GeneTherapy

Back to the Past
The Fall and Rise of Gene Therapy
Elena Busso
Mathieu Coustan
Stéphanie Penaud
Advanced Master in Biotechnology & Pharmaceutical Management
May 2016

1Back to the Past
ACKNOWLEDGEMENTS
We would have not been able to write this report without the help of several people. We would like to
thank Mark Chanel for his help and guidance throughout the whole project and Arsia Amir-Aslani and
Mickael Dubourg for the useful discussions about the financial part and the company valuation.
We also are specifically grateful for the people that gave us time to answer our questions. We would like to
thank Karine Charton, project manager at Genoscope (France) for her insights on the technology promises
and challenges. Her interview was priceless and enabled us to assess the promising therapeutic areas and
thus the success chance of the selected companies.
A special thank also to Elizabeth Wolffe, Vice President of Corporate Communication at Sangamo
Biosciences (US), for answering our questions about the competitive advantages of their gene editing
technology and other insights on the gene therapy field.
We would like also to thank Katherine A. High, M.D., Co-Founder, President and Chief Scientific Officer of
Spark Therapeutics who has gave us some of her precious time to discuss about the Gene Therapy field, the
challenges faced, her vision on future, and her insights on the AAV technology.
Finally, a last special thanks about the American Society of Gene & Cell Therapy: ASGCT which has kindly
tried to bring us some contacts to help us on this project.

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CONTENTS
Acknowledgements ............................................................................................................... 1
Contents ................................................................................................................................ 2
INTRODUCTION ..................................................................................................................... 4
REVIEW AND COMPARISON OF THE DIFFERENT GENE THERAPY TECHNOLOGIES................ 5
Viral vectors in Gene Therapy ............................................................................................... 5
Adeno-Associated Virus (AAV)............................................................................................... 5
Lentivirus ............................................................................................................................... 7
Summary comparison between these two viral vectors........................................................ 9
Ex-vivo gene therapies ........................................................................................................ 10
TCR (T cell receptors) ........................................................................................................... 10
CAR-T (chimeric antigen receptor): a breakthrough technology ......................................... 11
The two technologies in comparison ................................................................................... 14
Stem cell gene therapy ........................................................................................................ 16
Gene editing technologies................................................................................................... 16
Meganucleases.................................................................................................................... 17
Zinc-Finger Nucleases (ZFNs)............................................................................................... 18
TALEN .................................................................................................................................. 19
MegaTAL.............................................................................................................................. 20
CRISPR/Cas9 ........................................................................................................................ 21
Comparison of Different Programmable Nuclease Platforms. ............................................ 22
MARKET ANALYSIS............................................................................................................... 24
The actual market, general facts ......................................................................................... 24
Market Analysis ................................................................................................................... 24
Methodology ....................................................................................................................... 24
Promising markets in Short-Mid term ................................................................................. 26
Long term markets .............................................................................................................. 36

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Channels .............................................................................................................................. 40
FINANCIAL ANALYSIS ........................................................................................................... 43
Methodology ....................................................................................................................... 43
Companies Financial analysis .............................................................................................. 44
Cellectis................................................................................................................................ 44
Kite Pharma......................................................................................................................... 45
Sangamo.............................................................................................................................. 46
Bluebird bio.......................................................................................................................... 48
Juno therapeutics ................................................................................................................ 49
Spark.................................................................................................................................... 50
Uniqure................................................................................................................................ 52
Conclusions.......................................................................................................................... 54
Ethical discussion................................................................................................................. 54
General discussion............................................................................................................... 55
Bibliography......................................................................................................................... 58
ANNEXES.............................................................................................................................. 72
Questions for interviews: .................................................................................................... 72
Table 1. Conditions for which human gene transfer trials have been approved ................ 73

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INTRODUCTION
Gene therapy carries the promise to use genes to cure genetic diseases. But this promise appears much
more complicated to fulfill than expected. Since the first approved clinical trial in humans back in 19881
,
gene therapy has suffered difficult times, with the death of Jesse Gelsinger in 1999 in the USA and the death
of a boy treated with gene therapy in France after a treatment induced leukemia2
. These deaths were due
to two distinct issues: Jesse Gelsinger died from an immune response against the used virus (adenovirus).
It appeared that he should have been affected before the treatment and the injected dose was so high that
his immune system had a severe response, leading to the death of the patient. The French boy died from
another adverse effect of the former technologies. The virus used at that time was an integrative one,
meaning that the viral DNA integrates randomly in the host genome. Unfortunately, for four of the ten
treated patients, the virus integrated in oncogene regulators, provoking leukemias. One of the boys did not
survive to this induced disease. After these two events, gene therapy appeared to be too risky even if still
carrying big hopes for patients affected by genetic diseases for which no treatment exists.
During the following decade, research and clinical trials on gene therapy were carried out exclusively by
hospitals and academics as it was considered far too risky from a business point of view. Numerous clinical
trials were conducted (more than 1800 since the first ones)3
, allowing improvements in both viral vectors,
technologies and thus in safety for the patients. With these technological advances, gene therapy became
again compatible with business risk and in the last years, several companies emerged in this field.
Several big pharmaceutical companies have also rushed into the gene therapy field. For example GSK
recently received a positive recommendation for the marketing authorization for Strimvelis to treat patients
with a very rare disease called ADA-SCID (severe combined immunodeficiency due to adenosine deaminase
deficiency) from the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines
Agency (EMA) (ex-vivo autologous therapy)4
. Novartis is also involved in this field, more specifically through
the CAR-T technology with its first product probably on the market as soon as 20185
. But for these
companies, gene therapy represents only a very small part of their pipelines, so we’ve chosen to focus on
smaller biotechnological companies whose main assets are their advances in this field.
In this report, we will enlighten the new technologies that allow gene therapy to be back in the headlines,
the therapeutic areas where it has the best chance to succeed in the short term and finally, we will analyze
the valuation of seven companies involved in this field to assess whether or not these companies are over
or undervalued.
1
(Wirth, et al., 2013)
2
(Hacein-Bey-Abina, et al., 2003)
3
(Wirth, et al., 2013)
4
(GSK, 2016)
5
(Novartis, s.d.)

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REVIEW AND COMPARISON OF THE DIFFERENT GENE THERAPY TECHNOLOGIES
Two types of approaches can be used for gene therapy. The first one is the ex-vivo solution which consists
in withdrawing target cells from the patient and modifying them genetically before re-injecting them into
the patient. The second one is the in-vivo solution which consists in injecting directly the viral vector into
the patient (for now, mostly used for eye genetic disorders).
VIRAL VECTORS IN GENE THERAPY
To repair genetic disorders, gene therapy need to insert and integrate a new therapeutic gene into host
cells. For this step different techniques have been tried, some with Non-viral vectors (plasmids), but this
technique is very limited in term of time of expression of the therapeutic gene. So most of the actual clinical
trials on gene therapy are based on Viral vectors which are particularly efficient to deliver therapeutic genes
in host cells and then in host cell nucleus region for a more permanent solution in term of time of expression
of the therapeutic gene.
Below we will have a closer look on two viral vectors that are the most generic because they can transduce
both dividing and non-dividing cells.
ADENO-ASSOCIATED VIRUS (AAV)
It’s a single strand DNA virus of the parvovirus family. This virus presents a great advantage, it is non-
pathogenic, and so “most people treated with AAV will not build an immune response to remove the virus
and the cells that have been successfully treated with it”6
. About 90% of human population is infected by
this virus with no known symptoms.
To understand the mechanism of this virus, it’s important to know how it is built:
The genome is composed by two ITRs (Inverted Terminal Repeats) regions at the extremities that are playing
an essential role for the replication of the virus in the infected cells by making hairpin structures which
permit self-priming and so the synthesis of the second strand of DNA with an independent enzyme of the
host cell. These ITRs are also needed for the integration of viral DNA in the genome of the host cell. The last
role for these regions is for an efficient encapsidation of the AAV DNA.
The parts in the middle of the virus are ORFs (Open Reading Frames) which regroup genes coding for
proteins used in the virus lifecycle (Rep) and proteins needed to make the capsid (Cap). It’s also important
to note that the Rep proteins are needed for a good viral DNA insertion in the cell host genome.
Considering gene therapy, the idea is to replace the DNA contained between the two ITRs with the
therapeutic gene. For the production of recombinant AAV (rAAV), ITRs seem to be the only parts needed in
6
(Gene Therapy Net, s.d.)

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proximity of the therapeutic gene for its expression (the so-called cis elements), the proteins related to the
expression of genes Rep and Cap can be brought by another way in the host cell (so-called trans elements).
There is a wide variety of serotypes of AAV but the most commonly used is the AAV2. Other serotypes can
have little differences in term of expression and/or stability (expression efficiency in the long term), so
depending on the application another serotype can be used.
AAV is a virus who usually waits a stress on the host cell to express, otherwise it inserts in the host genome
(specifically in chromosome 19 for AAV2). So a second infection is needed to produce it in bigger scale, we
talk about “Helper Virus” (usually adenovirus or herpes helper). Two mechanisms of production exist for
the recombinant AAV:
 Host cells, with stabilized AAV inserted in the host genome, are infected with the adenovirus and
transfected with a plasmid containing the rAAV construct.
 Host cells are transfected with three types of plasmids, one containing the rAAV construct, one
with the Rep and Cap genes and a last one who carry the helper genes previously isolated from
the adenovirus.
As the wild type adenovirus is highly undesirable for the safety of the treatment, the second method is
usually preferred. But this virus presents disadvantages, the first one is that it’s small (4.7 kilobases long)
which can carry only a low capacity of DNA (so the therapeutic gene must be small), the second is the
difficulty to produce it. Indeed, this production method is expensive and labor-intensive.
Summary of recombinant AAV production.
Scheme from http://www.vectorbiolabs.com/vbs/page.html?m=261
“Recombinant AAV gives prolonged and stable expression in numerous animal models without notable
toxicity”7
. It’s important to note that as the rAAV doesn’t contain Rep genes but contains ITRs, the modified
DNA can insert into the host genome but only at a low rate. Furthermore, still because of the ITRs, the
therapeutic rAAV could exist either in circular form or linear concatemers in the nucleus of the host cells.
Then “the circular forms are thought to be responsible for vector persistence and long-term transgene
expression”8
.
7 (Vector Biolabs, s.d.)
8
(Vector Biolabs, s.d.)

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Fate of recombinant adeno-associated virus (rAAV) vector genomes
Figure from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2574934/
This gene therapy method can lead to a hazardous insertion of DNA and so to a deactivation of a host cell
gene, a phenomenon known as genotoxicity. According to different studies and especially the one from
Schultz et al. 9
, this integration rate is pretty low (usually inferior to 10%, but the assessments are not
precise) and can be very low (can be inferior to 3% but, again, the assessments are complicated and not
precise) if the infection occurs on non-dividing cells. Another factor which influences this integration rate is
the dose of the injected virus (the more virus is injected the more host cell genome integration can occur).
So this explains why many studies on this virus don’t report any issues and why the AAV technology is still
interesting. Furthermore, according to recent studies, many factors can also influence the integration site
of the virus, then helping to have a safer solution.
Still concerning the genotoxicity, Katherine A. High, CSO of Spark Therapeutics, indicates that “when you
inject DNA into a cell there is a risk to integrate. AAV don’t integrate very efficiently, so we know from
experience that if you inject mouse liver with AAV and you resect most of the liver, then the liver regrowth
and most of the renewed liver doesn’t carry the AAV anymore. So it wasn’t integrated. But in some low
level it does and it’s probably dose dependent. The way we address that problem, that is the way that we
use in many projects, is that you need to use the lowest effective dose. You should try to design your vector
in such a way that you could use it at a low dose” 10
.
Another challenge, highlighted by Karine Charton from Genethon, for this mainly non-integrative virus, is
the gene regulation. In the genome, genes are regulated not only by regulators but also by the chromatin
state. As the transgene is not integrated in the genome, it is not under the control of this kind of regulations.
If the organ targeting is not efficient enough, it may lead to the expression of the transgene in organs it is
not supposed to be expressed. For some genes, this unwanted expression could have very deleterious
effects, eventually leading to the death of animals11
.
LENTIVIRUS
9
(Schultz & Chamberlain, 2008)
10
(High, 2016)
11
(Karine Charton, 2016)

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It’s a single strand RNA virus of the retroviridae family. “Lentiviruses can deliver a significant amount of
genetic information into the DNA of the host cell”12
. Most recently used in gene therapy than the AAV, the
Lentiviral vector can respond to the issue of the low quantity of therapeutic DNA that the AAV can carry. A
well-known Lentivirus is the HIV (around 9.7 kilobases in size).
Lentiviral infections have also other advantages, “including high-efficiency infection of dividing and non-
dividing cells, long-term stable expression of a transgene, and low immunogenicity”13
.
To understand the mechanism of this virus, it is important to know how this virus is built:
The genome is composed by two LTRs (Long Terminal Repeats) regions and three main genes coding for
viral proteins which are Gag, Env (essentially coding for structural proteins, like capsid proteins) and Pol
(especially coding for Reverse Transcriptase (RT) and Integrase (IN)).
As for the AAV, the Lentivirus production process is quite complex because the host cells must receive four
different plasmids in order to generate safe modified Lentivirus. The genes are cut and integrated in
plasmids as shown below:
After all the plasmids are in the host cell, they can express and so create modified Lentivirus. Gag and Env
proteins are used to create the structure of the virus (especially capsid) then the Pol proteins, which are RT
and IN, and the therapeutic gene surrounded by the LTRs are integrated in the capsid (b and c steps in the
scheme below):
12
13

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Mechanism of production and infection of modified Lentivirus
Scheme from http://biology.kenyon.edu/slonc/gene-web/Lentiviral/Lentivi2.html
Reverse Transcriptase (RT) “uses the viral RNA genome as a template for the synthesis of a complementary
DNA copy. RT also has RNaseH activity for destruction of the RNA-template”14
(step d). “Integrase (IN) binds
both the viral cDNA generated by RT and the host DNA. Processing of the LTR by IN is performed prior to
insertion of the viral genome into the host DNA”15
(step e).
A recent study published in 2014 titled “Molecular mechanisms of retroviral integration site selection”16
indicates that integration of Lentivirus DNA in host cell is not fully random. For example, the HIV-1 prefers
to insert in active genes, but also some factors can help to vary the insertion site to provide safer solutions.
Research on this field seems to evolve pretty rapidly but, for now, there is a concern due to the potential
disruption of genes, and in particular of oncogenes, which can result in cancer development.
SUMMARY COMPARISON BETWEEN THESE TWO VIRAL VECTORS
AAV Lentivirus
Host Immune Reaction Low Low
DNA carry capacity Low (around 4Kb) Medium (around 9kb)
Production complexity High High
Infection efficiency High High
Long-term stable expression Yes Yes
Transduces dividing and non-dividing
cells
Yes Yes
14
15
16
(Kvaratskhelia, et al., 2014)

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Safety concern No
(non-pathogenic virus)
Yes
(same family as HIV-1)
Ex-vivo and in-vivo applications Yes Yes
These vectors are used both for in-vivo and ex-vivo gene therapy. They can be directly injected in the body
to trigger a specific organ (as seen before) or it can be used in cells extracted from the patient in order to
give them new properties. The viral vector is thus not directly infused in the body, since only the modified
cells are re-injected.
EX-VIVO GENE THERAPIES
The immune system is a natural defense against diseases, but cancer cells have the ability to escape it by
promoting an immunosuppressive response and by rendering immune cells ineffective. In particular, T cells
circulate through the bloodstream and detect dangerous cells by using special receptors on their surface,
called TCR (T cell receptor) which interact with antigens presented by MHC I and II (major histocompatibility
complex I and II) on the surface of target cells. If the T cell recognizes the antigen as “non-self”, thus
potentially dangerous, it will start a response to destroy it. Tumor cells, even if “self-derived”, present some
particular molecules called TAAs (Tumor Associated Antigens), expressed intact on the cell surface or as
peptide fragments bound to MHCs, which are used to discriminate tumor cells from normal tissues. New
promising gene therapy technologies exploiting the potential of immune cells are currently under
development. The two mainly used approaches are the TCR technology and the CAR-T technology, which
are quite similar to one another: they both involves harvesting T-cells from the patient (autologous
therapy), genetically engineering the collected cells in order to recognize specifically cancer cells and
reintroducing them back into the patient. In the following paragraph, the two approaches will be analyzed
separately, to underline their respective strengths and weaknesses. It should be noticed that, irrespective
of the approach considered, a proper TAA has to be identified17
.
TCR (T CELL RECEPTORS)
The TCR approach consists in re-
directing T cells against cancerous
targets through genetic
engineering of designer T-cell
receptors (TCRs) that recognize
tumor antigens whether inside or
outside the cell. Two main
approaches have been used to
generate TCRs that are specific for
other antigens that are expressed
by a wide variety of tumor cell
types. The former approach
consists in using allogenic T cells
from individuals who are not
tolerant for the patient TAA, while
17
(Kershaw, et al., 2013)
Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) mechanism scheme.
Source: (KitePharma, 2016)

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in the latter T cells from transgenic mice that express human MHCs (Major Histocompatibility Complexes)
are used. Then, the obtained cells can be incubated with tumor cells and the ones that react against the
tumor can be selected in order to clone the TCR genes (this procedure has been utilized to generate TCRs
for lymphoma, colon cancer and other malignancies). Alternatively, a library of many millions of TCR genes
can be generated in bacteriophages, and then select the bacteriophages that bind to tumor peptide
antigens (TCRs for virus-associated cancers have been isolated in this way)18
.
Several biotechnological start-ups are using this technology. Juno Therapeutics, for instance, selects its TCR
constructs by screening healthy donors for naturally occurring receptors with high affinity against a
MHC/peptide combination of interest. The selected receptors can be used directly or being furtherly
modified to improve their affinity for the target. Juno Therapeutics’ pipeline contains some TCR molecules
in clinical trials and, based on the limited number of patients who have been subjected to TCR treatment,
it seems that these cells behave like normal T cells when injected in the patient19
.
In addition, Immunocore and Adaptimmune are developing TCRs, and represent the biggest private biotech
fundraising in EU ever, ($300M), in May 201520
. Adaptimmune Therapeutics recently published a scientific
paper in which shows its last results of a Phase I/II trials on symptomatic myeloma patients, to evaluate the
safety and activity of engineered T-cells
expressing an affinity enhanced TCR that
recognizes the NY-ESO tumor antigen.
Encouraging clinical response was
observed in 16 out of 20 patients (80%),
with a complete recession of the disease,
and no strong adverse reactions such as
cytokine release were reported, although
the high IL-6 levels. When injected in the
patient, engineered T cells expanded,
trafficked to the bone marrow and
exhibited a cytotoxic phenotype.
Moreover, the study demonstrated that
a long-term persistence of engineered
cells in the peripheral blood was detectable in 90% of the patients who reached a two-year follow up. The
method of T-cell manufacture may be the key to unlock this persistence: Adaptimmune has induced a
double stimulation of the T-cell receptor (through CD3 and CD28) to select younger T-cells and also to
program them for a longer lasting expansion21
.
CAR-T (CHIMERIC ANTIGEN RECEPTOR): A BREAKTHROUGH TECHNOLOGY
18
19
(Juno Therapeutics, s.d.)
20
(Riquelme, 2015)
21
(Rapoport, et al., 2015)
Adaptimmune product candidate, recognizing a tumor antigen or peptide
associated with a HLA molecule. Source : (Labiotech, 2015)

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The CAR-T technology is a mix between gene therapy, cell therapy and immune oncology. The rationale at
the base is the same as for TCRs: to use the patient immune system to take on the disease. Unlike TCRs, this
approach consists in utilizing an engineered antibody fragment to “switch on” the T cell.
The procedure involves the
collection of the patient’s
white blood cells via
leukapheresis, and outside
the body certain T cells are
isolated and transfected,
usually using a viral vector,
with the genetic material to
make them express the
desired Chimeric Antigen
Receptor (CAR) on their
surface. When reinfused
back in the patient, the
resulting CAR-T cells
recognizes the antigen in
question, and by binding it
the CAR-T cell sends an
intracellular signal to the T
cell that activates a cytotoxic
response (destruction of the cancer cell)22
.
Through the years, CARs have been improved by additional modifications, and four main CARs generations
can be described. CARs are composed of an extracellular domain that recognizes the antigen, a
transmembrane domain and an intracellular domain, which send the intracellular signal in order to activate
the T cell. The first generation CARs are composed of a single intracellular domain derived from the CD3f
chain of the TCR/CD3 complex, a transmembrane domain and an extracellular domain most commonly
based upon scFv derived from monoclonal antibodies, which can or cannot contain a spacer domain to
modulate the physical location of the scFv. In the second generations CARs, additional intracellular domains
(such as CD28, CD137) have been added to provide costimulatory signals. The third generation CARs
included two costimulatory
domains in series in addition to
the intracellular domain. The
fourth generation CARs contain
also a CAR responsive promoter
activated by the intracellular
signal sent by the receptor,
leading to the transgenic
production of cytokines such as IL-
12 that further expand the overall
inflammatory anti-tumor
22
(van der Stegen, et al., 2015)
CARs evolution from 1st to 4th generation. Source: (Cheadle, et al., 2014)
Autolus CAR-T cell autologous engineering. Source : (Autolus, s.d.)

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response23
. Recently, the most modern CARs incorporate mechanisms to either dampen or amplify T cell
activation signals present on cancer cells or in the tumor microenvironment, in order to increase the
specificity of CAR-T for cancer cells versus normal cells. For example, a CAR- T cell can be engineered such
that it would be triggered in the presence of one target protein, but it would be inhibited if at the same
time a second signal is present, or the simultaneous presence of two signals can be required to maximal
activation of the CAR-T cells24
. Another approach to increase CARs selectivity towards cancer cells is called
“affinity tuning”, and exploits the fact that cancer cells express some specific receptors at much higher
levels. Lowering CARs’ affinity for these specific receptors (notably the epithelial growth factor receptor
(EGFR) and ErBb2) makes CAR-T cells preferentially recognize and eliminate tumor cells that have higher
amounts of EGFR or ErBb2, while sparing normal cells that express these receptors at lower levels. This
technique is useful especially when treating solid tumors25
.Since the generation of chimeric antibodies is
technically quite simple, a large number of CARs targeting different TAAs has been generated26
.
Juno Inhibitory CAR (iCAR) mechanism. Source (Juno Therapeutics, s.d.)
Many biotechnology companies are conducting trials for CARs, among them Juno Therapeutics,
KitePharma, Cellectis, Adaptimmune, Celgene and Celyad. The main therapeutic area is represented by
liquid tumors: Juno Therapeutics, for instance, is conducting clinical trials for using the CAR-T technology to
treat acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and chronic lymphocytic leukemia
(CLL), but is also trying to apply this technology to a number of solid cancers. The antigen targeted by most
of Juno’s CARs is CD19, an antigen present on B cell-related blood cancers27
.
23
(Cheadle, et al., 2014).
24
25
(Caruso, et al., 2015).
26
27

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THE TWO TECHNOLOGIES IN COMPARISON
TCRs and CAR-T are promising technologies, because they will target only the tumor alone, so having less
adverse events that chemo/monoclonal antibody therapies. Triggering of the immune system improves the
natural way that our body defends itself against tumor on a long-term basis, helping to reduce risk of
relapse, and prior trials results are very promising. Nevertheless, they both still have some limitations to
overcome. Many companies are conducting trials with both the approaches, which have some relative
advantages and disadvantages compared to each other. Firstly, it should not be forgotten that both
techniques require infection with viruses: to engineer the T-cells, scientists need to go inside the cell to
genetically modify its DNA genome. This is a key step, because T-cells are very refractory to the entry of
foreign DNA, consequently a very efficient gene transfer tool is needed28.
Currently, the most efficient way
to go inside T-cells is the use of Lentiviral Vectors, as discussed in the previous chapter. Many companies
are conducting trials with both the approaches, which have some relative advantages and disadvantages
compared to each other.
An advantage related to TCRs is that the TCRs are fully human, and thus they are less likely to elicit an
adverse response in the patients.
Moreover, the main advantage
related to this technology is that,
while CARs can only target
extracellular peptides, TCRs can
recognize both intracellular and
extracellular peptides, thus
significantly increasing the target
scope. On the other hand, this
technology also has some
limitations. Many studies failed to
demonstrate the long-term
persistence of the injected cells in
the patient, indicating that after
some time they are eliminated, thus
requiring an eventual second
injection. Moreover, it is impossible
to treat all patients with a specific
TCR: only a small proportion of patients can be treated with a TCR, because of the MHC-restricted nature
of TCR function, leading to a limitation in the applications of this technology. Furthermore, safety concerns
are not of secondary importance: the TCR injection may lead to a hyperactivation of the immune system,
causing high cytokine and interleukin levels (such as IL-6). This adverse reaction is known as “cytokine
storm” 18
.
Regarding CARs, a distinct advantage over TCRs is that CARs can recognize antigens in a non MHC-restricted
manner, thus they can be successfully used for all patients irrespective of their HLA type. However, some
production hurdles might prevent CARs to become easily accessible: many companies have made strong
28
(Eeckhout, 2015)
The two technologies in comparison. Source: (Adaptimmune, s.d.)

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involvement in making autologous CAR therapies (engineer the patient cells and then reinfusing them back
in the patient) work commercially, but this procedure is laborious and expensive. According to a Reuters
report29
, the production process takes at least two weeks per patient at a price of $450000 per treatment,
making it economically not realistic for healthcare payers. The biotech company Cellectis is trying to solve
this problem using an innovative allogenic approach: to use immune system cells taken from healthy donors
and to modify them to serve many cancer patients. Theoretically, a single donor could supply treatments
for more than 4,000 patients. However, to succeed and be safe, those engineered cells would have to be
further altered to ensure a patient’s body accepts them; otherwise, they may trigger a potentially deadly
immune system response against foreign invaders. These universal CAR-T cells (UCARTs) can be frozen and
shipped all over the world to serve
different patients, and might provide an
“off-the-shelf” option that will solve the
production hurdles of other
companies30
.
Cellectis’ allogeneic UCART19 has been
used at The Great Ormond Street
Hospital (UK) to successfully treat a baby
girl affected by refractory relapsed
Acute Lymphoblastic Leukemia (ALL)
who did not respond to any other
29
(Pierson, 2015)
30
(Cellectis, s.d.)
Cellectis allogeneic CAR-T manufacturing process. Source: (Cellectis, s.d.)
Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen
receptors. Source: (Kochenderfer & Rosenberg, 2013)

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treatment31
. Even if it is hard to say much based on one patient, everyone in the field is talking about CAR-
T as the miraculous cure for cancer and as the new biotech revolution after monoclonal antibodies.
Certainly, this is a growing and game-changing field, but now these technologies are at the beginning, and
many aspects have to be solved before they become easily accessible. According to André Choulika,
Cellectis’ CEO, there are four main challenges associated to CARs development32
. The first one is related to
cell administration: finding new ways of injecting repeated doses of the product (to ensure the therapy is
fully effective, given the fact that CAR-T cells have a limited lifespan) without causing immunogenic adverse
effects. Secondly, CAR-T are very sensitive and can target cells that express less receptors: on one hand,
this characteristic increases CAR-T targets, but on the other hand, they can start attacking healthy cells too.
One way to overcome this problem might be to induce “logic gates” where the cells will only act if a
combination of receptors are present. Another challenge will certainly be to find new TAA to target: most
of the CAR-T therapies today attack the CD19 antigen, present on B cells. In conclusion, this technology is
very promising, but some limitations still need to be solved, and probably combination with other therapies,
such as surgery and checkpoint inhibition, will be a more effective way to exploit all its potential.
STEM CELL GENE THERAPY
Stem cell gene therapy was one of the first technology to be used to correct genetic deficiencies and is the
technology used in the GSK gene therapy that received positive recommendation for ADA-SCID. The
principle is to collect stem cells from the patient, to insert a functional copy of the faulty gene and to
reinfuse the modified cells in the body. The stem cells that have mainly been used so far are hematopoietic
stem cells33
. Once re-injected, the modified stem cells will divide to create all blood cell types, allowing to
rescue the missing function. As stem cells are a renewed population, they divide all life long and the
therapeutic transgene is transferred to the hematopoietic cells in a very stable way.
This therapy proved to be efficient as immune-deficient children treated by gene therapy using stem cells
were cured, but adverse events occurred since the transgene integration caused leukemias34
. A strict quality
control of the integration site of the transgene for such therapy is very important as one faulty integration
event can lead to cancer development. The advances in gene editing could improve the safety of
hematopoietic stem cell gene therapy as we will see below.
Other types of stem cells are studied for use in gene therapy, including neural stem cells, but fir the moment
these technologies are still in a research stage.
GENE EDITING TECHNOLOGIES
The first gene therapy attempts aimed to add a functional copy of the faulty gene responsible for the
disease either on a transient or a more permanent way. In the transient way, the wild-type gene will not be
produced all life long and the disease will reappear in the future. The difficulties encountered in the past
31
(Pollack, 2015)
32
(Hemme, 2015)
33
(NIH, s.d.)
34
(Immune Deficiency Foundation, 2013)

17Back to the Past
with the permanent way were related to the fact that the new copy was integrated in the human genome
in random position, causing new diseases for the patient. It was the case of the trial conducted in 2000 at
the Necker hospital in Paris, where 4 out of 10 treated “bubble boys” developed leukemia induced by the
integration of the vector in their genomes35
. One of the children died and the gene therapy assays were
stopped.
In order to avoid random integration and to be able to modify directly the faulty copy of the gene, the cell
must achieve homologous recombination between the endogenous copy and the modified wild type copy
(transgene). In mammalian cells, homologous recombination rate is very low and the risk for non-
homologous recombination with the integration of the transgene elsewhere in the genome is very high.
The way to drastically improve the homologous recombination rate is by creating Double-Strand Breaks
(DSBs). In the past decade, new technologies capable of creating DSBs on targeted genes based on
engineered nucleases has emerged under the generic term “gene editing technologies”36
.
The introduction of DSBs in a specific location in the genome stimulates cellular DNA repair mechanisms,
including error-prone non-homologous end joining (NHEJ) and homology directed-repair (HDR). In the first
case, the NHEJ system will introduce random nucleotides to close the gap and hence inactivate the targeted
genes. The HDR system is used to modify the target gene, introduce a new gene or remove the full copy of
the gene, providing a donor template containing transgene.
Overview of possible genome editing outcomes using site-specific nucleases. From (Gaj, et al., 2013).
MEGANUCLEASES
Meganucleases are endonucleases found in microbial species with large (>14-bp) recognition sites, thus
they naturally are very specific. They were discovered in the late 1970s and were used as genome
engineering tool in eukaryotes since the middle of the 1990s. The best characterized meganucleases are I-
CreI from Chlamydomonas reinhardtii, I-SceI form Saccharomyces cerevisiae and I-DmoI from
Desulfurococcus mobilis. The DNA binding domains are also responsible for target sequence cleavage. This
35
(Hacein-Bey-Abina, et al., 2003)
36
(Cox, et al., 2015)

18Back to the Past
property makes meganucleases difficult to engineer as it is challenging to modify the target sequence
without affecting the cleavage activity.
Site recognition and cleavage activity of a chimeric meganuclease : this scheme shows the overlap between recognition and
cleavage activity from (Bertoni, 2014)
To create meganucleases with altered specificity, rational design coupled with randomization and high-
throughput screening format were used as well as highly sophisticated computational reprogramming.
Though another alternative strategy researchers have been able to fuse various natural meganucleases
domains to design sequence specific meganucleases37
. The construction of sequence specific enzymes for
all possible sequences is costly and time consuming. Furthermore, the efficiency of producing DSBs by
meganucleases is highly dependent of the methylation profile of the target. Meganucleases were the first
tool for genome editing in eukaryotic cells, with companies such as Cellectis (France) working on engineered
meganucleases since 1999. The small size of the genes coding for Meganucleases (~1kb) allows efficient
packaging in viral vectors or insertion in plasmids.
ZINC-FINGER NUCLEASES (ZFNS)
Zinc finger domains are among the most common types of DNA-binding motifs found in eukaryotes and are
usually found in transcription regulator proteins. Zinc Finger (ZF) domains are composed of around 30
amino-acids that recognize 3 DNA bases. Three different ZF domains are assembled to recognize a sequence
of 9 nucleotides. Synthetic enzymes have been engineered, using Zinc-fingers domains fused to the
cleavage domain of a restriction enzyme, FokI. FokI has physically separated recognition and cleavage
domains, the cleavage domain having no apparent sequence specificity38
. By fusing ZF domains to the
cleavage site of FokI, it is possible to direct the nuclease to a specific target in the genome. As the cleavage
activity of FokI requires dimer formation, a pair of ZFNs must be used, one targeting the upstream region
of the target, the other the downstream region. This property allows to have a recognition site of 18bp,
increasing the cleavage specificity. ZFN cleavage produces a gap of 5 to 7 bp (called spacer), that will be
filled up by NHEJ or HR, allowing gene disruption or gene replacement (given a donor DNA).
Zinc finger domains are quite easy to engineer compared to meganucleases and commercial kits are
available to customize the ZFNs in order to target specific sequences in the genomes (Sangamo). But protein
engineering is still challenging as all the combinations don’t show the same efficiency. The gene size of a
37
(Belfort & Bonocora, 2014)
38
(Carroll, 2011)

19Back to the Past
ZFN is around 1.1kb for a 9bp site recognition, thus requiring 2.2kb to be included in viral vectors or plasmids
for delivery.
Affinity for the target is not the same amongst the ZF domains and low affinity can induce off-target
cleavages.
Repair outcomes of a genomic double-strand break, illustrated for the case of ZFN cleavage. From (Carroll, 2011)
This technology is mainly used by Sangamo Biosciences for ex-vivo and in-vivo gene therapies. During an
interview with Elizabeth Wolffe, Vice President of Corporate Communication at Sangamo, she affirmed that
they decided to focus on this particular gene editing technology because they “believe that their ZFN
mediated genome editing has specific advantages over conventional forms of gene therapy mediated by
adeno-associated virus (AAV) or lentiviral vectors. In contrast to AAV-mediated gene addition, genome
editing provokes a permanent change in the genome which has the potential to provide a one-time life-
long treatment (since AAV is non-integrating). Unlike lentiviral approaches that elicit random integration of
therapeutic genes, gene addition mediated by ZFN-based genome editing is highly specific targeting an
investigator chosen site in the genome” 39
.
TALEN
Discovered in 1989 in Xanthomonas bacteria, Transcription Activator-Like Effectors (TALE) proteins contain
DNA-binding domains composed of a series of 33-35 amino acid repeats, each recognizing a single base
pair40
. More precisely, two amino acids in the repeats, the 12th
and the 13th
, known as repeat-variable
diresidues (RVDs) are responsible for the recognition specificity of one base41
. TALE specificity depends on
the repeated sequences number and order. Combining different DNA-binding domains in a same protein
enables to target every DNA sequences. TALE nucleases (TALEN) are composed of a gene specific TALE fused
to the cleavage domain of FokI (as for ZFNs). As ZFNs, TALEN are used in pair, each TALEN binding DNA on
both sides of the spacer, which will be eliminated during the cleavage. Commercial kits proposing TALEN
39
(Elizabeth Wolffe, 2016)
40
(Lamote & Lecampion, 2015)
41
(Gaj, et al., 2013)

20Back to the Past
synthesis are already available from several companies. TALEN are much easier to engineer than ZFNs and
a customized TALEN can be obtained in 2 weeks.
Model of a TALEN. A TAL Effector (TALE) polypeptide contains a series of typically 34-amino acid repeats, of which residues 12 and
13 [repeat variable diresidues (RVDs) shown in orange] are responsible for recognition of a specific base as shown in the box. FokI
nuclease is fused to the C-terminal end of the protein. From (Carlson, et al., 2012)
A clear disadvantage of TALEN compared to ZFN is the gene size, 2.5kb for a half TALEN targeting 13bp, thus
needing 5kb to be packaged and/or delivered. The repeated structure of TALENs can also be problematic
when introduced in retroviral vectors as when retro-transcription occurs, recombination and divergence in
the RVDs can be observed, modifying the targeted sequence. Talen are mainly used by Cellectis to produce
its allogeneic CAR-T. For ex-vivo gene therapy, the size of TALEN is less problematic as the coding RNA can
be introduced by other mechanisms than viral vectors, such as electroporation, ensuring a transient
expression and activities in the cells.
MEGATAL
In 201342
, a new kind of nucleases were synthetized that combine an engineered meganuclease fused to a
TALE. The goal was to address more efficiently the meganucleases to their target using a minimal TALE
recognition domain and by concentrating the meganucleases at the target site, to increase the site specific
cleavage activity. Combining the high cleavage specificity of meganucleases with the ease of engineering
TALENs, the recognition domain is very long and drastically decreases the risk of off-target DSBs that is the
main concern for the use of gene editing technologies in therapeutic areas.
This architecture allows the generation of extremely active and hyper-specific compact nucleases that are
compatible with all current viral and non-viral cell delivery methods.
Model of a MegaTAL. The megaTAL architecture involves fusion of a TAL effector (in green) through a short linker to the N-terminus
of a Meganuclease (in blue). From (Boissel, et al., 2014)
42
(Boissel, et al., 2014)

21Back to the Past
As the megaTAL works as monomer, the gene size is only around 2kb, making it easy to deliver. MegaTAL
can only contained 5 to 6 TALE repeats, decreasing the negative effects on too many repeats in delivery
systems as retrovirus.
CRISPR/CAS9
CRISPR/Cas9 was discovered in bacteria in 1987, but it was only in 2007 that the role of this cluster in the
acquired immunity of bacteria was demonstrated. CRISPR (Clustered Regularly Interspaced Palindromic
Repeats) are palindromic repeated sequences separated by unique sequences called spacers. They are
always associated with Cas genes (CRISPR associated) and tracrRNA. Spacers are transcribed in crRNA
(complementary to phage sequence) that in combination with tracrRNA will guide Cas9 to the phage
sequence that will be cleaved43
.
CRISPR themselves are actually not used for genome editing, so it is a common terminology misuse to talk
about CRISPR/Cas9 technology since only Cas9 (the nuclease), tracrRNA and guide RNAs (crRNA) are used
for genome editing44
.
The success of CRISPR/Cas9 gene editing tool is due to several characteristics:
 It doesn’t require protein engineering. Indeed, targeting of nuclease activity is only performed via
an RNA guide composed of a fusion between tracrRNA and crRNA. Only the crRNA part must be
target specific, while the others components remain constant.
 Targeting of specific sequence requires only classical and cheap cloning techniques of a specific
sequence of 20 bp.
 In a same expression vector, several tracrRNA/crRNA can be introduced to target several
genes/sequences in the same time.
 Cas9 protein cleavage activity is not sensitive to the methylation state of the target as ZFN and
TALEN.
The CRISPR/Cas9 system is thus very easy to use for academic laboratories and that explains the very high
number of studies published so far (more than 2000 publications since January 2013). Nonetheless, even if
the CRISPR/Cas9 system is very popular among the gene editing community because of its ease of use, it
has some caveats for gene therapy applications. The first one is that to be efficient, the targeting sequence
must be close to a PAM motif (the canonical PAM is 5'-NGG-3' for S. pyogenes Cas9 protein) thus limiting
the available targets32
. Furthermore, even if the guide RNA is 20bp long, only 8 to 12 nucleotides are used
for target recognition. This number is too low to prevent off-target cleavages. To prevent these unwanted
events, Cas9 nuclease were modified in order to cut only a single DNA strand. These engineered Cas9 are
called Cas9 nickases32
. These nickases must be used with two different guide RNAs that target close
sequences in the genome. The combined action of the two single strand breaks produces a DSB that will be
43
(Hsu, et al., 2014)
44
(Lamote & Lecampion, 2015)

22Back to the Past
repaired by HDR. A Cas9 nickase triggered in off-target position would only produce single strand breaks
that are not enough to increase the DNA repair pathway.
CRISPR/Cas9 mechanism of action From (Reis, et al., 2014)
Even if CRISPR/Cas9 is a very young technology (the idea to use it for gene editing came in 2012), one
company, Editas, which wants to apply this technology for gene therapy applications, went public this year,
with an IPO of $94M. Another one, Intellia therapeutics filed for a $120M IPO later this year. Editas already
claimed that CRISPR should be in human in 2017.
COMPARISON OF DIFFERENT PROGRAMMABLE NUCLEASE PLATFORMS.
Zinc Finger
Nuclease
TALEN Cas9 Meganuclease
Recognition site Typically 9 to 18 bp
per ZFN monomer,
18 to 36 bp per
ZFN pair
Typically 14 to 20 bp
per TALEN monomer,
28 to 40bp per TALEN
pair
22bp (20bp guide
sequence + 2bp PAM
sequence for S.
pyognes Cas9); up to
44 bp for double
nicking
Between 14 and 40
bp
Specificity Small number of
positional
mismatches
tolerated
Small number of
positional mismatches
tolerated
Positional and
multiple consecutive
mismatches tolerated
Small number of
positional
mismatches
tolerated
Targeting
constraints
Difficult to target
non-G-rich
sequences
5’ targeted base must
be a T for each TALEN
monomer
Targeted sequence
must precede a PAM
Targeting novel
sequences often
results in low
efficiency

23Back to the Past
Ease of
engineering
Difficult, may
require substantial
protein engineering
Moderate, requires
complex molecular
cloning methods
Easily re-targeted
using standard cloning
procedures and oligo
synthesis
Difficult, may
require substantial
protein engineering
Immunogenicity Likely low, as ZFs
are based on human
protein scaffold.
Fokl is derived
from bacteria and
may be
immunogenic
Unknown, protein
derived from
Xanthamonas sp.
Unknown, protein
derived from various
bacterial species
Unknown,
meganucleases
may be derived
from many
organisms
including
eukaryotes
Ease of ex vivo
delivery
Relatively easy
through methods
such as
electroporation and
viral transduction
Relatively easy through
methods such as
electroporation and
viral transduction
Relatively easy
through methods such
as electroporation and
viral transduction
Relatively easy
through methods
such as
electroporation and
viral transduction
Ease of in vivo
delivery
Relatively easy due
to small size of
ZFN expression
cassettes, allows
use in a variety of
viral vectors
Difficult due to the
large size of each
TALEN and repetitive
nature of DNA
encoding TALENs,
leading to unwanted
recombination events
when packaged into
lentiviral vectors
Moderate: The
commonly used Cas9
from S. pyogenes is
large and may impose
packaging problems
for viral vectors such
as AAV, but smaller
orthologs exist.
Relatively easy due
to small size of
meganucleases,
allows use in a
variety of viral
vectors.
Ease of
multiplexing
Low Low High Low
From (Cox, et al., 2015)
The development of these techniques brought high expectations for gene therapy, especially regarding the
CRISPR/Cas9 method. These techniques would be very important for diseases caused by mutations in very
long genes such as dystrophin, that is too long to be packed in a viral vector. In this case, only the faulty
part (one exon) would be introduced and corrected, allowing the packaging. But it means that all the
patients should be genotyped before the treatment, meaning that a personalized gene therapy approach
would be needed.
On the other hand, the off-target breaks issue is not completely resolved today and the efficiency must be
improved before the use of these techniques in vivo. Nonetheless, several companies have already used it
in humans but with ex-vivo modified cells. Based on the interview of Karine Charton, project manager at
Genethon (France), these techniques are very useful and powerful tools but needs to be developed further
before they can be used in vivo. The use for the ex-vivo gene therapy is more straightforward as the cells
must pass a quality control before being reinjected in the patient, allowing the nuclease inactivation and
unwanted event check. Leaving active nucleases in the organism without direct control is still challenging.
In her opinion, for lethal disease with no other alternatives, the risk-benefit ratio should be acceptable but
not for debilitating diseases, or at least not in the close future45
.
45

24Back to the Past
MARKET ANALYSIS
THE ACTUAL MARKET, GENERAL FACTS
According to Ginn et al46
,between 1989 and June 2012, 1843 clinical studies have been approved for Gene
Therapy (based mainly on official agencies and published articles but also on conferences and posters).
Clinical Trial (CT) data are difficult to exploit as many CTs are usually not well updated in case of encountered
difficulties, but they clearly show an enlightenment for Gene Therapy for the past 2 decades. Each year,
around 100 clinical studies are carried out since
the 2000’s.
Most of the studies carried out so far were phase
I or I/II studies (78.6%), showing that very few
clinical trials have succeeded in the first steps.
Having a deeper look on the indications (see
Annex 1), it appeared that a very wide range of
diseases is covered, from cancer to
cardiovascular diseases and neurological
disorders. Thus, the marketing analysis for
gene therapy as a whole seemed
impossible to perform. If we think that the
most common indications should be
monogenic diseases, as it seems the most
obvious and straightforward strategy
(only one gene to be targeted and
replaced), we’ll be surprised to see that
monogenic diseases account only for 8.7%
of all gene therapy CTs. To narrow down
the size of the market to analyze, we have
tried to answer two questions:
What are the most promising
indications based on the limitations of the technology?
 For which indications companies are carrying out CTs? Indeed, most of the CTs are carried out by
academic teams, with no drugs being developed afterwards.
MARKET ANALYSIS
METHODOLOGY
46
(Ginn, et al., 2013)
Phases of gene therapy clinical trials
Indications addressed by gene therapy clinical trials

25Back to the Past
To narrow the marketing analysis to the indications with the best chance of success in the coming years,
we have listed the companies involved in gene therapy. We have found 27 companies, excluded the big
pharmaceutical companies such as GSK or Novartis, that are also involved in the field but for which gene
therapy constitute a very small part of their pipeline. We decided to rate them following several
characteristics, as discussed below.
 IPO: the first criteria for our company classification is if they were public or not. As public
companies must communicate on their financials, pipeline, clinical results, we’ve decided to focus
mainly on that ones. Public companies received a score 1, while the private ones received a score
0. If they had filed an IPO but were not listed in the stock market, they had a 0.5 mark.
 Clinical stage: we decided to take into account the most advanced products in the pipeline and to
attribute the number of points based on the clinical phase (0 for preclinical, 1 for Phase 1, 2 for
Phase 2, 3 for Phase 3, 4 for regulatory, 5 for commercialization phase).
 Portfolio size: the whole pipeline was taken into account, including the preclinical products. In
order to reduce the weight of this criteria, as for most of the companies it included mainly
preclinical programs, we divided by 5 the number of programs.
 Diseases targeted (short term): Based on the interview with Karine Charton of Genethon, it
appeared that the challenges for gene therapy are not the same based on the targeted organ.
Deliver the treatment to the targeted cells being as specific as possible to avoid expression of the
transgene in off target cells is still a huge challenge for this technology. Based on these limitations,
we’ve thought that the most promising short-term gene therapy will focus on three “organs”: eye
because the treatment can be directly injected; blood, as most of the treatments target
lymphocytes that carry specific antigens and the treatment needs only to be intravenously inject;
liver, as hepatocytes are quite different from other cells and targeting them specifically is possible.
We decided to attribute one point for each program targeting these 3 therapeutic areas and for
which an IND had been filed,47
.
 Capacities: this includes, for example, the cash the company has to fund their project, the number
of employees, GMP manufacturing…
 Competitive advantages: This one is the only criteria that is a multiplier. It reflects the advantages
of the company through collaborations with big pharmaceuticals company, a breakthrough
technology compared to competitors allowing for example to produce at lower cost, the capacity
to rely on their own technologies rather than on collaborations…
The results of this analysis are shown below:
Company IPO
Clinical
stage
Portofolio
size (/5)
Diseases
targeted
(short term)
Capacities
Competitive
advantage
Total
Score
Bluebird Bio 1 3 2,0 2 1 3 27
Kite pharma 1 2 3,4 5 2 2 26,8
Sangamo Biosciences 1 2 2,6 1 2 3 25,8
UniQure 1 5 1,6 3 2 2 25,2
Cellectis 1 1 1,2 2 1 4 24,8
Juno Therapeutics 1 2 2,2 5 2 2 24,4
47

26Back to the Past
Spark Therapeutics 1 3 2,0 3 2 2 22
Celyad 1 3 1,2 1 2 2 16,4
Ziopharm oncology 1 2 2,0 1 2 2 16
Dimension 1 2 1,4 2 1 2 14,8
Adaptimmune 1 2 2,0 1 1 2 14
Applied Genetic
Technologies
1 1 0,4 2 2 2 12,8
RegenXBio 1 2 0,4 1 2 2 12,8
Editas 1 0 0,0 1 1 3 9
CRISPR Therapeutics 0 0 0,0 2 1 3 9
Voyager therapeutics 1 1 1,0 0 1 2 8
Baxalta 1 2 0,4 1 2 1 6,4
Bellicum 1 2 1,2 1 1 1 6,2
Intellia Therapeutics 0 0 0,0 1 1 3 6
Abeona Therapeutics 1 2 1,0 1 0 1 5
Lysogene 0 2 0,4 0 1 1 3,4
Avalanche
Biotehnologies
1 0 1,2 0 1 1 3,2
Renova 0 2 1,0 0 0 1 3
Juventas 0 1 0,6 0 1 1 2,6
Audentes
therapeutics
0,5 0 0,8 0 1 1 2,3
AveXis 1 1 0,2 0 0 1 2,2
Celladon 1 0 0,0 0 0 0 0
Based on this analysis, we decided to focus on the seven companies with the highest score: Bluebird Bio,
Kite pharma, Sangamo Bioscience, Uniqure, Cellectis, Juno therapeutics and Spark therapeutics.
The marketing analysis was mostly performed on the markets these companies want to address.
PROMISING MARKETS IN SHORT-MID TERM
Most of the Short-Mid-term markets are related to orphan diseases with unmet needs or restrictive and
costly treatments. Indeed, regarding unmet needs, companies aim at different things:
 Find patients ready to take the risk of a treatment with a breakthrough therapy.
 Prove the concept of the Gene Therapy and then increase the acceptance rate of this kind of
therapy for other indications.
 Use the orphan status to benefit of development advantages (50% tax credit on the cost of clinical
trials undertaken in the USA, a seven year period of marketing exclusivity following the marketing
approval, some written recommendations provided by the FDA concerning clinical and preclinical
studies to be completed in order to register the new drug48
).
48
(Orphanet, s.d.)

27Back to the Past
Developing a new therapy is very expensive and also the GT treatment production is expensive. In the case
of Orphan diseases, the limited number of patients tend to price at a high value this kind of treatments in
order to recover the developmental expenses.
Thus, there is a huge debate concerning the future price of such one-time treatments, while the cost for
each patient per year with usual medicines can be between $300 and $500K. The only49
precedent in Gene
Therapy is Glybera (for LPLD (Lipoprotein Lipase Deficiency) which has a very small prevalence of about 1-
2 cases per million people50
) developed by Uniqure and priced at near $1M. "The record-breaking price tag
came to light in November 2014, when Amsterdam-based Uniqure and its marketing partner Chiesi, of
Parma, Italy, filed a pricing dossier with German authorities to launch Glybera"51
. So we can wonder at
which level new treatments will be located on the price scale. A track on price could be the disease
prevalence, especially when there is a factor 2 or more between the potential number of patients, we could
expect a price difference.
OPHTHALMOLOGICAL DISEASES
The eye is one of the favorite targets for gene therapy, presenting some unique advantages such as being
an easily accessible organ, highly compartmentalized and immune-privileged. Gene replacement and gene
silencing technologies have been implicated as potentially efficacious therapies, since today the genetic
pathogenesis of ocular diseases is much more understood than in the past52
.
Glaucoma, is a chronic disease and one of the leading cause of blindness in the world. The elevated
intraocular pressure is the main risk. People with this disease need a lifetime treatment, or alternatively a
risky surgery can be performed. Thus, gene therapy that can provide a long-term effect with one single time
49
Four Chinese’s Gene Therapies have been made commercially available but only in Asian countries
50
(Camozzi, 2012)
51
(Morrison, 2015)
52
(Solinís, et al., 2015)
Treatment of ocular disorders by gene therapy. Source: (Solinís, et al., 2015)

28Back to the Past
injection could bring new interesting treatment solutions. “In 2013, the number of people (aged 40–80
years) with glaucoma worldwide was estimated to be 64.3 million, increasing to 76.0 million in 2020 and
111.8 million in 2040”53
.
Inherited Retinal Dystrophies (IRDs), regroup several rare blinding conditions with over than 220 genes
implicated, there is no pharmacologic treatment available for now.
 Retinitis Pigmentosa (RP) is the most common IRDs and represents a group of inherited disorders
in which different mutations on different genes implicated in the photoreceptors or in the retinal
pigments lead to progressive vision loss. RP can be inherited in an autosomal recessive, autosomal
dominant or X-linked manner. On a global scale, the prevalence of this disease is approximately
1:3,000 – 1:7,00054
, but in Europe and in the US “it is estimated to affect 1 in 3,500 to 1 in 4,000
people”55
.
Leber congenital amaurosis diseases is linked to at least 14 different genes and is a severe IRDs which
usually appears during the first year of life56
. This disease occurs in “2 to 3 per 100,000 newborns”. For this
disease, the most advanced gene therapy product being developed is SPK-RPE65, from Spark and currently
in phase 3. This is a treatment for RPE65 gene mediated blindness: “mutations in this gene account for 6 to
16 percent”57
of all cases of Leber congenital amaurosis. US and 5 major EU markets counted approximately
639 million affected people worldwide in 2014 (based on US 322 / Ger 82 / FR 64 / UK 63 / Ita 61 / Spain
4758
)). According to a calculation based on the prevalence, there are 12,780 to 19,170 people with Leber
congenital amaurosis, of whom 6 to 16% linked to REP65 mutations, so from 767 to 3067 people (6% of
12,780 and 16% of 19,170). As there is a birth rate of 12.5/1000 population for US and 10.1/1000 population
in EU, we can count 4.025 million children/year in US and 3.2017 million children/year in EU, thus 7.2267
million children in total. Then we can deduce that from 144 to 217 children have Leber congenital
amaurosis, of whom 8 to 35 REP65 mutation linked.
Choroideremia is another remarkable IRDs: it’s a monogenetic recessive disease that is due to mutations
on the CHM gene on the X chromosome, thus only males are affected. The disease leads to a progressive
vision loss and eventually to complete blindness. “The prevalence of choroideremia is estimated to be 1 in
50,000 to 100,000 people. However, it is likely that this condition is underdiagnosed because of its
similarities to other eye disorders”59
. Based on our calculation (US and EU5, 639 million people) it represents
between 6390 and 12780 people. Different companies are developing a gene therapy for this indication.
Considering the 7.2267 million children per year for US and EU5, we can deduce that 72 to 144 children per
year have choroideremia.
53
(Tham, et al., 2014)
54
(Fahim, et al., 2000)
55
(Genetics Home Reference, 2010)
56
(Francis, 2006)
57
58
(UN Economic and Social Affairs, 2014)
59

29Back to the Past
X-linked Juvenile Retinoschisis (XLRS) is an X-linked inherited retinal degenerative disease caused by
mutations in the RS1 gene. This disorder affects only males, while females can be carriers but usually do
not display symptoms, and is characterized by early onset, leading to complete vision loss during adulthood.
There is no treatment for this pathology60
. “The prevalence of X-linked juvenile retinoschisis is estimated to
be 1 in 5,000 to 25,000 men worldwide61
”.
Achromatopsia is an inherited autosomal recessive disorder characterized by defective vision, sensitivity to
light and absence of color vision. This disorder affects equally males and females, and occurs in
approximately 1 in 40,000 newborn children62
.
Age-Related Macular Degeneration (AMD)is a leading cause of vision loss in Europe and US that destroys
the macula, which is the part of the eye that provides sharp, central vision. AMD can be diagnosed as either
dry-AMD (non neovascular) or wet AMD (neovascular), which is the more advanced and severe stage of the
disease63
. Age-related macular degeneration has a higher prevalence among Caucasians, compared to
Asian, African and Hispanic populations. In the US, the prevalence of AMD ranges from 0,2% to 1,6%, while
in Europe ranges from 1,65% to 3,5%, and the
prevalence rate increase sharply with age. As
the proportion of people in the U.S. aged 65
and older increases, in the future more
people will develop age-related diseases such
as AMD. Some estimates indicate that “from
2000-2010, the number of people with AMD
grew 18 percent, [and the economic cost of
this disease increased] from 1.75 million to
2.07 million dollars64
”. According to a study
from the Eye Research Institute in
Singapore65
, in 2020 there will be 196 million
people affected by this disease, increasing to
288 million people in 2040.
Concerning the blindness and visual
impairments costs, a specific study was
conducted in the US in 1992, Chiang et al,
199266
, which indicate an average cost of $1,982 per year for children between 3 and 21 years. With the
inflation rate67
, it corresponds to $3,347 (approximately $60,246 for 18 years). For adults, the estimate is
at $11,896 per year: $20,088 per year in 2016 considering inflation (for people between 21 and 64 years
60
(Sieving, et al., 2003)
61
62
(AAPOS, 2015)
63
(Haddrill, 2016)
64
(National Eye Institute, s.d.)
65
(Wong, et al., 2014).
66
(Meads & Hyde, 2003)
67
(McMahon, 2014)
AMD prevalence increase with age. Source: (Anon., s.d.)

30Back to the Past
old). Then we have 20,088*43years = $863,784, and so 863,784+60,246=$924,000 for a blind person
between 3 and 64 years. In total, nearly $1Million in expenses (present value not discounted) for each blind
people since birth.
We can also note for ophthalmological diseases that, additionally to the lifetime cost of blindness which we
have estimated at around $1million per blind person, governments could make a Cost Utility Analysis (CUA)
to take into account the quality of life gain for blind people, providing favorable data to financially help
them accessing to this kind of treatment. Blind associations could also self-mobilized to collect funds for
these patients. These elements tend to indicate that the market penetration could be pretty high.
Furthermore, according to Katherine A. High, M.D., Co-Founder, President and Chief Scientific Officer of
Spark Therapeutics, this company expect to reach very substantial percentage of the patients for SPK-RPE65
because the treatment range is important, as most of the patients presented to the trial were qualified68
.
IMMUNE DEFICIENCIES AND BLOOD DISORDERS
We distinguish primary and secondary immunodeficiencies, the former are directly linked to a genetic
disorder, while the latter are consequences of something else like drug treatment or HIV infection. For our
purpose, we will focus only on primary immunodeficiency.
The Common Variable Immune Deficiency (CVID) is probably the most frequent primary immunodeficiency
with a prevalence of 1 in 25,000 persons69
. Referring to a publication of 2013 from Guani-Guerra E et al70
,
“symptomatic primary immunodeficiencies are now considered to range from 1:500 to 1:500,000 in the
general population in the USA and Europe”. In the same study, they also affirm that “a random digit dialing
telephone survey in 2007 (from Boyle JM, Buckley RH) estimated that one in 1200 people within the United
States are diagnosed with an immunodeficiency”71
.
Considering a world population above 7 billion in 2015, number of patients are at least around 14000 based
on the most pessimistic prevalence and potentially around 14 million with the highest prevalence.
Considering this wide range, it’s really difficult to obtain a pretty sure value, in all case, this primary
immunodeficiency has always been considered as a rare disease, so the more serious prevalence seems to
be 1: 25000 or 1: 500000, then between 14000 and 280000 people worldwide.
Hemophilia (type A and B) is a rare hereditary monogenic disorder that affects blood-clotting, leading to
dangerous internal and external bleeding episodes. The most prevalent form of the disease, hemophilia A,
is caused by a defect in clotting Factor VIII (FVIII), while defects in clotting Factor IX (FIX) lead to hemophilia
B. The most severe forms of hemophilia affect males, since this illness is an X-linked recessive disorder72
. It
appears worldwide and occurs in all racial groups. About 6,000 people are affected with hemophilia in the
UK, about 5400 people with hemophilia A and about 1100 with hemophilia B. According to the National
68
(High, 2016)
69
(Immune Deficiency Foundation, s.d.)
70
(Guaní-Guerra, et al., 2013)
71
immunodeficiency-primary-and-secondary
72 (WHO, s.d.)

31Back to the Past
Hemophilia Foundation (US) 73
and to the World Federation for Hemophilia74
, the worldwide incidence of
hemophilia is not well known, but estimated at more than 400,000 people (around 1 out of 10000 new born
children), and approximately 75% of people affected by this disease around the world still receive
inadequate treatment or have no access to it. Although it is the most common type of hemophilia, the type
A is still a rare condition, affecting about one in every 10,000 males, while only about 20% of people with
haemophilia have the B type, thus affecting about one in every 50,000 males. It is anticipated, however,
that the number of people with haemophilia in developed countries will increase steadily over the next few
decades75
: some estimations suggests that there are around 20,000 persons living with hemophilia in US76
,
and 22,000 in Europe77
. The standard of care today is very effective, since the missing clotting factor is
injected into the bloodstream using a needle, but complications for this treatment arise when the patient
starts developing antibodies against the injected protein. These clotting factors are mainly produced as
recombinant proteins by companies such as Pfizer (BeneFix, clotting factor IX), Baxter (Advate, clotting
factor VIII) and Bayer (Helixate FS and Xogenate FS, clotting factor VIII)78
.
World Federation of Hemophilia reports in a survey of 2012 around 28,000 cases around the world.
Depending of the mutations, the rate of coagulation factor protein is more or less important. The US
Hemophilia Foundation describes the different level of severity as below:"
 Severe (factor levels less than 1%) represent approximately 60% of cases
 Moderate (factor levels of 1-5%) represent approximately 15% of cases
 Mild (factor levels of 6%-30%) represent approximately 25% of cases"79
Presently, the treatments available are concentrated factor and can be from human (plasma-derived) or
from laboratory (through the use of DNA technology). Patients with severe hemophilia may be on a routine
treatment regimen (especially advised for children), we talked about prophylaxis. This prophylaxis
treatment can reduce or prevent joint disease, but it's far most expensive than injection on demand and
results naturally in more injections. The cost of hemophilia is not well known; it varies from patient to
patient. “Currently, hemophilia therapy is among the most expensive in the world with a total annual costs
per patient ranging from $60,000 to as much as $1,000,000 for some patients. Many patients are on a
prophylactic treatment plan requiring an intravenous(IV) infusion 3 times per week, for life. One infusion
for an adult weighing 150 lbs. is approximately $3000 for the medication alone. The average cost for adult
patients on a prophylactic regiments at this rate is currently $468,000 annually”80
. So, depending of the
gene therapy price, insurance payers should be mostly interested by the gene therapy approach for severe
affected people which represents around 16,800 patients (60% of 28,000). And, with 4,025 million
child/year in US and 49.5% of male; 3,2017 million child/year in EU5 and approximately 49% of male, then
it represents 3,561208 million of male child in total81
. By applying the 1/50,000 male epidemiology seen
73
(National Hemophilia Foundation, s.d.)
74
(World Federation for Hemophilia, 2012)
75
(The Hemophilia Society, s.d.)
76
(Centre for Disease Control and Prevention, 2015)
77
(efpia, s.d.)
78
(Canadian Hemophilia Society, s.d.)
79
(National Hemophilia Foundation, s.d.)
80
(Hemophilia Information, s.d.)
81
(Data World Bank, s.d.)

32Back to the Past
above, it represents approximately 71child per year who have hemophilia B and all are advised to be treated
in routine with current treatments, so are eligible for Gene Therapy.
Then a challenge on price and time efficiency is to address here to make a marketable product. But we can
notice that big pharma companies are interested by the GT approach and have signed collaborations, like
Pfizer with Spark Therapeutics. So this is a strategic investment and they have estimated this substitute
treatment by Gene Therapy commercially viable.
Hemoglobinopaties are inherited monogenic disorders that results in an abnormal structure of one of the
globin chains of the hemoglobin molecule. Common hemoglobinapaties include beta-thalassemia and sickle
cell disease. The alpha and beta thalassemia are the most common inherited single-gene disorders in the
world, and the burden of this disorder in many regions is of such a magnitude that it represents a major
public health concern. People with these disorders cannot make enough hemoglobin, which is found in red
blood cells, leading to organ dysfunction. Misshaped red blood cells, which lead to impaired blood flow and
anemia, characterize Sickle Cell Disease (SCD). According to WHO, approximately 5% of the global
population carries trait genes for these hemoglobinopaties, and over 300 000 and 500 000 affected children
are born each year. While SCD is the most frequent among these disorders (75% of all hemoglobinopaties)
and is not confined to a particular region of the world, beta thalassemia has higher prevalence in
Mediterranean countries, especially where malaria was or still is endemic. In Europe and US, is estimated
that respectively 1,500 and 15,000 patients are born each year with beta-thalassemia, and around 70% of
them are considered as the major form, that is transfusion-dependent82
. Regarding Sickle Cell Disease, there
are around 100,000 affected patients in US83
and less than 130,000 in Europe 84
. Thus, we can estimate that
around 8,000 (for beta-thalassemia) and 12,000 (for SCD) new patients might be available per year for an
eventual gene therapy treatment. The standard of care treatment for these disorders includes regular
blood transfusions and iron-chelation therapy. Thanks to these treatments, the patients’ life expectancy is
considerably increased. Moreover, allogeneic bone marrow transplantation can cure the diseases (although
this approach is limited by scarcity of donors and complications related to immune recognition)85
.
BLOOD CANCERS
Acute Lymphoblastic Leukemia (ALL) is a cancer that affects blood and bone marrow. It represents 75% of
the childhood leukemia. In the United States, the number of new diagnosed cases in 2015 was estimated
to 6,250 with a survival rate at 5 years of 67.5%86
. In European Union, we can estimate around 10,000 new
diagnosed cases by year. 40% of new cases are diagnosed in children but 80% of deaths occurred in adults87
.
Chemotherapy is the first treatment used for the cure of this disease but the relapse is quite frequent. Gene
therapy, because of its high cost, will probably be used for second line treatment, for relapse or for high
risk patients with poor prognosis such as those carrying the Philadelphia mutation (25% of adult ALL
82
(Bluebird Bio, s.d.)
83
(Center for Disease Control and Prevention, s.d.)
84
(European Medicines Agency, s.d.)
85 (Modell & Darlison, 2008)
86
(National Cancer Institute, s.d.)
87
(American Cancer Society, 2016)

33Back to the Past
patients)88
. For high risk patient or relapse ALL, bone marrow transplants are used. The cost of this
intervention is around $800,00089
. The best alternative for ALL is the CAR-T therapies with both autologous
and allogeneic approaches currently in development. Even with a cost around $450,00090
for autologous
CAR-T therapies, it is cheaper than a bone marrow transplant. It could be even truer for the allogeneic
approach. The number of patients that could be targeted by this gene therapy approach, in USA, UE and
Japan, can be estimated around 7,000 patients each year, including the unmet needs (deaths) and the high
risk patients.
Chronic Lymphocytic Leukemia is a type of leukemia (thus a blood cancer that affects leukocytes) where
the leukemia cells often build up slowly over time, and many people don't have any symptoms for at least
a few years. In time, the cells can spread to other parts of the body, including the lymph nodes, liver, and
spleen. Most people can live with this kind of leukemia for many years, but they are usually harder to cure
compared to the other types of leukemia91
. It is estimated that 18,960 new cases will be diagnosed in US in
2016, but 81,5% of the patients survive at least 5 years after the diagnosis92
. For US, Europe and Japan, we
can estimate around 10,000 available patients per year.
Multiple myeloma is a type of bone marrow cancer that arises from plasma cells. It is relatively uncommon,
representing 3.8% of all cancers. The number of new patients diagnosed with multiple myeloma in Europe
each year is estimated to be around 40,00093
and 22,000 in US94
. The current standard of care is treating
with chemotherapy and steroids, according to the severity of the disease and the age of the patient95
, thus
gene therapy might be a promising treatment for younger patients affected by more aggressive forms of
the disease. For this indication we estimated that around 10,000 patients per year could be available for
gene therapy treatments.
Non-Hodgkin Lymphoma (NHL) is a common cancer as it accounts for 4% of diagnosed cancers in the United
States96
. It starts in the lymphocytes and then spread in the body. According to the American Cancer
Society’s most recent estimates for non-Hodgkin’s lymphoma for 2016, about 72,580 people will be
diagnosed with NHL, so around 217,300 new cases in US, Japan and EU. The survival rate at 5 years is around
70.7%97
. Non-Hodgkin lymphomas can be of different subtypes. We can estimate that around 30,000
patients per year could be available for gene therapy treatment.
 Diffuse Large B-Cell Lymphoma (DLBCL)
88
(UCSF Medical Center, s.d.)
89
(National Foundation for Transplants, 2010)
90
(Pierson, 2015)
91
(American Cancer Society, s.d.)
92
(NIH, s.d.)
93
(European Cancer Observatory, s.d.)
94
(National Cancer Institute, 2014)
95
(Myeloma Patients Europe, 2016)
96
(American Cancer Society, 2016)
97
(Surveillance, Epidemiology and End Results program, s.d.)

34Back to the Past
This type of NHL is the most common as it accounts for 1 of 3 newly diagnosed NHL. It is also considered as
an aggressive form98
. Chemotherapy is used in combination with rituximab as the first line treatment and
cure the majority of the patients. Nonetheless, between 30 to 40% exhibit refractory or relapsed
DLBCL.99
For this patient, as for ALL, the rescue treatment can be stem cell or bone marrow transplant, so
given the price and the weak outcomes of these therapies, this population is the primary target for CAR-T
treatments. The number of patients that could be targeted is around 25,350 per year. DLBCL is a
heterogeneous class of lymphoma and several subtypes exist such as Primary Mediastinal Large B Cell
Lymphoma (PMBCL) or Transformed Follicular Lymphoma (TFL).
 Mantle Cell Lymphoma
Mantle Cell Lymphoma is a rare type of non-Hodgkin lymphoma as the prevalence is around 1/25,000100
,
with only 3,000 new cases per year in the United States101
. This disease represents a real unmet need as
only 30% of the treated patients have a complete response and the median survival rate is 2-5 years102
. For
the younger patients, a stem cell transplant is used as a treatment. This disease represents around 6,300
new patients per year with unmet needs in the United States, European Union and Japan.
RARE METABOLIC DISEASES
Lysosomal storage diseases are rare inherited monogenic metabolic diseases that are characterized by an
abnormal accumulation of various toxic materials in the body's cells, resulting from deficiencies in lysosomal
enzymes. There are nearly 50 of these disorders altogether, and they may affect different parts of the body,
including the skeleton, brain, skin, heart, and central nervous system. Individual LSD are very rare, but as a
group they affect about one in every 5,000 live births103
. Some of these disorders are mainly treated with
frequent enzyme replacements, but unfortunately, these strategies are costly and not completely effective,
and for many LSD there is no current treatment. There is increasing interest in the pharmaceutical field
98
(Lymphoma Research Foundation, s.d.)
99
(Sehn & Gascoyne, 2015)
100
(Ribrag, 2010)
101
(Lymphoma Research Foundation, 2010)
102
(Abbasi, 2015)
103
(National Organization for Rare Disorders, s.d.)

35Back to the Past
towards this type of rare disorders: 14 products for LSD treatment have been launched in US and EU until
2014.
 The Gaucher disease is the most common type of lysosomal storage disorder and is present in
approximately 1 in 20,000 newborns. Three distinct types of this disease have been identified,
based upon the absence (type 1) or presence and extent (types 2 and 3) of neurological
effects. 90% patients affected by Gaucher disorder have the type I, and for this type there is a
higher incidence among the Ashkenazic Jewish population, (1 in 450 births). No ethnic prevalence
is associated with Gaucher disease types 2 or 3. In all cases the symptoms start during childhood
and adolescence104
.
Overall launched products per LSD subtype. Source: (OrphanDrugs.org, 2014)
 The Fabry disease is another LSD that affects only males, and whose diagnosis is often delayed
because of the wide range of symptoms. The incidence is calculated at approximately 1 in 117,000
people105
.
 Mucopolysaccharide Storage Diseases (MPS) are a subgroup of LSD that include Hurler Disease
(also known as GM1 gangliosidosis), Hunter, Sanfilippo, Morquio, Maroteaux-Lamy and Sly
diseases. The MPS diseases are caused by mutations that interfere with the normal breakdown of
mucopolysaccharides. The prevalence of all forms of MPS is estimated to be one in 25,000 births106
.
Regarding Hurler’s disease alone, the prevalence is 1:200,000107
in Europe and around 1:100,000
in US108
. Considering a population of 742,5 million and 318,9 million, we can conservatively
estimate around 3700 and 3000 patients in Europe and US respectively.
Ornithine Transcarbamylase (OTC) Deficiency is a rare X-linked genetic disorder characterized by complete
or partial lack of the enzyme ornithine transcarbamylase (OTC), an enzyme involved in the urea cycle. The
deficiency of this enzyme leads to nitrogen accumulation in the patients’ blood, causing severe neurological
104
(National Gaucher Foundation, s.d.)
105
(National Tay-Sachs & Allied Disease Association, 2015)
106
(National Organization for Rare Diseases, s.d.)
107
(OrphaNet, 2014)
108
(National MPS Society, s.d.)

36Back to the Past
complications109
. Since this disease is X-linked, it affects male more than females, and is fully expressed in
male only. According to the Urea Cycle Disorder Consortium, it is estimated that approximately 10,000
patients are affected by OTC deficiency worldwide; however, many cases go misdiagnosed or undiagnosed,
making it difficult to determine the true frequency of this disorder. Nevertheless, the supposed frequency
of OTC deficiency is 1/50,000110111
.
The Glycogen Storage Diseases (GSD) are a group of rare disorders in which stored glycogen cannot be
metabolized into glucose to supply energy for the body. GSD Type I is an autosomal recessive genetic
disorder, caused by mutations in the G6PC gene (GSD type Ia) or the SLC37A4 gene (GSD type Ib). These
mutations cause accumulation of glycogen and fat in the liver and kidney, leading to growth retardation
and metabolic imbalances112
. The frequency of GSD Type I is approximately 1 in 100,000 births. This
condition affects males and females in equal numbers, but the prevalence is higher in Ashkenazi Jews
population (1 in 20,000). 80% of GSD type I disorders are GSD type Ia, while 20% are GSD type Ib113
.
Lipoprotein Lipase Deficiency (LPLD) is a rare genetic metabolic disorder characterized by a deficiency of
the enzyme lipoprotein lipase, leading to defective digestion of certain fats. In US, the prevalence of LPLD
is approximately 1 in 1,000,000. The disease has been described in all races, but the incidence is higher in
Quebec and Canada114
. In December 2015, Uniqure decided to abandon the ambition of a FDA approval for
the Glybera115
which is the GT for LPLD, so if we use our estimation of 317 million people for EU5, it
represents 317 people. Then based on our estimation of 3.2017 million children/year in EU5, it represents
3 new cases per year.
LONG TERM MARKETS
CANCER
According to the CDC116
study of 2012, 14.1 million new cancer are diagnosed each year and we can expect
19.3 million by 2025. This growth is expected as the global population increases and as our average lifetime
increase too. In 2012, 8.2 million people died from cancer and 32.6 million people survived with a cancer
diagnosed at least 5 years before. Cancer surely represents an unmet need and gene therapy could bring
solutions for it.
109
110
(Rare Diseases Clinical Research Network, s.d.)
111
(OrphaNet, s.d.)
112
113
(Froissart, et al., 2011)
114
(Brunzell, 1999)
115
(Taylor, 2015)
116
(Centers for Disease Control and Prevention, 2016)

37Back to the Past
Except for “liquid tumors”, addressed mainly by CAR-T therapies (and developed in the previous paragraph
about blood cancers), the cure of cancer by gene therapy is still challenging. The genetic background of
tumor cells can be multifactorial and different from one patient to another. For that reason, the main
technology involved in cancer cure in this field is the ex-vivo gene therapy, using CAR-T or TCR to target
cancer antigen. Nevertheless, identification of relevant targets and targeting of non-circulating tumor (solid
tumor) are still a big issue to be resolved. At the moment, the most advanced research in cancer is for
Glioblastoma, a brain cancer.
Glioblastoma (also known as astrocytoma grade 4) is a brain cancer with very poor outcomes. The first
treatment is surgery followed by radiotherapy or chemotherapy. The prevalence is 2-3/100,000 per year,
leading to a number of new cases in Europe, USA and Japan around 20,700. The survival rate for adults at
5 years is 6%, 20% for children. In general, many people live for less than a year after diagnosis117
. This
pathology represents a striking unmet needs as the survival rate is very low. Several genetic mutations have
been linked to glioblastoma, and in particular in EGFR gene for 57.4% of GBM. Around 50% of patients with
EGFR mutation carry a specific mutation, EGFRvIII. Patients carrying this mutation should thus represent
around 6,000 people in UE, USA and Japan.
CARDIOVASCULAR DISEASES
According to the World Health Organization, an estimation of 17.5 million people died from cardiovascular
diseases (CVDs) in 2012. It is the first cause of death in the world, representing around 31% of all deaths
per year. “An estimated 7.4 million were due to coronary heart disease and 6.7 million were due to
stroke”118
. Over than 75% of CVDs deaths occur in low and middle-income countries. CVDs represent indeed
a global unmet medical need, though gene therapy is not the only solution: in fact, “most cardiovascular
117
(Cancer Research UK, s.d.)
118
(World Health Organization, 2015)

38Back to the Past
diseases can be prevented by addressing behavioral risk factors such as tobacco use, unhealthy diet and
obesity, physical inactivity and harmful use of alcohol, using population-wide strategies”119
.
The economical cost of heart failure exceeded $30 billion in 2012120
.
Gene therapy can target three types of heart related diseases: coronary artery disease, heart failure and
arrhythmias121
. Before becoming an efficient target for gene therapy, the delivery systems to the heart need
to be improve as the targeting is at the moment not very efficient and requires a direct injection and are
too localized to be efficient in the whole heart.
Coronary artery disease accounts for 1 to 6 in the United States in 2009122
. Evenif pharmacological
treatments have improved the long-term survival, some patients are refractory to the available treatments
and can’t be cured even by surgery and developrefractory angina pectoris. This disease concerns 600 000
to 1.8 million Americans, with 50 000 to 100 000 new cases per year123
. These cases will be the most relevant
for gene therapy.
Arrhythmia issues were detected in 43% of sudden death cases analyzed, but the true prevalence is
unknown. It seems to be around 1% of the general population124
. The prevalence of atrial fibrillation in the
United States was around 6.1 million in 2010. No effective cure is available for main arrhythmias that cause
high morbidity and mortality. No arrhythmia gene therapy has yet entered human clinical trial.125
Heart failure: The clinical trial CUPID2 in phase 2b, carried out by Celladon, despite very encouraging results
in previous phases failed to meet their targets with no significant improvement compared to placebo in
April 2015126
. Following this failure, Celladon stopped its activities, showing that the targeting of organ such
as heart is very challenging for gene therapy treatments and finally merged with Eiger
BioPharmaceuticals127
.
NEURODEGENERATIVE DISEASES (LIKE PARKINSON'S AND HUNTINGTON'S DISEASE)
According to European commission and the National Institute of Neurological Disorders and Stroke (NINDS),
there are more than 600 neurological disorders. Roughly 50 million Americans are affected each year, still
according to the NINDS.
119
(World Health Organization, 2015)
120
(Perin, et al., 2015)
121
(Wolfram & Donahue, 2013)
122
(Go, et al., 2013)
123
(Povsic, et al., 2015)
124
(Thomas, 2014)
125
(Wolfram & Donahue, 2013)
126
(Stiles, 2015)
127
(Garde, 2015)

39Back to the Past
Alzheimer disease, “the prevalence rate is about 7% for individuals aged 65 or more, with the risk doubling
every 5 years after age 65 (McCullagh et al. 2001; McDowell 2001)”128
. In 2012, the Alzheimer’s Association
estimates that 5.4 million people have this disease in the US and they are expected to grow. By 2025, they
estimate the number of patients between 11 and 16 million for the US.
In May 15, 2012 the Obama Administration announced the release of the National Alzheimer’s Plan which
aims at finding solutions to prevent the disease.
For the Parkinson’s Disease, the number of patients is difficult to assess because the disease is usually
diagnosed at a late stage. According to a report of 2006, NINDS estimates to at least 500 000 cases in the
US. “In industrialized countries the prevalence of Parkinson’s disease is about 1% for people over 60, with
estimates of up to 4% for people in the highest age groups (de Lau & Breteler 2006)”129
.
Huntington’s disease (HD) is a monogenic autosomal dominant genetic disorder that causes the
progressive breakdown of nerve cells in a part of the brain called basal ganglia. This degenerative cell
damage impairs cognitive ability, movement and emotional control. Every child of a parent with HD has a
50/50 chance of carrying the faulty gene. HD affects males and females equally and noethnic or racial
correlation has been found. According to the WHO130
, in Western countries, the estimates about the
prevalence of this disorder indicate that HD affects five to seven per 100,000 people. According to the
Hungtington’s Disease Society of America (HDSA), today there are approximately 30,000 symptomatic
Americans and more than 200,000 at-risk of inheriting the disease131
. Nocurrent treatment isavailable.
Cerebral Adrenoleukodystrophy is the most aggressive form of all adrenoleukodistrophies, and is caused
by abnormalities on the ABCD1 gene on the X chromosome. This disorder affects children and adolescents
from 3 to 15 years old, and causes severe neurological deficiency and adrenal insufficiency. The only
available therapy is allogenic hematopoietic stem cell transplant, together with dietary and adrenal
insufficiency therapies132
. The incidence of ALD is estimated to be 1 in 17,000-20,000 males. This disorder
has the same prevalence among all ethnic groups 13360
. Considering a population of 350 million and 120
million males, we can conservatively estimate around 17,000 and 6,000 patients in Europe and US
respectively.
OTHER INDICATIONS (DIABETES, INFLAMMATORY DISEASES, VIRAL INFECTIONS)
Diabetes. According to the WHO estimation of 2000, the global number of people affected by diabetes “is
177 million. This will increase to at least 300 million by 2025” and they estimate the diabetes-linked death
128
(Brown, et al., 2005)
129
(McGovern Institute for Brain Research, 2014)
130
(WHO, s.d.)
131
(Huntington’s Disease Society of America, s.d.)
132
133
(Engelen & Kemp, 2015)

40Back to the Past
to be over 800,000, even if that this number is certainly underestimated. “A more plausible figure is likely
to be around 4 million deaths per year related to the
presence of the disorder”134
.
According to the American Diabetes Association, in
2012, 29.1 million Americans135
have diabetes.
Human Immunodeficiency Virus, in 2014, there
were 36,9 million people worldwide living with HIV,
and among them 2,6 million were children. The
majority of the people infected are living in low- to
middle-income countries, including Asia and
Africa136
. According to the WHO, approximately 1,2
million of people died from AIDS in the same year137
.
There are no currently available drugs to cure HIV
infection or AIDS, but many treatments are available that reduce viral replication through antiretroviral
drugs. There are over 30 antiretroviral drugs approved by the FDA and almost all are designed to inhibit the
viral replication pathway at certain stages. The major limit of these therapies is that they are not effective
towards the so-called viral reservoir, which is the portion of the virus that is integrated in the T-cells
genome. Thus, people with HIV need chronic treatments with antiretroviral therapy, causing an enormous
public healthcare cost138
. Nevertheless, given the high cost of gene therapy drugs (we can hypothesize that
a gene therapy drug against HIV could be sold at around 300,000$-400,000$) these kind of treatments will
only be available in high-income countries, notably US and Europe. In US, 1,2 million persons aged 13 and
older were living with HIV infection, but 156,300 of them (12.8%) were unaware of it139
, while some
estimations suggests that the number of infected persons in Europe is around 1,6 million 140
. Thus,
considering around 2,441,600 aware patients in Europe and US, we can estimate that around 10% of them
might be able to pay for a high cost gene therapy treatment. For our financial analysis we thus considered
250,000 new patients per year.
CHANNELS
As we have seen, TCR and CAR-T are having wide clinical success, which is reflected in the growing number
of strategic partnerships in this area that have attracted huge investments and involve large pharmaceutical
organizations. Nevertheless, before these products are made available for the patients, a number of
additional issues has to be addressed. Notably, the wide heterogeneity of the starting material and the
134
(WHO, s.d.)
135
(ClinicalTrials.gov, s.d.)
136
(AIDS.gov, 2015)
137
(WHO, s.d.)
138
(Bluebird Bio, 2015)
139
(Hall, et al., 2015)
140
(Who, s.d.)
Challenges in cell therapy, including material sourcing and
manufacture. Source: (Neves Gameiro, 2016)

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