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Gene Therapy
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Gene Therapy
Gene Therapy
By Samantha L. Ginn1,3 and Ian E. Alexander1,2,3
Keywords: gene therapy, clinical trials, gene delivery, biosafety, ethics, regulation
Abstract: Conducting a clinical trial poses a unique set of challenges that must be
addressed to ensure the safety of human subjects. In this article, we discuss issues that
relate in particular to gene-therapy clinical trials. These issues include the choice of gene
delivery system, the need for extensive preclinical testing to ensure the feasibility and
safety of the approach, and careful monitoring of subjects for short- and long-term
toxicity associated with the gene-transfer protocol. We consider the translational chal-
lenges specific for gene-therapy trials that comprise additional regulatory oversight,
unique ethical considerations, and the need for specialized personnel, infrastructure,
and reagents. We also provide a brief overview of current clinical activity, highlight the
main lessons learned from landmark gene-therapy trials, and conclude by discussing the
challenges facing the field as it moves into the future.
Gene therapy was conceived originally as an approach to the treatment of inherited monogenic dis-
eases and is defined by the United Kingdom’s Gene Therapy Advisory Committee as “the deliberate
introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic
purposes”[1] . Ongoing improvements in gene-transfer technologies and biosafety have been accompanied
by a growing appreciation of the broader scope of gene therapy. Pathophysiological processes such as
injury[2] and wound healing[3] , chronic pain[4] and arthritis[5] , cancer[6] , and acquired infections such as
human immunodeficiency virus type 1 (HIV-1)[7] are now becoming realistic targets for this promising
therapeutic modality.
The first authorized gene-transfer study took place at the National Institutes of Health (NIH) in 1989.
In this marker study (see Genetic Markers), tumor-infiltrating lymphocytes were harvested, genetically
tagged using a retroviral vector, and reinfused with the intention of examining the tumor-homing capacity
of these cells. This landmark study provided the first direct evidence that human cells could be genetically
modified and returned to a patient without harm[8] . Since then, over 2200 trials have been approved,
initiated, or completed worldwide, which are performed predominantly in the United States[9,10] . Most
studies have focused on cancer, with monogenic, cardiovascular, and infectious diseases the next
most frequent indications (Table 1). These predominantly early-phase trials have provided invaluable
proof-of-concept for gene therapy by confirming that desired changes to the target cell phenotype can
1 Gene Therapy Research Unit of the Children’s Medical Research Institute, Westmead, New South Wales, Australia
2 The Children’s Hospital at Westmead, Westmead, New South Wales, Australia
3 The University of Sydney, Westmead, New South Wales, Australia
Update based on original article by Samantha L. Ginn, Wiley StatsRef: Statistics Reference Online © 2014 John Wiley & Sons, Ltd.
Wiley StatsRef: Statistics Reference Online, © 2014–2016 John Wiley & Sons, Ltd. 1
This article is © 2016 John Wiley & Sons, Ltd.
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Gene Therapy
Table 1. Gene-therapy clinical trial indications.
Indication Gene-therapy clinical trials

Number Percent
Cancer diseases 1415 64.0

Monogenic diseases 209 9.5
Cardiovascular diseases 175 7.9
Infectious diseases 174 7.9
Healthy volunteers 53 2.4
Gene marking 50 2.3
Other diseases 46 2.1
Neurological diseases 43 1.9
Ocular diseases 31 1.4
Inflammatory diseases 14 0.6
Total 2210
Source: From www.abedia.com/wiley.
be achieved successfully. In most trials, however, an insufficient number of cells have been genetically
modified to achieve therapeutic benefit, although an increasing number of exceptions to the lack of
therapeutic efficacy are being reported. These include the treatment of several primary immunodeficiency
disorders[11 – 17] , X-linked adrenoleukodystrophy (X-ALD)[18] , metachromatic leukodystrophy[19,20] , Leber
congenital amaurosis[21 – 23] , and hemophilia B[24,25] .
1 Requirements for Successful Therapeutic Intervention
The prerequisites for successful gene therapy are complex and disease specific, but invariably include the
availability of an efficient gene delivery technology and an understanding of its properties, capacity, and
limitations, a detailed knowledge of the biology of the target cell population, and a precise understanding
of the molecular basis of the target disease (Figure 1). Although significant progress is being made in each
of these areas, the ability to achieve efficient gene delivery has been described as “the Achilles heel of gene
therapy[26] .”
1.1 Gene Delivery Technology
Gene delivery systems can be classified into two broad categories: nonviral physicochemical approaches
and recombinant viral systems. The comparative strengths of nonviral approaches include ease of chemical
characterization, simplicity and reproducibility of production, larger packaging capacity, and reduced
biosafety risks[27] . Gene delivery, however, is relatively inefficient and the effects are often transient.
Improvements to nucleic acid stability and potency as well as lipid and polymer delivery technology
are, however, advancing the field of nonviral gene delivery[28] . Examples of nonviral systems include
microinjection of individual cells, DNA-calcium phosphate coprecipitation, and the formulation of
DNA into artificial lipid vesicles, virus-like particles, or cationic polymers[29 – 40] . In contrast, viral
systems, which are commonly modified to render them replication incompetent, are markedly more
efficient and exploit favorable aspects of virus biology[26,41 – 43] . Viral vectors can be divided into two
main categories: nonintegrating and integrating, based on the intracellular fate of the vector genome.
These properties are important when considering the required duration of the treatment. Nonintegrat-
ing vectors are maintained extrachromosomally, whereas the genome of integrating vectors becomes
incorporated into the host-cell genome that provides the potential for stable long-term gene expression
2 Wiley StatsRef: Statistics Reference Online, © 2014–2016 John Wiley & Sons, Ltd.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
Gene delivery technology
Gene
therapy
Target cell Understanding of

biology the target disease
Figure 1. Requirements for successful therapeutic intervention.
(see Gene Expression Analysis). Limitations include increased biosafety risks caused by contamination
by replication-competent virus, the presence of potentially toxic viral gene products, and insertional
mutagenesis when integrating vectors are used[44 – 48] . Irrespective of the vector type, another important
limitation is the induction of unwanted immune responses directed against components of the delivery
system and/or the encoded transgene product. These responses may be either cell mediated or humoral
and depend on several variables that include the nature of the transgene, the vector and promoter
used, the route and site of administration, the vector dose, and host factors. Ultimately, host-vector
immune responses have the potential to influence clinical outcomes negatively[49 – 52] . Accordingly, the
optimization of gene delivery systems and strategies to evade deleterious immune responses remains
a fundamentally important challenge[53 – 56] . Difficulty in producing high-titer vector preparations and
constraints on packaging capacity is also a drawback. Hybrid systems that combine the advantageous
features of more than one virus or modified to include transposable elements have also been developed,
although their application has been largely in in vitro models[37,57 – 60] . Despite these limitations, the
relative efficiency of gene transfer has resulted in the predominant use of viral vectors in preclinical and
clinical gene-therapy studies up to the present time[10] . Although recombinant viral vectors have the
most immediate potential for clinical use, it is envisaged that these systems will be supplanted by hybrid
and derivative systems that combine the simplicity and safety of nonviral gene delivery with favorable
aspects of viral biology. Virosomes are a prototypic example of such a system and among these, the
Hemagglutinating Virus of Japan liposome has been the most extensively investigated[61 – 63] .
Finally, the use of gene-correction rather than gene-addition strategies, which include targeted
recombination[64 – 67] , antisense oligonucleotide-induced exon skipping[68,69] , and RNA interference[70,71] ,
is also being investigated. Such strategies will be particularly important in the context of dominant
disease processes in which the simple addition of a functionally normal gene is insufficient. Although
these approaches generally lack the efficiency required for human gene-therapy applications, promising
results are now being reported with the use of oligonucleotide-induced exon skipping for the treatment
of Duchenne muscular dystrophy[72,73] . It is now approaching two decades since the first targeted
nucleases were shown to bind DNA and create double strand breaks, a key requirement for site-specific
genome editing[74 – 78] . Although long-term follow-up is still required, promising reports for gene-editing
technology are now appearing in the literature with a zinc finger nucleases (ZFN) used in a clinical trial for
the treatment of HIV-1[79] and a transcription activator-like effector nuclease (TALEN) used to treat one
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Table 2. Properties of widely used viral vector systems.
Vector Adenovirus 𝛾-Retrovirusa) Lentivirusb) AAVc)
Genome dsDNA ssRNA ssRNA ssDNA

Insert capacity 7–30 kb 7 kb 10 kb 4.8 kb
Location in cell nucleus Extrachromosomal Integrated Integrated Extrachromosomal/integrated
Cell cycle-dependent gene transfer No Yes No No
Duration of transgene expression Transient Long-term Long-term Long-term
Functional titer (per mL) ≥1012 ≥107 ≥109 ≥109
Immunogenicity High Low Low Low
a) Most commonly derived from Moloney murine leukemia virus (MoMLV).

b) Most commonly derived from HIV-1.
c) AAV, adeno-associated virus.
child with advanced leukemia[80] . Recently, a system adapted from the bacterial immune system known as
the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR/Cas9)
nuclease[81 – 83] has improved the efficiency of genome editing significantly. Reportedly, in an attempt to
produce a nonhuman organ donor, the CRISPR/Cas9 gene-editing technology has been used to inactive
over 60 porcine endogenous retroviruses in pig embryos[84] . Further refinements, such as the use of viral
vector systems for delivery and the identification of alternate nucleases, are already showing potential for
gene-therapy applications[85,86] .
1.2 Target Cell Biology
Each viral vector system possesses a unique set of biological properties (Table 2), and their use is governed
largely by the biology of the target cell. For example, integration provides the molecular basis for stable
long-term gene expression as would be required for the treatment of genetic disease in replicating cell
populations. Integration also provides the potential for gene-modified cells to be expanded from a modest
number of progenitors. This attribute is of paramount importance when the genetic modification of cells
capable of enormous proliferative expansion is required. This feature has been powerfully illustrated in
the first successful treatment of a genetic disease by gene therapy[87,88] . In this study, a selective growth
and survival advantage was conferred on hematopoietic stem cells following transduction with a gamma-
retroviral vector based on the Moloney murine leukemia virus. Despite the advantages of viral integration,
nonintegrating vectors are potentially effective if the target cell is nondividing. Integration-deficient forms
of integrating vectors have also been developed in an attempt to increase biosafety[89 – 92] . Additional con-
straints, such as the replication state of the target cell or whether the target cell is amenable to ex vivo
manipulation, define vector choice and gene-transfer protocols even more. For example, vectors based
on lentiviruses and adeno-associated viruses (AAVs) can modify postmitotic and nondividing cells[93 – 96] ,
which makes them of particular interest for targets such as muscle and the nervous system.
1.3 Disease Pathophysiology
Before a gene-therapy approach can be considered feasible for a disease or physiological process, a precise
understanding of the underlying molecular basis is required. The requirement for transient or persistent
transgene expression must also be considered. For example, the extrachromosomal nature of adenoviral
vectors has the potential to limit the duration of gene expression by dilutional loss during cell division[97] .
In some contexts, in which only transient gene expression is required, such as anticancer gene therapy, this
may be a positive attribute. The pathophysiology of the target disease also defines the number of cells that
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Gene Therapy
must be successfully gene modified to achieve the desired therapeutic effect. For example, it was anticipated
that for the treatment of hemophilia B, levels of Factor IX as low as 1% of normal would be therapeutic[98] .
This hypothesis has now been demonstrated clinically, where 5% of normal activity resulted in a reduc-
tion of both bleeding episodes and the use of prophylactic factor IX concentrate[25] . Here, the transgene
product was a secreted protein, which possibly contributed to therapeutic efficacy. In cases where the
product acts in a cell-autonomous manner, however, gene transfer to a high percentage of target cells, in
contrast to a small number of cells expressing supraphysiological levels of transgene, would more likely
be required. For some more demanding disease phenotypes, expansion of gene-modified cells through
in vivo selection is one strategy by which the fundamental challenge of gene-modifying sufficient cells
to achieve therapeutic benefit may be overcome[99 – 101] . Ultimately, however, the development of more
efficient gene delivery and genome editing technologies is required to allow the effective treatment of
the many human diseases and pathophysiological processes that are potentially amenable to gene-therapy
approaches.
2 Preclinical Research
Before a clinical gene-therapy protocol can be considered for human application, extensive preclinical test-
ing is required (see Preclinical Treatment Evaluation). The data generated is vital in establishing whether
the target cell can be safely gene modified to produce the phenotypic changes required for disease correc-
tion. This involves years of preclinical experimental testing progressing from tissue culture models to small
animal models (most commonly mice) and finally to large animal models when feasible.
2.1 In Vitro Studies
An important first step in establishing proof-of-concept data for a clinical gene-therapy protocol is pro-
vided by in vitro studies. The manipulation of mammalian cells in culture can help define several impor-
tant experimental parameters. Taking into account the biology of the target cell, several gene-transfer
approaches could potentially be available. For example, if neuron-targeted gene transfer is required, sev-
eral recombinant viral vector systems are available, such as those based on AAV, herpes simplex virus, and
lentiviruses[102 – 107] . Using cells in culture, it is relatively easy to select the vector system that is most effec-
tive in genetically modifying the cell type of interest. Important parameters that can be determined in vitro
include the tropism of the virus for the relevant target cell population, the minimum vector dose required
to achieve the desired phenotypic changes, the level and the duration of transgene expression, and vector
toxicity. In vitro systems also allow aspects of expression cassette design to be examined such as the use
of tissue-specific promoters or regulated gene expression. Immortalized cell lines are commonly used for
such studies, but frequently they are transduced more readily than primary cells and do not consistently
model the challenge of transducing specific target cell populations in vivo. Culture of primary cells and
tissue explants where possible is therefore preferable, offering a more realistic representation of the target
cell population in its native state before proceeding to animal models.
2.2 Animal Models
Another prerequisite for successful gene therapy is the availability of an animal model that approx-
imates the human disease for preclinical testing. Indeed, successful phenotype correction in mouse
models (see Transgenic Mouse Model; Selection Study (Mouse Genetics)) of human disease is now
commonplace[12,108 – 111] . In 2015 alone, there were over 40 publications reporting successful phenotype
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correction of mice with diseases including Leber Congenital Amaurosis[112] , spinal muscular atrophy[113] ,
Pompe disease[114] , perforin deficiency[115] , and deafness[116] . Up to the present time, these successes have
rarely been replicated in large animal models or human clinical trials (see Clinical Trials, Overview).
The explanation for this is primarily quantitative. Success in larger subjects demands that proportionally
greater numbers of target cells be gene modified to reach a therapeutically meaningful threshold. The
average child, for example, is approximately 1000-fold bigger than a mouse and, therefore, presents a
correspondingly greater gene-transfer challenge. In addition to the challenge of size, other factors, such
as species-specific differences, exert potent effects in some contexts.
Animal models also provide valuable Safety data required by regulatory bodies (see Regulatory
Authorities) before approving early-phase human trials, but these models do not always accurately
predict adverse effects subsequently observed in human subjects (see Toxicity (Adverse Events)). For
example, in two therapeutically successful gene-therapy trials for the X-linked form of severe combined
immunodeficiency (SCID-X1), 5 of 20 patients developed a leukemia-like illness as a direct consequence
of retroviral insertion into chromosomal DNA[44,46,47] . Four of the five patients were successfully treated
with chemotherapy and are in complete remission and still retain the benefits of gene therapy. In
addition, after a decade of long-term follow-up, the survival rate for infants treated with gene therapy
is higher than that of children receiving conventional treatment who lack an human leukocyte antigen
(HLA)-identical sibling donor. The risk of insertional mutagenesis (see Mutagenicity Study) when using
integrating vectors had long been recognized, but formerly considered low because retroviral vectors had
previously been employed extensively without incident in animal models and in almost 300 documented
human clinical protocols[10] . Interestingly, concurrent with the first report of vector-mediated insertional
mutagenesis in humans, the first report of essentially the same phenomenon in mice was published[117] .
Collectively, these events illustrate the inherent challenge in predictive safety testing, whether in animal
models or in early-phase human clinical trials (see Phase II Trials; Phase I Trials). Preferably such
tests must be configured with specific adverse events in mind, and where possible, in a manner that
accommodates the possible contribution of the particular disease for which gene therapy is being
contemplated.
3 Translational Challenges of Gene-Therapy Trials
In comparison with drug-based clinical trials, several additional factors must be considered when under-
taking a human gene-therapy application. These include added layers of regulatory oversight, ethical con-
siderations (see Ethics of Randomized Trials) related to the genetic modification of the subject’s cells,
and availability of appropriate skills, infrastructure, and reagents. There is also considerable time period
from initial preclinical testing in the laboratory to a trial participant receiving therapy. Trials for SCID-X1
are a notable example, where, after almost a decade after the French-based trial was placed on voluntary
hold, a multinational trial utilizing a vector with improved safety features began accruing patients[118] . This
inertia, to some extent, could be overcome by more parallel activity between different research groups.
Results for the first nine patients have now been published; however, the long-term effect of this therapy
on leukemogenesis is currently unknown[119] .
3.1 Regulatory Oversight
Clinical research with genetic material poses safety and methodological challenges not shared by other
forms of human investigation. As a result, several regulatory requirements (see Regulatory Definitions)
must be satisfied before human studies that involve gene transfer can be initiated. In most countries,
compared with requirements for pharmaceutical products, these requirements are achieved through an
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additional layer, or layers, of regulation. Depending on the host country, regulatory oversight can be com-
plicated even more by the fact that existing regulatory frameworks have evolved for more conventional
therapeutic products. As for all human clinical research, gene-therapy trials must also be conducted
according to a set of standards referred to as Good Clinical Practice (GCP) that are based on the Dec-
laration of Helsinki[120] . In the United States, it is a federal requirement that clinical protocols involving
the administration of recombinant DNA products be reviewed and approved by filing an investigational
new drug (IND) application with the Food and Drug Administration (FDA). In addition, applications
must be approved by local institutional human ethics and biosafety committees (see Institutional Review
Boards (IRB)). The key regulatory issues for US-based clinical gene-therapy trials have been reviewed
by Cornetta and Smith[121] and Manilla et al.[122] In the United Kingdom, gene-therapy applications
are similarly regulated by the Medicines & Healthcare products Regulatory Agency (MHRA), requiring
a clinical trial authorization (CTA) for a clinical trial of a medicinal product and approval from local
institutional committees[123,124] . This regulatory complexity is particularly burdensome in the context of
multinational trials (see Multinational (Global) Trial) and is a major driver behind efforts for global
harmonization. Such efforts will not only facilitate international studies but also improve data quality and
participant safety[125] .
3.2 Special Ethical Considerations
In contrast to drug-based clinical trials, several special ethical issues must be considered for human
gene-transfer studies. These issues include the possibility of inadvertent germ-line transmission and,
depending on the type of delivery vehicle used, the ability to introduce life-long modifications to the
subject’s chromosomal DNA, the latter resulting in the need for long-term clinical follow-up. Currently,
only somatic cell gene-therapy protocols have been initiated. The use of germ-line manipulation, in
which the genomes of germ cells are deliberately modified with the likely effect of passing on changes to
subsequent generations, is opposed at this time. Although genetic manipulation of the human germ line is
illegal in many countries[126] , this consensus is not unanimous and its use remains the subject of vigorous
debate[127 – 133] .
For any research team who attempt to develop a new medical treatment, patient safety is of paramount
importance, and the decision to proceed with a gene-therapy approach requires a careful balancing of the
associated risks and benefits. For example, bone marrow Transplantation from an HLA-matched sibling
donor is the treatment of choice for diseases such as SCID-X1. Unfortunately, however, this option is avail-
able for approximately one in five affected infants. The alternative is transplantation from a haploidentical
or matched unrelated donor, and thus carries a substantial risk of severe immunologic complications and
of early mortality[134] . For these children, a gene-therapy approach may carry a lower risk even when pos-
sible adverse events associated with gene therapy, such as leukemia induction through insertional muta-
genesis, are taken into account[135 – 137] . Another ethical concern for gene-therapy trials is the enrollment
of infants and children given their inability to provide informed consent (see Informed Consent Process,
Forms, and Assent). Although it may be preferable to undertake early-phase clinical trials in adults, many
severe disease phenotypes are restricted to the pediatric age group, or where meaningful intervention
would only be possible early in the course of the target disease. Examples include SCID-X1[87,138] and cys-
tic fibrosis[139] . Accordingly, depending on the disease context, equally potent counter-balancing ethical
arguments are in favor of early-phase trials in the pediatric population.
Another important consideration is whether a need exists for long-term Monitoring of the subject after
the gene-transfer protocol. Parameters that include the ability of the delivery vehicle to integrate into the
genome, the site of integration, vector persistence, biology of the target cell, and transgene-specific effects
all influence the risk associated with the treatment. If no vector persistence exists, the risk is analogous to
that of any new drug, and long-term follow-up may not necessarily be required[140] .
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3.3 Skills, Infrastructure, and Reagents
To undertake a gene-therapy clinical trial, a research team requires access to specialized facilities as well
as appropriately trained staff to perform procedures in accordance with required standards. In most
countries, the rigor of these requirements increases in late-phase trials to the level of good manufacturing
practice (GMP). For each gene-therapy protocol, the set of skills required are governed largely by the
biology of the target cell. For example, in trials that involve gene transfer to hematopoietic stem cells, an
ex vivo approach is the method of choice. This approach requires a medical team to harvest the subject’s
bone marrow and personnel who can maintain the target cells in sterile culture for up to 5 days after
harvest[87] . This approach also requires the availability of an on-site cleanroom to perform the cellular
manipulations. To address these logistical challenges, emphasis has been placed on developing protocols
for the cryopreservation and transport of gene-modified cells. This allows the manipulation of patient
cells to occur at centralized locations and also provides methodological consistency throughout the trial.
Alternatively, it might be necessary to deliver the vector directly to the target cell in vivo. Examples of
this approach include gene transfer to organs such as the eye or brain or in gene-therapy protocols that
deliver oncolytic agents to a solid tumor. Finally, to facilitate clinical applications of virus-mediated gene
transfer, production must be scalable, reproducible, and yield vector at the highest possible titers. This
has led to the exploration of packaging systems such as baculovirus expression vectors and insect cells as
a potential method to produce rAAV economically[141,142] . In addition, vector stocks must be sterile and
free of detectable replication-competent virus, which requires independent testing at considerable cost.
Experimental products used for gene-transfer studies are often complex and difficult to characterize
completely in comparison with conventional pharmaceutical agents. This is true particularly for virus-
based gene delivery systems that are also challenging to produce on a large scale and cannot be sterilized
by autoclaving or radiation. Biological variability may also result from the packaging of empty virions, titer
differences between different production runs, and loss of titer during storage. The propensity of vectors to
undergo inadvertent sequence alteration during production through mechanisms such as recombination
or via transcription by error prone polymerases[143] must also be monitored.
4 Clinical Trials
Although originally conceived as a treatment for monogenic diseases, the major indication addressed by
gene-therapy trials to date has been cancer (64%, Table 1). This bias toward cancer trials in part reflects the
poor prognosis of many cancer phenotypes that make the risk/benefit ratio more favorable for experimen-
tal intervention. Although initial trials have been largely unsuccessful, positive outcomes are now being
reported. For example, the use of chimeric antigen receptors (CAR) brings together the expansion poten-
tial and persistence of cytotoxic T cells with the specificity of monoclonal antibodies. In 2011, researchers
from the Abramson Cancer Center in Philadelphia described the treatment of three patients with chronic
lymphocytic leukemia (CLL) with autologous T cells that were genetically modified to express a CAR with
specificity for the B-cell antigen CD19[144] . At the time of publication, two of the three patients were in
complete remission, at 10 and 11 months posttherapy, which has continued to this day. An increasing
number of Phase I clinical trials are being reported[145,146] with impressive remission rates observed in
patients with leukemia or lymphoma, especially acute lymphoblastic leukemia (ALL)[147 – 150] . Cardiovas-
cular, monogenic, and infectious diseases are the next most frequently addressed indications with 209
(9.5%), 175 (7.9%), and 174 (7.9%) trials, respectively, approved worldwide (Table 1).
Shortly after the first authorized gene-transfer trial was undertaken in 1989[8] , the first therapeutic trial
that involved two children who suffered from a severe combined immunodeficiency caused by adenosine
deaminase deficiency (ADA-SCID) was approved. This trial was unsuccessful for several reasons that
include maintaining the patients on polyethylene glycol-modified adenosine deaminase (PEG-ADA)
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Unknown 142
2015 45
2014 130
2013 121
2012 101
2011 87
2010 92
2009 81
2008 120
2007 90
2006 117
2005 112
2004 101
2003 85
2002 98
2001 108
2000 96
1999 116
1998 68
1997 82
1996 51
1995 67
1994 38
1993 37
1992 14
1991 8
1990 2
1989 1
0 20 40 60 80 100 120 140 160
Figure 2. Number of gene-therapy clinical trials approved worldwide from 1989 to 2015. Source:
Reprinted with permission from www.abedia.com/wiley.
therapy and using patient T lymphocytes as the target cell population[151] . Removal of PEG-ADA coupled
with a myeloablative conditioning regime and the targeting of hematopoietic stem cells with an improved
transduction protocol resulted in the successful treatment of patients in subsequent trials for ADA-
SCID[152 – 160] , which highlighted the need to allow the natural pathophysiology of the disease to confer
a positive growth or survival advantage to the transduced cells. After initiation of the first therapeutic
trial, a progressive increase occurred in the number of gene-therapy trials approved in the following years
(Figure 2). This increase slowed briefly in the mid-1990s after an NIH review committee cochaired by
Stuart Orkin and Arno Motulsky concluded that “Significant problems remain in all basic aspects of gene
therapy. Major difficulties at the basic level include shortcomings in all current gene-transfer vectors and
an inadequate understanding of the biological interaction of these vectors with the host[161] .” This trend
toward increasing numbers of gene-therapy trials leveled off after 1999 coincident with a report of the
first severe adverse event as a direct consequence of gene therapy[162] . In a Phase I dose escalation study
(see Dose Escalation and Up-and-Down Designs) that investigated the safety of an adenoviral vector
for the treatment of ornithine transcarbamylase deficiency, a young adult trial participant died as a result
of a severe systemic inflammatory reaction to the injected adenoviral vector. This incident resulted in the
suspension of all trials at the host institution by the FDA and a senate subcommittee investigation that
also looked more broadly at clinical trial safety and report across the United States[163] . In light of the
information that emerged, a renewed emphasis exists on critical evaluation of risk/benefit ratios for trial
participants and comprehensive reporting of trial outcomes and adverse events.
To date, gene-therapy trials have been performed in 36 countries (Figure 3) spanning five continents.
These trials have been recently reviewed by Ginn and Alexander[164] . Most trials have been conducted
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Multi country 4.2% (n=92)
USA 62.7% (n=1386)
UK 9.5% (n=209)
Germany 3.8% (n=84)
China 2.4% (n=54)
France 2.4 (n=52)
Switzerland 2.3% (n=50)
Japan 1.9% (n=41)
Netherlands 1.5% (n=34)
Australia 1.4% (n=32)
Canada 1.1% (n=25)
Other countries 6.8% (n=151)
Figure 3. Geographical distribution of gene-therapy clinical trials. Source: Reprinted with permission from
www.abedia.com/wiley.
in the United States with 1386 trials (62.7%) followed by the United Kingdom (209 trials, 9.5%). Viral
vectors are the most frequently used gene delivery system (Table 3) because of superior gene-transfer effi-
ciency over nonviral methods. Of the viral gene delivery systems available, adenoviral and retroviral vectors
(derived from murine retroviruses) have been most commonly used accounting for 21.7% and 18.9% of tri-
als, respectively (Table 3). Lentiviral vectors derived from HIV-1, after safety considerations relating to the
inherent properties of the parent virus were addressed, are now being used clinically and account for 5.2%
of trials. Because of differences in integration site patterns, lentiviral and foamy viral vectors may offer
a safer alternative to 𝛾-retroviral vectors derived from murine retroviruses[165 – 168] and lentiviral vectors
been used successfully in human trials for X-linked adrenoleukodystrophy (ALD)[18] , 𝛽-thalassemia[15] ,
Wiskott–Aldrich syndrome[17] , metachromatic leukodystrophy[19] , and cancer[144,169] .
5 Lessons Learned
Although results from predominantly early-phase gene-therapy trials have been largely unsuccessful in
providing clinical benefit to human subjects, they have (i) provided clear proof-of-concept for gene therapy,
(ii) demonstrated that gene therapy is relatively safe, and (iii) highlighted several important issues that
must be considered to advance the field. Careful target disease selection, being mindful of the limits of
existing gene delivery technologies, is one such example. It is now over 25 years since the first authorized
gene-transfer trial was undertaken and since then, over 2200 clinical trials have been initiated worldwide
(Table 1) with several notable successes since 2000, particularly in pediatric populations.
5.1 The Power of In Vivo Selection
The major reason gene therapy has been unsuccessful in providing clinical benefit in many disease contexts
is low gene-transfer efficiencies. Expansion of gene-modified cells, through in vivo selection, is one strategy
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
Table 3. Gene delivery systems in clinical trial use.
Vector Number of protocols
Adenovirus 480 (21.7%)

Retrovirus 417 (18.9%)
Naked/plasmid DNA 387 (17.5%)
Adeno-associated virus 137 (6.2%)
Vaccinia virus 121 (5.5%)
Lipofection 115 (5.2%)
Lentivirus 114 (5.2%)
Herpes simplex virus 73 (3.3%)
Poxvirus 68 (3.1%)
RNA transfer 39 (1.8%)
Poxvirus and vaccinia virus 33 (1.5%)
Other multisystem 33 (1.5%)
Other viral 22 (1.0%)
Listeria monocytogenes 15 (0.7%)
Other bacteria 10 (0.5%)
Sleeping beauty transposon 10 (0.5%)
Measles virus 9 (0.4%)
Saccharomyces cerevisiae 9 (0.4%)
Flavivirus 8 (0.4%)
Modified vaccinia Ankara (MVA) virus 7 (0.3%)
Antisense oligonucleotide 6 (0.3%)
Lactococcus lactis 6 (0.3%)
siRNA 5 (0.2%)
Gene gun 5 (0.2%)
Other nonviral 5 (0.2%)
Unknown 76 (3.4%)
Total 2210
Source: From www.abedia.com/wiley.
by which the fundamental challenge of gene-modifying sufficient cells to achieve therapeutic benefit can
be overcome[170] . The power of in vivo selection has been impressively illustrated in the SCID-X1 trial,
which is the first successful treatment of a genetic disease by gene therapy[87] . For most diseases, however,
the gene-corrected cells will not have a selective growth or survival advantage. Therefore, efforts to develop
strategies for providing modified cells with an exogenous selective advantage are being made. One such
strategy exploits mutant forms of the DNA repair enzyme methylguanine methyltransferase and target-
ing expression to hematopoietic progenitor cells using integrating vector systems. This strategy imparts
genetic chemoprotection to the gene-modified cells and has been successfully employed in large animal
models[171,172] and humans[173] .
5.2 Insertional Mutagenesis
Insertional mutagenesis is now recognized as a clinically established risk associated with the use of inte-
grating vector systems[44,46,47,174 – 176] and the use of such vector systems must be balanced against the
potential for therapeutic benefit. Although avoidance of integrating vector systems is not currently a viable
option for gene-therapy protocols targeting cell types that undergo rapid cell division, two broad strate-
gies by which the risk of insertional mutagenesis can be significantly reduced include (i) reduction in the
absolute number of integration sites to which patients are exposed and (ii) reduction of the risk associated
with individual integration events. Achievement of the former will depend on more sharply defining both
the desired target cell population and the minimum effective dose of gene-corrected cells to produce the
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
desired phenotypic effect as well as optimization of transduction conditions. Reduction of the risk asso-
ciated with individual integration events is theoretically achievable by careful choice of integrating vector
system and optimized expression cassette design that lacks strong viral promoter/enhancer elements.
6 The Way Forward
The capacity of gene therapy to cure human disease is now an established reality, but for now, most disease
phenotypes and pathophysiological processes potentially amenable to this exciting therapeutic approach
lie beyond the reach of existing technology. The major challenge for the future, therefore, is to address the
inherent shortcomings in the efficacy and safety of available gene delivery systems. Developments in this
area will be complemented by an improved understanding of target cell biology, in particular the capac-
ity to identify and manipulate stem cell populations coupled with improvements in systems for targeted
gene repair such as ZFNs[177,178] , TALENS[179] , and more recently the CRISPR/Cas9 nuclease[82,83] . The
landmark discovery by Takahashi and Yamanaka[180] that somatic cells can be reprogrammed to a state of
pluripotency through the ectopic expression of only four transcription factors[181,182] has the potential to be
a powerful tool for both gene and cellular therapies and the potential to revolutionize the field of regenera-
tive medicine by developing patient-specific treatments. These cells, termed induced pluripotent stem cells
(iPS), closely resemble embryonic stem (ES) cells in their morphology and growth properties and have also
been shown to express ES cell markers. Although a number of important issues need to be resolved before
these cells appear in the clinical setting, proof-of-principal for combining somatic cell reprogramming with
gene therapy for disease treatment already exist[183 – 186] and their use has immediate potential for basic
research, disease modeling, and drug screening as well as infinite possibilities for the future. These issues
include improvements in reprogramming efficiency, a more complete understanding of the development
potential and quality of the iPS cells produced, and establishing their safety profile in vivo, particularly with
respect to tumor formation. Refinements to the system using nonintegrating vectors, transient expression
systems, and small molecules[187 – 191] are also addressing safety concerns by eliminating unwanted long-
term expression of the encoded transcription factors and the possibility for insertional mutagenesis.
Unwanted host–vector interactions, such as immune responses directed against the vector and encoded
transgene product, must be better understood and avoided. Alternative strategies to prevent cell-mediated
destruction of the gene-corrected cells include modulation of the immune system and use of engineered
vectors to evade capsid-specific immune responses or transient immune suppression[192,193] . The latter
has been used successfully in a clinical trial for hemophilia B to limit hepatocellular toxicity and preserve
expression of transgenic factor IX, especially when treatment was initiated early[25] . Such progress is funda-
mentally dependent on sound basic and preclinical research coupled with iterative human clinical trials.
Finally, with an increasing number of therapeutic successes being reported and renewed investment in
gene-therapy technologies[194] , it is clear that the future of the field has never been more exciting.
Related Articles
Ethics of Randomized Trials; Ethics in Research; Regulatory Authorities; Safety; Preclinical Treat-
ment Evaluation.
References
[1] United Kingdom, Department of Health, Gene Therapy Advisory Committee (2006) http://webarchive.nationalarchives.
gov.uk/20060904200237/advisorybodies.doh.gov.uk/genetics/gtac/index.htm (accessed 19 January 2016).
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[2] Delalande, A., Gosselin, M.P., Suwalski, A., et al. (2015) Enhanced achilles tendon healing by fibromodulin gene transfer.
Nanomedicine: NBM, 11, 1735–1744.
[3] Peng, L.H., Wei, W., Shan, Y.H., et al. (2015) Beta-cyclodextrin-linked polyethylenimine nanoparticles facilitate gene
transfer and enhance the angiogenic capacity of mesenchymal stem cells for wound repair and regeneration. J. Biomed.
Nanotechnol., 11, 680–690.
[4] Guedon, J.M., Wu, S., Zheng, X., et al. (2015) Current gene therapy using viral vectors for chronic pain. Mol. Pain, 11, 27.
[5] Evans, C.H., Ghivizzani, S.C., and Robbins, P.D. (2013) Arthritis gene therapy and its tortuous path into the clinic. Transl.
Res., 161, 205–216.
[6] Gill, S. and June, C.H. (2015) Going viral: chimeric antigen receptor t-cell therapy for hematological malignancies.
Immunol. Rev., 263, 68–89.
[7] Ahlenstiel, C.L., Suzuki, K., Marks, K., et al. (2015) Controlling hiv-1: non-coding RNA gene therapy approaches to a
functional cure. Front. Immunol., 6, 474.
[8] Rosenberg, S.A., Aebersold, P., Cornetta, K., et al. (1990) Gene transfer into humans-immunotherapy of patients with
advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med.,
323, 570–578.
[9] Ginn, S.L., Alexander, I.E., Edelstein, M.L., et al. (2013) Gene therapy clinical trials worldwide to 2012 – an update. J. Gene
Med., 15, 65–77.
[10] Gene therapy clinical trials worldwide database (2015) Ed. John Wiley & Sons Ltd. http://www.wiley.co.uk/genmed/
clinical (accessed 19 January 2016).
[11] Gaspar, H.B., Cooray, S., Gilmour, K.C., et al. (2011) Long-term persistence of a polyclonal t cell repertoire after gene
therapy for x-linked severe combined immunodeficiency. Sci. Transl. Med., 3, 97ra79.
[12] Hacein-Bey-Abina, S., Hauer, J., Lim, A., et al. (2010) Efficacy of gene therapy for x-linked severe combined immunode-
ficiency. N. Engl. J. Med., 363, 355–364.
[13] Aiuti, A. and Roncarolo, M.G. (2009) Ten years of gene therapy for primary immune deficiencies. Hematol. Am. Soc.
Hematol. Educ. Program, 2009, 682–689.
[14] Gaspar, H.B., Cooray, S., Gilmour, K.C., et al. (2011) Hematopoietic stem cell gene therapy for adenosine deaminase-
deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction.
Sci. Transl. Med., 3, 97ra80.
[15] Cavazzana-Calvo, M., Payen, E., Negre, O., et al. (2010) Transfusion independence and hmga2 activation after gene
therapy of human beta-thalassaemia. Nature, 467, 318–322.
[16] Boztug, K., Schmidt, M., Schwarzer, A., et al. (2010) Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N. Engl. J.
Med., 363, 1918–1927.
[17] Aiuti, A., Biasco, L., Scaramuzza, S., et al. (2013) Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-
Aldrich syndrome. Science, 341, 1233151.
[18] Cartier, N., Hacein-Bey-Abina, S., Bartholomae, C.C., et al. (2009) Hematopoietic stem cell gene therapy with a lentiviral
vector in X-linked adrenoleukodystrophy. Science, 326, 818–823.
[19] Biffi, A., Montini, E., Lorioli, L., et al. (2013) Lentiviral hematopoietic stem cell gene therapy benefits metachromatic
leukodystrophy. Science, 341, 1233158.
[20] Hacein-Bey Abina, S., Gaspar, H.B., Blondeau, J., et al. (2015) Outcomes following gene therapy in patients with severe
Wiskott-Aldrich syndrome. JAMA, 313, 1550–1563.
[21] Bainbridge, J.W., Smith, A.J., Barker, S.S., et al. (2008) Effect of gene therapy on visual function in leber’s congenital
amaurosis. N. Engl. J. Med., 358, 2231–2239.
[22] Maguire, A.M., Simonelli, F., Pierce, E.A., et al. (2008) Safety and efficacy of gene transfer for leber’s congenital amaurosis.
N. Engl. J. Med., 358, 2240–2248.
[23] Maguire, A.M., High, K.A., Auricchio, A., et al. (2009) Age-dependent effects of rpe65 gene therapy for leber’s congenital
amaurosis: a phase 1 dose-escalation trial. Lancet, 374, 1597–1605.
[24] Nathwani, A.C., Tuddenham, E.G., Rangarajan, S., et al. (2011) Adenovirus-associated virus vector-mediated gene trans-
fer in hemophilia b. N. Engl. J. Med., 365, 2357–2365.
[25] Nathwani, A.C., Reiss, U.M., Tuddenham, E.G., et al. (2014) Long-term safety and efficacy of factor ix gene therapy in
hemophilia B. N. Engl. J. Med., 371, 1994–2004.
[26] Somia, N. and Verma, I.M. (2000) Gene therapy: trials and tribulations. Nat. Rev. Genet., 1, 91–99.
[27] Yin, H., Kanasty, R.L., Eltoukhy, A.A., et al. (2014) Non-viral vectors for gene-based therapy. Nat. Rev. Genet., 15, 541–555.
[28] Semple, S.C., Akinc, A., Chen, J., et al. (2010) Rational design of cationic lipids for sirna delivery. Nat. Biotechnol., 28,
172–176.
[29] Conwell, C.C. and Huang, L. (2005) Recent advances in non-viral gene delivery. Adv. Genet., 53, 3–18.
[30] Mehier-Humbert, S. and Guy, R.H. (2005) Physical methods for gene transfer: improving the kinetics of gene delivery
into cells. Adv. Drug Deliv. Rev., 57, 733–753.
[31] Simoes, S., Filipe, A., Faneca, H., et al. (2005) Cationic liposomes for gene delivery. Expert Opin. Drug Deliv., 2, 237–254.
[32] Louise, C. (2006) Nonviral vectors. Methods Mol. Biol., 333, 201–226.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[33] Lavigne, M.D. and Gorecki, D.C. (2006) Emerging vectors and targeting methods for nonviral gene therapy. Expert Opin.
Emerg. Drugs, 11, 541–557.
[34] O’Loughlin, A.J., Woffindale, C.A., and Wood, M.J. (2012) Exosomes and the emerging field of exosome-based gene
therapy. Curr. Gene Ther., 12, 262–274.
[35] Lee, Y., El Andaloussi, S., and Wood, M.J. (2012) Exosomes and microvesicles: extracellular vesicles for genetic informa-
tion transfer and gene therapy. Hum. Mol. Genet., 21, R125–R134.
[36] Ammar, I., Gogol-Doring, A., Miskey, C., et al. (2012) Retargeting transposon insertions by the adeno-associated virus
rep protein. Nucleic Acids Res., 40, 6693–6712.
[37] Di Matteo, M., Belay, E., Chuah, M.K., and Vandendriessche, T. (2012) Recent developments in transposon-mediated
gene therapy. Expert Opin. Biol. Ther., 12, 841–858.
[38] Argyros, O., Wong, S.P., Gowers, K., and Harbottle, R.P. (2012) Genetic modification of cancer cells using non-viral,
episomal s/mar vectors for in vivo tumour modelling. PLoS One, 7, e47920.
[39] Aronovich, E.L., McIvor, R.S., and Hackett, P.B. (2011) The sleeping beauty transposon system: a non-viral vector for gene
therapy. Hum. Mol. Genet., 20, R14–R20.
[40] Voigtlander, R., Haase, R., Muck-Hausl, M., et al. (2013) A novel adenoviral hybrid-vector system carrying a plasmid
replicon for safe and efficient cell and gene therapeutic applications. Mol. Ther. Nucleic Acids, 2, e83.
[41] Walther, W. and Stein, U. (2000) Viral vectors for gene transfer: a review of their use in the treatment of human diseases.
Drugs, 60, 249–271.
[42] Kay, M.A., Glorioso, J.C., and Naldini, L. (2001) Viral vectors for gene therapy: the art of turning infectious agents into
vehicles of therapeutics. Nat. Med., 7, 33–40.
[43] Verma, I.M. and Weitzman, M.D. (2005) Gene therapy: twenty-first century medicine. Annu. Rev. Biochem., 74, 711–738.
[44] Hacein-Bey-Abina, S., von Kalle, C., Schmidt, M., et al. (2003) A serious adverse event after successful gene therapy for
x-linked severe combined immunodeficiency. N. Engl. J. Med., 348, 255–256.
[45] Ott, M.G., Schmidt, M., Schwarzwaelder, K., et al. (2006) Correction of X-linked chronic granulomatous disease by gene
therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat. Med., 12, 401–409.
[46] Howe, S.J., Mansour, M.R., Schwarzwaelder, K., et al. (2008) Insertional mutagenesis combined with acquired somatic
mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest., 18, 3143–3150.
[47] Hacein-Bey-Abina, S., Garrigue, A., Wang, G.P., et al. (2008) Insertional oncogenesis in 4 patients after retrovirus-
mediated gene therapy of SCID-X1. J. Clin. Invest., 118, 3132–3142.
[48] Braun, C.J., Boztug, K., Paruzynski, A., et al. (2014) Gene therapy for Wiskott-Aldrich syndrome—long-term efficacy and
genotoxicity. Sci. Transl. Med., 6, 227ra233.
[49] Manno, C.S., Arruda, V.R., Pierce, G.F., et al. (2006) Successful transduction of liver in hemophilia by AAV-factor IX and
limitations imposed by the host immune response. Nat. Med., 12, 342–347.
[50] Mingozzi, F., Meulenberg, J.J., Hui, D.J., et al. (2009) AAV-1-mediated gene transfer to skeletal muscle in humans results
in dose-dependent activation of capsid-specific T cells. Blood, 114, 2077–2086.
[51] Ferreira, V., Twisk, J., Kwikkers, K., et al. (2014) Immune responses to intramuscular administration of alipogene tipar-
vovec (AAV1-LPL(S447X)) in a phase II clinical trial of lipoprotein lipase deficiency gene therapy. Hum. Gene Ther., 25,
180–188.
[52] Herzog, R.W. (2015) Hemophilia gene therapy: caught between a cure and an immune response. Mol. Ther., 23,
1411–1412.
[53] VandenDriessche, T. and Chuah, M.K. (2013) Vector decoys trick the immune response. Sci. Transl. Med., 5, 194fs128.
[54] Basner-Tschakarjan, E., Bijjiga, E., and Martino, A.T. (2014) Pre-clinical assessment of immune responses to adeno-
associated virus (AAV) vectors. Front. Immunol., 5, 28.
[55] Mingozzi, F., Chen, Y., Edmonson, S.C., et al. (2013) Prevalence and pharmacological modulation of humoral immunity
to AAV vectors in gene transfer to synovial tissue. Gene Ther., 20, 417–424.
[56] Masat, E., Pavani, G., and Mingozzi, F. (2013) Humoral immunity to AAV vectors in gene therapy: challenges and
potential solutions. Discov. Med., 15, 379–389.
[57] Huang, S. and Kamihira, M. (2013) Development of hybrid viral vectors for gene therapy. Biotechnol. Adv., 31, 208–223.
[58] Weber, C., Armbruster, N., Scheller, C., et al. (2013) Foamy virus-adenovirus hybrid vectors for gene therapy of the
arthritides. J. Gene Med., 15, 155–167.
[59] Sia, K.C., Chong, W.K., Ho, I.A., et al. (2010) Hybrid herpes simplex virus/epstein-barr virus amplicon viral vectors confer
enhanced transgene expression in primary human tumors and human bone marrow-derived mesenchymal stem cells.
J. Gene Med., 12, 848–858.
[60] Kubo, S., Haga, K., Tamamoto, A., et al. (2011) Adenovirus-retrovirus hybrid vectors achieve highly enhanced tumor
transduction and antitumor efficacy in vivo. Mol. Ther., 19, 76–82.
[61] Kaneda, Y., Saeki, Y., and Morishita, R. (1999) Gene therapy using HVJ-liposomes: the best of both worlds? Mol. Med.
Today, 5, 298–303.
[62] Kaneda, Y. (2000) Virosomes: evolution of the liposome as a targeted drug delivery system. Adv. Drug Deliv. Rev., 43,
197–205.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[63] Kaneda, Y. (2012) Virosome: a novel vector to enable multi-modal strategies for cancer therapy. Adv. Drug Deliv. Rev.,
64, 730–738.
[64] Capecchi, M.R. (1989) Altering the genome by homologous recombination. Science, 244, 1288–1292.
[65] Russell, D.W. and Hirata, R.K. (1998) Human gene targeting by viral vectors. Nat. Genet., 18, 325–330.
[66] Barzel, A., Paulk, N.K., Shi, Y., et al. (2015) Promoterless gene targeting without nucleases ameliorates haemophilia B in
mice. Nature, 517, 360–364.
[67] Sharma, R., Anguela, X.M., Doyon, Y., et al. (2015) In vivo genome editing of the albumin locus as a platform for protein
replacement therapy. Blood, 126, 1777–1784.
[68] Greer, K.L., Lochmuller, H., Flanigan, K., et al. (2014) Targeted exon skipping to correct exon duplications in the dys-
trophin gene. Mol. Ther. Nucleic Acids, 3, e155.
[69] Kinali, M., Arechavala-Gomeza, V., Feng, L., et al. (2009) Local restoration of dystrophin expression with the morpholino
oligomer avi-4658 in duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-
concept study. Lancet Neurol., 8, 918–928.
[70] Lundstrom, K. (2015) Special issue: gene therapy with emphasis on RNA interference. Viruses, 7, 4482–4487.
[71] Jiang, L., Frederick, J.M., and Baehr, W. (2014) RNA interference gene therapy in dominant retinitis pigmentosa and
cone-rod dystrophy mouse models caused by GCAP1 mutations. Front. Mol. Neurosci., 7, 25.
[72] Cirak, S., Arechavala-Gomeza, V., Guglieri, M., et al. (2011) Exon skipping and dystrophin restoration in patients with
duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label,
phase 2, dose-escalation study. Lancet, 378, 595–605.
[73] Voit, T., Topaloglu, H., Straub, V., et al. (2014) Safety and efficacy of drisapersen for the treatment of duchenne muscular
dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study. Lancet Neurol., 13, 987–996.
[74] Moscou, M.J. and Bogdanove, A.J. (2009) A simple cipher governs DNA recognition by TAL effectors. Science, 326, 1501.
[75] Boch, J., Scholze, H., Schornack, S., et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors.
Science, 326, 1509–1512.
[76] Beerli, R.R., Segal, D.J., Dreier, B., and Barbas, C.F., 3rd, (1998) Toward controlling gene expression at will: specific
regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building
blocks. Proc. Natl. Acad. Sci. U. S. A., 95, 14628–14633.
[77] Kim, Y.G., Cha, J., and Chandrasegaran, S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
Proc. Natl. Acad. Sci. U. S. A., 93, 1156–1160.
[78] Baker, M. (2012) Gene-editing nucleases. Nat. Methods, 9, 23–26.
[79] Tebas, P., Stein, D., Tang, W.W., et al. (2014) Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.
N. Engl. J. Med., 370, 901–910.
[80] Reardon, S. (2015) Leukaemia success heralds wave of gene-editing therapies. Nature, 527, 146–147.
[81] Jinek, M., Chylinski, K., Fonfara, I., et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity. Science, 337, 816–821.
[82] Cong, L., Ran, F.A., Cox, D., et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science, 339,
819–823.
[83] Mali, P., Yang, L., Esvelt, K.M., et al. (2013) RNA-guided human genome engineering via Cas9. Science, 339, 823–826.
[84] Reardon, S. (2015) Gene-editing record smashed in pigs. Nat. News, http://www.nature.com/news/gene-editing-record-
smashed-in-pigs-1.18525 (accessed 19 January 2016).
[85] Chen, X. and Goncalves, M.A. (2016) Engineered viruses as genome editing devices. Mol. Ther., 24 (3), 447–457.
[86] Yin, H., Xue, W., Chen, S., et al. (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and
phenotype. Nat. Biotechnol., 32, 551–553.
[87] Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., et al. (2000) Gene therapy of human severe combined immun-
odeficiency (SCID)-X1 disease. Science, 288, 669–672.
[88] Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., et al. (2002) Sustained correction of X-linked severe combined immunode-
ficiency by ex vivo gene therapy. N. Engl. J. Med., 346, 1185–1193.
[89] Yanez-Munoz, R.J., Balaggan, K.S., Macneil, A., et al. (2006) Effective gene therapy with nonintegrating lentiviral vectors.
Nat. Med., 12, 348–353.
[90] Torres, R., Garcia, A., Jimenez, M., et al. (2014) An integration-defective lentivirus-based resource for site-specific target-
ing of an edited safe-harbour locus in the human genome. Gene Ther., 21, 343–352.
[91] Kymalainen, H., Appelt, J.U., Giordano, F.A., et al. (2014) Long-term episomal transgene expression from mitotically
stable integration-deficient lentiviral vectors. Hum. Gene Ther., 25, 428–442.
[92] Tareen, S.U., Kelley-Clarke, B., Nicolai, C.J., et al. (2014) Design of a novel integration-deficient lentivector technology
that incorporates genetic and posttranslational elements to target human dendritic cells. Mol. Ther., 22, 575–587.
[93] Naldini, L., Blomer, U., Gallay, P., et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a
lentiviral vector. Science, 272, 263–267.
[94] Naldini, L., Blomer, U., Gage, F.H., et al. (1996) Efficient transfer, integration, and sustained long-term expression of the
transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. U. S. A., 93, 11382–11388.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[95] Adam, M.A., Ramesh, N., Miller, A.D., and Osborne, W.R. (1991) Internal initiation of translation in retroviral vectors
carrying picornavirus 5′ nontranslated regions. J. Virol., 65, 4985–4990.
[96] Monahan, P.E. and Samulski, R.J. (2000) AAV vectors: is clinical success on the horizon? Gene Ther., 7, 24–30.
[97] Ali, M., Lemoine, N.R., and Ring, C.J. (1994) The use of DNA viruses as vectors for gene therapy. Gene Ther., 1, 367–384.
[98] Nathwani, A.C. and Tuddenham, E.G. (1992) Epidemiology of coagulation disorders. Baillieres Clin. Haematol., 5,
383–439.
[99] Olszko, M.E., Adair, J.E., Linde, I., et al. (2015) Foamy viral vector integration sites in scid-repopulating cells after
MGMTP140K-mediated in vivo selection. Gene Ther., 22, 591–595.
[100] Cai, S., Ernstberger, A., Wang, H., et al. (2008) In vivo selection of hematopoietic stem cells transduced at a low
multiplicity-of-infection with a foamy viral MGMT(P140K) vector. Exp. Hematol., 36, 283–292.
[101] Paulk, N.K., Wursthorn, K., Haft, A., et al. (2012) In vivo selection of transplanted hepatocytes by pharmacological
inhibition of fumarylacetoacetate hydrolase in wild-type mice. Mol. Ther., 20, 1981–1987.
[102] Fink, D.J., Sternberg, L.R., Weber, P.C., et al. (1992) In vivo expression of beta-galactosidase in hippocampal neurons by
HSV-mediated gene transfer. Hum. Gene Ther., 3, 11–19.
[103] Xiao, X., Li, J., McCown, T.J., and Samulski, R.J. (1997) Gene transfer by adeno-associated virus vectors into the central
nervous system. Exp. Neurol., 144, 113–124.
[104] Blomer, U., Naldini, L., Kafri, T., et al. (1997) Highly efficient and sustained gene transfer in adult neurons with a lentivirus
vector. J. Virol., 71, 6641–6649.
[105] Glorioso, J.C., DeLuca, N.A., and Fink, D.J. (1995) Development and application of herpes simplex virus vectors for
human gene therapy. Annu. Rev. Microbiol., 49, 675–710.
[106] Hermens, W.T. and Verhaagen, J. (1998) Viral vectors, tools for gene transfer in the nervous system. Prog. Neurobiol., 55,
399–432.
[107] Fleming, J., Ginn, S.L., Weinberger, R.P., et al. (2001) Adeno-associated virus and lentivirus vectors mediate efficient and
sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons. Hum. Gene Ther., 12, 77–86.
[108] Chandler, R.J. and Venditti, C.P. (2007) Adenovirus-mediated gene delivery rescues a neonatal lethal murine model of
mut(0) methylmalonic acidemia. Hum. Gene Ther., 19, 53–60.
[109] Markusic, D.M., Herzog, R.W., Aslanidi, G.V., et al. (2010) High-efficiency transduction and correction of murine
hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines. Mol. Ther., 18, 2048–2056.
[110] van Til, N.P., Stok, M., Aerts Kaya, F.S., et al. (2010) Lentiviral gene therapy of murine hematopoietic stem cells ameliorates
the pompe disease phenotype. Blood, 115, 5329–5337.
[111] Uchiyama, T., Adriani, M., Jagadeesh, G.J., et al. (2012) Foamy virus vector-mediated gene correction of a mouse model
of Wiskott-Aldrich syndrome. Mol. Ther., 20, 1270–1279.
[112] Boye, S.L., Peterson, J.J., Choudhury, S., et al. (2015) Gene therapy fully restores vision to the All-Cone Nrl(−/−)
Gucy2e(−/−) mouse model of leber congenital amaurosis-1. Hum. Gene Ther., 26, 575–592.
[113] Bogdanik, L.P., Osborne, M.A., Davis, C., et al. (2015) Systemic, postsymptomatic antisense oligonucleotide rescues
motor unit maturation delay in a new mouse model for type II/III spinal muscular atrophy. Proc. Natl. Acad. Sci. U. S.
A., 112, E5863–E5872.
[114] Todd, A.G., McElroy, J.A., Grange, R.W., et al. (2015) Correcting neuromuscular deficits with gene therapy in pompe
disease. Ann. Neurol., 78, 222–234.
[115] Carmo, M., Risma, K.A., Arumugam, P., et al. (2015) Perforin gene transfer into hematopoietic stem cells improves
immune dysregulation in murine models of perforin deficiency. Mol. Ther., 23, 737–745.
[116] Askew, C., Rochat, C., Pan, B., et al. (2015) Tmc gene therapy restores auditory function in deaf mice. Sci. Transl. Med., 7,
295ra108.
[117] Li, Z., Dullmann, J., Schiedlmeier, B., et al. (2002) Murine leukemia induced by retroviral gene marking. Science, 296, 497.
[118] Herzog, R.W. (2010) Gene therapy for SCID-X1: round 2. Mol. Ther., 18, 1891.
[119] Hacein-Bey-Abina, S., Pai, S.Y., Gaspar, H.B., et al. (2014) A modified gamma-retrovirus vector for X-linked severe
combined immunodeficiency. N. Engl. J. Med., 371, 1407–1417.
[120] Declaration of Helsinki (2013) The World Medical Association, http://www.wma.net/en/20activities/10ethics/10helsinki/
DoH-Oct2013-JAMA.pdf (accessed 19 January 2016).
[121] Cornetta, K. and Smith, F.O. (2002) Regulatory issues for clinical gene therapy trials. Hum. Gene Ther., 13, 1143–1149.
[122] Manilla, P., Rebello, T., Afable, C., et al. (2005) Regulatory considerations for novel gene therapy products: a review of
the process leading to the first clinical lentiviral vector. Hum. Gene Ther., 16, 17–25.
[123] Cohen-Haguenauer, O. (2013) A comprehensive resource on EU regulatory information for investigators in gene
therapy clinical research and advanced therapy medicinal products. Hum. Gene Ther., 24, 12–18.
[124] Medicines, medical devices and blood regulation and safety—guidance (2014) Clinical Trials for Medicines: Apply
for Authorisation in the UK, https://www.gov.uk/guidance/clinical-trials-for-medicines-apply-for-authorisation-in-the-
uk#clinical-trial-application-form (accessed 19 January 2016).
[125] Dainesi, S.M. (2006) Seeking harmonization and quality in clinical trials. Clinics, 61, 3–8.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[126] Saey, T.H. (2015) Editing human germline cells sparks ethics debate. Sci. News Online, 187 (11. https://www.
sciencenews.org/article/editing-human-germline-cells-sparks-ethics-debate (accessed 19 January 2016)),
[127] Resnik, D.B. and Langer, P.J. (2001) Human germline gene therapy reconsidered. Hum. Gene Ther., 12, 1449–1458.
[128] Fuchs, M. (2006) Gene therapy. An ethical profile of a new medical territory. J. Gene Med., 8, 1358–1362.
[129] Fletcher, J.C. and Anderson, W.F. (1992) Germ-line gene therapy: a new stage of debate. Law Med. Health Care, 20,
26–39.
[130] Matthews, Q.L. and Curiel, D.T. (2007) Gene therapy: human germline genetic modification—assessing the scientific,
socioethical, and religious issues. South. Med. J., 100, 98–100.
[131] Pollack, R. (2015) Eugenics lurk in the shadow of crispr. Science, 348, 871.
[132] Baltimore, D., Berg, P., Botchan, M., et al. (2015) Biotechnology. A prudent path forward for genomic engineering and
germline gene modification. Science, 348, 36–38.
[133] Miller, H.I. (2015) Germline gene therapy: we’re ready. Science, 348, 1325.
[134] Antoine, C., Muller, S., Cant, A., et al. (2003) Long-term survival and transplantation of haemopoietic stem cells for
immunodeficiencies: report of the European experience 1968–99. Lancet, 361, 553–560.
[135] Cavazzana-Calvo, M., Thrasher, A., and Mavilio, F. (2004) The future of gene therapy. Nature, 427, 779–781.
[136] Touzot, F., Moshous, D., Creidy, R., et al. (2015) Faster T-cell development following gene therapy compared with
haploidentical HSCT in the treatment of SCID-X1. Blood, 125, 3563–3569.
[137] Kohn, D.B. (2015) Gene therapy outpaces haplo for SCID-X1. Blood, 125, 3521–3522.
[138] Thrasher, A.J., Hacein-Bey-Abina, S., Gaspar, H.B., et al. (2005) Failure of SCID-X1 gene theapy in older patients. Blood,
105, 4255–4257.
[139] Jaffe, A., Prasad, S.A., Larcher, V., and Hart, S. (2006) Gene therapy for children with cystic fibrosis—who has the right
to choose? J. Med. Ethics, 32, 361–364.
[140] Nyberg, K., Carter, B.J., Chen, T., et al. (2004) Workshop on long-term follow-up of participants in human gene transfer
research. Mol. Ther., 10, 976–980.
[141] Cecchini, S., Virag, T., and Kotin, R.M. (2011) Reproducible high yields of recombinant adeno-associated virus produced
using invertebrate cells in 0.02- to 200-liter cultures. Hum. Gene Ther., 22, 1021–1030.
[142] Negrete, A., Yang, L.C., Mendez, A.F., et al. (2007) Economized large-scale production of high yield of rAAV for gene
therapy applications exploiting baculovirus expression system. J. Gene Med., 9, 938–948.
[143] Federal Drug Authority (2013) Structural Characterization of Gene Transfer Vectors, http://www.fda.gov/downloads/
biologicsbloodvaccines/internationalactivities/ucm273199.pdf (accessed 19 January 2016).
[144] Kalos, M., Levine, B.L., Porter, D.L., et al. (2011) T cells with chimeric antigen receptors have potent antitumor effects
and can establish memory in patients with advanced leukemia. Sci. Transl. Med., 3, 95ra73.
[145] Sadelain, M. (2015) CAR therapy: the CD19 paradigm. J. Clin. Invest., 125, 3392–3400.
[146] Ramos, C.A., Heslop, H.E., and Brenner, M.K. (2016) CAR-T cell therapy for lymphoma. Annu. Rev. Med., 67, 165–183.
[147] Maude, S.L., Frey, N., Shaw, P.A., et al. (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia.
N. Engl. J. Med., 371, 1507–1517.
[148] Davila, M.L., Riviere, I., Wang, X., et al. (2014) Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell
acute lymphoblastic leukemia. Sci. Transl. Med., 6, 224ra225.
[149] Grupp, S.A., Kalos, M., Barrett, D., et al. (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.
N. Engl. J. Med., 368, 1509–1518.
[150] Brentjens, R.J., Davila, M.L., Riviere, I., et al. (2013) CD19-targeted T cells rapidly induce molecular remissions in adults
with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med., 5, 177ra138.
[151] Blaese, R.M., Culver, K.W., Miller, A.D., et al. (1995) T lymphocyte-directed gene therapy for ADA-SCID: initial trial results
after 4 years. Science, 270, 475–480.
[152] Aiuti, A., Vai, S., Mortellaro, A., et al. (2002) Immune reconstitution in ADA-SCID after PBL gene therapy and discontin-
uation of enzyme replacement. Nat. Med., 8, 423–425.
[153] Aiuti, A., Slavin, S., Aker, M., et al. (2002) Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloab-
lative conditioning. Science, 296, 2410–2413.
[154] Aiuti, A., Cattaneo, F., Galimberti, S., et al. (2009) Gene therapy for immunodeficiency due to adenosine deaminase
deficiency. N. Engl. J. Med., 360, 447–458.
[155] Ogino, S., Gulley, M.L., den Dunnen, J.T., and Wilson, R.B. (2007) Standard mutation nomenclature in molecular diag-
nostics: practical and educational challenges. J. Mol. Diagn., 9, 1–6.
[156] Kohn, D.B. (2010) Update on gene therapy for immunodeficiencies. Clin. Immunol., 135, 247–254.
[157] Engel, B.C., Podsakoff, G.M., Ireland, J.L., et al. (2007) Prolonged pancytopenia in a gene therapy patient with ADA-
deficient SCID and trisomy 8 mosaicism: a case report. Blood, 109, 503–506.
[158] Sokolic, R., Kesserwan, C., and Candotti, F. (2008) Recent advances in gene therapy for severe congenital immunodefi-
ciency diseases. Curr. Opin. Hematol., 15, 375–380.
[159] Gaspar, H.B. (2010) Bone marrow transplantation and alternatives for adenosine deaminase deficiency. Immunol.
Allergy Clin. North Am., 30, 221–236.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[160] Gaspar, H.B., Aiuti, A., Porta, F., et al. (2009) How I treat ADA deficiency. Blood, 114, 3524–3532.
[161] Orkin, S.H. and Motulsky, A.G. (1995) Report and Recommendations of the Panel to Assess the NIH Investment in Research
on Gene Therapy, http://osp.od.nih.gov/sites/default/files/resources/Orkin_Motulsky_Report.pdf (accessed 19 January
2016).
[162] Raper, S.E., Chirmule, N., Lee, F.S., et al. (2003) Fatal systemic inflammatory response syndrome in a ornithine transcar-
bamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab., 80, 148–158.
[163] Savulescu, J. (2001) Harm, ethics committees and the gene therapy death. J. Med. Ethics, 27, 148–150.
[164] Ginn, S.L. and Alexander, I.E. (2012) Gene therapy: progress in childhood disease. J. Paediatr. Child Health, 48, 466–471.
[165] Wu, X., Li, Y., Crise, B., and Burgess, S.M. (2003) Transcription start regions in the human genome are favored targets for
mlv integration. Science, 300, 1749–1751.
[166] Mitchell, R.S., Beitzel, B.F., Schroder, A.R., et al. (2004) Retroviral DNA integration: ASLV, HIV, and MLV show distinct target
site preferences. PLoS Biol., 2, E234.
[167] Hematti, P., Hong, B.K., Ferguson, C., et al. (2004) Distinct genomic integration of MLV and SIV vectors in primate
hematopoietic stem and progenitor cells. PLoS Biol., 2, e423.
[168] Erlwein, O. and McClure, M.O. (2010) Progress and prospects: foamy virus vectors enter a new age. Gene Ther., 17,
1423–1429.
[169] Porter, D.L., Levine, B.L., Kalos, M., et al. (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.
N. Engl. J. Med., 365, 725–733.
[170] Neff, T., Beard, B.C., and Kiem, H.P. (2006) Survival of the fittest: in vivo selection and stem cell gene therapy. Blood, 107,
1751–1760.
[171] Neff, T., Beard, B.C., Peterson, L.J., et al. (2005) Polyclonal chemoprotection against temozolomide in a large-animal
model of drug resistance gene therapy. Blood, 105, 997–1002.
[172] Neff, T., Horn, P.A., Peterson, L.J., et al. (2003) Methylguanine methyltransferase-mediated in vivo selection and chemo-
protection of allogeneic stem cells in a large-animal model. J. Clin. Invest., 112, 1581–1588.
[173] Adair, J.E., Beard, B.C., Trobridge, G.D., et al. (2012) Extended survival of glioblastoma patients after chemoprotective
HSC gene therapy. Sci. Transl. Med., 4, 133ra157.
[174] Stein, S., Ott, M.G., Schultze-Strasser, S., et al. (2010) Genomic instability and myelodysplasia with monosomy 7 conse-
quent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med., 16, 198–204.
[175] Persons, D.A. and Baum, C. (2011) Solving the problem of gamma-retroviral vectors containing long terminal repeats.
Mol. Ther., 19, 229–231.
[176] Klein, C. (2010) Effective Gene Therapy for Children with Wiskott-Aldrich Syndrome, https://idw-online.de/de/
news396307%3E (accessed 19 January 2016).
[177] Zou, J., Mali, P., Huang, X., et al. (2011) Site-specific gene correction of a point mutation in human iPS cells derived from
an adult patient with sickle cell disease. Blood, 118, 4599–4608.
[178] Cheng, L.T., Sun, L.T., and Tada, T. (2012) Genome editing in induced pluripotent stem cells. Genes Cells, 17, 431–438.
[179] Osborn, M.J., Starker, C.G., McElroy, A.N., et al. (2013) Talen-based gene correction for epidermolysis bullosa. Mol. Ther.,
21, 1151–1159.
[180] Takahashi, K. and Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast
cultures by defined factors. Cell, 126, 663–676.
[181] Takahashi, K., Okita, K., Nakagawa, M., and Yamanaka, S. (2007) Induction of pluripotent stem cells from fibroblast
cultures. Nat. Protoc., 2, 3081–3089.
[182] Yu, J., Vodyanik, M.A., Smuga-Otto, K., et al. (2007) Induced pluripotent stem cell lines derived from human somatic
cells. Science, 318, 1917–1920.
[183] Wernig, M., Zhao, J.P., Pruszak, J., et al. (2008) Neurons derived from reprogrammed fibroblasts functionally integrate
into the fetal brain and improve symptoms of rats with parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A., 105, 5856–5861.
[184] Hanna, J., Wernig, M., Markoulaki, S., et al. (2007) Treatment of sickle cell anemia mouse model with iPS cells generated
from autologous skin. Science, 318, 1920–1923.
[185] Raya, A., Rodriguez-Piza, I., Guenechea, G., et al. (2009) Disease-corrected haematopoietic progenitors from fanconi
anaemia induced pluripotent stem cells. Nature, 460, 53–59.
[186] Kazuki, Y., Hiratsuka, M., Takiguchi, M., et al. (2009) Complete genetic correction of ips cells from duchenne muscular
dystrophy. Mol. Ther., 18, 386–393.
[187] Warren, L., Manos, P.D., Ahfeldt, T., et al. (2010) Highly efficient reprogramming to pluripotency and directed differen-
tiation of human cells with synthetic modified mrna. Cell Stem Cell, 7, 618–630.
[188] Zhou, W. and Freed, C.R. (2009) Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent
stem cells. Stem Cells, 27, 2667–2674.
[189] Fusaki, N., Ban, H., Nishiyama, A., et al. (2009) Efficient induction of transgene-free human pluripotent stem cells using
a vector based on sendai virus, an rna virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B Phys.
Biol. Sci., 85, 348–362.
DOI: 10.1002/9781118445112.stat06926.pub2
Gene Therapy
[190] Kim, D., Kim, C.-H., Moon, J.-I., et al. (2009) Generation of human induced pluripotent stem cells by direct delivery of
reprogramming proteins. Cell Stem Cell, 4, 472–476.
[191] Yu, J., Hu, K., Smuga-Otto, K., et al. (2009) Human induced pluripotent stem cells free of vector and transgene sequences.
Science, 324, 797–801.
[192] Sack, B.K. and Herzog, R.W. (2009) Evading the immune response upon in vivo gene therapy with viral vectors. Curr.
Opin. Mol. Ther., 11, 493–503.
[193] Martino, A.T., Basner-Tschakarjan, E., Markusic, D.M., et al. (2013) Engineered aav vector minimizes in vivo targeting of
transduced hepatocytes by capsid-specific CD8+ T cells. Blood, 121, 2224–2233.
[194] Herper, M. (2014) Gene Therapy’s Big Comeback. In Forbes, online. http://www.forbes.com/sites/matthewherper/2014/
03/26/once-seen-as-too-scary-editing-peoples-genes-with-viruses-makes-a-618-million-comeback/2/ (accessed 19 Jan-
uary 2016).
Further Reading
Barrett, D.M., Grupp, S.A., and June, C.H. (2015) Chimeric antigen receptor- and TCR-modified T cells enter main street and wall
street. J. Immunol., 195, 755–761.
Bosley, K.S., Botchan, M., Bredenoord, A.L., et al. (2015) CRISPR germline engineering-the community speaks. Nat. Biotechnol.,
33, 478–486.
Cavazzana, M., Six, E., Lagresle-Peyrou, C., et al. (2016) Gene therapy for SCID-X1: where do we stand? Hum. Gene Ther., 27,
108–116.
Cicalese, M.P. and Aiuti, A. (2015) Clinical applications of gene therapy for primary immunodeficiencies. Hum. Gene Ther., 26,
210–219.
Cox, D.B., Platt, R.J., and Zhang, F. (2015) Therapeutic genome editing: prospects and challenges. Nat. Med., 21, 121–131.
Ginn, S.L. and Alexander, I.E. (2012) Gene therapy: progress in childhood disease. J. Paediatr. Child Health, 48, 466–471.
Gopinath, C., Nathar, T.J., Ghosh, A., et al. (2015) Contemporary animal models for human gene therapy applications. Curr. Gene
Ther., 15, 531–540.
Hastie, E. and Samulski, R.J. (2015) Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy
success—a personal perspective. Hum. Gene Ther., 26, 257–265.
Hill, A.B., Chen, M., Chen, C.K., et al. (2016) Overcoming gene-delivery hurdles: physiological considerations for nonviral vectors.
Trends Biotechnol., 34, 91–105.
Hotta, A. and Yamanaka, S. (2015) From genomics to gene therapy: induced pluripotent stem cells meet genome editing. Annu.
Rev. Genet., 49, 47–70.
Kotterman, M.A., Chalberg, T.W., and Schaffer, D.V. (2015) Viral vectors for gene therapy: translational and clinical outlook. Annu.
Rev. Biomed. Eng., 17, 63–89.
Lombardo, A. and Naldini, L. (2014) Genome editing: a tool for research and therapy: targeted genome editing hits the clinic.
Nat. Med., 20, 1101–1103.
Maeder, M.L. and Gersbach, C.A. (2016) Genome editing technologies for gene and cell therapy. Mol. Ther., 24, 430–446.
Maude, S. and Barrett, D.M. (2016) Current status of chimeric antigen receptor therapy for haematological malignancies. Br. J.
Haematol., 172, 11–22.
Naldini, L. (2015) Gene therapy returns to centre stage. Nature, 526, 351–360.
O’Reilly, M., Jambou, R., Rosenthal, E., et al. (2015) The National Institutes of Health oversight of human gene transfer research:
enhancing science and safety. Adv. Exp. Med. Biol., 871, 31–47.
Prakash, V., Moore, M., and Yanez-Munoz, R.J. (2016) Current progress in therapeutic gene editing for monogenic diseases. Mol.
Ther., 24, 465–474.
Sadelain, M. (2015) CAR therapy: the CD19 paradigm. J. Clin. Invest., 125, 3392–3400.
Salmikangas, P., Schuessler-Lenz, M., Ruiz, S., et al. (2015) Marketing regulatory oversight of advanced therapy medicinal
products (ATMPs) in Europe: the EMA/CAT perspective. Adv. Exp. Med. Biol., 871, 103–130.
Seymour, L.W. and Thrasher, A.J. (2012) Gene therapy matures in the clinic. Nat. Biotechnol., 30, 588–593.
Wolff, J.A. and Lederberg, J. (1994) An early history of gene transfer and therapy. Hum. Gene Ther., 5, 469–480.
DOI: 10.1002/9781118445112.stat06926.pub2
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Table 1. Gene-therapy clinical trial indications.

Indication Gene-therapy clinical trials

Cancer diseases 1415 64.0

Source: From www.abedia.com/wiley.

1 Requirements for Successful Therapeutic Intervention

1.1 Gene Delivery Technology

Gene delivery technology

Target cell Understanding of

Figure 1. Requirements for successful therapeutic intervention.

Table 2. Properties of widely used viral vector systems.

Vector Adenovirus 𝛾-Retrovirusa) Lentivirusb) AAVc)

Genome dsDNA ssRNA ssRNA ssDNA

a) Most commonly derived from Moloney murine leukemia virus (MoMLV).

1.2 Target Cell Biology

1.3 Disease Pathophysiology

2.1 In Vitro Studies

2.2 Animal Models

3 Translational Challenges of Gene-Therapy Trials

3.1 Regulatory Oversight

3.2 Special Ethical Considerations

3.3 Skills, Infrastructure, and Reagents

Multi country 4.2% (n=92)

USA 62.7% (n=1386)

Germany 3.8% (n=84)

China 2.4% (n=54)

France 2.4 (n=52)

Switzerland 2.3% (n=50)

Japan 1.9% (n=41)

Netherlands 1.5% (n=34)

Australia 1.4% (n=32)

Canada 1.1% (n=25)

Other countries 6.8% (n=151)

5.1 The Power of In Vivo Selection

Table 3. Gene delivery systems in clinical trial use.

Vector Number of protocols

Adenovirus 480 (21.7%)

Source: From www.abedia.com/wiley.

5.2 Insertional Mutagenesis

6 The Way Forward

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