Download Gene therapy of severe combined immunodeficiencies

PERSPECTIVES
OPINION
Gene therapy of severe combined
immunodeficiencies
Alain Fischer, Salima Hacein-Bey and Marina Cavazzana-Calvo
The concept that the outcome of a
devastating disease can be modified by
inserting a transgene into abnormal cells is
appealing. However, the gene-transfer
technologies that are available at present
have limited the success of gene therapy
so far. Nevertheless, severe combined
immunodeficiencies are a useful model,
because gene transfer can confer a
selective advantage to transduced cells.
In this way, a proof of concept for gene
therapy has been provided.
Adaptive immune responses can be impaired
in several ways. Several natural mutants have
been described in humans, all of which are
characterized by a complete block in T-cell
development1. They are known collectively as
severe combined immunodeficiences (SCIDs)
because, directly or indirectly, B-cell immunity is impaired also, and because the clinical
consequence is a devastating predisposition
to infections with many microorganisms.
SCID conditions can be classified into four
groups according to their pathophysiology
(FIG. 1). In most cases, the molecular mechanisms have been identified, which has provided, in several instances, insights into the
role of given gene products in lymphocyte
development. It was recognized quickly that
SCID conditions would be attractive models
for assessing the potential of gene therapy.
Indeed, there is a combination of factors that
make gene therapy for these immunodeficiencies favourable — namely, the identification of the genetic defects that are responsible for SCID conditions, the severity of the
NATURE REVIEWS | IMMUNOLOGY
conditions, the accessibility of haematopoietic stem cells for manipulation, the efficacy
of allogeneic haematopoietic stem-cell
transplantation (although its results are not
fully satisfactory in terms of survival rates)
and the correction of immune responses2–4.
This is why, as soon as the first gene to be
associated with a SCID condition — adenosine deaminase (ADA) — was identified, several laboratories used ADA deficiency as a
model for gene-therapy studies; this led to the
first gene-therapy trial in 1990 (REFS 5–9).
Although much information was gained
from this and other, subsequent studies, gene
transfer did not lead to a correction of the
immunodeficiency. Nevertheless, these pioneering studies provided the basis for successful gene therapy for another SCID condition
— X-linked SCID (SCID-X), common
cytokine receptor γ-chain deficiency (γc) —
as reported in 2000 (REF. 10).
Lessons from ADA gene-transfer trials
ADA deficiency was recognized as a cause of
SCID in 1972 (REF. 11), and the gene that is
responsible was cloned in 1984 (REF. 12). Given
the small size of the coding part of the gene, its
ubiquitous expression and its ‘house-keeping’
function, it was recognized immediately as a
possible candidate for gene transfer. Moloney
murine sarcoma virus-derived retroviral vectors were available to transport the ADA gene
into lymphopoietic progenitors or mature
lymphocytes when present. The use of retroviral vectors in this setting is based on their
ability to infect haematopoietic progenitors
and induce insertion of the transgene into the
target-cell genome (BOX 1). They are derived
from mouse oncoretroviruses13. The survival
and function of ADA-deficient T cells after
gene transfer were shown using a xenograft
model14. Almost simultaneously, an alternative therapy became available — an injectable
ADA enzyme that was stabilized by coupling
to polyethyleneglycol (PEG–ADA)15. Because
the accumulating toxic substrates of ADA
(adenosine and deoxyadenosine) diffuse freely
throughout the body, weekly injections of
PEG–ADA to ADA-deficient patients efficiently replaced the function of the missing
enzyme. Thereby, lymphocyte development
was restored to a level that was sufficient to
allow several patients to live free of infections.
These studies had several implications for
the development of gene therapy that
extended far beyond the rare condition of
ADA deficiency. First, transfer of the ADA
gene into T cells was shown to be a feasible
approach. This is an attractive method,
because T cells can easily be triggered to cycle
ex vivo so that they can be transduced efficiently using retroviral-mediated gene transfer. So, after feasibility tests, the first clinical
gene-therapy trial for ADA deficiency was
carried out5. Two ADA-deficient patients
were enrolled, and a third patient was treated
later in Japan6. This clinical trial produced
several interesting results (BOX 2). It was
shown that it was feasible to transduce ex vivo
a large number of T cells that were obtained
by BLOOD APHERESIS, and that despite the injection of numerous transduced cells, no
adverse events occurred. In particular, the
random pattern of transgene integration into
the genome did not induce any deleterious
events as a result of insertional mutagenesis.
This tells us — much more so than any kind
of experimental work — that the risk that is
associated with the use of retroviral vectors is
extremely low. Second, in at least one patient,
transduced T cells could still be detected 8–10
years after gene transfer. This is the best available demonstration of in vivo T-cell longevity
in humans, and it is an encouraging result for
the outcome of gene therapy that targets the
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PERSPECTIVES
Expansion 2
Expansion 1
TCRβ
rearrangement
T-cell precursor
pre-TCR
expression
Expansion 3
TCRα
rearrangement
Positive
selection
Survival
Mature
T cell
Adenosine deaminase
Purine phosphorylase
γc, JAK3,
IL7-Rα
RAG1/2,
Artemis
CD45, CD3,
ZAP70
RAG1/2,
Artemis
CD3, CD45,
ZAP70
γc, JAK3,
IL-7Rα
Defective
precursor
T-cell
expansion
Defective generation
of T- and B-cell receptors
Deoxynucleotideinduced T-cell
precursor apoptosis
Defective signalling
by γc-dependent
cytokines
Figure 1 | Defects in T-cell development that result in severe combined immunodeficiencies. T cells develop in the thymus from T-cell precursors, and pass
through distinct developmental stages to become mature T cells — namely, V(D)J recombination, pre-T-cell receptor (pre-TCR) expression and induced clonal
expansion, and TCR-triggered positive and negative selection. The common cytokine-receptor γ-chain (γc), Janus kinase 3 (JAK3), interleukin-7 receptor α-chain
(IL-7Rα), adenosine deaminase (ADA), recombination-activating gene 1 (RAG1) and RAG2, Artemis, CD3ε, CD3γ, CD45 and ζ-chain-associated protein, 70 kDa
(ZAP70) are all required for T-cell differentiation. Mutations of the genes that encode these proteins cause severe combined immunodeficiency (SCID) or profound
T-cell deficiency, which can be divided into four groups according to pathophysiology (represented here by the four blue boxes). Proteins are illustrated at the
approximate stage at which they exert their function. The stage at which ADA deficiency leads to the abortion of T-cell generation is highly speculative. TCR γδ T-cell
development, as well as the generation of CD4+ and CD8+ T cells, is not shown for the sake of simplicity. D, diversity; J, joining; V, variable.
lymphoid system. However, most of the circulating T cells were not transduced, and
because researchers were not allowed to withdraw PEG–ADA treatment, it was not possible to tell whether ADA gene transfer into
T cells was of any benefit for these patients.
Concurrently, several groups had the
ambitious goal to permanently correct ADA
deficiency by inserting the ADA gene into
haematopoietic progenitors7–9,16,17. Three trials were carried out: one that used two vectors
to infect peripheral T cells and bone-marrow
progenitors7; one that targeted cord-blood
progenitors of newborns8,16; and one that targeted bone-marrow cells from older patients9.
Here, again, all patients also received
PEG–ADA. Although the overall outcome
was disappointing (given the expected
hopes), some valuable lessons were learned.
In one trial, three patients received transduced autologous cord-blood cells8,16. A small
percentage of leukocytes was transduced, and
these leukocytes still persisted eight years
later16. There were many more detectable
tranduced T cells than transduced myeloid
cells, a finding that indicates that gene transfer might give a selective advantage to T-cell
precursors. This means that transgene
expression in haematopoietic progenitors
provides a selective survival, growth and,
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possibly, differentiation advantage to lymphocyte precursors, which emphasizes the
important role of ADA activity in the lymphocyte differentiation pathway, compared
with its dispensable role during myelopoiesis.
To detect a survival and/or growth advantage,
three conditions need to be met: transgene
expression should confer survival and/or
growth properties to transduced cells; transduced cells should undergo several cycles of
division before terminal differentiation to
produce a high number of functionally corrected cells; and the resultant differentiated
cells should be long-lived cells, such as T cells.
In this study, ADA gene expression was
switched off in resting T cells and an attempt
to interrupt enzyme supplementation with
PEG–ADA led to a reduction in T-cell counts
and immunity. ADA transgene expression
was not sufficient to restore lymphopoiesis.
In retrospect, ten years later, it is clear that
at that time, ADA deficiency was not the best
disease to choose as a model to investigate
gene therapy. PEG–ADA treatment is likely to
have abrogated the potential for a selective
growth advantage of transduced lymphocyte
progenitors, because untransduced cells could
survive and proliferate. The recent description
of a partial increase in T-cell count and function after PEG–ADA therapy had been discontinued in one patient who underwent ADA
gene transfer into peripheral-blood lymphocytes supports this hypothesis17. Another
obstacle was that because ADA deficiency
Box 1 | Characteristics of retroviral vectors
• Mouse oncoretrovirus (RNA)
• Binding to and entry into human haematopoietic progenitors
• Provirus integration into the genome of dividing cells
• Transgene replication with cell division
• Transgene expression under control of the viral long terminal repeat (LTR) or an
enodogeneous promoter
• Feasibility of the production of replication-incompetent viral particles
• No viral protein expressed
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PERSPECTIVES
Box 2 | Lessons from gene-therapy trials for adenosine-deaminase deficiency
• Transducing billions of T cells was harmless
• Need for preclinical testing in an animal model
• Longevity of transduced T cells
• In the absence of a selective advantage provided to transduced cells, the available gene-transfer
technology does not result in a sufficiently high level of correction
growth advantages as these proteins have a
role only in the function of differentiated
cells. Also, granulocytes have a very short halflife (~2 days), so any transduced cell will be
present in the periphery for only a short time.
Survival, proliferation and longevity of differentiated cells are required to achieve efficient
gene transfer.
Gene therapy of SCID-X
is lethal at birth in mice, no animal model
was available at that time18. It is only recently
that an ADA-deficient mouse model has
become available by using a two-stage
genetic-engineering strategy to rescue these
mice19. Finally, ADA deficiency is also toxic
for the liver, the lung and the central nervous system. It is still unclear whether efficient ADA gene transfer into lymphoid
progenitors would be sufficient to avoid the
metabolic consequences of the disease.
Gene therapy of phagocyte disorders
Innate immunity can be defective in humans
owing to inherited mutations in genes that
encode proteins that are involved in important
functions of phagocytic cells (granulocytes
and monocytes/macrophages). Deficiency in
the expression and/or function of β2-integrin
renders granulocytes unable to migrate into
tissues, which exposes patients to unabated
bacterial infections. This condition is known
as leukocyte adhesion deficiency (LAD)20.
Deficiency in the NADPH oxidase complex,
which is necessary to deliver oxygen radicals
into phagosomes that contain engulfed
microorganisms, also results in susceptibility to
bacterial and fungal infections. This condition
— which is known as chronic granulomatous
disease (CGD) — can be caused by mutations
in four genes that encode the α- and βsubunits of cytochrome b (P22PHOX and
GP91PHOX, respectively) and two cytosolic
proteins (neutrophil cytosolic factor 1
(P47PHOX) and neutrophil cytosolic factor 2
(P67PHOX))21. Given the severity of these
conditions, correction by gene transfer into
haematopoietic progenitors has been
attempted. Successful gene transfer has been
achieved in mouse models using retroviral
vectors22–24. However, clinical trials — which
consisted of ex vivo gene transfer into
haematopoietic progenitor cells that had been
positively selected for CD34 cell-surface
expression and that were mobilized from the
bone marrow into the blood, without the use
of myeloablative treatment — resulted in
only the transient detection of transduced
mature myeloid cells, at a frequency of ≤2%.
Repeated injections of transduced cells did
not improve these results25–28. An immune
NATURE REVIEWS | IMMUNOLOGY
reaction against the transgene product was
not detected and, so, cannot account for this
observation. Here, again, important conclusions can be drawn. These trials were carried
out using the most advanced technology
available — including, high retroviral titres;
pseudotyping with the Gibbon ape leukaemia
virus (GALV) envelope in one setting, the use
of which increases the frequency of target-cell
infection29; the use of cytokines that induce
the division of CD34+ cells; and transduction
in the presence of a fibronectin fragment,
CH-296, which markedly enhances the transduction rate30. Therefore, despite these
advanced techniques, the limited efficiency of
gene transfer in human haematopoietic stem
cells is clear. In the absence of a selective
advantage, if very few stem cells are transduced, the detection of transduced differentiated cells will be very low.
In our opinion, these inherited phagocytic
disorders are poor candidates for gene therapy because the expression of the genes that
encode β2-integrin or NADPH-oxidase components is unlikely to provide any survival or
A subset of SCID conditions is characterized
by faulty T-cell (with or without natural killer
(NK)-cell) development (FIG. 1). These deficiencies occur because signalling downstream
of receptors that contain γc (which includes
the receptors for IL-2, IL-4, IL-7, IL-9, IL-11,
IL-15 and IL-21) is defective. Binding of these
receptors to the appropriate cytokine normally results in the activation of Janus kinases
(JAKs). Then, JAKs phosphorylate cellular
substrates, including signal transducer and
activator of transcription (STAT) proteins,
which translocate to the nucleus and control
downstream signalling. These SCID conditions can occur as a result of the lack of
expression of the γc cytokine-receptor subunit or the expression of a non-functional
form (SCID-X)31 — owing to a defect in the
JAK3 tyrosine kinase that is associated with
the γc tail32,33 — or due to the lack of expression of the IL-7 receptor α-chain (IL-7Rα)34
(FIG. 2). In the latter case, IL-7 cannot deliver
the survival and proliferative signals that are
required for the clonal expansion of T-cell
precursors before T-cell receptor (TCR)β
Mutant or
absent γc
α
Cytokine
JAK
STAT Y P Y
PY
γc
α
Cytokine
JAK
IL-7R
α-chain
absent
α
Cytokine
Cytokine
γc
γc
JAK
Y P Y STAT
YP
Defective
JAK
JAK
JAK
JAK
JAK
Target-gene transcription
and downstream signalling
Figure 2 | Schemes of γ-chain-containing cytokine receptors. Severe combined immunodeficiency
(SCID) results from defects in signalling downstream of cytokine receptors that contain the common
cytokine-receptor γ-chain (γc). γc is a component of the receptors (R) for interleukin-2 (IL-2), IL-4, IL-7,
IL-9, IL-15 and IL-21. Of these receptors, the IL-2R and IL-15R are composed of three subunits (α,β
and γ), whereas the IL-4R, IL-7R, IL-9R and IL-21R are composed of two subunits (α and γ). In the
absence of γc, normal cytokine signalling — which results in the activation of Janus kinases (JAKs) and the
phosphorylation (P) of signal transducer and activator of transcription (STAT) proteins — and resultant
downstream signalling fail to occur. Causes of SCID that have been identified so far include mutations that
result in a deficiency of γc, the expression of a dysfunctional γc, the expression of a dysfunctional JAK
protein (that cannot be activated to phosphorylate STAT molecules) or deficiency of the IL-7R α-chain.
Here, a two-subunit receptor (for example, IL-7R) is shown for simplicity.
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PERSPECTIVES
CD34+ haematopoietic
progenitor cell
γc gene
LTR
Cytoplasm
RNA
DNA
Virus
Nucleus
Cytokine
γc
Chromosome
γc
γc gene
Chromosome
α
Cell proliferation
γc mRNA
Figure 3 | Principle of ex vivo gene therapy of the SCID-X condition. CD34+ cells are incubated
ex vivo with supernatant that contains retrovirus encoding the common cytokine-receptor γ-chain (γc)
gene. Binding of the virus to a cell is followed by viral entry. Viral RNA is retrotranscribed into DNA, which,
as a preintegration complex, can recombine with the cell’s genome. The γc gene can be transcribed,
being under the control of the viral long terminal repeat (LTR), which leads to protein synthesis, membrane
expression and function. mRNA, messenger RNA.
gene rearrangement35. Given the function of
γc, JAK3 and IL-7Rα, any lymphocyte precursor that expresses these proteins should
have a tremendous growth advantage over
non-corrected cells, because the clonal
expansion of T-cell precursors at this stage of
development, at least in mice, is massive35.
This assumption has received strong experimental support from two sets of data. First,
we observed a partial correction of the
immunodeficiency of one SCID-X patient by
means of a ‘natural’ form of gene therapy. This
patient had a small number of normal T cells
(in other words, that expressed a functional γc
protein) in his blood, whereas all other cells
carried the inherited γc gene mutation36. The
only possible explanation for this finding is
that a spontaneous reversion of the mutation
had occurred in a T-cell precursor and, so, had
corrected the γc gene defect. This single event
led to the generation of T cells that comprised
approximately 1% of the TCR Vβ repertoire,
as shown by TCR Vβ–Jβ frequency usage and
37
CDR3-SEQUENCE DETERMINATIONS . These T cells
could be detected over a two-year period. So,
at least 10–11 division cycles occurred
between gene-mutation reversion and TCRβ
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gene-rearrangement events. Therefore, the
extraordinary capacity of T-cell precursors
to proliferate has been shown. Second, Jak3
gene transfer using a retroviral vector into
haematopoietic stem cells (HSCs) from Jak3−/−
mice led to the correction of immunodeficiency38,39. Interestingly, it was shown that
although the frequency of transduced T cells
was 100%, the frequency was much lower for
peripheral myeloid cells, which showed, for
the first time, the experimental validity of
the selective-advantage concept. Meanwhile,
as a prerequisite for clinical trials, the γc gene
was transferred successfully into HSCs from
γc-deficient mice40–42 (FIG. 3). The in vitro correction of γc expression and function in
B cells43–45, T cells and NK progenitor cells46,47
was also shown using ex vivo retroviralmediated gene transfer.
A SCID-X gene-therapy clinical trial was
started in 1999. Patients were enrolled
under the following criteria of eligibility:
the diagnosis of SCID-X, the lack of an
HLA-identical donor and the informed consent of the parents. The protocol consisted of
the EX VIVO TRANSDUCTION of CD34-enriched
bone-marrow cells that were harvested from
the iliac crest (FIG. 4). This trial provided data
that confirmed that the selective-advantage
concept was valid. If we consider patients
who were followed for longer than one year,
the following conclusions can be made10,48.
In four out of five patients, the infusion of
transduced CD34+ cells led to the generation, within 6–12 weeks, of peripheral transduced T cells that had characteristics similar
to those of age-matched controls (in terms
of cell count, subset distribution, TCR diversity and antigen-driven activation). Newly
formed T cells that were emigrating from the
thymus could be detected. So far, correction
of the T-cell immunodeficiency has been
sustained for a period of up to 3.3 years.
Despite a low rate of B-cell transduction,
immunoglobulin production was, at least in
part, restored10,44. Correction of the immunodeficiency was sufficient to prevent the clinical manifestations of SCID in these patients,
such as protracted diarrhoea, and it restored
the normal response to childhood infections.
These four children are, so far, enjoying a
normal life. All T cells and NK cells that are
detectable in the blood of the children carry
and express the γc transgene, as shown by
quantitative PCR analyses and immunofluorescence studies, respectively, whereas not
more than 0.1–1% of myeloid cells and
1–5% of B cells carry and express the γc
transgene. So, a similar gene-transfer technology that was used to transduce human
CD34+ cells led to the efficient gene therapy
of SCID-X, despite the fact that it had no
effect in CGD and LAD. Interestingly, the
fact that a few transduced myeloid cells, as
well as bone-marrow precursor cells, are
detectable up to two years after treatment,
Glossary
BLOOD APHERESIS
The ex vivo selection of blood cells. Unwanted cells are
infused back into the donor.
CDR3-SEQUENCE DETERMINATION
Sequencing of the complementarity-determining
region 3 (CDR3) of rearranged T-cell receptor genes of
individual T-cell clones.
COMMON LYMPHOID PROGENITOR
(CLP). A progenitor that is committed to the lymphoid
lineage that can give rise to all lymphocyte subsets,
including T cells, B cells and natural killer cells.
EX VIVO TRANSDUCTION
The ex vivo insertion of a transgene into cells. Cells are
then placed back into the host.
V(D)J RECOMBINATION
Somatic rearrangement of variable (V), diversity (D)
and joining (J) regions of antigen-receptor-encoding
genes, which leads to the repertoire diversity of both
T- and B-cell receptors.
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PERSPECTIVES
FACS
SCF
MGDF
FLT3L
IL-3
Patients BM is harvested
(30–100 ml)
Anti-CD34
antibody
CD34+ cells
positively
selected
One-day culture
IV cell
infusion
MFG-γc
vector
×3
Infection
Figure 4 | Scheme of the transduction protocol for the SCID-X gene-therapy trial. The patient’s bone marrow (BM) was harvested and the CD34+ cells
were selected. These cells were first incubated with cytokines that can provide survival and proliferative signals, before they were infected (three one-day cycles of
infection) with the retroviral vector. Cells were contained in bags coated with a fibronectin fragment (CH-296), which facilitated cell–virus interaction. After
completion of the four-day procedure, cells were washed and injected back into the patient intravenously (IV) without additional therapy. FACS, fluorescenceactivated cell sorting; FLT3L, FLT3 ligand; IL-3, interleukin-3; MFG, retroviral vector; MGDF, megakaryocyte growth and development factor; SCF, stem-cell factor.
indicates that some transduced pluripotent
progenitors do persist in the bone marrow of
these children. Combined with T-cell
longevity, this finding raises the hope that
the correction of the immunodeficiency
could be fairly stable. Nevertheless, γc gene
silencing or the loss of precursors could temper this optimistic view (BOX 3).
revertants, it seems that one T-cell precursor
can restore ~1% of the T-cell repertoire37. If
we assume a linear additive effect, a few hundred cells might be sufficient to provide, at
least for some years, a full T-cell repertoire. If
correct, this assumption raises the hope that
the other T-cell immunodeficiencies that are
the result of faulty T-cell development should
be amenable to gene therapy.
The future of gene therapy for SCIDs
These results provide proof of principle for
the potential efficacy of gene therapy, provided that a significant selective advantage is
conferred to transduced cells. The studies
need to be confirmed in a larger series of
patients to assess their precise medical potential. They also constitute a basis from which
extension to the treatment of other immunodeficiencies or genetic defects of the
haematopoietic system can be envisaged.
Remaining questions
How many transduced cells are required? The
answer to this question might be determined
by competitive experiments in animal models
by administering a given number of untransduced cells with an increasing number of
transduced cells. According to the observations that have been made in spontaneous
What about NK- and B-cell development? In
the SCID-X gene-therapy trial, it has been
observed that, although transduced NK cells
can be detected, their actual counts are fairly
low. This is comparable to observations in
SCID recipients of allogeneic stem-cell transplantation. The reasons for this are unknown,
and it remains to be analysed whether it is due
to limitations in NK-precursor cell division
and/or survival.
Transduced B cells account for a lower
percentage of circulating B cells in SCID-X
patients after γc gene transfer into CD34+
cells than is true for the T-cell population.
This could be the consequence of competition for space with the large number of
untransduced B cells. Alternatively, it could
be owing to the much lower ability of B-cell
precursors to proliferate and/or a weaker
Box 3 | The outcome of gene therapy for SCID-X10,48
Benefits
• Development of diversified T cells
• Sustained correction of the T-cell deficiency (up to three years)
• Multiple integration sites in T cells, which indicates the polyclonal origin of T cells
• Disappearance of patients’ susceptibility to opportunistic infection
Limitations
• Low natural-killer-cell counts (as seen after allogeneic haematopoietic stem-cell transplantation)
• Low percentage of transduced B cells (although antibody production does occur)
• Long-term outcome is unknown (potential gene-silencing effects or exhaustion of transduced
progenitors)
NATURE REVIEWS | IMMUNOLOGY
selective advantage conferred by the expression of γc on B cells compared with T cells.
The treatment of B-cell-deficient SCIDs,
such as recombination-activating gene 1
(RAG1)/RAG2 or Artemis deficiencies, should
tell us how feasible is the correction of B-cell
deficiency after gene transfer into CD34+ cells.
What is the optimal target for gene transfer?
For the treatment of inherited lymphocyte
deficiencies, efficient gene transfer into selfrenewing haematopoietic stem cells would be
the obvious solution. However, the integration
complex of the vectors that can be used in the
clinic at present cannot cross the nuclear
membrane. Stem cells, which are mostly noncycling, have an intact nuclear membrane and
are, therefore, refractory to gene transfer13.
However, for SCIDs, given the potentially
small number of transduced cells that is
required for efficacy (see above), it is possible
that the small number of cycling stem cells can
be transduced, eventually ensuring disease
correction. This optimistic assumption can be
tested by seeing whether transduced lymphoid
and myeloid cells are persistently detected
over time. In mice, it is generally accepted that
COMMON LYMPHOCYTE PROGENITORS (CLPs) give
rise to the different lymphocyte lineages in
appropriate environments49. If CLPs exist in
the lymphocyte-lineage differentiation pathway in humans, these would be an attractive
target for lymphoid-specific gene therapy to
avoid expression of the transgene in myeloid
cells. As CLPs do potentially express markers
for selection, this approach might be possible50. The key question concerns the lifespan
of such cells, which, in the mouse, do not have
self-renewal capacity 49.
Which diseases to consider for gene therapy?
Every condition for which the wild-type
product of a defective gene can confer a selective advantage to transduced lymphocyte
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PERSPECTIVES
precursors is a possible model for gene therapy. This applies to all forms of SCID (FIG. 1).
Above all, the JAK3 and IL-7Rα deficiencies
share the same mechanism and should, in
principle, be as responsive to gene therapy as
SCID-X. The fact that a gene reversion also
partially corrected an ADA-deficiency phenotype strongly indicates that ADA deficiency
could be on the list as well, provided that supplementation with PEG–ADA is omitted51.
During thymocyte development, defects in
V(D)J RECOMBINATION lead to thymocyte death at
a step that follows γc-dependent cell proliferation. It is tempting to speculate that the
induction of expression of RAG1, RAG2 or
Artemis in lymphocyte precursors would
indirectly provide the expected selective
advantage by enabling the survival of cells
after TCR (or B-cell receptor) rearrangements52,53. Sustained expression of the transgene, at least for RAG1 and RAG2, is not
necessary in mature T and B cells, which
makes these conditions even more attractive.
Experiments are required to test this hypothesis. The later the block in T-cell development,
the less likely it is that gene transfer will lead
to T-cell-precursor survival and development.
Nevertheless, the recent observation of a partial correction of T-cell immunodeficiency in
an adult patient with Wiskott–Aldrich syndrome (WAS) extends this concept54. That the
immunodeficiency status of the patient
improved markedly is very promising for the
development of gene therapy for WAS. WAS
is caused by mutations of the gene that
encodes WASP, a permanently expressed protein that is involved in actin-cytoskeleton
rearrangement in haematopoietic cells55.
Lymphocyte development is normal in WAS
patients, whereas T-cell activation, migration
and survival are impaired55. The consequences of WASP gene reversion are crucial,
as this is the first demonstration that a
peripheral T-cell deficiency can be overcome,
at least in part, by gene correction in a T-cell
precursor. This might lead to a marked
widening of the scope of gene therapy in the
field of primary immunodeficiencies.
How to improve gene transfer? The limitation
of available vectors, in terms of crossing the
nuclear membrane, hampers the further
application of gene therapy. Nevertheless,
there are two potential tracks to follow. One
consists of creating a positive selection pressure for transduced cells. This can be achieved
by transducing a second gene that encodes a
selectable marker — such as a drug-resistance
product (multidrug resistance 1, MDR1; or
dihydrofolate reductase, DHFR) or a membrane protein (IL-2 receptor α-chain or
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CD24) — which enables enrichment for
transduced cells, using a vector with an internal ribosomal entry site (IRES) element that
allows independent translation from a common messenger RNA56–58. Preliminary experiments show that this is feasible, although
proof of in vivo efficacy is awaited.
The alternative approach consists of
improving the technology of gene transfer
into HSCs. Much work is being devoted to
this important task. Approaches that are
being tested include the use of viral envelopes
— such as the RD114 envelope of a feline
retrovirus59 — that bind to specific receptors
that are expressed by HSCs with higher affinity, the prevention of gene silencing by deleting inhibition sequences60 or the use of
lentiviral vectors that can transduce noncycling cells61. Indeed, the pre-integration
complex of lentiviruses, such as HIV-1, can
cross the nuclear membrane and allow for
efficient integration. This characteristic has
been used successfully to show that human
haematopoietic stem cells can be efficiently
transduced58. If the safety issues that are
raised by HIV-derived vectors can be
resolved, the use of such vectors should be an
important development in this field.
Combined with the use of restricted lineage
expression, as shown for haemoglobin
expression in erythroid cells62, this technology could make the treatment of many more
genetic disorders feasible.
Analysis of what has been achieved and a
look to the future lead to the same conclusion
— that efficient gene therapy requires both an
in-depth understanding of the mechanism of
the targeted disease and the development of
new, adapted technologies. The design of vectors that allow the transduction of non-cycling
stem cells is, in this respect, a source of hope.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Note added in proof
Recently, Aiuti et al.63 have reported a sustained
and significant correction of the immunodeficiency that is caused by ADA deficiency by
retroviral gene transfer into marrow progenitor cells of patients who did not receive enzyme
supplementation. These results confirm the
validity of the selective-advantage concept as
applied to gene therapy.
Alain Fischer, Salima Hacein-Bey and Marina
Cavazzana-Calvo are at INSERM U429, Hôpital
Necker, 149 rue de Sèvres, 75015 Paris, France.
Correspondence to A.F.
e-mail: [email protected]
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Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www/ncbi.nlm.nih.gov/Entrez/
GALV | HIV-1 | Moloney murine sarcoma virus
LocusLink: http://www/ncbi.nlm.nih.gov/LocusLink/
ADA | Artemis | β2-integrin | CD3ε | CD3γ | CD24 | CD34 |
CD45 | DHFR | fibronectin | FLT3L | γc | GP91PHOX | IL-2 | IL-3 |
IL-4 | IL-7 | IL-7Rα | IL-9 | IL-11 | IL-15 | IL-21 | IL-2 receptor
α-chain | JAKs | JAK3 | Jak3 | MDR1 | MGDF | NADPH
oxidase | P22PHOX | P47PHOX | P67PHOX | RAG1 | RAG2 |
SCF | STATs | WASP | ZAP70
OMIM: http://www/ncbi.nlm.nih.gov/Omim/
ADA deficiency | CGD | LAD | SCID-X | WAS
FURTHER INFORMATION
American Society of Gene Therapy: http://asgt.org/
European Society for Immunodeficiencies:
http://www.esid.org/
European Society of Gene Therapy: http://www.esgt.org/
Access to this interactive links box is free online.
VOLUME 2 | AUGUST 2002 | 6 2 1