Available online at www.sciencedirect.

com

Gene therapy for primary immunodeficiencies: part 1

Marina Cavazzana-Calvo1,2,4,5, Alain Fischer1,3,4,
Salima Hacein-Bey-Abina2,4,5 and Alessandro Aiuti6,7
Over 60 patients affected by SCID due to IL2RG deficiency
(SCID-X1) or adenosine deaminase (ADA)-SCID have received
hematopoietic stem cell gene therapy in the past 15 years using
gammaretroviral vectors, resulting in immune reconstitution
and clinical benefit in the majority of them. However, the
occurrence of insertional oncogenesis in the SCID-X1 trials has
led to the development of new clinical trials based on
integrating vectors with improved safety design as well as
investigation on new technologies for highly efficient gene
targeting and site-specific gene editing. Here we will present
the experience and perspectives of gene therapy for SCID-X1
and ADA-SCID and discuss the pros and cons of gene therapy
in comparison to allogeneic transplantation.
Addresses
1
U768 INSERM, Paris, France
2
Departement de Biotherapie, AP-HP, Hopital Universitaire Necker
Enfants Malades, Paris, France
3
Unite dImmuno-Hematologie Pediatrique, Hopital Universitaire Necker
Enfants Malades, Paris, France
4
Universite Paris Descartes, Sorbonne Paris Cite, IMAGINE Institute,
Paris, France
5
CIC Biotherapie GHU Ouest, INSERM-APHP, Paris, France
6
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan,
Italy
7
Department of Pediatrics, University of Rome Tor Vergata, Childrens
Hospital Bambino Gesu`, Rome, Italy

replacing the diseased hematopoietic lineages with normal ones. These results prove that transplantation of
normal hematopoietic stem cells (HSC) or their progenitors can correct a variety of PIDs of the adaptive and
innate immune systems. However, HSCT is associated
with several serious adverse events, including the toxicity
of myeloablative chemotherapy and, above all, the consequences of the potential immune conflict between
donor and recipient. In the absence of an appropriate
donor, the addition of a corrected copy of a gene into
autologous HSC is an appealing strategy. The hematopoietic system displays optimal characteristics for the ex
vivo gene therapy purposes.
Its accessibility, ability to survive in ex vivo cell culture
and transplantability are extremely favorable parameters
for their use in gene therapy. In fact, over twenty years of
research on ex vivo manipulations of this stem cell compartment in association with several years of intense
research in virology has enabled rapid advances in this
field. We will try to summarize here where we are in
comparison with HSCT and the expected future progress

SCID-X1: the proof of principle

Introduction

The most frequent form of SCID is SCID-X1. The

condition is caused by a lack of the common gc cytokine
receptor subunit, which causes a complete block in T-cell
and natural killer (NK) cell development [1]. It was
thought that SCID-X1 was the most accurate model for
assessing gene therapy (GT) because spontaneous reversion of the mutation in the gc-encoding IL2RG gene led
to significant correction of the immune deficiency. This
observation supported the hypothesis whereby transduced lymphocyte progenitors carry a selective advantage
over their non-transduced counterparts [2]. Furthermore,
IL2RG is expressed by all hematopoietic cells and is not
tightly regulated thus reducing the risk of toxicity
related to aberrant expression. It was thus expected that
gene correction of a proportion of lymphoid progenitors
would be enough to restore at least the T-cell compartment and that the effect would be long-lasting.

Many primary immunodeficiencies (PIDs) are lethal diseases. In the absence of treatment, severe combined
immunodeficiencies (SCIDs) cause death within the first
year of life. Other PID variants combine deficiencies of
adaptive immunity (such as WiskottAldrich syndrome
[WAS]) and diseases causing hemophagocytic lymphohistiocytosis [HLH] that can also be fatal in young
children. Allogeneic hematopoietic stem cell transplantation (HSCT) can cure many of these disorders by

Thus, following in vitro and in vivo preclinical tests in gcdeficient mice, several clinical trials were performed. The
vectors were based on the use of retroviral (RV) [3,4]
constructs in which the common gc cytokine receptor
subunit was placed under the transcriptional control of
the long terminal repeat (LTR), with the use of either an
amphotropic envelope or the gibbon ape leukemia virus
envelope. Between 1999 and 2006, 20 subjects with

Corresponding author: Cavazzana-Calvo, Marina

(m.cavazzana@nck.aphp.fr)

Current Opinion in Immunology 2012, 24:580584

This review comes from a themed issue on Immunogenetics and
transplantation
Edited by Alain Fischer and Matthew Porteus
For a complete overview see the Issue and the Editorial
Available online 12th September 2012
0952-7915/$ see front matter, # 2012 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.coi.2012.08.008

Current Opinion in Immunology 2012, 24:580584

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Gene therapy for primary immunodeficiencies Cavazzana-Calvo et al. 581

SCID-X1 were treated in two trials in Paris and then

London [5,6]. All the subjects lacked an HLA-identical
donor and received ex vivo-transduced CD34 cells in the
absence of any additional therapy.
The efficacy of gc gene transfer has been clearly demonstrated. Between 5 and 12 years after GT, 17 of the 20
treated subjects are alive and display full or nearly full
correction of the T-cell immunodeficiency [5,6], including normal T-cell subset counts, sustained detection
of nave T cells (even in the subjects who had been
treated for leukemia), a diversified T-cell repertoire and
normal T-cell-mediated immune functions. The gc gene
transfer led to clear clinical benefits because patients first
recovered from ongoing infections with a poor prognosis
(such as disseminated BCG and VZV infections) and were
then able to live in a normal environment without any
evidence of particular susceptibility to infection.
The same protocol has been applied to treat patients with
atypical SCID-X1 (caused by hypomorphic mutations)
and those who displayed limited T-cell reconstitution
following HSCT. Effective CD34-cell transduction
resulted in little or no improvement in T-cell production
probably as a consequence of thymic function loss [7,8].
The extent of correction of the NK cell deficiency in the
two trials for the conventional SCID-X1 form was not as
impressive. Despite an early rise, NK cell counts eventually fell to very low values. Remarkably, this was also
observed in non-myeloablated SCID-X1 patients who
had undergone allogeneic HSCT. A partially persistent
NK cell deficiency does not appear to be harmful. This
finding indicates that the dynamics of NK populations
(i.e. cell development, expansion and survival) differ
significantly from those of T cells. The B cells were
poorly transduced (<1%) and were not detected in the
blood 23 years after GT. This is somehow expected in
the absence of pre-conditioning, which is required for
effective HSC engraftment. Despite the apparent lack of
persistent gc + B cells, most patients do not require
immunoglobulin substitution. Overall, these results constitute the first proof of principle of long-term, effective
GT. The persistence of T-cell receptor excision circles
(TREC)+ naive T cells and their reappearance in patients
who had undergone chemotherapy 36 years after GT
suggests strongly that hematopoietic progenitor cells with
self-renewal capacity persist and are performing sustained
thymopoiesis. Vector integration studies are helping us to
characterize the biology and life span of T-cell progenitors and their long-lasting progeny [9]. Thus, more than
10 years later and despite the low numbers of patients
treated by GT (by comparison with the greater numbers
of HLA-genoidentical allogeneic HSCT), the biological
and clinical results of these two types of therapy are
highly similar to the point of being indistinguishable
[5,10]. Nevertheless, severe adverse events have
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occurred, with five of the 20 subjects having developed

T-cell leukemia 2.55 years after GT [1113]. In four
cases, chemotherapy was easily able to destroy abnormal
clones; these children are currently in remission and
doing well. However, the remaining subject died from
chemotherapy-refractory leukemia [11]. The occurrence
of these serious complications prompted discontinuation
of these trials.
Considerable efforts have since been made to understand
the mechanism by which this unexpectedly frequent
complication had occurred. It was found that proliferative
clones carried RV integrations within oncogene loci
[10,11]. One oncogene in particular (LMO-2) had been
targeted in four out the five cases. It became clear that
RV-mediated gene transfer could deregulate proto-oncogene expression through the LTRs enhancer activity. It
is nevertheless likely that additional factors are involved
because none of the subjects treated successfully with a
similar gene-therapy approach for another SCID (adenosine deaminase [ADA] deficiency [14,15]; see below)
developed leukemic complications despite the fact that
a similar RV integration pattern was found [16,17]. No
definitive evidence to explain this difference has been
presented. These data pave the way for further use of GT
in patients with SCID-X1; indeed, an ongoing international trial (running in the United Kingdom, United
States and France) is using a self-inactivating (SIN) RV
vector which should reduce the risk of insertional mutagenesis. Alternative approaches based on gene correction
and targeted integrations using nucleases have achieved
so far relatively low levels of correction in hematopoietic
progenitors in vitro, but these could be compensated by
the selective advantage for successfully corrected cells
[18]. Studies to thoroughly assess the potential impact of
these nucleases on the genome will be required before
moving to clinical application for SCID [19,20].

The experience of ADA-SCID gene therapy

ADA-deficiency has been an attractive target of gene
therapy approaches since the early 1990s. ADA is a purine
salvage enzyme expressed in all tissues of the body, which
catalyses the irreversible deamination of 20 -deoxyadenosine and adenosine to 20 -deoxyinosine and inosine
respectively. ADA-SCID is autosomal recessive disorder
constituting the cause of approximately 1520% of all
cases of SCID. In addition to the typical SCID manifestations, the ubiquitous nature of ADA expression leads to
a number of significant non-immunological abnormalities
in several organs as a result of the accumulation of the
purine toxic metabolites, including bone, neurological,
and lung defects [21]. Allogeneic HSCT from matched
sibling and family donors is the treatment of choice for
ADA-SCID while survival is significantly reduced in
transplant from matched unrelated (66%) or haploidentical donors (43%) [22]. For patients who survive transplant, long-term cellular and humoral immune recovery is
Current Opinion in Immunology 2012, 24:580584

582 Immunogenetics and transplantation

achieved. Enzyme replacement therapy (ERT) with

PEG-ADA is another option for ADA-SCID patients.
Although PEG-ADA therapy is well tolerated and can
restore immune function to protective levels, long-term
follow-up suggests an incomplete immune recovery with
progressive decline over time [21]. Gene therapy with
ADA-transduced autologous stem/progenitor cells
represents an alternative option for ADA-SCID patient.
Since year 2000, 40 patients have been treated in Italy,
UK, and USA, with CD34+ cells transduced with gammaretroviral vector encoding the ADA gene, achieving
substantial clinical benefit in the majority of them.
Patients received low intensity conditioning with Busulfan (Italy and USA) or Melphalan (UK) which was
required to allow engraftment of multilineage stem/progenitor cells. In addition they stopped enzyme replacement therapy to take advantage of the selective growth
advantage for gene corrected over defective cells. At
present, all patients are alive and in 29 of them ERT
is no longer required ([14,15,23] and unpublished observations). Gene therapy resulted in persistent engraftment
of transduced cells, increase of lymphocyte counts, improvement of cellular and humoral responses, and adequate metabolic detoxification [14,15]. Gene corrected
cells were detected in myeloid and lymphoid subsets, the
latter being more represented due to their survival
advantage [14,15]. Studies on the kinetics of immune
reconstitution in the Milan trial showed that T-cell
homeostatic expansion contributes substantially to
immune recovery, similarly to HSCT, but is not associated with senescence in the stem cell compartment [24].
We also found that functional defects in Treg cells [25]
and alterations in B-cell tolerance [26] detected at baseline were corrected after gene therapy. Moreover, childrens growth and bone age improved following
treatment, although was not normalized in all patients
[27].
The cumulative experience of these studies did not
reveal leukemic or oncogenic events, unlike other PID
trials with gammaretroviral vectors indicating that ADASCID gene therapy has a better risk/benefit profile.
Indeed, vector integration analyses did not reveal in vivo
skewing or expansion of potentially dangerous clones
[17,28]. The use of self-inactivating lentiviral vectors
may further reduce the risk of insertional mutagenesis
and at the same time increase gene transfer efficiency into
hematopoietic stem/progenitor cells and their progeny.

Gene therapy for Artemis: towards clinical

application
Among the SCID forms, the T-B-SCID due to Artemis
gene mutation is another excellent candidate for a GT
approach and a strong rationale supports its clinical application. First, a survey performed at the Necker hospital
on the long-term outcome (i.e. 30 years) of 90 SCID
patients clearly show some specific treatment difficulties
Current Opinion in Immunology 2012, 24:580584

of these patients affected by an increased cell sensitivity

to irradiation. The survival probability at 2 years after
HLA partially incompatible HSCT is 50% for the V(D)J
defects. For the Artemis patients, we have discerned an
increased post-transplantation morbidity characterized by
auto-inflammatory complications, long-term need of parenteral nutrition, severe and persistent signs of chronic
GvHD, recurrent and severe infections due to an unsatisfying immunological reconstitution. The unusual persistence of severe clinical signs due to cGvHD is due to
their incapacity to repair the GvHD induced organ lesions
depending on the importance of this gene in the DNA
repair mechanisms [29]. In addition, extensive preclinical
work using an Artemis K.O. mice model and the Artemis
neg CD34+ human samples has well demonstrated the
biological efficacy of this approach without any evidence
of severe adverse event [30,31]. Thanks to initiative of
European networks (i.e. CELL-PID) and the manufacturing capacity of Genethon, we hope to initiate a new
European multicentric study in the early 2013.

Tentative comparison: hematopoietic stem

cell transplantation versus gene therapy
clinical results
For several PIDs that are lethal within the first year of life
or in childhood, allogeneic HSCT represents the treatment of choice. At present, HSCT from an HLA-matched
sibling donor confers at least a 90% chance of cure for
children suffering from a SCID and about an 80% chance
of cure for children affected with non-SCID PIDs [10].
The survival rate is significantly lower when a related but
HLA-mismatched donor (i.e. the mother or father) is used
(66% and 55% for SCID and non-SCID patients, respectively. Surprisingly, for SCID patients, the 3-year survival
rate reported in 2010 for matched unrelated donors
(MUDs) by the Inborn Errors Working Party of the
European Group for Blood and Marrow Transplantation
and the European Society for Immunodeficiency yielded
values similar to those observed in a haploidentical
setting [10].
There are several possible explanations for the latter
unexpected results. Firstly, the two transplanted groups
(MUDs vs. haploidentical donors) differed in terms of the
distribution of different SCID forms a key parameter
that may well interfere with the outcome. The low
number of patients in the MUD group makes it difficult
to draw firm conclusions as to the donor choice issue. In a
recently published international collaborative study of
106 adenosine deaminase (ADA)-deficient patients who
underwent 119 transplants, the donor type turned out to
be the most important factor influencing the outcome.
The survival data showed that MUD transplants were
much better than those for haploidentical transplants,
being HLA-identical sibling donor the best option
when available [22]. Lastly, an important contribution
by Brown et al. [32] has enriched the debate on the role of
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Gene therapy for primary immunodeficiencies Cavazzana-Calvo et al. 583

alternative therapeutic choices (i.e. HSCT vs. GT) for

SCID patients. The authors showed clearly that SCID
babies transplanted at birth because of early diagnosis due
to positive family history have an overall survival rate of
over 90% irrespective of the donor profile, conditioning
regimen and underlying genetic diagnosis. Thus, a
personalized choice of therapy for SCID patients must
include the age at transplant, the presence of ongoing
opportunistic and/or severe infections, the SCID diagnosis and recipient/donor compatibility. Based on the
long-term results of both allogeneic HSCT and the first
SCID-X1 gene therapy trials [5,6,10,15] this latter
option should be reserved for SCID patients aged over
1 month and for whom an HLA-identical sibling donor or
a perfectly matched unrelated donor (of bone marrow or
cord blood) will not be available in the coming three
months. For ADA-SCID, the availability of enzyme
replacement therapy and the reduced survival of MUD
transplant should be carefully considered in the decision
[21].
The experience in the SCID-X1 and ADA-SCID confirmed the initial assumption that gene therapy
approaches carry a low risk of early complications as
compared to allogeneic transplant due to the lack of graft
versus host disease (GvHD) and reduced risk of infections in the absence of immune suppression and when no
or mild conditioning is administered. However, it is also
important to note that GT faces obstacles that are common to all HSCT settings, such as (i) the need to
condition the patient when high engraftment of genecorrected hematopoietic stem/progenitor cells is required;
(ii) the time required to achieve restoration of adaptive
immune restoration (which, to date, does not appear to be
significantly shorter than that observed after CD34+immunoselected allotransplantation); and (iii) the need
for appropriate evaluation of thymic function on a per
disease basis. These shared challenges are even more
important for non-SCID conditions, such as Wiskott
Aldrich syndrome, chronic granulomatous disease and
familial lymphohistiocytosis. For all the latter conditions,
the 4-year survival rate showed a marked improvement
over the period 20002005 for patients transplanted with a
MUD and was exactly the same as that observed with a
matched sibling (i.e. 79%) [10].
In addition GT presents specific, open issues firstly the
long-term safety and secondly the use of patients bone
marrows stem cell and precursor cells which may have an
abnormal composition or could be more susceptible to
mutagenesis. In terms of safety, it is important to note
that all but one of the first-generation clinical trials (based
on the use of gamma oncoretrovirus-derived vectors)
encountered severe adverse events (SAE) due to insertional mutagenesis [11,33,34]. No clear explanation has
been found to the absence of any SAE in the ADA-SCID
gene therapy trial. The specific physiopathology of this
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disease and the function of the ADA transgene could

participate to this observation. The long-term clinical
results of ongoing trials using self-inactivated vectors
are eagerly awaited, with the expectation that they will
better define the role of GT with respect to conventional
HSCT. These constructs are specifically designed to
reduce the probability of vector-mediated transactivation
of neighboring genes by the elimination of the enhancer
sequences from viral LTRs. The stable, long-term prevention of severe adverse events (or a significant
reduction in their frequency) will guide us in broadening
the indications for GT in SCID patients. To date, no
adverse events due to insertional oncogenesis have been
reported for the new generation of self-inactivated retroviral or lentiviral vectors.

Acknowledgements
Supported by grants from: the European Commission: Advanced Cell-based
Therapies for the treatment of Primary ImmunoDeficiency (Cell-PID),
HEALTH F5-2010-261387; the Italian Telethon Foundation; the European
Research Council (ERC): Stem cell and gene therapy approaches for
inherited diseases with unsatisfying or no therapeutic option, ERC-2010AdG_20100317.

References and recommended reading

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