Review
Gene Therapy
1. Introduction
2. Molecular basis of severe
combined immunodeficiency
3. Haematopoietic stem cell
transplantation for severe
combined immunodeficiency
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4. Enzyme replacement therapy
for ADA-SCID
5. Gene therapy for SCID-X1
6. Gene therapy for ADA-SCID
7. Gene therapy for other severe
combined immunodeficiencies
8. Insertional mutagenesis and
risks of gene therapy
9. Expert opinion and conclusion:
future prospects for severe
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combined immunodeficiency
gene therapy
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10.1517/14712598.5.9.1175 © 2005 Ashley Publications Ltd ISSN 1471-2598
Gene therapy for severe combined
immunodeficiencies
H Bobby Gaspar† & Adrian J Thrasher
†Institute of Child Health, Molecular Immunology Unit, 30 Guilford Street, London, WC1N 1EH, UK
Severe combined immune deficiencies (SCIDs) are a group of monogenic
diseases resulting in profound disturbances of lymphocyte development and
function. Affected individuals are prone to life-threatening infections and
without treatment do not survive beyond the first year of life. Haematopoi-
etic stem cell transplantation from a well-matched donor offers high rates
of survival, but in the absence of a suitable matched donor, parental haplo-
identical transplants are associated with greater complications, lower
success rates and in some instances poor long-term immune recovery.
Alternative therapeutic options based on correction of the defective gene
by retroviral gene delivery have been used to correct X-linked SCID
(SCID-X1) and adenosine deaminase-deficient SCID (ADA-SCID). A number
of clinical trials have established that ex vivo gene transfer into haemato-
poietic progenitor cells allows effective recovery of immune defects and
that gene therapy can offer a successful alternative to transplantation. The
development of leukaemia as a result of insertional mutagenesis in one trial
of gene therapy for SCID-X1 has raised concerns regarding the toxicity of
retroviral vector-based gene delivery. These side effects are now being
studied in detail and measures to prevent such events through alternative
vectors delivery systems are in development at present.
Keywords: adenosine deaminase, gamma chain, gene therapy, insertional mutagenesis,
retroviral vector, severe combined immunodeficiency
Expert Opin. Biol. Ther. (2005) 5(9):1175-1182
1. Introduction
The most severe forms of primary immunodeficiency are known as severe combined
immunodeficiencies (SCIDs). These are a group of diseases in which T lymphocyte
development and in some cases function are severely disrupted and associated with
diverse disorders of development and functionality of B lymphocytes and natural
killer (NK) cells [1]. Affected infants often become seriously unwell in the first few
months of life with opportunistic infections, chronic diarrhoea and failure to thrive,
and without treatment most will die in the first year of life. To date, the mainstays of
treatment have been supportive care, antibiotic therapy and immunoglobulin replace-
ment, followed in almost all cases by allogeneic haematopoietic stem cell transplanta-
tion (HSCT) [2,3]. Over the past 20 years, improved understanding of the molecular
basis of these conditions and advances in gene transfer technology have resulted in the
development of successful gene therapy strategies for two forms of SCID.
2. Molecular basis of severe combined immunodeficiency
In recent years the genetic basis of almost three-quarters of all primary immune
deficiencies have been established [4]. X-linked SCID (SCID-X1) accounts for
∼ 40 – 50% of all SCIDs and is caused by mutations in the gene encoding the
common cytokine receptor gamma chain (γc). This is a subunit of the cytokine
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 Gene therapy for severe combined immunodeficiencies
receptor complex for interleukins (ILs) 2, 4, 7, 9, 15 and
21 [5,6]. In the absence of γc signalling, many aspects 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
(T-B+NK- SCID) [7], although individuals with partial T cell
development have been identified. A much rarer autosomal
recessive form of T-B+NK- SCID arises from mutations in
Janus kinase (JAK)3, an intracellular tyrosine kinase that is
activated through γc binding and subsequently activates the
transcription factor signal transducer and activator of
transcription 5 [8].
Deficiency of adenosine deaminase (ADA) accounts for up
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to 20% of SCID cases [9]. The ADA gene maps to 20q13.11
and is an enzyme that is expressed in all tissues of the body,
although at variable levels, with the highest activity in the
thymus and lymphoid tissues. ADA plays an essential role in
purine metabolite salvage pathways, and an absence of ADA
activity results in the accumulation of the substrates deoxy-
adenosine (dAdo) and deoxyadenosinetriphosphate (dATP),
which through a variety of mechanisms lead to inhibition of
DNA synthesis, impaired cell division and apoptosis. These
defects have a profound effect on lymphocyte development
and a T-B-NK- is seen in the most severely affected infants
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with preservation of NK cell numbers in some cases. Most
ADA-SCID patients (85 – 90%) present in the first year of
life, although delayed presentation as a result of residual ADA
activity is seen in a minority of cases.
Defects of antigen receptor gene rearrangement, causing a
T-B-NK+ phenotype, account for most other cases of SCID.
The failure to produce functional T or B cell receptors arises
either due to mutations in the recombinase activating genes
(RAG 1 and 2) or the Artemis gene [10,11]. These encode
lymphoid cell-specific factors required during the recombina-
tion of variable, diverse and junctional (VDJ) regions of
antigen-specific receptor genes. Approximately 10% of
T-B-NK+ SCIDs remain undiagnosed at a molecular level,
suggesting that other genes involved in VDJ recombination
may be involved. Table 1 details the molecular basis of SCID
disorders and highlights the lymphocyte subset pattern
usually associated with each condition.
3.Haematopoietic stem cell transplantation
for severe combined immunodeficiency
Until recently, HSCT has been the only curative therapy for
SCID. Data on transplant outcome is now available from a
wide number of sources and centres, the largest data source
being the combined efforts of a European Stem Cell Trans-
plantation registry [2,12]. If a genotypically matched family
donor is available, bone marrow transplantation is a highly
successful procedure with recent results indicating a long-term
survival rate of ∼ 90% for all forms of SCID. The high
survival rates are partly due to the fact that the absence of
T and/or NK cells allows engraftment in the absence of
1176 Expert Opin. Biol. Ther. (2005) 5(9)
myelosuppressive conditioning. Under these circumstances,
tissue damage caused by myeloablative chemotherapy is
avoided and mature T cells from the unmanipulated graft
provides rapid protection from viral infection. Matched
unrelated donor transplants are increasingly used in major
centres, and with the advent of reduced intensity conditioning,
results are steadily improving and in some cases are equivalent
to those achieved using matched sibling donors [13,14].
For a significant proportion of patients, a well-matched
related or unrelated donor is unavailable and in these cases
transplant can be undertaken from a mismatched (halploidenti-
cal) parental donor. Host/donor human leukocyte antigen
disparity necessitates rigorous depletion of donor T cells to
avoid graft-versus-host disease, and the donor graft is rich in
haematopoetic progenitor cells, but lacks mature T cell popula-
tions. Thus, T cell immune recovery is delayed for 6 months or
more during which time the recipient remains susceptible to
viral infection. Controversy remains as to whether myeloblation
with chemotherapy is required prior to the return of donor
cells. The lack of chemotherapy avoids short-term tissue
toxicity, but there is an increased risk of rejection and most
probably poor engraftment of early donor haematopoietic
progenitors leading to continuing humoral defects and
impaired long-term immune reconstitution. Nevertheless, the
overall survival for haploidentical transplants with or without
chemotherapy is in the order of 70 – 80% for SCID-X1 [2,3],
but may be significantly less for other forms of SCID
(ADA-SCID, 29% 3-year survival; T-B-NK+ SCID, 32% 3-year
survival) [2,3,15]. Therefore, there is clearly a need for an alterna-
tive to haploidentical transplant to minimise toxicity, enhance
long-term immune recovery and improve overall survival.
4. Enzyme replacement therapy for ADA-SCID
An alternative modality of treatment for the ADA-deficient
form of SCID (ADA-SCID) is exogenous enzyme replace-
ment with polyethylene glycol-conjugated bovine ADA
(PEG-ADA). Regular intramuscular injections of PEG-ADA
result in rapid systemic detoxification, with reduction of dAdo
and dATP to near normal levels within a few weeks of starting
therapy. Immune recovery follows and although no formal
data exists, reports suggest that ∼ 70% of children show an
improvement in lymphocyte counts to near normal
levels [16,17]. Of these 50% remain on immunoglobulin
replacement due to continued humoral impairment. The
long-term prognosis for children on PEG-ADA without any
corrective procedure being undertaken is unclear (M Hersh-
field, ESID 2002). PEG-ADA has been used in conjunction
with gene therapy although this may have had a detrimental
effect on the success of initial clinical trials (see section below).
5. Gene therapy for SCID-X1
Many incremental advances in gene transfer technology have
now been translated into successful gene therapy for SCID-X1.

Table 1. Severe combined immunodeficiencies.
Disorder Mutated gene
SCID-X1 Common γc
JAK-3 deficiency JAK3
IL-7 receptor deficiency IL-7 receptor α
RAG 1,2 deficiency RAG1 and 2
Artemis Artemis
Adenosine deaminase deficiency ADA
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T cell receptor deficiencies CD3 γεζδ
CD45 deficiency CD45
γc: Cytokine receptor gamma chain; ADA: Adenosine deaminase; IL: Interleukin; JAK: Janus kinase; NK: Natural killer; RAG: Recombinase activating gene;
SCID: Severe combined immunodeficiency; VDJ: Variable, diverse and junctional.
These have included activation of cells with high concentra-
tions of cytokines (thereby making them susceptible to
gammaretrovirus vector-mediated gene transfer), and trans-
duction in containers coated with a recombinant fibronectin
fragment (RetroNectin™) that is believed to facilitate
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co-localisation of virus particle and the target cell [18]. In the
first landmark study, a conventional amphotropic retroviral
vector encoding the human γc cDNA (regulated by Moloney
murine leukaemia virus long-terminal repeat sequences), was
used to transduce 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 been
reported in the scientific literature [19,20]. To date, 11 infants in
total have been treated, with good immunological reconstitution
in the majority of patients. One child showed no evidence in the
periphery of genetically modified cells and probably seques-
trated the graft into an enlarged spleen. A further patient had
poor immune recovery, in this case due to a low genetically
modified cell dose (< 1 × 106 γc transduced CD34+ cells). Both
these patients subsequently underwent a successful unrelated
donor HSCT. In nearly all patients, NK cells appeared between
2 and 4 weeks after infusion of cells, followed by new thymic
T lymphocyte emigrants at 10 – 12 weeks. With some variation,
the number and distribution of these T cells normalised rapidly
(more rapidly than observed following haploidentical transplan-
tation). Functional analysis in terms of proliferative responses to
mitogenic, T cell receptor (TCR) and specific antigen stimula-
tion was normal. By phenotypic analysis of TCRVβ usage and
TCR spectratyping, a complex and diverse T cell population
was demonstrated. Analysis of naive T cell markers, CD45RA+
and of T cell receptor excision circles (TRECs – by-products of
TCR rearrangement and, thus, surrogate markers of thymic
activity) suggests increased thymic activity and the emergence of
thymus educated T lymphocytes. Functionality of the humoral
Expert Opin. Biol. Ther. (2005) 5(9)
Gaspar & Thrasher
Molecular defect Phenotype
Absence of functional receptors T-B+NK-
for IL-2, 4, 7, 9, 15 and 21
Defect of signalling via IL-2, 4, 7, T-B+NK-
9, 15 and 21
Absence of IL-7 receptor α T-B+NK+
Defective VDJ recombination T-B-NK+
Defective VDJ recombination; T-B-NK+
radiation sensitivity
Block in purine salvage T-B-NK+/-
metabolism
Defective T cell signalling T-B+NK+
Abnormal T cell signalling T+B+NK+
system was also restored, maybe not quite as effectively, but to a
sufficient degree that discontinuation of immunoglobulin
therapy was possible in a number of patients.
The authors have also initiated a similar study, using an
MFG-based retroviral vector packaged in PG13 cells and
pseudotyped with a gibbon ape leukaemia virus (GALV) enve-
lope [21]. Patients who lacked a matched sibling or unrelated
donor were enrolled and similar to the French study, autolo-
gous CD34+ cells were purified and transduced in a 96-h
transduction protocol and returned to the patient without the
use of cytoreductive conditioning. To date, seven children (six
of whom were under the age of 1 year and one aged 3 years)
have been enrolled. In all cases there has been evidence of
immune recovery with kinetics of T and NK cell recovery
similar to the previous study. Recovery into the normal range
has been seen in 3 patients, all of whom have discontinued
prophylactic antibiotic and antibody replacement therapy. In
two patients immune recovery is suboptimal and patients
remain on prophylactic treatment, and in the last two patients
with the shortest follow-up, there is continued improvement of
immune parameters. Clinically, all patients have cleared viral
infections, demonstrate improved growth parameters and are
living at home with no restrictions on socialisation or contact.
The levels of gene marking in all lineages are also similar in
both studies. All T cells show evidence of transgene integra-
tion with a copy number of 1 – 2 transgene copies per cell.
Persistent long-term marking in myeloid cells (between 0.1
and 1%) suggests that long-lived stem or progenitor cells have
been successfully transduced. It is difficult to know whether
thymopoiesis arises from late prethymic T cell precursors or
from less committed progenitors that arise earlier in the hier-
archy of haematopoietic stem cells. This will have important
implications for the longevity of T cell reconstitution and can
really only be resolved by longitudinal study of naive T cell
production, and by isolation of common integration sites
between myeloid and lymphoid populations. Ultimately, the
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 Gene therapy for severe combined immunodeficiencies
longevity of functional reconstitution can only be determined
by clinical monitoring.
Two older patients (aged 20 years and 15 years), one in each
of the above studies, were also treated and in both cases, despite
effective gene transfer to bone marrow CD34+ cells, there was
no evidence of immune recovery in response to gene therapy
and only limited and transient presence of the transgene was
observed [22]. The reasons for failure are difficult to define, but
there may be intrinsic host-dependent restrictions to efficacy.
In particular, in older individuals in whom there may have
been thymic involution over many years, there is likely to be a
limitation to initiation of normal thymopoiesis. In such cases,
early intervention is likely to achieve greater success.
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6. Gene therapy for ADA-SCID
The first human gene therapy studies were conducted on
patients with ADA deficiency in the early 1990s [23-26].
Despite continued and in some cases significant presence of
the transgene in patient cells over many years, it is generally
agreed that these initial studies were unsuccessful in correcting
the immune defect in ADA-SCID. A major factor was the
continued use of PEG-ADA enzyme replacement therapy,
which in itself improved immune function, but may also have
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abrogated the survival advantage of gene-modified cells. Over
a decade on, detailed analysis of the patients originally treated
provides important information about the longevity and effi-
cacy of gene transfer [27]. In one patient treated > 10 years ago
by repeated gammaretroviral vector-mediated ADA gene
transfer into stimulated peripheral blood lymphocytes, there
is > 10% gene marking in peripheral blood mononuclear cells
(PBMC), and ADA activity in PBMCs remains at ∼ 25% of
normal. In a later study, umbilical cord blood CD34+ cells
harvested from antenatally diagnosed ADA-SCID patients
were transduced and reinfused in the first week of life. Again
little clinical benefit was seen, and the level of gene marking
was low (1 – 10% of T lymphocytes) in all three children [28].
Recent clonal integration analysis using linear amplification
method-polymerase chain reaction (LAM-PCR) demonstrates
that transgene-containing T lymphocytes are monoclonal or
oligoclonal (with 1 – 5 different integration sites) > 8 years
after gene therapy, and that single prelymphoid clones
contribute between 25 and 100% of genetically corrected
lymphocytes, whereas marking in other lineages is
negligible [29]. These findings clearly demonstrate that human
T cells have a lifespan in the peripheral circulation of
> 10 years, and also that transgenes regulated by gammaretro-
viral sequences continue to be expressed in peripheral T cells
and resist in vivo silencing.
The continuation of PEG-ADA throughout these early
studies almost certainly compromised the efficient engraft-
ment of transduced cells. This assumption is supported by an
important observation in one patient participating in another
gene therapy study who demonstrated that a survival
advantage for gene transduced cells did exist in the absence of
1178 Expert Opin. Biol. Ther. (2005) 5(9)
PEG-ADA [30]. In this individual, who 10 years after gene
therapy had 1 – 3% gene transduced T cells, the cessation of
PEG-ADA treatment led to a rapid increase in the proportion
of gene-modified T cells, eventually reaching nearly 100% of
all T lymphocytes. Absolute CD3+ T cell counts also
increased and T cell proliferative responses were restored.
These data suggest that significant gene marking can be
achieved by retroviral gene transfer, but that the withdrawal
of enzyme replacement is a prerequisite for the proliferation
of gene marked T cells. Clinical observations also highlight
the systemic nature of ADA deficiency [31,32] and suggest
that systemic detoxification by high levels of engraftment of
gene-modified cells in all haematopoietic lineages may be
beneficial. Two studies have now incorporated significant
changes into ADA-SCID gene therapy protocols to achieve
these goals. In both trials, patients received a mild
non-myeloablative conditioning regimen prior to the infu-
sion of gene-modified cells, thereby allowing engraftment of
a greater number of modified cells (PEG-ADA was either
not used or withdrawn prior to treatment). In the first of
these trials, patients who had not previously received
PEG-ADA were treated with Busulphan at a dose of 4 mg/kg
prior to return of gene transduced autologous CD34+
cells [33]. Five children have now been treated and in all cases
there has been substantial reconstitution of T, B and NK cell
numbers; although similar to the SCID-X1 studies, patients
receiving low levels (< 1 × 106/kg) of gene-modified CD34+
cells showed decreased benefit. Molecular analysis shows a
diverse TCR repertoire and an increase in TREC activity
levels, indicating the successful engraftment of prethymic
progenitor populations. Lineage-specific transgene analysis
by quantitative polymerase chain reaction shows high-level
marking in T, NK and B cells, and persistence of
gene-modified granulocytes, monocytes and megakarocytes
at levels between 5 – 20%, again suggesting that multipotent
progenitors have engrafted. In a second study using an opti-
mised GALV pseudotyped gammaretroviral vector, one
patient has been treated following cessation of PEG-ADA
one month before gene therapy and using a single dose of
Melphalan (140 mg/m2) preconditioning before the return
of autologous gene-modified cells (Gaspar et al.,
ASGT 2005). T, B and NK cell recovery with metabolic
correction of dATP levels have now been sustained for
> 1 year following the procedure. The results from both
studies are extremely encouraging. The key to correction of
the immunological and metabolic abnormalities in
ADA-SCID appears 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.
 Gaspar & Thrasher

Table 2. Clinical trials of gene therapy for SCID (previous and current).

Defect Cellular target Conditioning Vector/envelope No. of patients

*Common γc BM CD34 None MFG-ampho 12
*Common γc BM CD34 None MFG-GALV 8
*Common γc PBSC CD34 None MFG-GALV 2
ADA Peripheral T cells None LASN-ampho 2
ADA BM/peripheral lymphocytes None DCA-ampho 7
ADA UCB CD34+ None LASN-ampho 3
ADA BM CD34+ None LgAL-ampho 3
ADA Peripheral T cells None LASN-ampho 1
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*ADA BM CD34+ Busulphan 4 mg/kg GIADA1-ampho 5

*ADA BM CD34+ Melphalan 140 mg/m2 SFFV-GALV 1
*ADA BM CD34+ None GCsap-M-GALV 4
MND-ADA-GALV
*ADA BM CD34+ None GCsap-M-GALV 2
JAK-3 BM CD34+ None MSCV 1
*Currently active study.
γc: Cytokine receptor gamma chain; ADA: Adenosine deaminase; Ampho: Amphotropic envelope; BM: Bone marrow; GALV: Gibbon ape leukaemia virus envelope;
JAK: Janus kinase; PBSC: Peripheral blood stem cells; UCB: Umbilical cord blood.
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7.Gene therapy for other severe combined
immunodeficiencies
The molecular basis of autosomal recessive T-B+NK- SCID is
mutation of the receptor tyrosine kinase gene JAK-3 [8]. The
dependence of γc 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.
One patient has been treated by retroviral vector-mediated
transduction of haematopoietic stem cells, but no immune
recovery was seen, although no published data exists
(Sorrentino et al., unpublished data). Patients with muta-
tions of the RAG-1 and -2 genes characteristically present
with absence of both B and T cells. Moloney-based gamma-
retroviral vectors have recently been shown to effectively
reconstitute RAG-2-deficient mice in the absence of detecta-
ble toxicity, even though gene expression was not tightly
regulated [34]. One way to obviate 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. It has recently been
shown that RAG-2-/- mutant embryonic stem cells, repaired
by standard homologous recombination technology, can be
grown in vitro to provide sufficient haematopoietic progeni-
tors for engraftment and correction of RAG-2 mutant
mice [35]. This is the first example of gene therapy combined
with a therapeutic cloning strategy, and clearly has important
implications for the future treatment of many genetic
disorders. Other severe immunodeficiencies are also targets
for treatment by gene therapy and are at various stages of
development. Preclinical studies in ZAP-70 deficiency show
that reconstitution of ZAP-70-mediated signalling in T cells
can be restored by retroviral gene transfer [36,37]. In
Wiskott-Aldrich syndrome, where mutations in the WASP
gene result in a heterogenous phenotype of combined
immunodeficiency, thrombocytopaenia and eczema, a
number of studies including correction of a murine model
show considerable promise and may allow development of
clinical trials in the near future [38-41].
8.Insertional mutagenesis and risks of
gene therapy
For retroviruses, which depend on chromosomal integration
for stability of transduction, the most prominent safety
concern has been for insertional mutagenicity. Prior to the
inception of the clinical studies detailed above, data from
numerous animal studies and > 300 clinical trials in which
patients have received retroviral vectors, and from theoretical
considerations, the risk of clinically manifesting insertional
mutagenesis had been judged to be low. However, in one
SCID-X1 gene therapy study, 3 of 11 patients who had shown
successful immune recovery following gene therapy developed
T cell leukaemia 3 years after treatment ([42,43] and
Cavazzana-Calvo et al., ASGT Genotoxicity Retreat, St. Louis,
June 2005). Detailed study of the leukaemic clones shows that
in two cases vector insertion into the LMO-2 proto-oncogene
locus led to upregulation of LMO-2 protein expression, most
probably as a result of retroviral long terminal repeat (LTR)

Expert Opin. Biol. Ther. (2005) 5(9) 1179

 Gene therapy for severe combined immunodeficiencies
enhancer activity. Analysis of the third patient most recently
described is ongoing. This is the first demonstration of inser-
tional mutagenesis in human gene therapy, and follows the
recent description of a similar event leading to myeloid
leukaemia in mice, in which the oncogene was identified as
Evi-1 [44]. From more recent studies it is clear that retroviruses
show preference for integration into transcriptionally active
genes and gammaretroviruses (as used in the SCID-X1 and
ADA-SCID trials) in particular integrate preferentially into a
5-kb region around the transcription start site [45,46]. Retro-
viral vector dose escalation studies in murine models also
demonstrate that the potential for leukaemogenesis is related
to increased vector dose and thereby increased transgene copy
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number [47]. These studies also suggest that combinatorial
insertional events may be important in leukaemogenesis.
Thus, the use of gammaretroviral vectors per se may represent
an inherent risk, but the lack of serious adverse events (SAEs)
in other retroviral vector-based clinical studies would suggest
that other factors may be equally as important. The high
incidence in this SCID-X1 study, compared, for example,
with ADA-SCID gene therapy where similar numbers of
patients have been treated for equivalent and in some cases
longer periods of time, may imply that the γc transgene itself
may play a synergistic role with oncogene activation. At
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present there is no direct evidence for a contribution of
dysregulated γc expression in lymphoid cells, although the
very nature of γc as a lymphoid growth factor makes this a
possibility and is being studied carefully. Interestingly, analysis
of one leukaemic tumour arising from infection with replica-
tion-competent retrovirus shows clonal integrations in both
LMO-2 and γc [48]. The chances of finding coincident inte-
grations into both genes within the same tumour are exceed-
ingly small and provide genetic evidence for a synergistic
mechanism. Cells with high proliferative potential such as
thymocytes are also likely to be more susceptible to transfor-
mation 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 quanti-
fied. 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 leukaemogenesis
unless recurrent hotspots associated with clinical disease
become evident. The accurate characterisation of adverse
events, the utilisation of protocols to test toxicity in a rigorous
way and the development of methods to minimise risks are
therefore essential.
The applicability of any novel therapy, including gene
therapy, ultimately depends on the balance of risks against
those of alternative treatments. Of eighteen SCID-X1 patients
now treated in two clinical trials, three have developed SAEs,
two of which have been treated and are in remission, and one
of whom has died due to leukaemic relapse. The alternative
for this group of patients is mismatched allogeneic HSCT,
which carries a 20% 1-year mortality and is also associated
1180 Expert Opin. Biol. Ther. (2005) 5(9)
with significant short- and long-term morbidities. Carefully
monitored clinical studies and analysis of larger numbers of
patients will be required to address these important issues.
9.Expert opinion and conclusion:
future prospects for severe combined
immunodeficiency gene therapy
The results from the SCID-X1 and ADA-SCID studies show
that haematopoietic stem cell gene therapy can offer excellent
immune system and clinical recovery. The use of low intensity
preconditioning coupled with gene therapy, as used for
ADA-SCID, offers much hope for many other inherited
haematological and immunological disorders where initial
cytoreduction may be a prerequisite for successful outcome. It
is likely that these first few studies will allow further techno-
logical development and initiation of clinical trials in other
conditions over the next decade.
It is clear that clinical progress can only be achieved in
combination with improvements in basic vector design to
promote both safety and efficacy. For gammaretroviral
vectors, where insertional events may pose a consistent risk,
it is necessary to limit LTR enhancer activity in order to
prevent transcription of non-target genes. This may be
achieved by the use of specialised DNA sequences, termed
enhancer blockers (or insulators), that can interfere with an
enhancers ability to communicate with a target promoter
when positioned between the two [49]. Targeting of retroviral
vectors to ‘safe’ regions in the genome is another possibility,
but it is likely that the next generation of gammaretroviral
vectors to be developed for clinical use will be self-inactivating
(SIN) vectors, where transgene expression is controlled by a
non-viral promoter with limited or no enhancer activity.
Alternative SIN 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. Methods to minimise 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. In particular, lentiviral
vectors provide greater capacity for incorporation of more
complex and physiological regulatory sequences. The rela-
tive 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 has 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 existing technologies have until recently been
inefficient. Recent innovations, such as zinc-finger technology
whereby double-strand breaks induced by zinc-finger nucle-
ases can create specific sequence alterations by stimulating
 Gaspar & Thrasher

homologous recombination between the chromosome and an
extrachromosomal DNA donor, show efficient correction of
γc-deficient cells, at least in in vitro studies, and may hold
considerable promise if gene delivery to haematopoietic
progenitor cells can be demonstrated [50]. 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 thera-
peutic modalities, an increased understanding of mechanisms
and increased sophistication of technology will translate into
even more effective and safe application.

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Affiliation
H Bobby Gaspar†1 & Adrian J Thrasher
†Author for correspondence
1Institute of Child Health,
Molecular Immunology Unit, 30 Guilford Street,
London, WC1N 1EH, UK
Tel: +44 (0)207 905 2319;
Fax: +44 (0)207 905 2810;
E-mail: h.gaspar@ich.ucl.ac.uk