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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.
Chapter 2
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
was born.
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
Gene Therapy
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
28 Denèfle
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
(Table 1).
Factually, one can also notice a sustained input of about a
hundred new clinical trials per year since 1999 (7). This seems in
Table 1
Number of gene therapy trials worldwide (7)
Gene therapy clinical trials
Number %
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
Total 1,537
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
golden age.”
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:
and Basic
30 Denèfle
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:
Clinical Trials
and Case Studies
3.1. Systemic Delivery
Has Not Been
Introduction to Gene Therapy: A Clinical Aftermath
Table 2
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
compartment (intramuscular
(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
Locoregional: Duchenne’s
dystrophy with plasmids or AAV
Stereotactic: Parkinson’s
disease with AAV
Comments Autologous cells are handled in a dedicated
processing center
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
Local immune
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
practical translational
approaches, from mouse-
based experiments to human
clinical development
32 Denèfle
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
phase I.
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
34 Denèfle
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
3.1.2. Duchenne’s
Muscular Dystrophy
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
Potential Promise
to Disease Treatments
3.2.1. Parkinson’s Disease
36 Denèfle
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
Immunologic Disorders
38 Denèfle
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
function (54).
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/
metabolic disorders.
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
and disability.
3.3. Two Clear-Cut
Examples of Products
Successfully Reaching
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
4. Future
and Prospects
40 Denèfle
addressing acquired complex disorders, e.g. cancer, cardiovascular,
neurodegenerative diseases.
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
diseases (PAD).
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
administration protocols.
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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... Viral yapı aktarılan hücrede kısa sürede fark edilmekte ve gen etkisiz hale getirilebilmektedir. Eğer UPKH insanda kullanılacaksa gen transferi ile ilgili sorunların da çözülmesi gereklidir (14,15). Bu nedenlerle son yıllarda virus olmadan (virus free) gen aktarımı veya gen aktarılmadan (integration free) UPKH elde edilmesi yöntemleri denenmektedir (15,16). ...
... Mutasyon taşımayan hücrelerin kök hücre benzeri yapıya dönmesinin ardından elde edilen UPKH'ler hasta fareye kemik iliği nakli ile aktarılmıştır. Bu gen tedavi uygulaması ile hasta farede orak hücre anemisine ait bulguların çoğunun düzeldiği gözlenmiştir (14,27,31). ...
... Birçok çalışmanın ancak çok azında hücreler UPKH yapılanması kazanmaktadır. Daha etkin ve problem oluşturmayan gen aktarım metodlarının bulunması çok önemlidir (10,11,14). ...
Full-text available
Induced pluripotent stem cells with self renewal and differentiation abilities were established from mature cells by transcription factors. These cells may be used in regenerative medicine as embryonic stem cells. Recently a mutated gene in a cell obtained from a mouse which had a genetic disease was corrected by gene targeting techniques. These cells were converted into induced pluripotent stem cell by using transcription factor genes and transferred into the same mouse. As a result, the clinical findings of genetic disease mostly disappeared in the mouse. In this manuscript, the new uses of induced pluripotent stem cells in medicine were discussed.
... There is now a growing number of phase I and II clinical trials on gene therapies for a variety of hematological diseases such as Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD) and metachromatic leukodystrophy (MLD). Thus, over 1700 gene therapy clinical trials have been or are in progress in areas such as the treatment of cancer, infectious diseases, and genetic diseases, with an average of about 100 new clinical trials of gene therapy opened each year [61]. ...
Stem cell-based therapies are increasingly recognized as a new way to treat various diseases and injuries, with a wide range of health benefits. The goal is to heal or replace diseased or destroyed organs or body parts with healthy new cells provided by stem cell transplantation. The current practical form of stem cell therapy is the hematopoietic stem cells transplant applied for the treatment of hematological disorders. Additionally, there are over 2100 clinical studies in progress concerning hematopoietic stem cell therapies. All of them are using hematopoietic stem cells in the treatment of various diseases like: cancers, leukemia, lymphoma, cardiac failure, neural disorders, auto-immune diseases, immunodeficiency, metabolic or genetic disorders. Several challenges are to be addressed prior to develop and apply large scale cell therapies: 1) to elucidate and control the mechanisms of differentiation and development toward a specific cell type needed to treat the disease, 2) to obtain a sufficient number of desired cell type for transplantation, 3) to overcame the immune rejection and 4) to demonstrate that transplanted cells fulfill their normal functions in vivo after transplants.
... With the insertion of the gene in cells containing the mutated version you can reset function thus curing disease. But also with gene therapy you can inactivate a mutated gene that is not working properly and even introduce genes that help to fight a particular disease [1][2][3][4][5]. ...
... Nanocarriers offer unique possibilities for overcoming cellular barriers in order to improve the delivery of various drugs and gene nanomedicines123. A fundamental step towards the design of nanocarriers is the complete knowledge of the delivery mechanisms such as cellular uptake, cytoplasmic transport, endosomal escape, and nuclear localization. ...
Full-text available
Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method for characterizing the mode of motion of nanocarriers and for quantifying their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP–DOPC/DNA (DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOPC: dioleoylphosphocholine) and DC-Chol–DOPE/DNA (DC-Chol: 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; DOPE: dioleoylphosphatidylethanolamine) lipoplexes in CHO-K1 (CHO: Chinese hamster ovary) live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient: D ≈ 0.003 μm2 s−1). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity v ≈ 0.03 μm2 s−1 and random motion. The method permitted us to generate an intracellular transport map showing several regions of concerted motion of lipoplexes.
The personalized medicine movement pivots on the integration of patients’ genetic and genomic data for prevention, diagnosis, and treatment. However, it does not refer to a single test or type of data like a gene sequence, but rather assessment of a range of molecular-based changes that are impacted by and can impact the macro- and micro-environment. At this point in time, several areas of research are actively under investigation, yielding new clues about human health and disease, and informing development of new clinical interventions. These areas of research and clinical applications also raise a host of issues that will need to be addressed to enable the continued pace of research and clinical implementation. This chapter aims to provide an overview of several areas of research that are driving the personalized medicine movement as well as some of the ethical, legal, and clinical issues that have been raised.
The Ad5/11 chimeric oncolytic adenovirus represents a promising new platform for anticancer therapy. Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells and is the most common malignant myeloid disorder in adults. Myeloid and other hematopoietic cell lineages are involved in the process of clonal proliferation and differentiation. In the present study, we aimed to ascertain whether chimeric oncolytic adenovirus-mediated transfer of the human interleukin-24 (IL-24) gene induces enhanced antitumor potency. Our results showed that the Ad5/11 chimeric oncolytic adenovirus carrying hIL-24 (AdCN205‑11-IL-24) produced high levels of hIL-24 in AML cancer cells, as compared with the Ad5 oncolytic adenovirus expressing hIL-24 (AdCN205-IL-24). AdCN205-11-IL-24 specifically induced a cytotoxic effect on AML cancer cells, but had little or no effect on a normal cell line. AdCN205-11-IL-24 induced higher antitumor activity in AML cancer cells by inducing apoptosis in vitro. This study suggests that transfer of IL-24 by an Ad5/11 chimeric oncolytic adenovirus may be a potent antitumor approach for AML cancer therapy.
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Regenerative medicine has begun to define a new perspective of future clinical practice. The lack of basic data regarding to basic stem cell biology-survival, migration, differentiation, integration in a real time manner when transplanted into damaged tissue remains a major challenge for design stem cell therapies. So, visualization of injected stem cells provides additional insight into the future therapeutic benefits. Although current imaging modalities including magnetic resonance imaging, positron emission tomography, single photon emission computed tomography, bioluminescence imaging, and fluorescence imaging offer some morphological as well as functional information, they lack the ability to assess and track in vivo biological phenomenon, a pivotal link for greater mechanistic understanding following cell-based intervention. This review will therefore discuss currently available in vivo imaging modalities and image processing techniques which may potentiate this field of research. - See more at:
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A human immunodeficiency virus (HIV)-based vector pseudotyped with the Ebola Zaire (EboZ) viral envelope glycoprotein (GP) was recently shown to transduce murine airway epithelia cells in vivo. In this study, the vector was further redesigned to improve gene transfer and also to increase safety. We used mutant EboZ envelopes for pseudotyping, which resulted in higher titers and increased transduction of airway cells in vivo compared to vectors pseudotyped with wild-type EboZ GP. As these envelopes lack regions associated with toxicity of the wild-type EboZ GP, they should also be safer to use for pseudotyping of lentiviral vectors. In addition, lentiviral vectors were created based on feline immunodeficiency virus and shown to have similar efficiency of transduction compared to HIV-based vectors. The creation of lentiviral vectors with highly engineered EboZ envelopes improved the performance of the system and should also increase its safety since only minimal regions of the EboZ envelope, which lack the toxic domain, are used.
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We previously demonstrated that direct intramuscular injection of rAAV2 or rAAV6 in wild-type dogs resulted in robust T-cell responses to viral capsid proteins, and others have shown that cellular immunity to adeno-associated virus (AAV) capsid proteins coincided with liver toxicity and elimination of transgene expression in a human trial of hemophilia B. Here, we show that the heparin-binding ability of a given AAV serotype does not determine the induction of T-cell responses following intramuscular injection in dogs, and identify multiple epitopes in the AAV capsid protein that are recognized by T cells elicited by AAV injection. We also demonstrate that noninvasive magnetic resonance imaging (MRI) can accurately detect local inflammatory responses following intramuscular rAAV injection in dogs. These studies suggest that pseudotyping rAAV vectors to remove heparin-binding activity will not be sufficient to abrogate immunogenicity, and validate the utility of enzyme-linked immunosorbent spot (ELISpot) assay and MRI for monitoring immune and inflammatory responses following intramuscular injection of rAAV vectors in preclinical studies in dogs. These assays should be incorporated into future human clinical trials of AAV gene therapy to monitor immune responses.
Critical limb ischemia (CLI) represents the most severe stage of atherosclerotic lower extremity peripheral artery disease (PAD), and CLI prevalence is expected to increase as the population ages. The current standard of care for CLI relies on direct revascularization, either by endovascular techniques or open surgical approaches, as there are few effective medical treatments for this condition. Therapeutic angiogenesis is a novel approach to improving limb outcomes for these patients. Experimental preclinical studies and phase I/II clinical trials of therapeutic angiogenesis using gene transfer in patients with CLI unsuitable for revascularization have shown promising results. In this review, we describe the potential clinical impact of this new approach as an adjunct in our therapeutic armamentarium.
We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as γ chain (γc) deficiency] in 9 out of 10 patients by retrovirus-mediated γc gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with γδ+ or αβ+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.
The field of gene therapy has evolved through several stages, from its origins in molecular biology, to virus-mediated transfer of foreign genetic material, to the rapid ascent of early human clinical trials and more recently to a period of self-examination. By 1995, it became apparent that gene therapeutics was going to be far more complicated than originally hoped or perhaps anticipated. The field had gone through at least five years of highly visible and publicized clinical studies but the hype and hope clearly exceeded the accomplishments. Responding to concerns of investigators and the public alike, NIH director Harold Varmus established an advisory committee; their report ( had a remarkably positive effect on the field. Renewed efforts were made to understand the basic biology of gene transfer, the molecular biology of vectors and the cellular mechanisms of vector uptake and gene expression. The stampede to clinical trials abated. The field had undergone a 'market correction'.
Restenosis remains the main limitation of interventional cardiology. Restenosis occurs when angioplasty-induced intimal hyperplasia as well as arterial remodelling result in flow-limiting renarrowing of the arterial lumen at the angioplasty site. Intimal hyperplasia is an important candidate for gene therapy since it is related to smooth muscle cell proliferation, which is an inviting target for molecular antiproliferative strategies. To date, adenoviral vectors are, by far, the most efficient vectors to perform in vivo arterial gene transfer. These vectors, as well as others, have been recently used to demonstrate that therapeutic genes encoding cytotoxic (herpes virus thymidine kinase) or cytostatic (hypophosphorylatable Rb, Gax, endothelial nitric oxide synthase) products successfully inhibit smooth muscle cell proliferation and related intimal hyperplasia. Despite substantial progress, major technical issues, including the toxicity of first-generation adenoviral vectors, inefficient transduction of atherosclerotic arteries, and the risk of extra-arterial transfection remain to be addressed before gene therapy is applied to clinical restenosis.
Preclinical studies have indicated that angiogenic growth factors can stimulate the development of collateral arteries, a concept called "therapeutic angiogenesis." The objectives of this phase 1 clinical trial were (1) to document the safety and feasibility of intramuscular gene transfer by use of naked plasmid DNA encoding an endothelial cell mitogen and (2) to analyze potential therapeutic benefits in patients with critical limb ischemia. Gene transfer was performed in 10 limbs of 9 patients with nonhealing ischemic ulcers (n=7/10) and/or rest pain (n=10/10) due to peripheral arterial disease. A total dose of 4000 microg of naked plasmid DNA encoding the 165-amino-acid isoform of human vascular endothelial growth factor (phVEGF165) was injected directly into the muscles of the ischemic limb. Gene expression was documented by a transient increase in serum levels of VEGF monitored by ELISA. The ankle-brachial index improved significantly (0.33+/-0.05 to 0.48+/-0.03, P=.02); newly visible collateral blood vessels were directly documented by contrast angiography in 7 limbs; and magnetic resonance angiography showed qualitative evidence of improved distal flow in 8 limbs. Ischemic ulcers healed or markedly improved in 4 of 7 limbs, including successful limb salvage in 3 patients recommended for below-knee amputation. Tissue specimens obtained from an amputee 10 weeks after gene therapy showed foci of proliferating endothelial cells by immunohistochemistry. PCR and Southern blot analyses indicated persistence of small amounts of plasmid DNA. Complications were limited to transient lower-extremity edema in 6 patients, consistent with VEGF enhancement of vascular permeability. These findings may be cautiously interpreted to indicate that intramuscular injection of naked plasmid DNA achieves constitutive overexpression of VEGF sufficient to induce therapeutic angiogenesis in selected patients with critical limb ischemia.
Findings in the first clinical trial in which an adeno-associated virus (AAV) vector was introduced into the liver of human subjects highlighted an issue not previously identified in animal studies. Upon AAV gene transfer to liver, two subjects developed transient elevation of liver enzymes, likely as a consequence of immune rejection of transduced hepatocytes mediated by AAV capsid-specific CD8(+) T cells. Studies in healthy donors showed that humans carry a population of antigen-specific memory CD8(+) T cells probably arising from wild-type AAV infections. The hypothesis formulated at that time was that these cells expanded upon re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer, and cleared AAV epitope-bearing transduced hepatocytes. Other hypotheses have been formulated which include specific receptor-binding properties of AAV-2 capsid, presence of capsid-expressing DNA in AAV vector preparations, and expression of alternate open reading frames from the transgene; emerging data from clinical trials however fail to support these competing hypotheses. Possible solutions to the problem are discussed, including the administration of a short-term immunosuppression regimen concomitant with gene transfer, or the development of more efficient vectors that can be administered at lower doses. While more studies will be necessary to define mechanisms and risks associated with capsid-specific immune responses in humans, monitoring of these responses in clinical trials will be essential to achieving the goal of long-term therapeutic gene transfer in humans.