the toes. This might be explained by inherent differences in
sensitivity to the natural opioids called endorphins
Earlier this year, Nektar Therapeutics (San Francisco,
that are released during exercise. Not everyone
CA) announced positive Phase III results of their
experiences the “runner’s high”— feelings of calm and
addiction-resistant opioid, NKTR-181. Like traditional
mild euphoria – in much the same way that not everyone
opioids, NKTR-181 works by entering the brain and
experiences euphoric feelings from pain medications.
activating mu-opioid receptors. However, the molecule is
These differences may help to explain why some people
designed to enter the brain much slower than other mu-
enjoy exercise and others don’t, and why some people
receptor activators, reducing the feelings of euphoria
get addicted to opioids—while others can take them or
while offering pain relief.
leave them.
Researchers at Tulane University (New Orleans, LA)
With several of the opioid alternatives outlined here
have developed a compound that is derived from the
already showing success in clinical studies, there is hope
endogenous opioid, endomorphin. Endogenous opioids
on the horizon for patients who suffer from chronic pain
are chemicals produced naturally by the body that bind
but want to avoid the risk of addiction. Next week, we’ll
to and activate the mu-opioid receptors, resulting in pain
explore the use of medical devices in pain management.

Bye-Bye Opioids? Introducing
Electroceuticals
MEDICINE-FREE PAIN MANAGEMENT
Migraine relief without drugs? No “digestive issues” due
to pain meds after surgery? Better still, no worry about
addiction after that appendectomy or hip replacement?
Sounds a bit science-fictiony, does it not?
The news reminds us nearly every day of the profound
need for pain management without opioids. As you read
last week, alternatives to analgesics such as morphine,
codeine, or fentanyl exist. But science also takes us
beyond medicine: researchers have developed devices
that control pain and inflammation electronically. This
drug-free approach is called electrostimulation–using Groups of neurons form nerves outside the central
low levels of electricity to zap pain. nervous system. Nerves transmit sensory information
such as touch, temperature, and pain. Different kinds
THE BODY ELECTRIC of nerves respond according to the specific receptor on
How does one feel pain in the first place? Pain, and more their surface. Pain receptors detect chemicals that tissue
pleasant sensations (the taste of chocolate, for instance) damage releases. The pain signal then zooms along the
come courtesy of the nervous system. This system nerve to the central nervous system for interpretation
serves as the wiring that enables the machines of our and response by the brain. Pain can also be caused
bodies to move, experience, and interpret the world. by direct damage to the nerves themselves (think
pinched nerve).
Our nervous system has two main parts that work
in tandem: the central and peripheral nervous
SLAMMING THE GATE ON PAIN
systems. The central nervous system functions as the
“switchboard” that sends and receives information from As you no doubt noticed, pain perception is complex.
peripheral nervous system’s local lines. The peripheral Remember the last time you bashed your knee? Did you
nervous system is a vast network of nerves found rub it? The Gate Theory of Pain says this rubbing action
everywhere in body. does temporarily reduce pain. In that moment, signals
from your non-pain nerves block signals from your pain
The central nervous system is made up of neurons.
nerves. In other words, non-pain nerves prevent pain
These cells send and receive signals electrochemically,
from reaching your brain.
through signaling molecules called neurotransmitters.
These chemicals are converted into electrical signals Electrostimulation relies on the body’s ability to run
in the neuron. Here’s how: when a neurotransmitter interference on pain. It uses electricity to treat chronic
reaches one edge of a neuron, the dendrite, it opens pain by stimulating a patient’s peripheral nerves or
ion channels in the cell membrane. These tiny openings spinal cord. In either case, a small pulse generator sends
allow the positively charged sodium ions to pass electrical pulses to the nerves or spinal cord, “shutting
through. These positive ions travel down the other side the gate” on pain.
of the neuron through an extension called an axon. Several companies already have electrostimulation-
When the electrical charge reaches the end of the based devices on the market, including:
axon, it signals the release of other neurotransmitters,
• Medtronic (Fridley, MN). Its Intellis spinal cord
which then switch on other neurons and the chain of
neurostimulation system gained FDA approval
sensation unfurls.
in September and European Union approval for
managing chronic pain earlier this month. This
small, implantable neurostimulator communicates

wirelessly with a Samsung tablet that a doctor uses Institutes of Health (Bethesda MD) has established
to adjust pain relief and monitor patients’ activity. the Stimulating Peripheral Activity to Relieve Conditions
More activity indicates that the patient’s level of pain (SPARC) program to fund the basic exploration of how
is lessening. peripheral nerves’ electrical signals control the function
• Stimwave (Pompano Beach, FL). This company’s of internal organs. This effort may lay the groundwork
latest device, the StimQ Peripheral Nerve Stimulator, for electroceuticals–the next generation of tiny
was approved in August for chronic pain. Tiny neurostimulation devices.
enough to be inserted by needle, it communicates In the private sphere, GlaxoSmithKline (GSK; Middlesex,
wirelessly with an external transmitter that U.K.) leads the charge. The corporation is heavily
delivers electrical pulses. The device is also safe involved in creating a Nerve Atlas to map the nervous
to wear in MRI machines. This innovation makes system’s role in disease. GSK is also investing in start-
neurostimulation available to people whose ups such as SetPoint Medical (Santa Clarita, CA),
treatment requires ongoing imaging. which has already produced clinical data supporting
• Abbott (North Chicago, IL) also offers the use of electroceuticals on rheumatoid arthritis .
neurostimulation systems. Their most recent is the Another of GSK’s notable investments in electroceuticals
BurstDR system. Developed by St. Jude Medical in St. was the launch of Galvani (Stevenage, England) in
Paul, MN, this proprietary technology delivers bursts 2016, in partnership with Verily (San Francisco, CA).
of electrical stimulation in a way that mimics the Galvani is developing electroceuticals for chronic
body’s natural response to pain. conditions including Type 2 diabetes, autoimmune, and
endocrine disorders.
THE LATEST BUZZ: NEW

COCKTAIL FODDER: THE DOWNSIDE
APPLICATIONS AND NEW TECH
OF FEELING NO PAIN
While neurostimulation can treat chronic pain, the
technology may provide other therapies as well. Feeling no pain sounds great, doesn’t it? Yet people born
Because nerve signals permeate the entire body, with congenital analgesia suffer greatly from injury and
modulating them may work for other medical illness, because they never feel the pain associated with
conditions. For example, neural signaling partially them. In a BBC interview, sufferer Steven Pete talked
controls inflammation, the body’s response to injury. about breaking a limb roller skating as a boy, noticing it
This suggests that neurostimulation may treat only when friends pointed out the bloody wound caused
inflammatory disorders such as Crohn’s disease and by the bone protruding from his leg! Fortunately, this
rheumatoid arthritis. inherited nervous system disorder is extremely rare,
with only 20 or so cases known in the medical literature.
Federal agencies, large pharmaceutical companies,
and small start-ups are taking note. The National

Zinc Finger Nucleases
CATCHING THE RIGHT BREAK genome and the nuclease cuts the specific location. The
double-stranded break (DSB) or cut activates the non-
How are ZFNs made? To start, zinc finger proteins (ZFP)
homologous end joining (NHEJ) repair pathway, this most
are sequence-specific, DNA-binding proteins that
often results in a disruption of a gene, useful for gene
activate gene expression. They are engineered to
“knockouts”. If a repair template is delivered at the same
recognize unique sites within the genome. While ZFPs do
time as the break, the homologous directed repair (HDR)
not have the ability to cut DNA on their own, scientists
pathway kicks in. This method is useful to “knockin” a
can fuse a ZFP together with a DNA-cutting enzyme
gene. This video explains a potential application for zinc
called nuclease—the “N” in ZFN. The marriage of ZFP to
finger nucleases in HIV.
nuclease creates ZFN.
How do ZFNs work? Zinc finger proteins bring the
zinc finger nuclease to the engineered location of the

Plants That Heal
NATURE’S MEDICINE CABINET Doctors also prescribe ephedrine to prevent low blood
pressure during spinal anesthesia.
Where does medicine come from? Before it gets to
your medicine chest? Before you purchase it from Indian snakeroot (Rauwouofia serpentina) is also
your neighborhood drugstore? Next time you’re hiking featured prominently in early Chinese and Indian
through a forest or gazing at your pretty screensaver medicine. It contains an indole alkaloid, reserpine.
of the Olympic Peninsula, think of this: the magic that Studying this chemical helped neuroscientists
relieves a throbbing headache or lowers your dad’s learn how neurotransmitters such as serotonin or
blood pressure may well have started with a plant. dopamine govern depression. Reserpine is prescribed
for hypertension and psychosis. Mahatma Gandhi
Historically, humans have looked to the world around us
reportedly relied on reserpine to calm his nerves.
for what we need. Plants in particular offer the power to
feed, heal or potentially harm us. They produce an array
of bioactive compounds to survive and thrive. Some of FROM FOREST TO PHARMACY
these substances are also biologically active in people Plants beyond those used in traditional medicine
too. Sometimes, years of hard work and testing turn one continue to influence modern medicine. In fact, for the
of these compounds into drug we can use. past 25 years, approximately one quarter of prescribed
medicines in the US contain plant derivatives.
DOCTOR MOM IN THE FOREST PRIMEVAL According to some historians, the modern
Imagine early humans, scrabbling for survival out on pharmaceutical industry began as early as the mid-
the savanna somewhere. One of the toddlers develops a 1800s. Others say the early 1900s. Regardless, the first
burning fever. Desperate, the mother remembers those commercial drugs derived largely from plant extracts.
berries along that new stretch of riverbank. She asks They include these old favorites:
herself, “What if?” Let’s say the berries cure the baby’s • Digitalis. Cardiac glycosides from the foxglove plant
malady. Voila—the first medicine, courtesy of Dr. Mom. stimulate cardiac muscles and are used to treat
congestive heart failure. Careful! Every part of these
THE DIVINE FARMER funky, beautiful flowers is poisonous to dogs, cats
As long as humans have eaten plants, we’ve and humans.
experimented with their health effects. The Chinese • Aspirin. Aka acetylsalicylic acid. Salicin, the bioactive
were likely the first to formalize their knowledge into ingredient behind this most basic medicine comes
what we call Traditional Chinese Medicine (TCM). from willow tree bark. Many of us use it to manage
According to Chinese lore, the mythical emperor pain, fevers and inflammation. My mom uses it to
Shennong, sometimes known as the Divine Farmer, help prevent stroke.
commanded his herbalists to record the effects of • Quinine. This alkaloid is a product of bark from
medicinal plants approximately 5,000 years ago. the native South American cinchona tree. In the
Ethnobotanists and others have examined many TCM past, quinine was famously used against malaria.
plants for use in pharmaceuticals. Some have yielded It is currently used to treat patients with lupus or
ingredients regularly used in western medicine. rheumatoid arthritis. For malaria, a derivative of
They include: another plant ousted the cinchona—artemisinin,
Mahuang or Ephedra (Ephedra sinica). Mahuang was which comes from sweet wormwood.
used to relieve asthma, or at least wheezing and Through the 1930s, pharmacological research remained
congestion. Scientists now know that mahuang contains focused on screening natural products. Plants, bacteria
an alkaloid called ephedrine. Ephedrine has a long and fungi were tested for their potential anti-cancer,
history in western medicine as well. It’s the active anti-inflammatory or anti-bacterial properties.
ingredient in many cold medicines, including Sudafed.

In 1955, the National Cancer Institute began screening rule is that these products are considered safe until
natural products for cancer treatment. This program proven unsafe.
yielded a potent anti-cancer compound, taxol. Taxol Some supplements are whole plant extracts, containing
came from the bark of the pacific yew, an evergreen multiple bioactive ingredients. If a pharmaceutical
native to the Pacific Northwest. In 1993, the FDA company wants to develop a drug from a supplement,
approved a modified version of taxol, Paclitaxel, for it generally needs to isolate and test a single compound
chemotherapy. Today, Paclitaxel is on the World Health from the whole plant extract. Companies such as
Organization’s List of Essential Medicines. Sanofi (Paris, France), Novartis (Basel, Switzerland) and
Scientists these days often synthesize new drugs using Pfizer (New York, NY) are investigating plant-derived
organic chemistry or recombinant DNA techniques. compounds in their drug discovery pipeline. One smaller
These medicines specifically target areas in the company, Napo Pharmaceuticals (San Francisco, CA),
human body where diseases originate. Many in the isolated a compound from the tropical croton plant
pharmaceutical and medical communities no longer (Croton lechery). It won FDA approval as an anti-diarrheal
consider natural products a primary source of new for HIV patients in 2013.
therapeutic compounds. Here are a couple plants and one fungus currently
However, the plant kingdom remains a biological marketed as supplements. Remember, caveat emptor—
treasure trove. A 2016 study by England’s Royal Botanic guinea pigs are us.
Gardens Kew estimates our amazing planet supports White mulberry. Dried fruit from the white mulberry
about 390,900 species of plants. So far, researchers have tree tastes a bit like raisins. The fruit contains fiber,
screened only about six percent for biologic activity in protein and vitamins and deoxynojirimycin, or DNJ.
humans. Who knows what pharmaceutical miracles may DNJ is a glucosidase inhibitor that acts upon the sugar
originate in this immense diversity? metabolism pathway to prevent a spike of blood-sugar
levels after eating. DNJ may prove useful for diabetics.
A SUPPLEMENT IS NOT A DRUG
Devil’s claw. This small desert plant is native to
More and more, people use plant-based supplements Southern Africa. In the early 1800s, Europeans
like echinacea to combat their colds or valerian to introduced this plant at home, where its root became
help them sleep. Scientist are investigating some a popular remedy for pain and osteoarthritis in France
supplements for their medical potential. However, and Germany. Harpagide, a glycoside from devil’s
it’s vital to remember that the FDA does not consider claw, appears to control inflammation. Devil’s claw has
supplements drugs. So, what are the differences also gained some traction with dog and horse owners
between a supplement and a drug? because those animals also experience relief after taking
The FDA evaluates and monitors the development of the herb.
prescription medicine in the US. The agency defines Chaga. Fungal tea, anyone? Strange but true—look
drugs as substances intended to diagnose, treat or for a chaga concoction on the chalkboard of your local
prevent disease. They must pass clinical trials before coffee shop soon. This fungus grows on hardwood
being approved for consumer use. The process often trees in chilly, northern forests (Maine, Alaska, Canada,
takes twelve years or more and incredible resources— Siberia). Packed with antioxidant enzymes, phytosterols
intellectual and monetary. and other bioactive compounds, preliminary research
In contrast, the FDA has no power to regulate suggests the ability of chaga extract to reduce
supplements. Under the Dietary Supplement Health inflammation, reduce fatigue and boost immunity. No
and Education Act, the FDA must treat supplements like verdict on taste though!
food. The law doesn’t require supplements be tested for Next week, we’ll move from the forest and the garden
safety, purity or quality. They include minerals, vitamins, to the lab—explaining key steps researchers take to
amino acids and other biological substances. The general identify and move potential medicinal compounds into
clinical trials.

Drug Discovery 101
ON THE ROAD TO NEW MEDICINES X MARKS THE MOLECULE
For most of the 20th century, we discovered new drugs Researchers identify their molecular targets by
by trial and error. Scientists investigated countless answering one incredibly complex question: How does
unrelated compounds in animals to see which improved diseased tissue differ from healthy tissue? Scientists look
disease symptoms. For instance, in the 1950s and 60s, at two biological clues: differences in gene sequences
British scientists at Boots Laboratories tested hundreds (mutations), and changes in the levels of specific
of unrelated chemicals on guinea pigs searching for an proteins. For example, does a lung tumor biopsy show
alternative to aspirin for treating pain and inflammation. mutations absent in healthy lungs? Are levels of protein
This scattershot approach consumed vast amounts of expression higher or lower in the diseased tissue? If
time and resources, with limited success. the answer to either question is yes, scientists have a
potential target for their ‘as yet to be developed’ drug.
PICKING UP THE PACE—
RATIONAL DRUG DISCOVERY
Beginning in the 1980s, scientists began to take a new
tack in developing drugs. They adopted an approach
known as rational drug discovery or mechanism-based
drug design. In this method, researchers first seek to
understand a disease at the cellular level, and ideally,
identify its mechanism. Once the cellular mechanism
becomes clear, scientists can identify a drug target:
the molecule (usually a protein) involved in the illness
that the ‘as yet undeveloped drug’ will hopefully act on.
Although still labor and resource intensive, rational drug
discovery tends to produce more highly-effective drug
therapies than the old trial and error approach. TESTING, TESTING
In the quest for new medicines, as in the rest of life, few
PROBLEMS WITH PROTEINS things are as simple as we hope. Just because a protein
plays a fundamental role in an illness doesn’t always
Identifying a target molecule for a drug to act upon
make it a good drug target.
requires years of basic research on a disease’s biology.
Most diseases stem from issues related to how our Consider the following scenario: Researchers discover
bodies produce proteins. Significant underproduction a specific protein target that affects the course of a
of a protein causes disease; for instance, little to no disease—great! They then design a drug to inhibit
insulin production leads to Type 1 diabetes. In contrast, said target—also great! Problem solved, right? No so
overproduction of a protein can also produce disease; fast. Sometimes a different protein can quickly take
too many of the growth factor receptor HER2 can result over the target’s function, nullifying the new drug’s
in certain breast cancers. Finally, errors, or mutations, hoped-for effect. It’s back to the drawing board for the
sometimes affect protein function. This is the case when disappointed scientists.
a mutated tumor suppressor protein no longer controls In other cases, inhibiting a specific target achieves a
cell division and cancer develops. Understanding which goal – say halting cancer cell growth – but results in
proteins correspond to a particular disease means unexpected side effects, such as irregular heartbeat.
researchers can design drugs to go after that disease- It’s crucial that scientists validate that when a target
causing protein specifically. is altered, that change to the target is both safe
and effective.

Researchers verify the suitability of target molecules by TERM OF THE WEEK: BIOMARKERS
answering two important questions: does the target play
One important tool in drug discovery and development
a key role in the disease? and will targeting the molecule
are biomarkers. Biomarkers are molecules that doctors
likely be safe and effective? Validation most often
and others use to measure normal or abnormal
includes cell-based (in vitro) and animal (in vivo) testing.
biological processes in the body. They can help show the
The goal of many drug therapies is to stop or slow a
effects of a disease or therapeutic intervention.
target’s activity. Consequently, many tests, or validation
assays, seek to find out what happens when a drug puts LDL cholesterol is a classic biomarker familiar to
the brakes on a molecular bad actor. anyone over the age of 35. Numerous studies have
linked it to heart disease. LDL cholesterol makes an
One of the most popular testing methods is RNA
ideal biomarker because of these three reasons: it
interference (RNAi). This technique can rapidly block
circulates in our blood and can be obtained by a simple
production of a particular protein. It does so by
blood draw; it can indicate heart disease early on, since
destroying the messenger RNA that codes for the
someone can have elevated cholesterol long before
protein. This destruction can quickly demonstrate what
developing atherosclerosis; and it changes as the
happens to a cell when protein production ceases, thus
disease progresses. In developing drugs, researchers
mimicking the effects of a strong inhibitor drug.
have used changes in LDL as a surrogate endpoint.
If a cell model shows promise, researchers move on Surrogate endpoints are outcomes that researchers can
to an animal model. Often, scientists use “knockout” measure to determine relatively quickly whether the trial
mice – in which they have disrupted a target gene that intervention benefits the participants.
codes for the target protein. Researchers pose similar
In the case of heart disease, researchers establish
questions to those asked in cell-based testing, but on
a target cholesterol level in lieu of waiting years or
the larger scale of the whole animal. Do the mice get
even decades to demonstrate a change in disease
cancer or Parkinson’s disease or heart disease when the
progression. In many cases, measuring biomarkers has
target gene is tampered? Because in vivo testing allows
turned out to be key to running shorter, less expensive
researchers to examine whole-body effects, it provides
clinical trials. We know now that statins, which lower
valuable information about target safety that in vitro
cholesterol, can increase longevity because patients
testing simply cannot.
have taken them for more than 30 years. Initial trials,
however, simply showed a decrease in patients’
blood cholesterol.
Identifying and validating drug targets are only the first
steps on the long road to safe, effective new medicines.
Tune in next week to find out how researchers identify
therapeutics that might make it into clinical trials.

Drug Discovery 201
WE WANNA NEW DRUG GLOW
“One that won’t make me sick/ One that won’t make me Imagine “Enzyme Z” causes “Disease Z.” Its cure lies in
crash my car/ and make me feel three feet thick…” Huey shutting down, inhibiting “Enzyme Z.” Enzymes feature
Lewis is singing about love, but he voices very human active sites, a kind of pocket that binds various small
concerns when it comes to the medicines that heal molecules. Researchers manipulate these small molecule
bodies and minds. inhibitors to light up--emit a fluorescent signal if they
bind (shut down) the enzyme.
Last time, the Weekly explored how researchers identify
drug targets—the molecules in our bodies that make
good candidates for therapeutic action. It’s a painstaking
but strategic process, and only the first step toward
Huey’s mythical remedy.
THERAPEUTIC CHOICES
There are small molecule drugs and large molecule
drugs. Small molecule drugs include medicines most
of us know, such as aspirin and over-the-counter cold
remedies. In contrast, large molecule drugs, also known
as biologics, are often specialty drugs, delivered through
injection by a healthcare professional.
NO GLOW
Therapeutic choice hinges on a disease’s origins. If an
illness originates outside the cell, a biologic is a logical Scientists use similar colorimetric tests for large
choice since it is too big to enter the cell. Though more molecule drugs such as antibodies. Let’s say that
expensive to produce, biologics typically cause fewer activated “Receptor X” turns on a particular gene that
adverse reactions than their smaller counterparts. in turn causes cancer. Scientists can engineer cells so
when Receptor X is activated, the cell produces a green
Small molecule drugs attack disease from within the
fluorescence and when it is not activated no green
cell. The downside? Their small size means they have
appears. Researchers are hoping for “no glow” or in lab-
the potential to also activate many off-target molecules.
speak, a “blank.” Blanks are the winners that go on to the
This collateral damage increases the chance of
next stage of drug development.
adverse reactions.
ASSAY DEVELOPMENT:
GLOW AND NO GLOW
To find the best drug candidate, scientists need to
design easy-to-perform, large-scale, fast, and accurate
assays. Think automation. Think high throughput
screening. Scientists often work with tests that produce
a fluorescent signal or color change because color is easy
to measure, relatively inexpensive to design, and safe.
Depending on the assay, a color change may or may not
indicate a drug candidate works.

HIT TO LEAD LEAD TO CANDIDATE
High throughput screening ideally results in several Biopharma companies typically put lead compounds
promising drug candidates or “hits.” To become “leads” through several rounds of optimization before they can
that merit animal testing, hits must pass rigorous in become drug candidates for preclinical testing. Lead
vitro testing. On this leg of the drug discovery voyage, optimization is the process by which a drug candidate
researchers ask three questions: is designed via iterative rounds of synthesis. For small
1. Is the hit safe? molecule drugs, this tweaking may include creating
derivatives of the lead. Often made using computer-
2. Is the hit specific to the target? aided design (CAD), derivatives can help ensure the lead
3. Does the hit show promise to treat the disease? fits well with its target. Researchers then test each for
If a potential lead is supposed to inhibit an enzyme—it improved potency, fewer side effects, solubility (making
should act on that particular enzyme. A lead with effects sure that the drug stays liquid and won’t form clumps)
that are too general may result in a drug with dangerous and shelf life, otherwise known as stability.
adverse reactions. Therefore, researchers aim to develop Of course, optimization for large molecule drugs differs
a drug that inhibits the target at a concentration low because biologics are genetically encoded. Researchers
enough to avoid interfering with related enzymes. This have developed techniques to create mutations in
approach provides a therapeutic window within which proteins at specific locations. Again, the goal is to see if
the drug is effective and safe. changes produce a better drug—maybe even one up to
Huey Lewis’s rigorous standards. If so, researchers have
at long last an official drug candidate, one ready for the
rigors of preclinical testing in animals.

Gene Therapy Cures
THE PROMISE OF GENE THERAPY UNFOLDS ASTOUNDING IMPROVEMENT
In many ways, 2017 was the year of gene therapy in the Clinical trial participants’ vision loss ranged from mild
United States. Patients and pharmaceutical companies to severe. The trials included patients from ages four
celebrated the approval of not one, but three treatments to forty-four. For 93% of them (27 out of 29), Luxturna
for otherwise untreatable health conditions. Researchers treatment improved patients’ visual function, as shown
have been working on developing safe, effective gene by a “multi-luminance mobility testing (MLMT) score.”
therapies for three decades. Early trials were plagued The MLMT measures the ability to navigate an obstacle
with safety issues. Consequently, the field remained on course in low light.
hold until researchers could address those problems
with new and improved gene-delivery vehicles. This COST OF A ONE-TIME CURE
Weekly explores these innovative approaches to
Luxturna is priced at $425,000 per eye. Most patients
intractable illness and disease.
need treatment in both eyes. Its expense comes in part
from the nature of the treatment as a one-time, cure.
THE FIRST APPROVALS Spark Therapeutics is discussing payment options with
The FDA approved the first gene therapies in the United insurers to help allay the sticker shock. One plan in
States last year --Chymera and Yescarta--both chimeric the works with Pilgrim Health, a large non-profit New
antigen receptor T-cell (CAR-T) therapies. They deliver England-based insurer, hinges on outcomes. Should
a gene to cancer patients’ white blood cells in order the treatment fail to meet the intended outcome at
to program them to attack specific cancer cells. CAR-T certain intervals post-treatment, Luxturna will refund
treatments involve removing a patient’s white blood the treatment’s cost. Another proposal in the works
cells, programming them to contain a cancer-destroying would allow insurers to spread payments out over
gene, and then re-administering them to the patient. multiple years.
TWO FIRSTS IN ONE TERM OF THE WEEK: VECTOR

Just before year’s end, the FDA approved a therapy Scientists have adapted some viruses to transport
for a rare form of inherited blindness. Developed by therapies by tweaking them to target disease instead of
Spark Therapeutics (Philadelphia, PA), Luxturna is the causing illness. These souped-up microbes are known
first gene therapy to target a genetic disorder. It’s also as viral vectors. They are the vehicles that make genetic
first in another significant way. Unlike the new cancer therapy like Luxturna possible. The virus itself is simply a
treatments, patients receive Luxturna directly, via sub- segment of genetic material— RNA or DNA—surrounded
retinal injection. by a protein coat. Proteins on the surface of the vector
The blindness treated by Luxturna is known as “biallelic (the modified virus) target proteins on a patient’s specific
RPE65 mutation-associated retinal dystrophy.” diseased or malfunctioning cell surface. These viral
vectors then incorporate DNA into the patient’s genome
• Biallelic: Pertaining to both copies of a particular by “tricking” the patient’s cells into producing the DNA it
gene (allele) delivered. Ultimately, this enables a patient to make the
• RPE65: A protein in the retina that helps convert light functional protein that he or she lacked.
into the electric signals the brain interprets as sight Over a decade ago, gene therapy stalled because of
• Retina: Light-sensitive tissue in the eye safety concerns. Researchers could not initially control
• Dystrophy: Wasting of tissue the insertion point of modified genes. In some cases,
the introduced genes disrupted patients’ other genes,
The corrected version of the RPE65 gene helps repair causing serious illness. Researchers have now developed
patients’ retinal health and vision. vectors that allow them to more precisely target where
a therapeutic gene goes into a person’s genome, making

the treatment much safer because it doesn’t interfere tissues. Lentiviral vectors have a higher carrying capacity
with the function of critical genes. as well, so may be preferred for very large genes or in
cases where the goal is to deliver more than one gene.
LOOKING AHEAD
Luxturna’s success raises hopes of cures for numerous
other diseases caused by a defect in a single gene,
including hemophilia, sickle cell anemia, Huntington’s
disease, and other types of hereditary blindness. As
the table below indicates, many companies have gene
therapy treatments in clinical trials.
Types of Gene Therapy Vectors
The most commonly used type of viral vector for gene

therapy applications are adeno-associated viruses (AAV).
However, AAVs aren’t the only vector in town. Lentiviral
vectors are also being tested in gene therapy clinical
trials. The choice of vector depends in part on the target
tissue and the size of the genetic payload. AAV vectors so
far seem to be best at targeting eye and muscle tissues,
while there is some evidence that lentiviral vectors are
better at targeting blood and central nervous system 2018 may see more groundbreaking approvals as these
treatments make their way through clinical testing.

Drug Discovery 301
DRUG DISCOVERY 301 • The time it takes to absorb a drug into the body;
Biotech Primer Weekly wrapped up last year by exploring • Maximum concentration (Cmax) of a drug in plasma
the first two stages of drug discovery. We looked at and target tissues;
how pharmaceutical companies identify drug targets, • Bioavailability (where in the body and at what
or the molecules (usually proteins) involved in an illness concentration the drug ends up);
that an ‘as yet undeveloped drug’ will hopefully act on.
• Half-life, or how long a drug takes to reach half its
Next, we examined how researchers develop those
maximum concentration in the body;
pharmaceutical candidates. Now we turn to what comes
next for fledgling drugs on the arduous journey to FDA • Clearance, or the time for a drug to reach
approval. First, though, a smidgeon of Latin. undetectable levels through excretion.
Pharmacodynamic analyses involve observing the
PRIMUM NON NOCERE biological repercussions of increasing amounts of a drug.
This maxim from Hippocrates means “First, do no harm.” Negative side effects include nausea, loss of appetite,
Before testing a drug on people, researchers must make fatigue, skin sensitivity, and changes in blood pressure
sure it’s safe in two important ways. First, they assess a or heart rate. Animal PK and PD studies give scientists an
substance’s safety in vitro. This Latin phrase means “in idea of what a safe and effective dose of a drug might be
glass”—that is, lab-grown cells. The cell type varies, but in people.
if possible, researchers use one that is relevant to the
disease in question. For example, they may use lung cells
to test a drug for respiratory syncytial virus (RSV).
In vivo testing (“in a living thing”) comes next. This area
of preclinical testing assesses a drug candidate’s toxicity
in at least two different species of animals, such as mice
and guinea pigs. The animals receive more of the drug
for longer than would human volunteers. Meanwhile,
lab technicians watch the animals for adverse effects.
Preclinical testing must follow the FDA’s Good
Laboratory Practice (GLP) guidelines. These regulations
help ensure scientific integrity and humane treatment of
laboratory animals.
HUNTING FOR MUTANTS
HOMING IN ON THE SWEET SPOT Most drug candidates also undergo mutagenicity
testing, which determines their likelihood of triggering
Almost any substance–even water!–can be toxic in
mutations. Causing mutations indicates that a fledgling
very high amounts. Consequently, drug developers
drug may be carcinogenic, hence often consigning it to
aim for just the right dose–one that gives the desired
the graveyard of pharmaceutical failure. One common
effect with minimal unwanted consequences. Finding
screening for mutagenicity is the Ames test, which
the sweet spot is the domain of pharmacokinetic
identifies chemicals that cause increased rates of
(PK) and pharmacodynamic (PD) analyses. Think of
mutations in bacteria.
pharmacokinetics as “what a body does to a drug” and
pharmacodynamics as “what a drug does to a body.” Researchers can also assess a drug’s carcinogenic
potential by examining test animals for tumors.
PK analyses typically measure:

Q/T TESTING intended drug target. More off-target interactions
mean a greater chance of undesirable side-effects.
Heart problems aren’t just bad for people, they’re
bad for baby drugs too. So, companies try to rule out
anything with potential cardiac side effects as early as THE PAPER CHASE
possible. To find drugs that may be heart-unhealthy, Once researchers amass enough safety data to ensure
researchers look at the “QT interval.” This is the time that a drug candidate will be safe for people, they submit
between the start of the Q wave and the end of the an Investigational New Drug (IND) application to the FDA.
T wave in the heart’s electrical cycle. A lengthened The IND application contains short-term safety data,
QT interval suggests improper activity in a person’s information on manufacturing the drug, and details
ventricles and is a risk factor for sudden death. If an about the methodology and design of clinical testing.
experimental drug binds to proteins on heart cells that If the FDA blesses the application, the drug candidate
enable the flow of calcium ions into the heart, they may may enter Phase 1 clinical testing. Animal testing often
extend a QT interval. Longer intervals generally seal a continues in parallel with clinical (human) studies to
drug’s doom. collect long-term safety data, especially for medicines
that treat chronic diseases.
ALTERNATIVES TO ANIMAL TESTING
Currently, drug development necessitates in vivo testing
to best understand how drugs act in a human body.
In vitro testing simply can’t replicate Home sapiens’
complex physiology. However, two innovations may
help reduce the animal testing involved in the search for
new treatments:
• 3D tissue arrays: Companies such as Organovo (San
Diego, CA) create tissue arrays that better mimic
human physiology than flat layers of cells in tissue
culture flasks.
• Computer modeling: Researchers at the University
of California, San Francisco, and SeaChange COCKTAIL FODDER
Pharmaceuticals (San Francisco, CA) have
For every 5,000 drugs tested preclinically, only about
developed software that reliably predicts small
five show enough promise to justify submitting an IND
molecule drugs’ interaction with “off-target”
application to the FDA. Once a drug clears the preclinical
molecules. These are cellular proteins other than the
testing hurdle, it can move on to human testing. Tune in
next week to learn more about this crucial process.

From Drug Development
to Approval: Phase I/II
PHASE I AND II CLINICAL TRIALS trials may use patients rather than healthy volunteers.
For example, cancer drugs have a level of toxicity that we
Every drug in clinical use today, from the latest CAR-T
would not want to expose healthy volunteers to, but that
treatment to older cholesterol-lowering statins,
toxicity is an acceptable risk for patients who may have
share one thing in common: they have all successfully
no other options.
navigated the rigorous clinical trials process. This is no
small feat, as only ~10% of the drugs that enter Phase This first stage involves a number of different tests.
I testing successfully emerge as marketed products. First, volunteers take escalating doses of the drug under
Those few drugs that show remarkable success in early close observation. If and when they experience adverse
clinical trials make headlines, and deservedly so. Recent effects, they stop taking the drug. This establishes the
drugs that have made a big splash include Amgen’s Maximum Tolerated Dose, or MTD, which becomes a
oncolytic virus drug Imlygic, which is restoring faith in benchmark for the remaining trials. The MTD helps
therapeutic cancer vaccines, and AbbVie’s Venetoclax, assure investigators and subjects that the treatment is
which seems to melt away tumors in chronic lymphocytic unlikely to be toxic.
leukemia patients. Other studies look at pharmocodynamics (PD) and
This week, we’ll take a look at the first two phases of pharmacokinetics (PK). The first examines what the drug
clinical trials. does to the body; the second, what the body does to the
drug. These investigations help determine drug dosage.
TERM OF THE WEEK: ENDPOINT Variations in how people of different sizes, ages, and
genetic backgrounds etc., will likely respond to a new
Clinical trials measure endpoints, that is, major health drug make testing in different populations critical.
outcomes. There are generally two types:
• Clinical endpoints refer to benefits such as PHASE II
survival, decreased pain, the absence of disease, or
Phase II examines drug efficacy as well as continuing
greater mobility.
safety tests. Phase II trials involve larger groups of
• Surrogate endpoints substitute for clinical participants, all of whom are patients. Group size varies,
endpoints when they are impossible or impractical depending on the target market. A drug being developed
to measure. For instance, a clinical benefit, such for Type 2 diabetes needs far more participants than one
as survival, hopefully, takes decades to observe. for a rare disease such as ALS.
Researchers may instead look to shorter-term
Phase II studies are usually randomized, double-blind
phenomena. For example, in studying a drug
studies. This means that patients are randomly assigned
designed to prevent heart disease, they can monitor
the drug or the placebo and even the researchers
cholesterol levels instead of decreased fatality from
involved don’t know who receives which. This helps
heart attacks. Similarly, in some cancer treatments,
guard against bias in determining who goes into which
reduced tumor size stands in for longer life.
group. Otherwise, investigators may naturally want
The FDA requires that clinical protocols clearly define to ensure the sickest patients receive the promising
endpoints. They are front and center in the application experimental treatments.
companies submit to the FDA that request permission
A placebo is also known as the “standard of care group”.
to study a new drug. This application is called an
According to the National Institutes of Health (NIH),
Investigational New Drug application or IND.
the standard of care is a treatment accepted by medical
experts as proper for a certain disease and widely used
PHASE I by medical professionals. For example, in clinical trials
The FDA divides clinical studies into three main phases. for Amgen’s Repatha, researchers compared its safety
Phase I usually tests drug safety in healthy volunteers, and efficacy against statins, the current standard of care
typically one hundred or fewer. In some cases, Phase I for cholesterol reduction. Repatha was demonstrated to

be as effective as statins, and had a better safety profile COCKTAIL FODDER
in some patients, which led the FDA to approve Repatha.
The first antibiotic, penicillin, was discovered in 1928
when microbiologist Alexander Fleming noticed the mold
THE POWER OF POSITIVE THINKING Penicillium secreted a substance that killed his bacterial
Investigators measure drug efficacy by monitoring cultures. Inspired by this story, biochemist Akira Endo,
predefined endpoints. They always look at effectiveness who was working at Sankyo Pharmaceuticals (Tokyo)
relative to a control group--people who receive either in 1972, decided to screen Penicillium cultures in hopes
standard of care treatment or a placebo. of finding a chemical that would inhibit cholesterol.
Endo’s quest paid off – he discovered a compound that
The “placebo effect” may stem from an expectation or
eventually went on to be one for the first statin drugs
belief that the “medicine” will work. The placebo control
approved in 1987.
is very important since scientists have firmly established
that placebos can significantly affect patient health. Why would mold cells secrete an anti-cholesterol drug?
Recent evidence shows that people dealing with chronic For the same reason they secrete penicillin – to kill the
pain can produce natural opioid painkillers in response bacteria that they are competing with for resources.
to a placebo. More rarely, the reverse of the placebo Cholesterol is required for bacteria to build functional
effect – the “nocebo effect” – occurs. In other words, cell walls.
some patients expect a study drug will either not help or Next week, we’ll continue along the pathway to approval
in fact harm them. as we zoom in on drug development Phase III and IV.

To Approval: Phase III
PHASE III IS NO GUARANTEE A crossover study’s strength lies in its ability to capture
differences in response to a drug versus a placebo in the
Our last Biotech Primer WEEKLY explored the riskiest
same patient. This eliminates the inevitable individual
part of the human clinical trials pathway: Phase II.
variations among subjects in the experimental and
About 70% of drugs that enter Phase II never make it
control groups in the more traditional parallel study.
out. Most often, it’s because they fail to demonstrate
effectiveness. Even making it to Phase III is no guarantee
of success – about 40% of drugs fizzle out during this
period. In today’s WEEKLY, we look at the final stage of
clinical testing and the innovative clinical designs that are
pushing for faster drug approvals.
CHOICES, CHOICES, CHOICES

Phase III clinical trials continue to test a treatment’s
efficacy and safety, but in still larger groups of patients.
Bigger groups mean more statistically significant
results. As in Phase II, the trials have traditionally been
randomized, double-blind studies. If the investigational
drug appears to work, patients are allowed to continue
THE NEW KID IN TOWN: ADAPTIVE DESIGN
taking the medication after the trial ends, before
regulatory approval. This is an example of what doctors Adaptive studies offer more flexibility than traditional
and other medical professionals call compassionate use. designs, and are gaining in popularity as more
efficient in bringing new drugs to the market. They
Choices, choices, choices... In addition to randomized,
allow investigators to modify the trial design as they
double-blind trials, researchers have other study designs
go, rather than spending time and money pursuing
at their disposal, which we will explore below.
drug formulations or dosages that ultimately prove
ineffective. For example, researchers may separate
THE PATRIARCHS: PARALLEL participants into different dosage groups. At a pre-
VS CROSSOVER DESIGNS specified time, they may note how patients respond to
Parallel studies use the classic experimental design. different doses. If one dose seems more effective, the
Participants are divided into two groups. The treatment researchers will conduct the rest of the trials using only
group receives the experimental drug. The control group that dosage. Researchers set the trial protocol, which
receives either the current standard of care or a placebo. lays out the adaptation schedule and processes before
the trial begins.
Investigators sometimes decide to conduct a crossover
study. Here, both groups receive the medicine and then One prominent example of adaptive design is I-SPY 2
the placebo or vice versa. The treatments are separated study (Investigation of Serial Studies to Predict Your
by a washout period in which patients are taken off the Therapeutic Response with Imaging and Molecular
study medicine (or placebo) to eliminate any effects. Analysis). This trial, being conducted by 20 different
cancer research centers across the US, examines up to
Both parallel and crossover studies include a baseline
12 different breast cancer therapies at once. Drugs that
period before patients take the drug or placebo. It allows
show promise proceed to Phase III, allowing space for
researchers to gather the initial health information
other drugs to enter the trial. “Graduates” of the I-SPY 2
against which they will compare changes observed
study include AbbVie’s (North Chicago, IL) Veliparib and
during or after the study. For example, when assessing
Merck’s (Kenilworth, NJ) MK-2206.
a new cholesterol medication, researchers might take
baseline cholesterol levels. The 21st Century Cures Act promotes the use of
adaptive trial design. This benefits volunteers, as

researchers assign more patients to study groups taking NEW APPROACH TO FIGHTING
medicine that demonstrates promise. It’s good news for CANCER: UMBRELLA DESIGN
non-study patients too, who stand to benefit from new
treatments getting to market faster. These trials reflect the fact that scientists classify most
cancers as specific subtypes based on the mutation
involved. An umbrella study may look at patients with
NEW APPROACH TO FIGHTING
one type of cancer, for example, lung cancer. However,
CANCER: BASKET DESIGN researchers divide participants into subgroups based
The design of clinical trials for new oncology drugs is on the particular mutation behind their lung cancer, and
changing to reflect our increasing understanding of treat them with drugs designed to target the subtype
cancer as a disease driven by mutations. Traditionally, specifically. This allows researchers to identify patients
trials test drugs on a specific illness, say, lung or most likely to benefit from the new drug.
breast cancer. In contrast, basket trials classify cancers One ongoing umbrella study is the National Cancer
according to the mutation they exhibit, not the tissue Institute’s Alchemist Lung Cancer Enrichment Marker
they affect. For example, the FDA initially approved the Identification and Sequencing Trials (ALCHEMIST) study.
drug Zelboraf for melanoma patients with a mutation In it, researchers sequence patients’ tumors and assign
in their “BRAF” gene. Researchers at Memorial Sloan them to different treatment groups according to the
Kettering Cancer Center (New York, NY) explored subtype mutation.
whether Zelboraf could work against other cancers with
the same genetic signature – the BRAF mutation. The
results indicated that Zelboraf also effectively treated
BRAF-mutated non-small cell lung cancer, Erdheim-
Chester disease, and Langerhans cell histiocytosis. These
last two, rare blood cancers, illustrate a key benefit of
basket trials: they may offer patients with rare cancers
better access to clinical trials, depending on their tumor’s
genetic signature.
Phase III is the final hurdle for drug candidates. If a

drug “survives,” things are looking good for developers
and patients alike. It’s not a slam dunk yet though, so
next week we look at the approval process and what
comes next.

To Approval: Phase IV
PHARMA FINISH LINE: FDA APPROVAL Accelerated Approval allows drugs to go forward
using surrogate endpoints instead of clinical endpoints.
Last week, we focused on the final stage of clinical
Surrogate endpoints, such as lowered blood pressure
testing, Phase III trials, where drug developers assess the
or reduced tumor size predict rather than demonstrate
safety and efficacy of their drug in large patient groups.
clinical benefits. In these cases, pharmaceutical
At the end of Phase III, drug developers face the moment
companies must run post-market studies to verify the
of truth: does the study data support claims that the
anticipated effect.
new drug is both safe and effective? If the answer is yes,
then it’s time to submit either a New Drug Application Priority Review means the FDA will aim for a decision
(NDA) for small molecule drugs, or a Biologics Licensing within six months.
Application (BLA) for large molecule drugs. This week, Fast Track is based on preclinical or clinical data that
we’ll examine the approval process, including various suggests the product addresses a specified unmet
pathways for expedited approval, and touch on post- medical need. The designation enables developers to
approval safety studies also known as Phase IV. communicate more often with the FDA. The agency
provides guidance on clinical trial design and process,
UNDER THE FDA MICROSCOPE which helps resolve questions or issues quickly. These
Once a company submits either an NDA or BLA, the designees also qualify for Accelerated Approval, Priority
FDA takes about a year for review–which seems Review, and Rolling Review—which allows developers
governmentally slow unless you consider that most to submit each section of an NDA or BLA as they finish,
applications run 100,000 pages or longer. There are rather than all at once.
three possible responses: Breakthrough Therapy designates drugs that may
• Approval letter: Ta dah! greatly improve patient health. The bar is set high
to join this privileged group, though. It requires
• Approvable letter: Close, and this is how to get the preliminary clinical evidence of effectiveness. Once
cigar! FDA requests correction of minor deficiencies, granted, Breakthrough designees receive Fast Track
labeling changes, or post-approval studies. advantages, as well as intensive guidance on their
• Not approvable letter: Thanks for playing! FDA development program as early as Phase I. They also get
elaborates on deficiencies in the application and why an “organizational commitment involving senior FDA
the drug is not approved. managers,” according to the FDA website.
For many drugs, the development, testing, and Orphan Drugs are often found in the above categories.
approval journey is straightforward. Difficult, Companies develop them for rare diseases, those which
but straightforward. affect fewer than 200,000 Americans. These include
hemophilia and Gaucher’s Disease, a genetic disorder
THAT SPECIAL SOMETHING that causes skeletal and neurological issues. Prior to
Certain drugs are given special consideration throughout the Orphan Drug Act of 1983, the industry had no
the FDA approval process. These particular therapies are financial incentives to work on therapies for such small
eligible for regulatory designations that speed up the populations. Orphans lacked commercial sponsors,
review and get a product to market more quickly. They “parents,” to shepherd them through lengthy and
must first meet two criteria. A drug must target a serious expensive trials. The orphan drug legislation provides
condition that likely results in death or significantly the following incentives:
impairs daily living. Examples include cancer and • Federal tax credits of up to 50% off research costs
Alzheimer’s disease. The drug also needs to address a
• Increased protection from generic competition
major, unmet medical need. That is, either no medicine
exists, or current therapy has safety issues. • Waivers of FDA application and product fees
(amounting to hundreds of thousands of dollars)
The FDA’s special designations include:

COCKTAIL FODDER: PDUFA the Plavix label carry a black box warning – a warning
outlined in a black box, which is the strictest warning put
The Prescription Drug User Fee Act (PDUFA) requires
on the labeling of prescription drugs or drug products by
drug developers to pay a fee to the FDA to help fund the
the FDA.
work necessary for timely approvals. The “PDUFA date”
of a drug is the date by which the FDA has committed to The rigorous path from preclinical testing to approval
review its application. For 2018, the PDUFA fee required usually takes a decade or longer, with no promise of
when submitting an NDA or BLA that requires clinical success. According to the Biotechnology Innovation
data is $2,421,495. Organization, approximately one in ten drugs entering
clinical testing ultimately make the grade. The highest
FINISHED, NOT DONE rate of failure occurs in Phase II; only 31% of drugs
proceed to Phase III. Of those that make it to the final
Phase IV generally refers to post-market studies, which Phase III, a little more than half - 58% - are considered
companies undertake after a drug is approved and at the safe and effective enough for an NDA or BLA submission.
pharmacy. Drugs that were approved using a surrogate From start to finish, only 10% of the drugs that begin
endpoint are monitored to confirm clinical efficacy. For Phase I reach the market.
all drugs, safety is monitored and confirmed. Phase IV
includes informal studies of doctor and patient reports,
which sometimes reveal unanticipated side effects.
Such “surprises” aren’t uncommon in brand new drugs,
because so many more people take them once out
on the market versus the small number of patients in
clinical trials.
For instance, after the anti-clotting medicine Plavix
(Bristol-Myers Squibb; New York, NY) was approved,
doctors found it less effective in some people than
expected. Further probing found that the drug wasn’t
fully activated in about 14% of patients because their
bodies produced a less active form of the liver enzyme
Despite the low odds, innovative companies continue to
that activates Plavix. Fortunately, a genetic test can
bring new drugs to market every year. Stay with us as we
quickly reveal whether a patient can benefit from Plavix.
continue following their stories in 2018.
This unlooked-for result spurred the FDA to require that

Market Access
MARKET ACCESS PRIMER England-based insurer Harvard Pilgrim’s contract
with Amgen (Thousand Oaks, CA) provides a “pay-for-
For the last few weeks we here at Biotech Primer have
performance” rebate to Harvard Pilgrim if Amgen’s
tracked the progression of a drug candidate from the
new cholesterol drug Repatha, a PCSK9-inhibitor drug,
lab to the marketplace, where only the fittest survive.
doesn’t perform as well as in clinical trials. The benefit to
Winning at clinical trials means earning an official
Amgen is preferred positioning on the Harvard Pilgrim
regulatory approval. Congratulations! But as any
formulary as well as access to real world and post-
seasoned drug developer will tell you, the game has only
marketing data.
just begun. Ensuring newly-approved drugs are available
to all patients is the next hurdle. With other novel high-impact, high-cost drugs such
as CAR-T treatments and gene therapies, payors are
In this WEEKLY we’ll explore market access for
questioning how to best afford these medicines. For
innovative products such as cancer-fighting drugs and
example, Spark Therapeutics (Philadelphia, PA) recently
gene therapies.
received FDA approval for Luxturna, a gene therapy
treatment for hereditary blindness. The treatment is
TERM OF THE WEEK: MARKET ACCESS remarkable, in that it promises a lifetime cure with one
Market access is the process of proving a product’s administration - at a cost of $425,000 per eye. Spark
value to ensure reimbursement with commercial payors, Therapeutics is working on an outcome-based payment
government payors and integrated delivery systems. scheme in collaboration with Harvard Pilgrim Health
These payor stakeholders are often referred to as and with Centers for Medicare and Medicaid Services
“organized customer groups.” (CMS). If Luxturna fails to meet the intended outcomes
According to Linda Lander, President of Inside Out at certain intervals post treatment, Spark would refund
Market Access, “market access aligns incentives between some or all the treatment’s cost.
payors, pharmaceutical companies, providers, and These types of agreements allow patients the chance to
patients to lead to cost-effective models.” benefit from a new therapy that a payor might otherwise
be reluctant to take a chance on.
DRUG APPROVAL DOESN’T
EQUAL REIMBURSEMENT AN INDEPENDENT VOICE
Launching a new drug, especially a high-cost one, Organized customer groups are increasingly using
requires proving its value early in the process. Just independent analysis to guide reimbursement decisions.
because a drug is approved doesn’t mean it will be These independent agencies, which include the
reimbursed for coverage. Without reimbursement California Technology Association (CTA), the National
patients will not benefit, nor will the drug’s sponsor Comprehensive Care Network (NCCN), and Institute
recover the significant resources it invested of Clinical and Economic Review (ICER), evaluate and
in development. determine the “reasonable price/value” of drugs in the
context of an overall healthcare budget.
Organized customer groups are now demanding
evidence of value. Market access involves a discussion According to Steve Pearson, Executive Director of ICER,
and negotiation between the payor and the drug the organization asks four key questions:
sponsor to define and demonstrate value. This is • How well does the drug work?
increasingly achieved via outcome-based reimbursement
• How much better is it than what we already have?
and real world evidence, in which patient health and
outcomes data gathered outside of clinical trials drives • How much could it save us?
downstream reimbursement. • How much would it cost to treat everyone who
Outcomes-based contracts include some sort of refund needs it?
if the new drug does not perform as advertised. New

In conducting its analysis, ICER reaches out to patients, acceptance for their initial drug prices; for example,
doctors, and drug makers. The end goal is to calculate Spark Therapeutics met with ICER prior to the Luxturna
a value-based price benchmark that reflects the launch to help guide pricing strategy.
drug’s value to patients and to the healthcare system. As new technologies create highly innovative treatments,
According to the American Managed Care Pharmacy’s the issue of pricing must be addressed. By deftly
annual survey of payors, more than 50 percent now navigating market access, companies can ensure more
employ ICER evaluations in their reimbursement reviews. patients have access to these advances.
As a result, biopharma companies increasingly work with
ICER and other independent organizations to gain payor

to Approval: A Recap
FROM THE LAB TO THE PATIENT Biopharma companies typically put lead compounds
through several rounds of optimization before they can
In this issue of the Biotech Primer WEEKLY we will
become drug candidates for preclinical testing. Lead
recap the past seven issues that highlight the journey a
optimization is the process by which a drug candidate is
molecule takes from the lab to the patient.
designed via iterative rounds of synthesis.
Beginning in the 1980’s, scientists took a new tack in
Drug Discovery 201, published December 2017
developing drugs. They adopted an approach known
as rational drug discovery. Using this methodology,
researchers first seek to understand a disease at the DRUG DISCOVERY: SAFETY
cellular level to identify its mechanism. Once the cellular AND SUBMISSION
mechanism becomes clear, scientists can identify a drug In vivo testing (“in a living thing”) comes next. This area
target: the molecule involved in the illness that the ‘as of preclinical testing assesses a drug candidate’s toxicity
yet undeveloped drug’ will hopefully act on. in at least two different species of animals, such as mice
Fast forward many years and hundreds of millions of and guinea pigs. The animals receive more of the drug
dollars and drum roll please... the ‘as yet undeveloped for longer than would human volunteers. Meanwhile,
drug’ may just become the drug that cures your once lab technicians watch the animals for adverse effects.
incurable illness. Preclinical testing must follow the FDA’s Good
Laboratory Practice (GLP) guidelines. These regulations
DRUG DISCOVERY: IDENTIFY AND VERIFY help ensure scientific integrity and humane treatment of
laboratory animals.
Researchers identify their molecular targets by
answering one incredibly complex question: How does Once researchers amass enough safety data to ensure
diseased tissue differ from healthy tissue? that a drug candidate will be safe for people, they submit
an Investigational New Drug (IND) application to the FDA.
Researchers verify the suitability of target molecules by
If the FDA blesses the application, the drug candidate
answering two important questions: does the target play
may enter Phase 1 clinical testing.
a key role in the disease? and will targeting the molecule
likely be safe and effective? Drug Discovery 301, published January 2018
Identifying and validating drug targets are only the first

steps on the long road to safe, effective new medicines.
PHASE I/II: MAY THE ODDS
BE IN YOUR FAVOR
Drug Discovery 101, published December 2017
Every drug in clinical use today, from the latest CAR-T
treatment to older cholesterol-lowering statins,
DRUG DISCOVERY: HIT TO LEAD
share one thing in common: they have all successfully
To find the best drug candidate, scientists need to design navigated the rigorous clinical trials process. This is no
easy-to-perform, large-scale, fast, and accurate assays. small feat, as only ~10% of the drugs that enter Phase
Think automation. Think high throughput screening. I testing successfully emerge as marketed products.
High throughput screening ideally results in several Those few drugs that show remarkable success in early
promising drug candidates or “hits.” To become “leads” clinical trials make headlines, and deservedly so.
that merit animal testing, hits must pass rigorous in Phase I/II, published January 2018
vitro testing. On this leg of the drug discovery voyage,
researchers ask three questions:
PHASE III: CHOICES, CHOICES, CHOICES
1. Is the hit safe?
Phase III clinical trials continue to test a treatment’s
2. Is the hit specific to the target? efficacy and safety, but in still larger groups of patients.
3. Does the hit show promise to treat the disease? Bigger groups mean more statistically significant
results. As in Phase II, the trials have traditionally been

randomized, double-blind studies. If the investigational submit each section of an New Drug Application (NDA) or
drug appears to work, patients are allowed to continue Biologics License Agreement (BLA) as they finish, rather
taking the medication after the trial ends, before than all at once.
regulatory approval. This is an example of what doctors Breakthrough Therapy designates drugs that may
and other medical professionals call compassionate use. greatly improve patient health. The bar is set high
Choices, choices, choices… In addition to randomized, to join this privileged group, though. It requires
double-blind trials, researchers have other study designs preliminary clinical evidence of effectiveness. Once
at their disposal, which we will explore below. granted, Breakthrough designees receive Fast Track
Phase III, published February 2018 advantages, as well as intensive guidance on their
development program as early as Phase I. They also get
an “organizational commitment involving senior FDA
PHASE IV: THAT SOMETHING SPECIAL
managers,” according to the FDA website.
The FDA’s special designations include:
Phase IV, published February 2018
Accelerated Approval allows drugs to go forward
using surrogate endpoints instead of clinical endpoints. GETTING MEDICATIONS TO
Surrogate endpoints, such as lowered blood pressure
PATIENTS: MARKET ACCESS
or reduced tumor size predict rather than demonstrate
clinical benefits. In these cases, pharmaceutical Market access is the process of proving a product’s
companies must run post-market studies to verify the value to ensure reimbursement with commercial payors,
anticipated effect. government payors and integrated delivery systems.
These payor stakeholders are often referred to as
Priority Review means the FDA will aim for a decision
“organized customer groups.”
within six months.
According to Linda Lander, President of Inside Out
Fast Track is based on preclinical or clinical data that
Market Access, “market access aligns incentives between
suggests the product addresses a specified unmet
payors, pharmaceutical companies, providers, and
medical need. The designation enables developers to
patients to lead to cost-effective models.”
communicate more often with the FDA. The agency
provides guidance on clinical trial design and process, Market Access, published February 2018
which helps resolve questions or issues quickly. These Despite the low odds, innovative biopharma companies
designees also qualify for Accelerated Approval, Priority continue to bring new drugs to market every year. Stay
Review, and Rolling Review—which allows developers to with us as we continue following their stories in 2018.

Who’s Your Daddy? The
Science of 23andMe
HEY—CHECK OUT THOSE GENES! “STR” stands for “short tandem repeat.” These are short
sequences of DNA that repeat from five to fifty times.
There’s an old saying, “If you don’t know where you’ve
The number of times a particular STR repeats varies
come from, you can’t know where you’re going.” We used
from person to person.
to rely on paper birth certificates, marriage licenses and
memory to help discover where we’ve come from; but
paper gets damaged, people are fallible, and memories THIS DNA IS NOT THAT DNA
fade. Leave it to biotech to come up with a better way. SNPs and STRs provide different types of information
New heritage-hunting techniques come courtesy of one depending on their DNA origin. We humans have
our greatest scientific achievements: the sequencing of different types of DNA:
the human genome.
• The sex chromosomes are the X and the Y
We now have tools to genetically examine ourselves chromosomes. Men have one Y chromosome and
and our past. The ability to decode genetics at modest one X chromosome; women have two Xs only. Thus,
expense allowed the DNA testing industry to develop. the Y chromosome contains DNA information about
Companies such as 23andMe, Ancestry.com, and paternal ancestry.
National Geographic sell genetic tests online. These
• Mitochondrial DNA (mtDNA) is found in tiny
kits, consisting of a plastic tube, a baggy, and a postage-
compartments called mitochondria that convert
paid mailer, promise biological insight into the past. But
sugar to energy in cells. Only woman pass on mtDNA
what about the science? This Biotech Primer Weekly
because during fertilization the DNA in sperm
overviews commercial DNA testing and how it works.
mitochondria is quickly destroyed. Mitochondrial
DNA can help trace maternal ancestry.
A DASH OF DIFFERENCE
• Autosomal DNA is all the genetic material that is not
Every human being alive shares an astounding 99.9% found in the sex chromosomes and in mitochondrial
of their DNA! Only one tenth of one percent of genetic DNA. Autosomal DNA is found in the pairs of
“stuff” accounts for a planetful of differences. That’s autosomal chromosomes numbered one through
because our genome consists of an astonishing three 22. These 22 pairs come from both mom and
billion-plus building blocks—the famous As, Cs, Ts dad – one copy from each. Autosomal DNA offers
and Gs. generic information about both the maternal and
Areas of variation in genes are called genetic markers. paternal lineages.
Geneticists and others use genetic markers to assess the
probability that two people share a common ancestor. DEEP ANCESTRY
The more genetic markers two people share, the more
Not only are types of DNA inherited differently – some
likely they are related. The only people with the same
from mom, some from dad, some from both – but
exact genetic markers are identical twins.
the rate at which their DNA sequence changes from
generation to generation also varies. This is because
ALPHABET SOUP: SNPS AND STRS mtDNA and Y chromosomal DNA change only through
The two major types of genetic markers are SNPs and random mutation of their As, Cs, Ts and Gs. Change can
STRs. In the alphabet soup of genetics, “SNP” is short take centuries before a noticeable difference appears.
hand for “single nucleotide polymorphism.” It simply By comparing genetic markers from mtDNA and Y
means that one of the three billion building blocks that chromosomal DNA with those of indigenous peoples
make up a person’s DNA has been switched. In other from various parts of the world, it’s possible to estimate
words, an A has been swapped with a G. where your ancestors hailed from way back when.

MAKING FAMILY CONNECTIONS each egg a woman produces only carries half her DNA
only some will carry Mongolian markers. Therefore, one
Each person shares half their genetic markers with
sibling could show Mongolian ancestry, while another
siblings, parents, and any children they have. We share
doesn’t.
about a quarter with grandparents or grandchildren,
aunts and uncles, nieces and nephews, and half-siblings.
The amount of DNA we have in common with relatives OUR POSSIBLY HAIRY PAST
diminishes by half in each successive generation. By What’s one of the most intriguing secrets genetic
seven generations back, the amount of DNA shared testing can reveal? Just how much of a Neanderthal you
among relatives is less than one percent. really are! Our smaller, sturdier cousins’ genome was
So, autosomal DNA is useful in trying to make published in 2010, based on some very well-preserved
connections between living relatives or determining how DNA taken from bones found in a Croatian cave. The
far back you share an ancestor with someone. However, evidence suggests that early humans mated with
it doesn’t shed light on “deep ancestry,” or the region of Homo neanderthalensis. Thanks to DNA testing, we
the world our ancestors came from centuries or even can find out just how cozy our distant relatives were
millennia ago. with Neanderthals. The average person is about five
percent Neanderthal.
COCKTAIL FODDER: SAME FAMILY, Today’s technology makes it possible for the curious
DIFFERENT ANCESTOR? to shed light on their DNA. The databases of genetic
markers are growing apace, making it easier to uncover
Can siblings’ ancestral origins be different? The long-lost relatives, for better or for worse. So how
surprising answer is yes! Imagine that a small about it? Are you ready to find out who you really are,
percentage, say 10 percent, of a woman’s DNA contained biologically speaking?
genetic markers indicating Mongolian ancestry. Because

Meatless Meat: Biotech
Burger Ain’t No Bean Patty
MISSION IMPOSSIBLE: MEATLESS MEAT? compare their speeds to a baseline spectrum,
which allows them to detect the molecules in their
Imagine biting into a juicy cheeseburger: the flavor,
sample burger.
the texture, the smell. Now, imagine the cheeseburger
meatless. Impossible? No. Impossible Foods, a Redwood
City, CA-based company has used biotechnology to THE HAMBURGER MOLECULE
create a plant-based burger amazingly similar to the One key molecule Impossible Foods scientists identified
bovine original. In this edition of the Biotech Primer as responsible for a hamburger’s meaty flavor is heme.
Weekly, we examine how they did it. This iron-carrying molecule is associated with one of the
major muscle proteins, myoglobin. Since meat is pretty
UNCOVERING THE MYSTERY OF MEAT much muscle, it’s no surprise that it’s pretty much heme
that accounts for meat’s taste.
Impossible Foods founder Patrick Brown knew he
needed to understand America’s favorite sandwich at Heme isn’t merely some muscle-bound molecule. It’s
its most basic level. Discovering the specific molecules also found in the roots of soy plants, attached to the
that imbue hamburgers with their unique deliciousness, protein leghemoglobin. Soy contains far less heme
he reasoned, would enable him to build a kinder, more than cow muscle. That’s why soy-based veggie burgers
planet-friendly burger. lack that signature meaty taste. Soy contains so little
vegetarian heme that using the plant to meatify veggie
So how to get at what makes a burger a burger?
burgers just won’t work.
Step one: acquire hamburger meat.
There’s more than one way to skin a soybean though—
Step two: cook. Researchers at Impossible Foods heated those savvy Impossible Foods people genetically
ground beef, essentially vaporizing it into a usable engineered yeast cells to contain the soy leghemoglobin
sample. These gaseous burger components include gene. Voila! A sustainable, high-volume production
some of the same molecules responsible for those platform for leghemoglobin is suddenly possible.
mouth-watering smells that waft through neighborhoods Heme is harvested from the yeast to become a major
and parks in warm weather. ingredient the secret Impossible Burger recipe. The
Step three of the molecular dissection is a little more recipe also includes wheat and potato proteins for
complicated than meat and heat. It requires a gas texture, and coconut oil as a stand in for fat.
chromatogram mass spectrometer, which is really two
gadgets in one. Not standard kitchen equipment. ENGINEERING THAT BLOODY TASTE
Chromatography is the process of separating Impossible Foods set out to recreate not only the taste,
components of a mixture. Burger “gas,” like the earth’s but other parts of the burger-eating experience. That
atmosphere, is composed of molecules with different leads to the question: does a medium rare Impossible
physical and chemical properties. Think different sizes, Burger ooze a little “blood?”
shapes, and charges. Scientists pass the burger fumes
Of course. The faux blood looks strikingly similar to the
through a long, narrow tube that contains a material that
real thing. That’s because heme not only makes meat
various molecules “stick to” to a greater or lesser degree,
taste meaty, it makes blood red. Heme binds iron, which
based on their makeup. The assorted molecules leave
in turn binds oxygen. The chemical bond between iron
the tube at different rates, separating.
and oxygen reflects light so that it appears crimson.
The exiting molecules then move on to the spectrometer
While the editorial staff of the Weekly has yet to
which “ionizes” or gives them an electric charge.
sample the Impossible Burger (hint hint, Dr. Brown),
Negatively charged molecules then pass through a
eater-on-the-street reports are favorable. How does
magnetic field toward a negatively charged panel.
it stack up nutritionally? Protein, fat, and iron content
Molecules with different masses travel at different
are comparable to a lean ground beef patty – with the
rates, creating a graph or mass spectrum. Researchers
added benefit of zero cholesterol. The Impossible Burger

is currently available in restaurants at select locations Several companies are working to make lab-grown
around the country. beef cheaper and easier to produce. These include
SuperMeat (Tel Aviv, Israel); Mosameat (Maastricht, The
BEYOND IMPOSSIBLE: LAB-GROWN MEAT Netherlands); and Memphis Meats (San Francisco, CA).
Memphis Meats expects to have a supermarket-ready
But wait, there’s more! Another type of biotech burger is
product by 2021; Mosameat estimates a similar timeline.
on the horizon: lab-grown meat. Basically, researchers
extract stem cells from cow muscle and grow them in Going vegetarian can be tough, but many find it a
the lab. pathway to better health. Moreover, beef production
uses significantly more natural resources than the
This approach to making “cultured” or “clean” meat still
equivalent amount of veggie or cultured meat. And that’s
has a way to go before it lands on your dinner plate.
not to mention all that beefy methane. Global livestock
Growing cells in the lab isn’t cheap. Similar to animals,
production accounts for about 15% of this greenhouse
cultured meat cells need care and feeding. The health,
gas, which contributes to global warming. For those
safety and technological requirements of culturing
who want to cut back or eliminate meat consumption,
meat result in great expense. One pound of lab-grown
but still crave the taste of a good old-fashioned burger,
beef runs thousands of dollars. Miraculously, Kobe beef
biotechnology just might have the answer.
seems almost affordable.

Alzheimer’s Disease: A
Tough Nut To Crack
AFFECTING 5.1 MILLION TANGLES
Alzheimer’s disease (AD) ranks as one of the toughest Another brain protein associated with Alzheimer’s is tau,
nuts to crack within drug discovery and development. which is concentrated in the neurons and is primarily
Current treatments merely manage symptoms, so understood as a component in stabilizing nerve cell
finding a better solution becomes more and more urgent structure. Abnormal aggregates of tau form “tangles”
as the aging population grows. within nerve cells. These tangles are correlated with
Approximately 70 percent of dementia cases are caused the onset of Alzheimer’s along with the characteristic
by AD. It is a neurodegenerative disorder— neurons plaque formations.
progressively lose structure and function. As the disease
continues and more neurons are damaged and die, GENETICS PLAYS A ROLE
symptoms get worse. Neurons in the hippocampal About 70% of Alzheimer’s cases are thought to have
region of the brain associated with memory formation at least some genetic association, with different genes
are among the first affected. By 2025, the number of being implicated depending on the type of Alzheimer’s.
people age 65 and older with AD is projected to reach
A gene found on chromosome 19 called the
7.1 million—a 40 percent increase from the 5.1 million
apolipoprotein E gene (APOE) influences the
affected in 2015 (Alzheimer’s Association).
development of late-onset Alzheimer’s. Individuals
A number of different companies are working to develop with different variants of the APOE gene have different
treatments, with a few already in clinical trials. In this risk profiles:
two-part series, we’ll first explain what is known about
• Variant ε2 (APOE2) is rare and possibly lessens or
the molecular causes of AD, then explore Alzheimer’s
delays Alzheimer’s onset
drugs that are in development.
• Variant ε3 (APOE3) is neutral
PLAQUES AND TANGLES • Variant ε4 (APOE4) is associated with a significantly
increased risk of Alzheimer’s
PLAQUES The APOE proteins plays a role in clearing Aβ from
Alzheimer’s disease is associated with the build-up the brain, with APOE4 carrying out this function less
of amyloid-beta (Aβ) plaques in patients’ brains. But efficiently than the other variants. There is also some
what, exactly, are Aβ plaques? Aβ plaques derive from evidence that APOE4 contributes to the breakdown
the cleavage of a protein called the amyloid precursor of the blood-brain barrier seen in patients, resulting
protein, which is thought to play a role in the formation in increased brain inflammation—another marker of
of synapses. Individual Aβ molecules clump together to Alzheimer’s. A better understanding of APOE4’s role
form the plaques associated with Alzheimer’s. in Alzheimer’s onset may lead to the development of a
whole new class of drug.
Until recently, the mechanism by which Aβ plaques
might cause Alzheimer’s was not known. Recent research
from Stanford University suggests the plaques bind IF AT FIRST YOU DON’T SUCCEED…
to a receptor on nerve cells, disrupting their function. A number of drug developers have attempted to use
However, there is no absolute consensus on whether monoclonal antibodies (mAbs) to disrupt the formation
these clumps of protein are the origins of AD or a of the AD-associated Aβ plaques. Unfortunately,
symptom of the underlying cause. this approach has yet to experience clinical success.
However, this doesn’t mean that the approach is not
viable – different mAbs attach to different parts of Aβ,
meaning that the outcome of one mAb trial does not
necessarily predict the outcome of another. Biogen
(Cambridge, MA) and Roche (Basel, Switzerland) both

have mAb therapies targeting Aβ plaques in Phase III UNTANGLING TAU
clinical development. In earlier phase studies, these
AbbVie (North Chicago, IL) is targeting tau, the other
mAbs showed the most promise in patients with less
major protein associated with AD. The company
advanced forms of the disease.
currently has an anti-tau mAb therapy in Phase II clinical
The prevention of Aβ plaques is also the focus of testing, and last month announced a partnership with
growing interest in creating an AD vaccine. Leading this Voyager Therapeutics (Cambridge, MA) to develop a gene
effort is Novartis (Basel, Switzerland), whose CAD106 therapy treatment that targets tau. The treatment will
vaccine contains fragments of the Aβ protein and has deliver a gene encoding an anti-tau antibody directly to
been shown to be safe in Phase I trials. The goal of the cells in patients’ brains – enabling those cells to make the
vaccine is to activate an immune response against Aβ, antibody. If successful, this would bypass altogether the
thereby reducing its accumulation and potential to form blood-brain barrier that makes it difficult for some drugs
plaques in the brain. CAD106 is currently in Phase II/III to even enter the brain.
studies of efficacy, in which the vaccine is being tested
in cognitively normal individuals between the ages of THE FUTURE?
60 and 70 who are at high risk of developing the disease
based on their APOE4 status. Amyloid-beta and tau remain tantalizing targets for AD
drug development because of their close association
with the disease. However, new approaches are
springing up as frustration over the lack of progress in
treating this devastating disease grows. Stay tuned for
the next Weekly, where we will describe some of these
new approaches.

Exploring Different Strategies
to Fight Alzheimer’s
TAKE THAT, ALZHEIMER’S to reverse previously observed cognitive declines. The
hippocampus is one of the first regions of the brain
Alzheimer’s pernicious amyloid-beta plaques and
to show damage in AD and is thought to play a role in
tau tangles, discussed last week, remain important
memory formation and spatial navigation. Neuronascent
targets for the biotech industry. In the past few years,
is preparing for Phase I trials of NNI-362.
however, companies have begun to search more broadly
for new treatments. This Weekly looks at products in
development that use different strategies to fight this NEUROINFLAMMATION
heartbreaking illness. Neuroinflammation is one of the drivers of
neurodegeneration in Alzheimer’s disease, multiple
REVIVING THE BRAIN? sclerosis and other brain disorders. Research conducted
at Stanford University (Palo Alto, CA) suggests that
Loss of neurons is Alzheimer’s signature, devastating
the protein c1q is present at higher levels in people
effect. What if we could jump start the development of
with Alzheimer’s disease. C1q accumulates at neuronal
new brain cells? Two companies are trying to do just that.
synapses, the key points of communication between
Neurotrope Biosciences (New York, NY) is developing brain cells. This protein also signals other immune cells,
bryostatin, a drug that activates protein kinase C such as macrophages—which then chomp up cellular
epsilon (PKCꞓ). This protein plays a key role in forming debris present in affected brains. The accumulation
memories. In animal models of stroke, traumatic brain of c1q could account for the loss of synapses and
injury, and Alzheimer’s disease, bryostatin appears to accompanying mental decline.
restore deficits in synapses (connections between brain
South San Francisco-based Annexon is working
cells) and decrease cell death. These results suggest that
on a promising therapy that centers on controlling
bryostatin could help to prevent the loss of neurons and
inflammation in the brain. ANX005, now in preclinical
restore and generate new synapses.
development, is a monoclonal antibody that mops up
Phase II clinical studies of late-stage Alzheimer’s excess c1q.
patients demonstrated improved cognitive function as
Another company homing in on neuroinflammation
measured by the Severe Impairment Battery Scale (SIB),
is vTv Therapeutics (High Point, NC). Their drug,
a standard tool for evaluating treatment response in
Azeliragon, now in Phase III clinical development, is a
advanced Alzheimer’s. Their improvement was greater
small molecule inhibitor of the receptor for advanced
than that seen in patients given the placebo, but the
glycation endproducts (RAGE). RAGE is present on many
difference was not statistically significant. A Neurotrope
neurological cell types. Its activation may promote
Biosciences spokesperson says that it considers the
amyloid-beta production and transport, tau aggregation,
Phase II study exploratory, designed to determine
and chronic inflammation. Preventing any of these
correct dosing. The company is planning a larger
developments could improve Alzheimer’s symptoms.
confirmatory trial in hopes of demonstrating statistically
significant efficacy.
AND THAT AND THAT AND THAT!
Neuronascent (Clarksville, MD) also aims to develop
small molecule activators of neurogenesis. By screening Rather than target Aβ plaques directly, Yumanity
large chemical libraries, the company has identified Therapeutics (Cambridge, MA) is trying to identify
compounds that show promise of sparking neurogenesis the problems they cause. Yumanity scientists have
from adult neural stem cells in both tissue culture and engineered yeast cells to overproduce the Aβ protein
mouse models. and monitor its detrimental effects, such as disrupting
the action of other important cellular proteins.
The company’s lead compound, NNI-362, promoted
the growth of new hippocampal neurons in mice. Surprisingly, yeast share many molecular pathways with
The new cells migrated to the correct location and humans. This similarity means researchers can use the
differentiated. Moreover, they survived long enough little fungi to screen for potential drugs that address

protein disruption. Promising candidates are then Oryzon researchers identified an enzyme, lysine-specific
tested in Alzheimer’s patient-derived cells. By tackling histone demethylase 1 (LSD1), which makes epigenetic
a completely different disease mechanism, the new modifications to genes that results in “turning them
compounds may achieve greater success than seen so down” so they produce less of the corresponding
far with drugs that act directly on amyloid beta or tau. protein. LSD1 makes these changes to genes that
Yumanity is currently in the lead-optimization phase of support neuronal survival. Oryzon scientists have
pre-clinical development. designed a drug, ORY-2001, that inhibits LSD1. Inhibiting
In partnership with Biogen (Cambridge, MA), Cambridge- LSD1 could mean that more neurons survive in AD
based Proteostasis Therapeutics is targeting patients, leading to improved cognitive function. ORY-
AD-associated protein aggregates by activating 2001 recently entered Phase II clinical trials.
proteasomes. These cellular components get rid of
damaged proteins and dysfunctional protein aggregates A TWIST ON TAU
by dismantling their chemical bonds. The protein USP14 Finally, the elusive AD treatment may lie in pursuing a
inhibits proteasomes. Proteostasis is working on the well-established target after all—but at a new angle.
preclinical development of a USP14 inhibitor that allows That’s where Ionis Pharmaceuticals (Carlsbad, CA) is
proteasomes to fully activate in AD patients. This makes headed in the Phase I/IIA clinical studies of their drug,
it more likely that the proteasomes will recognize and IONIS-MAPT. This antisense drug targets the source of
destroy amyloid plaques and tau tangles. the tangles associated with AD. Like other antisense
Oryzon Genomics (Barcelona, Spain) is taking an drugs, IONIS-MAPT destroys tau mRNA, thereby
epigenetic approach to Alzheimer’s. Epigenetic diminishing tau protein production.
modifications are chemical changes to gene sequences It’s encouraging to know how many therapies are in the
that don’t change the information content but instead Alzheimer’s treatment pipeline. With more hard work
affect how much that content is used – in other words, and investment, perhaps one of the many introduced
the amount of a particular protein that the body makes. above will lead to a cure.

Cancer Diagnostic In A Drop
THE LATEST IN CANCER DIAGNOSTICS Genomic Health (Redwood City, CA) currently markets
tissue-based genomic tests for the detection and
Hearing the words “it might be cancer” paired with
classification of breast and prostate cancer, and is
your doctor’s perplexed look is enough to send shock
developing cfDNA-based tests for breast (blood sample)
waves through your body. Getting to the heart of a
and bladder (urine sample) cancers.
diagnosis usually requires a surgical biopsy—removal
and examination of the suspected tissue for visible signs Qiagen (Hilden, Germany) is developing cfDNA liquid
of cancer. biopsy diagnostics in partnership with pharmaceutical
companies such as AstraZeneca (London, U.K.), Tokai
Less invasive diagnostic tests—called liquid biopsies—
Pharmaceuticals (Boston, MA), Novartis (Basel,
might just bring more choices to doctors and patients.
Switzerland), and Eli Lilly (Indianapolis, IN).
They are becoming today’s reality thanks to our
ability to isolate molecules from body fluids. These
diagnostic innovations pair technology with the latest EXTRACTING EXOSOMES
in biomarkers, and are rapidly gaining acceptance as a Exosomes are lipid-encased vesicles that contain cellular
reliable way to screen for cancer and to monitor disease protein, DNA, and RNA and typically have surface
progression and response to treatment. This week we’ll proteins specific to their native cell. These attributes,
examine the different types of liquid biopsies and how combined with the fact that they are found in many
they work. different body fluids, make exosomes a very attractive
TERM OF THE WEEK: LIQUID BIOPSY possibility for liquid biopsy. The idea is to capture
exosomes based on tumor-specific surface markers or to
A liquid biopsy is a test that is able to detect the
collect exosomes and identify them as cancer-associated
presence of cancer using blood, urine, saliva, or other
by examining the enclosed DNA or RNA.
bodily fluid as the sample rather than tissue from
a specific organ. The technique is possible because Aptly named Exosome Diagnostics (Cambridge, MA) has
cancerous tissues shed cells, DNA, and tiny lipid-encased an exosome-based urine and blood tests for prostate
compartments called exosomes. Liquid biopsies detect cancer on the market, and another in development for
the presence of these cancer-associated biomarkers. lung cancer. Qiagen has a partnership with Exosome
Diagnostics to help develop additional exosome-
based cancer diagnostics to complement their work
DISCOVERY BY CELL-FREE DNA
with cfDNA.
When cells in the body die, they release cell-free DNA
(cfDNA)—this includes dying tumor cells. cfDNA-based
CONSIDER CIRCULATING TUMOR CELLS
tests are a type of liquid biopsy because they seek out
a biomarker – in this case, tumor DNA - in body fluids The final category of liquid biopsy is perhaps the
and then identify cancer-specific mutations using PCR or most obvious—circulating tumor cells (CTCs), or
next-generation sequencing analysis. cells splintered from a tumor and circulating in the
bloodstream. The challenge lies in detecting CTCs: some
Trovagene (San Diego, CA) analyzes cfDNA found in
estimates classify them as rare as one circulating tumor
urine samples, which patients collect at home. Currently,
cell per billion normal cells!
Trovagene has tests detecting mutations associated with
melanoma, colon cancer, and non-small cell lung cancer, Janssen Diagnostics (Raritan, NJ) currently markets
as well as the presence of viral DNA for the diagnosis of CellSearch, the single FDA-approved test that allows
human papilloma virus. Exact Sciences Laboratories physicians to identify early CTCs from blood samples.
(Madison, WI) uses at-home collection in their colon Monoclonal antibodies (mAbs) capable of recognizing
cancer test, which analyzes cfDNA in stool samples for proteins on the surface of migrating tumor cells are
cancer-associated DNA. chemically linked to magnetic nanoparticles and then
added to a patient’s blood sample. These tumor-specific
mAbs grab hold of the CTCs, and a strong magnetic field

is then applied to the sample, isolating the captured cells THE FUTURE
for identification and analysis. CellSearch is currently
Ultimately, the best liquid biopsies may contain a
used to monitor the efficacy of treatments for breast,
combination of all the above approaches. Biocept
prostate, and colorectal cancer. A higher number of CTCs
(San Diego) is leading the way by developing liquid
detected may indicate a higher incidence of metastasis,
biopsies that analyze both cfDNA and CTCs. Biocept
or a less than effective treatment route if used to
currently markets liquid biopsy tests for the detection
quantify cancer therapy success.
of lung cancer, breast, colorectal, gastric, prostate, and
Another way to identify CTCs may be cell size—CTCs melanoma. Biocept also has its eye on combination
tend to be significantly larger than other cells in the liquid biopsies for both colon cancer and melanoma.
blood, and this size differential may be exploited in
Today, liquid biopsies are mainly used for monitoring
a microfluidics-based approach to cell separation.
the progress of or response to treatment of already-
Researchers at National University in Singapore
diagnosed cancers rather than as initial diagnostic tests.
(Singapore) and MIT (Cambridge, MA) have developed a
A major goal in the field is to develop tests that can be
microfluidics chip that routes cells from a blood sample
used routinely to detect cancer in seemingly healthy
into different channels based upon cell size. Although
people, which should translate to better treatment
still in the preclinical research phase, this approach
outcomes. Research published by a group at Johns
shows promise for capturing a wide range of CTCs.
Hopkins University (Baltimore, MD) in January 2018
Epic Sciences (San Diego, CA) adopts a “no cell left suggests that a liquid biopsy test that detects both
behind” game plan thanks to technology developed by cancer-associated cfDNA and proteins known to be
the Scripps Research Institute (La Jolla, CA). Automated characteristic of certain types of cancer may be better at
fluorescence-microscopy identifies the CTCs in blood detecting cancer early on than those that look at just one
samples placed on microscope slides. A detailed analysis biomarker. Dubbed Cancer-SEEK, the tool is capable of
of three million cells per slide is performed, each blood detecting a number of different cancers, including ovary
sample yielding approximately twelve slides. This and liver, and may soon begin testing as a screening tool.
technology may potentially hone in on the presence of a
As the technologies to detect cfDNA, CTCs, and cancer-
single CTC. Epic Sciences’ CTC-detecting platform is used
specific exosomes progresses, we can expect to see
by Genomic Health in their Oncotype-Dx AR-V7 Nucleus
an increasing number liquid biopsies available, making
Detect test, which determines whether or not prostate
the detection and treatment of a range of cancers less
cancers patients’ tumors have developed mutations that
invasive and more manageable.
make them resistant to common types of treatment.

Turning On Cellular
Garbage Disposals
PROTEASOMES TO THE RESCUE “bifunctional small molecules” – which simultaneously
bind to two different proteins.
Many drugs work by stopping overactive proteins that
cause disease. The leukemia drug Gleevec, for example, With PROTAC, one end binds to the target, the other
is a small-molecule inhibitor (antagonist) of the protein to E3 ligase. This interaction transfers ubiquitin to the
Bcr-Abl, whose over-activity promotes excessive target protein for eventual disposal. PROTAC doesn’t
cell division. Humira treats a range of autoimmune necessarily have to recognize a specific part of the
diseases by stopping TNF-alpha, a protein that target, such as the active site of an enzyme. That allows
activates inflammation. researchers to focus on a wider range of proteins than
possible with existing technologies, such as small
Such antagonists can be powerful. However, it’s not
molecule inhibitors, which must fit precisely in an
always possible to develop a strong inhibitor of a
enzyme’s active site to work.
disease-associated protein. And when scientists do
develop them, resistance often emerges, rendering a Arvinas has released preclinical data suggesting that
drug ineffective. PROTAC successfully lowers levels of the protein BRD4
in lymphoma, multiple myeloma, and prostate cancer
What if, instead of merely inhibiting a protein, we
cells. BRD4 plays a role in cell division, and mutated
could totally get rid of it? It turns out that our cells
versions are associated with various cancers. In the past
already have that ability. In this Biotech Primer
year, Arvinas announced collaborations with Pfizer and
WEEKLY, we look at a new class of drugs scientists are
Genentech, which should speed the progress of getting
developing to take advantage of our bodies’ microscopic
these molecules to the clinic.
sanitation departments.
C4 Therapeutics (Cambridge, MA) is developing a
A CELLULAR GARBAGE DISPOSAL similar small molecule platform that connects disease-
associated proteins with cellular ubiquitination enzymes.
If allowed to accumulate, proteins can interfere with Dubbed “degronimids,” the molecules are still in
normal cell function. Therefore, all cells contain preclinical development. They have already attracted
proteasomes, compartments that break apart unneeded the attention of Google-backed Calico, with whom C4
or damaged proteins. Proteasomal degradation also recently signed a five-year deal to work together to treat
provides a way to recycle the amino acid building blocks diseases of aging.
of proteins. Once a protein is broken down, a cell can use
the leftover amino acid “bits” to rebuild new proteins. Kymera (Cambridge, MA) is also utilizing small molecules
to activate target-specific proteasome degradation,
Proteins are targeted for degradation through the action focusing first on oncology and autoimmunity. In March,
of E3 ligase. This enzyme attaches another protein, Celgene (Summit, NJ) announced a project with Vividion
ubiquitin, to the targeted protein. Ubiquitin then guides (La Jolla, CA) in the hopes of discovering ubiquitin-
the target into a proteasome, where it’s broken down. proteasome system interacting drugs.
If scientists figure out how to “tag” disease-associated
proteins with ubiquitin, they can activate our cellular
NOW WAIT A SECOND…
garbage disposal to fight illness. Several companies are
working on clever ways to do just that. It’s clear that many drug developers place great faith in
tapping proteasome power to advance human health.
SMALL MOLECULES, BIG RESULTS? In what could seem completely contradictory, other
companies are taking the opposite approach: squelching
Researchers at Arvinas (New Haven, CT) are developing the proteasome.
a platform to target disease-causing proteins based on
ubiquitination/proteasome systems. Dubbed PROTAC The process of apoptosis, or programmed cell death,
(Proteolysis-Targeting Chimera), the platform consists of occurs naturally in cells as a protective mechanism. For
example, cells that sustain large amounts of damage to

DNA activate apoptosis to prevent them from seeding Francisco), is another proteasome inhibitor approved as
a tumor. a second-line treatment for multiple myeloma. Both are
Many pharmaceutical companies are trying to co-opt small molecule drugs.
apoptosis to treat cancer. One way to induce the process A better understanding of how our cells process
is to inhibit the action of proteasomes. The resulting unwanted proteins has opened up an entirely new
buildup of damaged proteins signals the cell that approach to treating diseases. Manipulating the world’s
something is seriously amiss, setting off cell death. tiniest garbage disposals may hold the key to healing
The FDA has approved Velcade, marketed by Millennium otherwise untreatable conditions.
Pharmaceuticals(Cambridge, MA), to treat multiple
myeloma. Krypolis, developed by Onyx (South San

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How are companies developing drugs to target the ubiquitin-proteasome system?

How are companies developing drugs to target the ubiquitin-proteasome system?

What are proteasome inhibitors and how are they used therapeutically?

What are proteasome inhibitors and how are they used therapeutically?

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2018 BIOTECH PRIMER WEEKLY COMPENDIUM

3 Taking A Swing At Allergies 63 Eye Of The Cytokine Storm

7 GMO Apples Hit Marketplace 67 From Fantasy To Reality: Xenotransplantation

9 Stem Cell Snapshot 69 Picturing Disease

11 Therapeutic Antibody Primer 71 Circadian Rhythm & Disease

13 The Mechanics Of Melanoma 73 Vaccines: Powerful Simplicity

15 The Intrigue Of HIF 75 DNA Vaccines Explained

17 The PARP Race Is On 77 Natural Born Cancer Killers

19 The Science Of CRISPR/CAS9 79 Off-Color: The Science

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SILENCING THE GENE ON THE MARKET

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• A monoclonal antibody that is highly specific for a

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EASILY CONFUSED: DNA DAMAGE

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As these and other potential treatments move through

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TERM OF THE WEEK: NUCLEIC ACID

There are two types of nucleic acids: DNA and RNA.

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Duchenne’s muscular dystrophy (DMD) and spinal

The complex yet fascinating world of antisense marches

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Each square represents one gene, or gene variant, within

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THE GENESIS OF GENELUX

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