Introduction to Gene Therapy: A Clinical Aftermath
Patrice P. Denèﬂe
Despite three decades of huge progress in molecular genetics, in cloning of disease causative gene as well
as technology breakthroughs in viral biotechnology, out of thousands of gene therapy clinical trials that
have been initiated, only very few are now reaching regulatory approval. We shall review some of the
major hurdles, and based on the current either positive or negative examples, we try to initiate drawing
a learning curve from experience and possibly identify the major drivers for future successful achievement
of human gene therapy trials.
Key words: Gene therapy, Clinical trials, Viral and nonviral approaches, Systemic delivery, Local
delivery, Ex vivo gene therapy
The invention of recombinant DNA technology (1) consequently
led to the immediate inception of engineered gene transfer into
human cells, aiming at reversing a cellular dysfunction or creating
new cellular function. The concept of direct therapeutic beneﬁt
based on a gene defect correction in human cells or on gene therapy
Exactly 30 years ago, Martin Cline made a ﬁrst early and cer-
tainly premature human gene therapy attempt in 1979 at treating
severe thalassemia patients through an ex vivo b-globing gene
transfer protocol in the bone marrow of two patients in Italy and
Israel (2). As the protocol had not received any otherwise manda-
tory approval by regulatory bodies, the study was promptly ter-
minated and Cline was forced to resign his department
chairmanship at UCLA (University of California, Los Angeles)
and lost several research grants. Subsequently, the Recombinant
1. Three Decades
of Human Clinical
Otto-Wilhelm Merten and Mohamed Al-Rubeai (eds.), Viral Vectors for Gene Therapy: Methods and Protocols,
Methods in Molecular Biology, vol. 737, DOI 10.1007/978-1-61779-095-9_2, © Springer Science+Business Media, LLC 2011
DNA Advisor y Committee (RAC) at the National Institute of
Health (NIH) was urged in 1980 to expand its regulatory function
beyond recombinant DNA experiments so as to include human
gene therapy studies.
In 1982, a seminar was held at the Branbury Conference
Center of Cold Spring Harbors Labs. A group of scientists, led by
Ted Friedmann and Paul Berg, came together to build the foun-
dations of gene therapy and to draw what its future might be. As
an outcome, the ﬁrst book on gene therapy (3) was and is still a
landmark reference to this ﬁeld.
In 1989, Rosenberg et al. initiated the ﬁrst RAC-approved
gene therapy clinical trial, which was actually a “gene-labeling”
study targeting a neomycin-resistance gene transfer into tumor-
inﬁltrating lymphocytes using a retroviral construct, for the treat-
ment of metastatic melanoma with Interleukin-2 (4).
Effectively, a therapeutic gene clinical trial took place in 1990
to treat severe combined immunodeﬁciency (SCID) by transfer-
ring the adenosine deaminase (ADA) gene into T-cells using a
retroviral vector. No signiﬁcant clinical beneﬁt was observed,
albeit the protocol appeared to be safe for the patients (5, 6).
These pioneer clinical studies, as well as some others, land-
marked the inception in the 1990s of a major burst of academic,
clinical, biotechnological, and sustained ﬁnancial efforts lasting
for more than two decades (7). Even today, there are thousand
clinical trials registered as ongoing. Among which, 65 trials that
are declared in late stage (i.e., phase II–phase III) have proven to
be safe and would be in the clinical beneﬁt evaluation phase
Factually, one can also notice a sustained input of about a
hundred new clinical trials per year since 1999 (7). This seems in
Number of gene therapy trials worldwide (7)
Gene therapy clinical trials
Phase I 928 60.4
Phase I/II 288 18.7
Phase II 254 16.5
Phase II/III 13 0.8
Phase III 52 3.4
Single subject 2 0.1
Introduction to Gene Therapy: A Clinical Aftermath
clear contrast with the commonly held opinion that gene therapy
would be no longer active because of disengagement, especially
from certain large pharmaceutical industries, after a “1990s
Despite this constant entry ﬂow into clinical trials, the quasi-
absence of a registered drug after 20 years is quite compelling and
worth revisiting from a pure clinical development strategy
Most of the initial failures were most probably due to very
naive “science-driven” approach to clinical practice, but even
today, many projects are simply blocked because of fundamental
absence of translational research practice and still a strong under-
estimation of some key technical challenges. The rest of this book
addresses the fundamentals to be considered at the molecular
biology and the bioengineering level, but one should also pay
attention to the most standard clinical development parameters,
which sometimes are simply lacking in the project development
In the late 1990s, a news & views section in a major journal
was entitled: “Gene therapy has been keeping for long pretending
to be 5 years from the clinics.” With more than a thousand clinical
trials launched, the goal is no longer to enter man study for the
sake of a nice publication. The goal is set to complete successfully
human clinical trials and get to product registration, which we are
closer now than ever.
As a source of major hope for many incurable human diseases, the
concept of human gene therapy was immediately perceived as
the highest promise for curative treatment: a therapy acting at the
root of the genetic dysfunction.
The concept of gene therapy is relying on gene intervention.
From a pure pharmacokinetic point of view, nucleic acid has a
poor cell penetration capacity. For the past 30 years, an incredible
armada of viral and nonviral vectors has been engineered to for-
mulate the nucleic-acid-based “active principle.” Therefore, virus-
derived gene delivery vectors were thought from the beginning
to be optimized biomimetic vehicles. However, since they have
also evolved under a very high selection environment of infec-
tious agents, humans are also naturally equipped with very sophis-
ticated defense systems. These defense systems, which are often
speciﬁc to higher primates, cannot be ignored in the context of a
gene therapy clinical development plan, especially when it comes
to use of a natural human-derived virus. Other hurdles are the
active virus loads and the amount of virus particles to be used to
achieve therapeutic effects, which combined with the administration
2. Gene Therapy:
route are very difﬁcult to predict in terms of clinical pharmacology
and drug safety, imposing extremely careful clinical development
As foreign DNA cannot stay freely in a dividing cell, it does
not get associated with the host DNA replication machinery. On
one hand, one has engineered integrative vectors enabling the
“therapeutic gene” to be integrated into the host DNA, thereby
enabling long-term expression potential (e.g. use of oncoretroviral
or lentiviral vectors). A major drawback is the random insertion
into the host genome that can lead to serious adverse effect (SAE)
(8). On the other hand, one has tailored “nonintegrative vectors,”
which are mainly used to transfer DNA into quiescent cells but
which will be lost after a few replication cycles in dividing cells
(e.g. adenoviral or adeno-associated viral vectors).
The nature of target tissue/cell and the length of desired
therapeutic effect have, therefore, to be taken into consideration
in the gene therapy project charter.
In addition, the routes of administration of a therapeutic
principle can have major consequences both in terms of efﬁcacy
and safety. Routinely, one classiﬁes gene therapy protocols into
three main categories: ex vivo, local in vivo, and systemic in vivo
administrations (see Table 2).
In other words, the ﬁeld has been facing major challenges,
from novelty to translational research, which have often been
complicated by speciﬁc ethical concerns (9) led by the subjective
perception of gene therapy practice as a “Sorcerer’s apprentice.”
For the sake of clarity, we now focus on speciﬁc sets of exam-
ples, including dead ends, mixed successes to the most promising,
clinical studies that are intended to contribute to the frame into
which the ﬁeld should continue to contribute to the improve-
ment of human health.
After several years of clinical attempts, lack of clinical efﬁcacy,
major SAEs, and often unsurmounted industrial bioproduction
issues, one should ask the question of clinical plausibility of
systemic gene therapy protocols. The treatment of human diseases
often requires systemic administration procedures, and most often
oral or intraparenteral routes. Using viral or nonviral approaches
via the oral route, no protocol has yet been able to achieve satis-
factory results in preclinical studies; therefore, most studies have
focused on parental routes. Given the classical multiplicity of
infection (MOI) in the range of 10–10,000, authors are considering
a routine dose ranging from 108 to 1015 viral particles per kg of
body weight. This effective dose deﬁnition immediately triggers
3. Current Status:
and Case Studies
3.1. Systemic Delivery
Has Not Been
Introduction to Gene Therapy: A Clinical Aftermath
Routes of administration used in gene therapy protocols
Routes of administration Ex vivo Local in vivo Systemic
Deﬁnition Gene transfer is performed out of the
living organism; the therapeutic agent
is the “reinfused cells”
Gene transfer product is injected
into a local,
and possibly isolated body
(IM), intratumoral, locoregional,
Product is administered
through oral or intraparen-
teral route so that it can
reach all parts of the body
Examples SCID-ADA protocol IM: NV1FGF
dystrophy with plasmids or AAV
disease with AAV
Comments Autologous cells are handled in a dedicated
The efﬁcacy of treatment
is related to the ability of transduced cells
to perform sustainable effects
Local administration is preferred
if therapeutic beneﬁt can be
reaction can be speciﬁc
Product leakage has to be
Dose-limiting rate and major
reaction to large viral load
are commonly encountered,
generally limiting the
approaches, from mouse-
based experiments to human
several major technical, pharmacological, and immunological
hurdles to consider. We can schematically classify them as
Mastering an industrial bioprocess that is scalable to the Good
Manufacturing Practice (GMP)-compliant production of
clinical and eventually commercial batches
Deﬁning a puriﬁcation process and a formulation that is on
line with the vector physicochemical properties and the
desired volume to be injected
Documenting the pharmacokinetics and ADMET (adsorp-
tion, desorption, metabolism, elimination, and toxicity) prop-
erties of vectors in human at such high doses
Documenting, in terms of long-term potential side effects,
the immunoreactivity against the vector itself or the thera-
peutic cells, and the fate of the product if it needs to be
Below are two examples of gene therapy concepts that have
emerged more than 20 years ago, for which clinical realization is
desperately kept on being delayed, i.e., in cystic ﬁbrosis (10) and
Duchenne’s muscular dystrophy (DMD) (11).
Although predominantly used in the pioneering days of CF gene
therapy, adenovirus-based vector usage has dropped in the last
decade due to poor transduction efﬁciency in human airway epi-
thelial cells and the inability for readministration. In addition, a
study by Tosi et al. raised concerns that antiadenovirus immune
responses, in particular cytotoxic T-lymphocyte-mediated (CTL)
responses and major histocompatibility complex class I antigen
(MHC-I) presentation, may be further enhanced if the host has a
preexisting Pseudomonas infection (12). These data highlighted
potential problems for adenovirus-based vectors in CF gene therapy
and deﬁnitely conﬁned the use of adenovirus-based vectors for
CF gene transfer to upstream research studies.
As a potential alternative to adenovirus, adeno-associated
virus (AAV) (13) was assessed for lung transduction in clinical
cystic ﬁbrosis gene therapy trials. However, the feasibility of
repeated AAV administration is still unresolved, and the limited
capacity of AAV to carry the full-length cystic ﬁbrosis transmem-
brane conductance regulator (CFTR) gene and a suitably strong
promoter remains a signiﬁcant problem. However, Lai et al. (14)
have recently shown that the efﬁciency of AAV trans-splicing can be
greatly improved through rational vector design and may, therefore,
allow the CFTR cDNA to be split between two viral vectors.
So far, two human gene therapy phase I/II protocols have
been undertaken with incremental and repeat doses of AAV, up to
2 × 1012 and 2 × 1013 DNase-resistant particles, respectively (13, 15).
3.1.1. Cystic Fibrosis
Introduction to Gene Therapy: A Clinical Aftermath
In both studies, viral shedding and increases in neutralizing
antibodies were observed, but no serious adverse event (8) was
associated to the virus administration. Importantly, a signiﬁcant
reduction in sputum IL-8 and some improvement in lung func-
tion were noted after the ﬁrst administration, but not after the
second or third administration.
On the basis of these studies, Targeted Genetics Corporation
initiated a large repeat-administration multicentric phase IIb
study (100 subjects), sufﬁciently powered to detect signiﬁcant
changes in lung function. Eligible subjects were randomized to
two aerosolized doses of either AAV-CF or placebo 30 days apart.
The subjects underwent pulmonary function testing every 2
weeks during the active portion of the study (3 months) and were
followed for safety for a total of 7 months. No publication is avail-
able 4 years after the study was completed, but the company
announced that the trial had not met its primary end point and,
therefore, the CF program has been discontinued (16).
There may be several reasons for these new disappointing
outcomes: (1) As for adenoviral vector, AAV-2 was still too inef-
ﬁcient in reaching airway epithelial cells via the apical membrane,
(2) the inverted terminal repeat (ITR) promoter used to drive
CFTR expression was not strong enough, and (3) repeat admin-
istration of AAV-2 to the lung was actually not possible owing to
the mounting of an antiviral immune response. Finally, on the
back of previously published AAV-2 aerosolization studies,
Croteau et al. (17) evaluated the effects of exposure of healthy
volunteers to AAV2. Based on airborne vector particle calcula-
tions, the authors estimated exposure to 0.0006% of the adminis-
tered dose. At such an infradose, no deleterious health effects
were detectable, but this underlies the strong requirement in
improving the general ADMET properties of the vector system
and the necessity to perform these studies even before going into
Studies are currently underway to assess the feasibility of
repeated administration of lentivirus-based vectors into airways
by several groups (18, 19), and further data will be needed before
the relevance of such viruses for CF gene therapy can be decided.
In addition, the safety proﬁle of virus insertion into the genome
of airway epithelial cells will have to be carefully monitored.
With the concept that bone marrow-derived hematopoietic
or mesenchymal stem cells may have the capacity to differentiate
into airway epithelial cells (20), some groups have entered this
very challenging and controversial approach for the treatment of
CF (21, 22).
On the nonviral side, parallel work had been made regarding
the formulation of vectors (23), and the United Kingdom (UK)
CF Gene Therapy Consortium clinical trial program has been
carefully comparing these agents and is now assessing whether the
most efﬁcient currently available nonviral gene transfer agent is
able to alter CF lung disease. As the extension of gene transfer
achieved is still too small and transient to drive any clear thera-
peutic beneﬁt, most research for CF gene therapy has returned to
the laboratory. In UK, there are no more trials ongoing at present,
but it remains the goal of the UK Consortium to work together
to meet the challenges and enhance progress to a phase III (large-
scale) study this year.
Finally, electroporation and some emerging physical delivery
methods such as ultrasound and magnetofection have shown
encouraging results in vitro and in rodent models, and again,
translational research into larger animal models, such as sheep,
and hopefully in the clinic is challenging (24, 25).
In perspective as of today, one can expect the promise for a
curative therapy for CFTR may not rely on gene therapy, but on
“protein-decay” therapy, with the phase II clinical development
of a small molecule, miglustat, by Actelion, which has been shown
to slow down the mutated protein degradation and enables it to
be exported to the membrane (26).
DMD is an X-linked inherited disorder that leads to major systemic
muscle weakness and degeneration. Muscle ﬁber necrosis is related
to the dystrophin gene deﬁciency itself (27). Becker muscular
dystrophy (BMD) has clinical picture similar to that of DMD but
is generally milder than DMD, and the onset of symptoms usually
occurs later. The clinical distinction between the two conditions
is relatively easy because (1) less severe muscle weakness is
observed in patients with BMD and (2) affected maternal uncles
with BMD continue to be ambulatory after age 15–20 years. The
cloning of the dystrophin gene opened the door for gene therapy
(27–30). However, as in systemic disorders, there are major
roadblocks including (1) the large amount of skeletal muscle
(basically half the body weight of a healthy human being), (2) the
involvement of cardiac and the peritoneal muscles in the disease,
and (3) the extremely large size of the dystrophin protein,
427 kDa, encoded by a 79 exons gene (28, 31, 32).
In one study, nine DMD/BMD patients were injected with a
naked dystrophin gene-carrying plasmid into the radialis muscle.
Patients were divided into three cohorts, each injected with one
of following three doses: 200 mg once, 600 mg once, or 600 mg
twice (2 weeks apart). Biopsies were then retrieved 3 weeks
postinjection, and amplicon DNA could be detected only in 6/9
patients. Patients from the ﬁrst cohort and one patient from the
second cohort exhibited 0.8–8% of weak, complete sarcolemma
labeling (29), while 3–26% of muscle ﬁbers showed incomplete/
partial labeling. The third group showed 2–5% complete sarco-
lemma labeling and 6–7% showed partial labeling. There were no
observed adverse effects to the treatment. The study concluded
Introduction to Gene Therapy: A Clinical Aftermath
that the expression of dystrophin was low (29), and thus, the
study was not pursued. One may question why the study was ini-
tiated despite the product obviously failed to meet basic efﬁcacy
requirements to reach future clinical application and even worse
was facing major industrial bioproduction pitfalls given the clini-
cal doses that could be inferred from preclinical studies.
For several years, several preclinical studies have been initi-
ated, and ﬁnally several concurrent clinical trials were initiated
using various pseudotyped adeno-associated viruses (33) as a
vehicle to deliver either truncated versions of the gene (mini or
microdystrophin) or an exon-skipping RNA structure, all thought
to achieve truncated albeit functional dystrophin protein expres-
sion (28, 34, 35). The AAV vector, whatever the serotype, pro-
vides superior transduction efﬁciency to the skeletal muscle but is
also a source for potential immune response that remains to be
carefully understood (36–38). No conclusive result has been
drawn yet from the current clinical studies. However, the intra-
muscular high-dose pharmacokinetic proﬁle in relevant preclinical
models and eventually in humans is yet to be thoroughly docu-
mented prior to launching any efﬁcacy clinical gene therapy.
However, the last 5–7 years, reviewed elsewhere (11), have
seen unrivaled progress in efﬁcient systemic delivery of synthetic
and chemically modiﬁed oligonucleotides again used to enforce
mutated exon splicing (39). This progress has led to several more
clinical trials, which are labeled as “small molecule” trials, i.e., out
of the boundaries of gene therapy. The most advanced clinical
trial, led by a company called Prosensa in Holland, is completing
a phase IIb and has led to ﬁnalize a collaborative agreement with
GSK in October 2009, marking the return of large pharmaceutical
companies in the plain ﬁeld.
The above examples clearly illustrate how gene therapy has pro-
gressively moved from “systemic” administration routes toward
more pragmatic local administration regimen or to alternative
small molecule innovative therapeutics. We now review the most
promising local gene therapy clinical protocols.
Parkinson’s disease is primarily due to the local degeneration of
nigrostriatal neurons projecting into striatum, and a subsequent
shortage of dopamine in this target region. Predisposing and risk
factors are numerous but disease mechanism remains unclear.
More than a million patients are affected both in Europe and the
USA. So far, the main treatment has been oral administration of
l-DOPA, a dopamine precursor, but patients generally encounter
motor complications after 5 years of treatment. Deep stimulation
surgery, therefore, becomes the second phase of disease manage-
ment for 0.5% of patients in France each year.
3.2. Gene Therapy
to Disease Treatments
3.2.1. Parkinson’s Disease
The therapeutic challenge is then to trigger continuous release
of dopamine into striatum neurons. Gene therapy is a plausible
approach, as far as cellular therapies could be. In addition to be
continuous, dopamine release should remain local, to avoid dys-
kinesia effects observed in systemic administration of the precursor
in the pharmacologic treatment.
Several clinical trials have been undertaken (40, 41). In California,
Avigen, later taken over by Genzyme, initiated a trial with an
AAV-vector to express the l-DOPA converting enzyme, and
another biotechnology company, Ceregene, conducted a phase I
open label study with 12 patients, then a phase II trial with an
AAV-based vector expressing neurturin (CERE-120), a neuron
survival factor (42). Very recently, Ceregene has reported addi-
tional clinical data from a double-blinded, controlled phase II
trial of CERE-120 in 58 patients with advanced Parkinson’s disease.
The company, however, announced that the phase II trial did not
meet its primary end point of improvement in the Uniﬁed
Parkinson’s Disease Rating Scale (UPDRS) motor off score at 12
months of follow-up, although several secondary end points sug-
gested a modest clinical beneﬁt. An additional, protocol-prescribed
analysis reported focused on further analysis of the data from the
30 subjects who continued to be evaluated under double-blinded
conditions for up to 18 months, which indicate increasing effects
of CERE-120 over time. A clinically modest but statistically sig-
niﬁcant treatment effect in the primary efﬁcacy measure (UPDRS
motor off; p = 0.025), as well as similar effects on several more
secondary motor measures (p < 0.05), was seen at the 18 months
end point. Not a single measure similarly favored sham surgery at
either the 12 or 18 months time points. Additionally, CERE-120
appears safe when administered to advanced Parkinson’s disease
patients, with no signiﬁcant concerns related to the neurosurgical
procedure, the gene therapy vector, or the expression of neur-
turin in the Parkinson’s disease brain. Long-term safety was also
performed in a primate model and was satisfactory (43). The
company also reported the results of an analysis of neurturin
gene expression in the brains from two CERE-120 treated sub-
jects who died of causes unrelated to treatment. These analyses
revealed that CERE-120 produced a clear evidence of neurturin
expression in the targeted putamen but no evidence for transport
of this protein to the cell bodies of the degenerating neurons,
located in the substantia nigra. In addition to the known cell loss
in Parkinson’s disease, and in agreement with the perspectives
deﬁned elsewhere (44), these ﬁndings suggest that deﬁcient
axonal transport in degenerating nigrostriatal neurons in advanced
Parkinson’s disease impaired transport of CERE-120 and/or
neurturin from putaminal terminals to nigral cell bodies, reducing
the therapeutic effect of CERE-120.
In parallel to this study, Oxford Biomedica, in collaboration
with a group in Hospital H. Mondor in France, has built an
Introduction to Gene Therapy: A Clinical Aftermath
equine lentivirus-based vector to express three genes involved in
dopamine synthesis. The product (ProSavin) is administered
locally to the region of the brain called the striatum, converting
cells into a replacement dopamine factory within the brain, thus
replacing the patient’s own lost source of the neurotransmitter.
A phase I/II study was initiated in December 2007 in France
with patients with mid- to late-stage Parkinson’s disease who are
failing on current treatment with l-DOPA but have not pro-
gressed to experiencing drug-induced movement disorders called
dyskinesia. After a ﬁrst cohort of three patients who showed no
side effect or an antibody response (42), the dose-escalation stage
of the study has progressed to the second dose level. The 6-month
data from the ﬁrst dose level suggest ProSavin is safe and well
tolerated and showed encouraging evidence of efﬁcacy (42).
Another successful albeit often controversial is the case of ex vivo
gene therapy. This is the case of severe combined immunologic
disorders (SCID) treatment. Soon after the ﬁrst US trial led by
Blaese and colleagues (5), a network of European groups led by
A. Fischer in France, A. Trascher in the UK, and M. Roncarolo in
Italy initiated similar protocols for the treatment of SCID. The
successful treatment of the ﬁrst patients was greeted with a lot of
enthusiasm when it was ﬁrst reported in 2000 and 2002 (45–47).
However, this euphoria turned to a serious alert at the end of
2002 when two of the ﬁrst ten children treated in France devel-
oped SAE, described as leukemia-like conditions (48). As demon-
strated later, the insertion of the therapeutic DNA into the patient
cells had occurred next to one speciﬁc locus LMO2 (the proto-
oncogene LIP domain only two locus) (49–51). With the news of
this devastating event, most SCID-X1 gene therapy trials were
placed on hold worldwide. However, in view of patient overall
and lack of alternative treatment, some ADA and SCID-X1 trials
were pursued, with extremely careful monitoring and better
vector types designed so as to reduce the odds of such adverse
effect. Work is now focusing on correcting the gene without trig-
gering an insertional oncogenic event.
Between 1999 and 2007, gene therapy has restored the
immune systems of at least 26 children with two forms [ADA-
SCID (nine children) and SCID-X1 (ten children)) of the disorder,
and four of the ten SCID-X1 patients had developed leukemia-
related SAE (52). As of today, 20 children have been treated, four
of them have developed leukemia-like adverse effects and one
patient has unfortunately died from leukemia. From a clinical point
of view, patients, who have been able to lead a normal life for
periods up to 3 years, should be considered cured by this pioneer-
ing gene therapy treatment. Otherwise, 10 years later, none of
these 20 children would be alive today without gene therapy.
Based on this clinical success, several important protocols are
now entering the clinical stage. A major example is that of the
3.2.2. Severe Combined
Wiskott–Aldrich syndrome (WAS), which is a complex primary
immunodeﬁciency disorder associated with microthrombocy-
topenia, autoimmunity, and susceptibility to malignant lymphoma.
At the molecular level, WAS is caused by mutations in the gene
encoding the Wiskott–Aldrich syndrome protein (WASP). WASP
is a cytosolic adaptor protein mediating the rearrangement of the
actin cytoskeleton upon surface receptor signaling, which in turn
is instrumental for cognate and innate immunity, cell motility,
and protection against autoimmune disease (53). WASP confers
selective advantage for speciﬁc hematopoietic cell populations
and serves a unique role in marginal zone B-cell homeostasis and
The success of such blood stem cell transplantation is related
to the patient’s age, the conditioning regimen precell infusion,
and the extent of reconstitution postcell reinfusion. Since WASP
is expressed exclusively in hematopoietic stem cells, and because
WASP exerts a strong selective pressure, gene therapy is expected
to cure the disease (55). Cumulative preclinical data obtained
from WASP-deﬁcient murine models and human cells indicate a
marked improvement of the impaired cellular and immunological
phenotypes associated with WASP deﬁciency. A ﬁrst clinical trial is
currently being conducted with a retroviral construct (55, 56)
with a careful monitoring of insertional events (57). However,
capitalizing on experience with SCID-ADA and establishing a
solid European network, A. Galy and colleagues have engineered,
validated, and GMP-produced a very potent lentiviral product
(58) and a three-site clinical study is due to start in 2010 (59).
As stated above, the most promising gene therapy clinical results
are obtained with local delivery procedures. In addition to the
above examples, two key examples of successful development of
candidate drugs up to the phase III are in the ﬁeld of vascular/
The ﬁrst example is that of lipoprotein lipase gene for the treatment
of familial lipoprotein lipase deﬁciency. The product initially
cloned into adenovirus and retroviruses by us in the 1990s
(60–62) is now carried onto an AAV vector (63). Very encourag-
ing data have been obtained through a direct multiple intramus-
cular (IM) injection in the inner limb with corrective expression
obtained for several weeks postinjection (64), and the product
registration has been started by European Medical Agency (EMA)
in January 2010 as a centralized procedure, which is the standard
route for all advanced therapies.
The second example is that of peripheral vascular disease (PVD),
which is predominantly affecting the lower extremities. PVD has
a relatively low mortality but results in considerable morbidity
3.3. Two Clear-Cut
Examples of Products
3.3.1. Lipoprotein Lipase
3.3.2. Peripheral Vascular
Introduction to Gene Therapy: A Clinical Aftermath
Even though angioplasty and reconstructive surgery are
somewhat effective treatment options for many patients with
peripheral arterial insufﬁciency, these procedures are associated
with considerable risks, notably restenosis after peripheral angio-
plasty. In addition, the severity and progressive nature of this dis-
ease often limit these treatment options, resulting in persistent,
disabling symptoms or limb loss. PVD, therefore, represents an
attractive target for a gene therapy approach to restoration of
effective limb perfusion in selected patients (65).
Dr. Jeffrey Isner and his colleagues have taken a novel
approach (66) to the problem of peripheral artery insufﬁciency
with encouraging results. This group has been at the forefront of
angiogenic gene therapy for peripheral artery insufﬁciency, pub-
lishing several studies over the past 15 years that have set the
ground (65–69) for the clinical study by Sanoﬁ-Aventis.
Fibroblast growth factor 1, FGF1, is a proangiogenic factor
acting on various cellular subtypes, and more particularly involved
in preexisting microvessels sprouting, microcapillary network
genesis, and arteriolic maturation. Pharmacodynamic studies of
an FGF-encoding plasmid (70, 71) in two animal models con-
ﬁrmed the therapeutic potential of such an vector (70, 71). Several
preclinical toxicity studies were also performed to document vector
lack of integration as well as lack of neither oncogenic nor retin-
opathic potential of the product.
Two human clinical trials (phase I–IIa) were performed and
have documented good tolerance to NV1 FGF as well as local
angiogenesis effects limited to the injection point, conﬁrming
product safety (72, 73). Consequently, a ﬁrst phase II double-
blinded clinical study was performed with 125 patients, to docu-
ment product efﬁcacy and has achieved a remarkable twofold
reduction of amputation in the treated group vs. placebo (74).
As of today, a large-scale pivotal phase III trial, called
TAMARIS, is ongoing (75) since November 2007 (490 patients,
130 clinical centers) to document reduction of amputation and
increase of life span. The study is aimed to be completed by July
2010 (76). These results, if proven positive, will most probably
result in a long-awaited milestone, i.e., the registration of the ﬁrst
gene therapy product for a large clinical indication.
Several lines of observations can be drawn from these past 20
years of clinical trials.
First, yet the primordial concept was meant to tackle inher-
itable genetic disorders, seen as low-hanging fruits for a fast
clinical proof of concept, most of the clinical protocols have been
addressing acquired complex disorders, e.g. cancer, cardiovascular,
Second, even though the science was sort of intuitively genuine,
clinical gene therapy is now understood as a “difﬁcult” clinical
development ﬁeld, and there is still a trend from private investors
to stay away from this area, although major clinical successes are
now emerging, such as for the SCID and now peripheral artery
Third, the driving force has remained often too long in the
hands of academic research, and thus, clinical development has
been failing repeatedly because of translational research issues,
such as good laboratory practice (GLP) preclinical, clinical devel-
opment, and GMP lack of expertise.
Fourth, although viral vector are considered as best in class to
achieve efﬁcacy in men, major adverse effects have been encoun-
tered such as vector-related oncogenesis in some trials and complex
immunologic responses to the virus in most of systemic and local
However, watching the drug pipeline from the market approval
end, several investigational new drugs are by now registered or
close to approval, namely, RTV-ADA treated cells from the treat-
ment of SCID-ADA in Italy (52), the AAV-LPL product in Europe
(64), and NV1FGF for the treatment of PAD (76, 77).
In the new perspective of true clinical realization and positive
learning experience, the mastering and practical application of the
right set of tools such as vector design and scale-up production
will become true strategic advantages for future gene therapy
In memory of Prof. J. M. ISNER, who has pioneered gene ther-
apy and has opened the avenue to the ﬁrst ever gene therapy
product registration for cardiovascular diseases.
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