Gene Therapy (2003) 10, 19992004

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REVIEW

Gene therapy progress and prospects: gene therapy

for severe combined immunodeficiency
HB Gaspar, S Howe and AJ Thrasher
Molecular Immunology Unit, Institute of Child Health, London, UK

Severe combined immunodeficiencies have long been

targeted as a group of disorders amenable to gene therapy
because of their defined molecular biology and pathophysiology, and the prediction that corrected cells would have
profound growth and survival advantage. Recently, several

clinical studies have shown that conventional gene transfer

technology can produce major beneficial therapeutic effects
in these patients, but, as for all cellular and pharmacological
treatment approaches, with a finite potential for toxicity.
Gene Therapy (2003) 10, 19992004. doi:10.1038/sj.gt.3302150

Keywords: ; SCID-X1; ADA; PEG-ADA; LMO-2; insertional mutagenesis

In brief
Progress

Sustained correction of X-severe combined immunodeficiency (SCID) has been observed in clinical trials.

Cells in patients with adenosine deaminase (ADA)
deficiency, treated by lymphocyte or stem cell gene
therapy, persist and maintain transgene expression
for many years.

Withdrawal of PEG-ADA from patients treated by
lymphocyte gene therapy for ADA-deficient SCID
results in enhanced immunological reconstitution.

Successful gene therapy for ADA-deficient SCID can
be achieved in the absence of PEG-ADA and in
combination with myelosuppression.

Animal models of RAG-2- and JAK-3-deficient SCID
have been corrected using similar strategies.

Insertional mutagenesis has been observed in human
studies, reinforcing the need to develop methods for
optimization of protocol safety.

Sustained correction of X-severe combined

immunodeficiency (SCID) has been observed
in clinical trials
The most severe forms of primary immunodeficiency are
known as SCIDs. These are a group of diseases in which
Correspondence: Dr AJ Thrasher, Molecular Immunology Unit, Institute
of Child Health, 30 Guilford Street, London WC1N 1EH, UK

Prospects

As a group of well-defined disorders, SCIDs are
amenable to treatment by gene therapy, and an
extended range will enter clinical study over the next
few years.

The durability of immunological reconstitution will
determine the effectiveness and the need for repeated
administration.

The absolute risk of clinically manifesting mutagenesis using retroviral vectors is at present unknown,
and will only be determined by the extended
observation of more patients, and by the development of clinically relevant models to test for toxicity
in a rigorous way.

The characteristics of retroviral and lentiviral integration in human patients will be determined by
mapping integration sites.

Risks of mutagenesis will be reduced by improved
design of vectors that restrict promoter activity to
relevant cell types and within the domain of the
therapeutic transgene.

Development of targeted integration or of stable
episomal vector systems will also enhance safety.

Gene-repair strategies may have particular efficacy in
SCID because of the profound growth and survival
advantage conferred to corrected cells.

T-lymphocyte development is invariably interrupted,

and associated with diverse disorders of development
and functionality of B lymphocytes, and natural killer
(NK) cells.1 X-linked SCID (SCID-X1) accounts for
approximately 5060% of all SCIDs, and is caused by
mutations in the gene encoding the common cytokine
receptor gamma chain (gc). This is a subunit of the
cytokine receptor complex for interleukins (IL) 2, 4, 7, 9,
15 and 21.2 In the absence of gc signaling, many aspects

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HB Gaspar et al

2000

of immune cell development and function are compromised. The classical immunophenotype of SCID-X1 is the
absence of T and NK cells, and persistence of dysfunctional B cells (TB NKSCID). If a genotypically
matched family donor is available, bone marrow
transplantation is a highly successful procedure with a
long-term survival rate of over 90%. The high survival
rates are partly due to the fact that the absence of T and
NK cells in SCID-X1 patients allows engraftment in
the absence of myelosuppressive conditioning. For the
majority of individuals, this is not possible, and the
survival from mismatched family (usually parental
donors) transplants is less good, and is associated with
predictable toxicity.
Many incremental advances in gene transfer technology have recently been translated into successful gene
therapy for SCID-X1.3 These have included the activation
of cells with high concentrations of cytokines (thereby
making them susceptible to gamma retrovirus vectormediated gene transfer), and transduction in containers
coated with a recombinant fibronectin fragment (RetroNectint) that is believed to facilitate the colocalization of
the virus particle and the target cell.4,5 In the first
landmark study, a conventional amphotropic retroviral
vector encoding a gc cDNA (regulated by Moloney
murine leukaemia virus long-terminal repeat sequences)
was used to transduce ex vivo autologous CD34 cells
(separated by conventional magnetic bead technology
from a bone marrow harvest). The cells were reinfused
into the patients in the absence of preconditioning. The
results obtained from the first five patients have recently
been reported in the scientific literature.6 To date, 10
infants in total have now been treated, with good
immunological reconstitution in all but one, in whom
the graft appears to have become sequestered in a
pathologically enlarged spleen6,7 In nearly all patients,
NK cells appeared between 2 and 4 weeks after infusion
of cells, followed by new thymic T-lymphocyte emigrants at 1012 weeks. With some variation, the number
and distribution of these T cells normalized rapidly
(more rapidly than observed following haploidentical
transplantation). They also appeared to function normally in terms of proliferative response to mitogens, Tcell receptor (TCR), and specific antigen stimulation, and
to have a complex phenotypic and molecular diversity
of TCR. Functionality of the humoral system was also
restored, maybe not quite as effectively, but to a sufficient
degree that discontinuation of immunoglobulin therapy
was possible. Persistent long-term marking in myeloid
cells (between 0.1 and 1%) suggests that long-lived stem
or progenitor cells have been successfully transduced.
More recently, we have initiated a similar study for the
treatment of SCID-X1. The transduction protocols and
vector are very similar although we have used a gibbon
apeleukaemia virus (GALV) pseudotype, and conditions that obviate the requirement for foetal calf serum.
Although the follow-up period for four children treated
in our study is short, all have cleared viral infections, and
immunological reconstitution has followed a similar
pattern8 (Thrasher et al, manuscript in preparation).
The contribution to the initial burst of thymopoiesis
from relatively late T-cell precursors in the original
transduced CD34 cell population versus that from cells
earlier in the haematopoietic differentiation hierarchy,
which have engrafted in the bone marrow, has not yet

Gene Therapy

been determined. This may have important implications

for the durability of immunological reconstitution, and
for sustained production of new T cells. These issues may
be resolved by longitudinal study of naive T-cell
production, and by the isolation of common integration
sites between myeloid and lymphoid populations.
Ultimately, the longevity of functional reconstitution
can only be determined by clinical monitoring, but it
should also be feasible to repeat gene therapy on
multiple occasions. Unknown, however, are the time
frames within which this will be clinically effective,
particularly bearing in mind that there may be agerelated restrictions to the reinitiation of thymopoiesis in
these patients.

Cells in patients with ADA deficiency treated

by lymphocyte or stem cell gene therapy
persist and maintain transgene expression for
many years
Deficiency of the purine salvage enzyme adenosine
deaminase (ADA) accounts for approximately 1020%
of all SCIDs. ADA catalyses the deamination of deoxyadenosine (dAdo) and adenosine to deoxyinosine and
inosine, respectively, and the lack of ADA leads to the
build of the metabolites deoxyATP (dATP) and dAdo,
which have profound effects on lymphocyte development and function through a number of cellular
mechanisms. There is variation in the severity of the
condition but most ADA patients have very low
numbers of T and B lymphocytes. Bone marrow
transplantation is highly successful in the genotypically
matched setting, but human leucocyte antigen (HLA)mismatched transplants have poor survival outcomes.
An alternative modality of treatment is exogenous
enzyme replacement with polyethylene glycol-conjugated bovine ADA, which as regular intramuscular
injections can result in the correction of metabolic and
immunological abnormalities, albeit only partially in a
significant number of cases.9
The first human gene therapy studies were conducted
on patients with ADA deficiency in the early 1990s. It is
generally agreed that these initial studies were unsuccessful in correcting the immune defect in ADA-SCID.
This was in part due to the continued use of PEG-ADA
enzyme replacement therapy, which in itself improved
the immune function but may also have blunted the
survival advantage of gene-modified cells (discussed
further below). However, a decade on, more detailed
analysis of the patients originally treated does provide
important information about the longevity and efficacy
of gene transfer.10 In the first human clinical gene
therapy study, two patients were treated following
repeated gammaretroviral vector-mediated ADA gene
transfer into stimulated peripheral blood lymphocytes.
Patient 1 still shows over 15% of gene-marked cells in
peripheral blood mononuclear cells (PBMCs) and ADA
activity in PBMCs remains at B25% of the normal. The
level of gene marking (0.1% of PBMCs) and ADA activity
(o5% of normal) is considerably less in patient 2, which
may reflect the smaller number of gene-transduced cells
initially infused, but may also be due to the development
of an immune response against the retroviral envelope

Gene therapy for severe immunodeficiency

HB Gaspar et al

and lipoprotein components of the foetal calf serum used

for culturing the cells. However, these findings clearly
demonstrate that human T cells have a lifespan greater
than 10 years in the peripheral circulation, and also that
transgenes regulated by gammaretroviral sequences
continue to express in peripheral T cells and resist in
vivo silencing. Molecular analysis of TCR diversity,
combined with transgene integration analysis, reveals
that within individual Vb family clones, each cell
contains multiple unique integration sites. This suggests
that, in the initial phase, cells were repeatedly sampled
and transduced.
In a later study, umbilical cord blood CD34 cells
were harvested from antenatally diagnosed ADA-SCID
patients, transduced and reinfused in the first week of
life. In these patients, little clinical benefit was seen,
and the level of gene marking was low (110% of T
lymphocytes) in all the three children. Recent clonal
integration analyses demonstrate that transgene-containing T lymphocytes are monoclonal or oligoclonal (with
15 different integration sites) more than 8 years after
gene therapy, and that single prelymphoid clones
contribute between 25 and 100% of genetically corrected
lymphocytes.11 Marking in other lineages is consistently
less than 1%. Again, the continuation of PEG-ADA
throughout these early studies almost certainly compromised the efficient engraftment of transduced cells.

Withdrawal of PEG-ADA from patients

treated by lymphocyte gene therapy for
ADA-deficient SCID results in enhanced
immunological reconstitution
Matched sibling donor transplants for ADA-SCID
performed without conditioning result in rapid engraftment and persistence of donor T lymphocytes, strongly
suggesting that T lymphocytes expressing ADA have
a powerful proliferative and survival advantage. The
failure of initial gene therapy studies to demonstrate the
proliferation and expansion of gene-modified cells
seemed to question this premise, although the continued
administration of PEG-ADA in all these patients may
have abrogated this advantage. Recently, one patient
participating in another study demonstrated convincingly that a survival advantage for gene-transduced
cells did exist in the absence of PEG-ADA. In this
individual, who had received multiple infusions of
autologous transduced peripheral blood lymphocytes
(PBLs), and who had reached a plateau of 13% genetransduced T cells, PEG-ADA-associated immune dysregulatory problems led to a gradual discontinuation of
enzyme replacement. As the dose was decreased and
finally stopped, the percentage of transduced T cells as
assessed by PCR quantification increased, eventually
reaching nearly 100% of all T lymphocytes.12 Absolute
CD3 T-cell counts also increased and stabilized at
levels higher than prediscontinuation values. T-cell
proliferative responses were also restored, and analysis
of ADA metabolites showed a rise in intracellular PBL
ADA activity.
The blunting effect of PEG-ADA may also be
responsible for the disappointing observations made in
a more recent study that employed enhanced vectors,

and transduction conditions similar to those used for

SCID-X1 trials.13 As before, transduced CD34 cells
were returned to the patients without conditioning and
patients continued PEG-ADA treatment. ADA gene
marking was only seen at low levels (in a range from
0.0001 to 3.6%). On the basis of previous observations,
the investigators plan to withdraw PEG-ADA 1 year after
treatment.

2001

Successful gene therapy for ADA-deficient

SCID can be achieved in the absence of
PEG-ADA and in combination with
myelosuppression
Results of a new study have clearly demonstrated that
ADA-SCID can be successfully treated by gene therapy.14
In this protocol, CD34 cells were transduced with an
amphotropic gammaretroviral vector (originally used in
PBL transduction studies) under current optimal conditions. Two important changes were incorporated into the
protocol. Firstly, for economic reasons, patients were not
commenced on PEG-ADA and, secondly, patients received a mild dose of conditioning (4 mg/kg of
busulphan as 2 mg/kg on two successive days) prior to
the return of gene-modified cells. A neutrophil and
platelet nadir were seen at approximately 3 weeks, but
neither child required blood product support. One
patient is now 20 months. Over 2 years, postgene
transfer one patient has normal numbers of peripheral
T, B and NK cells. This patient has normal immunoglobulin production and is not receiving any prophylactic
therapy. There has also been impressive correction of the
metabolic defects, with dATP levels falling to 10% of that
at diagnosis (comparable to that achieved following
successful BMT). The second patient is now 12 months
postgene therapy, but was an older child (approximately
2.5 years) at the time of treatment and also received a
lower cell dosage. In this patient, recovery has been
slower and the T-cell reconstitution at present is
suboptimal, but significantly improved from pretransplant levels. There is evidence of some immunoglobulin
production, but the patient remains on replacement
therapy. A third child has been treated more recently
using the same protocol,15 and is showing a recovery
similar to that in patient 1. Molecular analysis of the first
two patients shows a diverse TCR repertoire and an
increase in TCR excision circle (TREC) levels, indicating
the successful engraftment of prethymic progenitor
populations. Lineage-specific transgene analysis by
quantitative PCR shows high level marking in T, NK
and B cells, and the persistence of gene-modified
granulocytes, monocytes and megakaryocytes at levels
between 5 and 20%, again suggesting that multipotent
progenitors have engrafted. Somewhat surprising is the
level to which marking has persisted in myeloid cells,
particularly at the dosage of conditioning employed.
This may suggest that a survival advantage is not
restricted to lymphoid cells, and that it also extends to
myeloid and haematopoietic multipotent progenitor
cells.
The results from this study are extremely encouraging.
At present, it is difficult to determine whether the
success of the procedure is due to the lack of PEGGene Therapy

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HB Gaspar et al

2002

ADA or the use of nonmyeloablative conditioning, but it

is likely that the combination is important. The key to
correction of the metabolic abnormalities in ADA-SCID
seems to be the delivery of large amounts of ADA
enzyme, whether exogenously in the form of PEG-ADA
or intracellularly as gene-modified cells. The use of
conditioning may facilitate the initial engraftment of a
greater number of gene-modified cells. Certainly, if
immune function in these patients is sustained, and
further patients show a similar safety profile and
immune response, this strategy holds great promise for
ADA-SCID and potentially other haematopoietic conditions.

Insertional mutagenesis has been observed

in human studies, reinforcing the need to
develop methods for optimization of protocol
safety
For retroviruses, which depend on chromosomal integration for the stability of transduction, the most
prominent safety concern has been for insertional
mutagenicity.19 On the basis of numerous animal studies
and over 300 clinical trials in which patients have
received retroviral vectors, and from theoretical considerations, the risk of clinically manifesting insertional
mutagenesis has been judged to be low. However, in a
recent murine HSC retroviral transduction study, insertion of the vector into the oncogene Evi-1 led to
development of myeloid leukaemia.20 This has been
followed by the reported development of uncontrolled
clonal T-cell proliferation in two patients in the Paris
SCID-XI clinical trial (Table 1).7,21 Having initially
achieved successful immunological reconstitution, both
developed lymphoproliferation approximately 3 years
after the gene therapy procedure. In both patients,
retroviral vector insertion into or near the LMO-2
proto-oncogene resulted in high-level expression of
LMO-2 in the clones, almost certainly as a result of
retroviral enhancer-mediated activation of transcription.
Activation of LMO-2 is known to participate in human
leukaemogenesis by chromosomal translocation, and
results in the development of T-cell lymphoproliferation
and leukaemia in mice, albeit with a long latency. It is
therefore likely that other contributing factors are
required for these events to manifest. At present, there
is no evidence for the contribution of dysregulated gc
expression in lymphoid cells, although this remains a
possibility and is being studied carefully. Cells with high
proliferative potential, such as thymocytes, are also likely
to be more susceptible to transformation following an
insertional event than quiescent cells, if they acquire
additional adverse mutations unrelated to the gene
therapy itself. This increased risk cannot yet be quantified. The integration of the vector into LMO-2 in both
cases strongly suggests that there is some preference for
the survival of these clones or less likely, for integration
at this site (Hacein-Bey-Abina et al, submitted for
publication). The detailed molecular analysis of insertion
events in patients undergoing gene therapy will greatly
assist in the delineation of integration points within the
genome, but is unlikely to be able to predict potential for

Animal models of JAK-3- and RAG-2deficient SCID have been corrected using
similar strategies
The molecular
basis of autosomal recessive
TB NKSCID is mutation of the receptor tyrosine
kinase gene JAK-3. The dependence of gc on signalling
through JAK-3 is responsible for a clinical and
immunological phenotype identical to that of SCID-X1,
and the rationale for gene therapy is therefore similar.
Correction of a murine model of JAK-3-deficient SCID
has been achieved using both myelosuppresssive,
and more relevant to clinical studies, conditioning-free
protocols.16 Patients with mutations of the recombinaseactivating genes RAG-1 and RAG-2 characteristically
present with the absence of both B and T cells. Moloneybased
gammaretroviral
vectors
have
recently
been shown to effectively reconstitute RAG-2-deficient
mice, and in the absence of detectable toxicity, even
though gene expression was not tightly regulated.17 One
way to obviate the toxicity arising from dysregulated
gene expression in any condition, and to achieve
physiological activity, is to correct genetic mutations by
gene repair or homologous recombination. Recently, it
has been shown that RAG2/ mutant murine embryonic stem (ES) cells, repaired by standard homologous
recombination technology, can be grown in vitro to
provide sufficient haematopoietic progenitors for engraftment and correction of RAG-2 mutant mice.18 This is
the first example of gene therapy combined with a
therapeutic cloning strategy and, clearly, has important
implications for future treatment of many genetic
disorders.

Table 1

Current clinical trials of gene therapy for SCID

Disease

Gene

Retroviral vector

Envelope

ADA-D
ADA-D

ADA
ADA

A
A

ADA-D
SCID-X1
SCID-X1
AR-SCID

ADA
gc
gc
JAK-3

GIADA1
GCsap-M-ADA
MND-ADA
SFFV-ADA-WPRE
MFG
MFG
MSCV

G
A
G
G

Cell type treated

*BM CD34+
BM CD34+
UCB CD34+
*BM CD34+
BM CD34+
BM CD34+
BM CD34+

Reference/PI
14
11,13

AJ Thrasher/HB Gaspar
3,6,7

AJ Thrasher
BP Sorrentino

Summary of clinical trials for SCID using gammaretroviral vectors ongoing from 2000. A, amphotropic envelope; G, gibbonapeleukaemia
virus envelope; ADA, adenosine deaminase deficiency; SCID-X1, X-linked SCID; BM, bone marrow; UCB, umbilical cord blood; gc, common
cytokine receptor gamma chain. *Denotes the use of preconditioning.
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Gene therapy for severe immunodeficiency

HB Gaspar et al

mutagenesis unless recurrent hotspots associated with

clinical disease become evident.2224 In combination with
conventional monitoring of lymphocyte numbers and
distributions, longitudinal monitoring of integration sites
will provide an important way of monitoring for
pathological clonal expansions. The applicability of any
novel therapy, including gene therapy, ultimately depends on the balance of risks against those of alternative
treatments. The accurate characterization of adverse
events, the utilization of protocols to test toxicity in a
rigorous way, and the development of methods to
minimize risks are therefore essential.

Future prospects for SCID

Recent clinical trials have shown that at least some forms
of SCID can be effectively treated by gene therapy. Much,
however, can be done to improve the efficiency and
safety of current protocols. The design of vectors used for
gene delivery is clearly important and modifications may
be possible, which will limit the risks of mutagenesis,
for example by the incorporation of DNA and RNA
insulator sequences in integrating vectors, by the use of
self-inactivating vectors or by targeting safe regions in
the genome.25,26 Alternative vectors based on lentiviruses
or foamy viruses that obviate prolonged ex vivo culture
may allow the preservation of larger numbers of multipotential progenitor cells, but at the same time may
produce higher numbers of insertion events in each
cell.2731 Methods to minimize the number of integration
events per cell, and to limit the number of engrafting
clones, for example by more stringent purification of
stem cell (or defined target cell) populations, may
therefore also be beneficial. Probably, the most straightforward way to improve safety is to dispense with the
powerful viral enhancer sequences that can dysregulate
gene expression over large chromatin domains. Lentiviral vectors, in particular, provide greater capacity for
the incorporation of more complex and physiological
regulatory sequences. The relative risk for each type of
vector modification needs to be determined in clinically
relevant animal-model systems, and the effectiveness of
these models to predict side effects in humans will have
to be validated. The development of homologous
recombination or gene repair to correct mutations, or
the construction of mitotically stable extrachromosomal
vectors, would obviate many of these problems, but
current technologies are inefficient.32 Once again, SCID
may be a perfect initial target for this strategy, as even
limited efficiency will be sufficient to provide clinical
benefit. The future for gene therapy of SCID is exciting,
but has been clouded by the occurrence of toxicity. As for
all novel therapeutic modalities, increased understanding of mechanisms, and increased sophistication of
technology will translate into even more effective and
safe application. There is no better paradigm for this
process than allogeneic bone marrow transplantation.

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