Adrian J. Thrasher, Salima Hacein-Bey-Abina, H. Bobby Gaspar, Stephane Blanche, E. Graham Davies, Kathryn Parsley, Kimberly Gilmour,
Douglas King, Steven Howe, Joanna Sinclair, Christophe Hue, Fre ´de ´rique Carlier, Christof von Kalle, Genevie `ve de Saint Basile,
Franc ¸oise le Deist, Alain Fischer, and Marina Cavazzana-Calvo
Gene therapy has been shown to be a
highly effective treatment for infants with
typical X-linked severe combined immu-
patients in whom previous allogeneic
attenuated disease who may present later
in life, the optimal treatment strategy in
the absence of human leukocyte antigen
(HLA)–matched donors is unclear. Here
we report the failure of gene therapy in 2
such patients, despite effective gene
transfer to bone marrow CD34?cells,
suggesting that there are intrinsic host-
dependent restrictions to efficacy. In par-
ticular, there is likely to be a limitation to
initiation of normal thymopoiesis, and we
therefore suggest that intervention for
these patients should be considered as
early as possible. (Blood. 2005;105:
© 2005 by TheAmerican Society of Hematology
X-linked severe combined immunodeficiency (SCID-X1) is
caused by mutations in the gene encoding the common cytokine
receptor gamma chain (?c), inducing the complete absence of
mature T and natural killer (NK) cells.1Attenuated forms of
?c-deficiency have been described, and due to atypical clinical
and immunological phenotypes, may well be underrecognized.2
Human leukocyte antigen (HLA)–genoidentical hematopoietic
stem cell transplantation (HSCT) confers a 90% chance of
disease-free survival in the first 10 to 20 years. In contrast,
haploidentical HSCT is associated with an overall 75% survival
rate at 5 years and a significant risk of graft-versus-host disease
(GVHD).3,4In addition, over a long term, there is often evidence
for persistent B-cell deficiency and, in some cases, progressive
defects of cellular immunity as a result of an exaggerated
decline in thymic output.5-8
Recently, 2 clinical studies of somatic gene therapy for
SCID-X1 have demonstrated sustained correction of immunode-
ficiency in 15 out of 16 infants with classical disease despite the
occurrence of a clonal proliferation in 2 of them.9-12However, it
remains unclear whether this strategy can be effectively applied
to patients in whom the disease phenotype is attenuated, and
also to patients with residual defects or failing immunity
following previous HSCT.
Approval was obtained from the review boards of the Institute of Child
Health and the Great Ormond Street Hospital for Children (London), the
Ho ˆpital Necker and the INSERM (Paris), and the Cincinnati Children’s
Research Foundation (OH) for these studies. Informed consent was
provided according to the Declaration of Helsinki.
Patient 1. Patient 1 (P1) was the first child of consanguineous
parents and was 20 years old at the time of gene therapy. He was
diagnosed soon after birth and found to have a 16 base pair (bp)
deletion within exon 5 of the ?c gene leading to a frameshift at
F221 and abolition of cell-surface protein expression. He received
a whole HLA phenoidentical paternal HSCT at the age of 3 weeks
without any prior myeloablative conditioning. Over the following
losing enteropathy. Reassessment of his immune status revealed
marked T-cell deficiency, absent NK cells, and low numbers of B
cells. A magnetic resonance imaging (MRI) of his chest confirmed
bronchiectasis, and absence of thymic tissue.Attempts to locate his
estranged father failed, ruling out the possibility of a supplemental
HSCT. An unrelated donor search was also unsuccessful. Follow-
ing ethical and regulatory approval, and with his fully informed
consent, he was treated by gene therapy.
From the Molecular Immunology Unit, Institute of Child Health, London, United
Kingdom; Department of Biotherapy, Unite ´ d’Immunologie et d’He ´matologie
Pe ´diatriques, Institut National de la Sante ´ et de la Recherche Me ´dicale
(INSERM) Unit 429, and Laboratoire d’Immunologie Pe ´diatrique, Ho ˆpital
Necker, Paris, France; Immunology Unit, Great Ormond Street Hospital for
Children, London, United Kingdom; and Cincinnati Children’s Research
Foundation, Cincinnati, OH.
Submitted December 20, 2004; accepted January 14, 2005. Prepublished
online as Blood First Edition Paper, February 1, 2005; DOI 10.1182/blood-
Supported by grants from INSERM, lst Association Franc ¸aise contre les
Myopathies, Le Programme Hospitalier de Recherche Clinique of the
French Health Ministry, the Assistance Publique-Ho ˆpitaux de Paris, EC
contract no. QLK3-LT 2001 (coordinator, G. Wagemaker), the Jeffrey Modell
Foundation, The Welcome Trust (A.J.T.), the Primary Immunodeficiency
Association, the Jeans for Genes Appeal, and the Chronic Granulomatous
Disease Research Trust.
A.J.T. and S.H.-B.-A. contributed equally to this work.
An Inside Blood analysis of this article appears in the front of this issue.
Reprints: Marina Cavazzana-Calvo, Department of Biotherapy, Ho ˆpital Necker,
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by TheAmerican Society of Hematology
4255 BLOOD, 1 JUNE 2005?VOLUME 105, NUMBER 11
Patient 2. P2 was born in 1985 to unrelated parents. From 5
years of age, he developed recurrent respiratory tract infections,
protracted diarrhea, and extensive molluscum contagiosum. Immu-
nologic investigations revealed a profound CD4?lymphopenia,
although T, NK, and B cell counts were normal. Serum levels of
immunoglobulin (Ig) G, IgA, and IgM were normal or increased.
IgG2, IgG3, and IgG4 levels were low and antibodies to immuniza-
tion antigens were undetectable. In vitro T-cell responses showed
normal proliferation to mitogens and very low or absent prolifera-
tion to specific antigens. Over the subsequent 10-year period, his
clinical status deteriorated and he developed bronchiectasias,
chronic pulmonary failure, protracted diarrhea, and recurrent
herpes zoster virus infection. The search for a ?c gene mutation
revealed a 664 C to A, R222S mutation in exon 5. While ?c
expression was weakly detected on T cells, CD34?cells did not
express the protein. At the age of 15 years, he was considered for
HSCT but no genoidentical or HLA-matched unrelated donors
were available. Following ethical and regulatory approval, and
with his fully informed consent, he was treated by gene therapy.
Gene transfer protocols
Details of gammaretroviral vectors and transduction protocols have been
In vitro investigation
Lymphoid and hematopoietic cell subsets were phenotypically character-
ized by the labeling of whole blood with combinations of directly
conjugated monoclonal antibodies, all purchased from Becton Dickinson
Biosciences (San Jose, CA). Proliferation assays, T-cell receptor (TCR)
spectratypes, T-cell receptor signal joint excision circles (TRECs), and
provirus integration (MFG-B2-?c) were measured as previously de-
scribed.10,11The copy number of the ?-chain transgene was determined by
real-time polymerase chain reaction (PCR) after extracting DNAfrom cells
described. Insertion site analysis for visualization of the clonal contribution
mediated PCR (LAM-PCR) as previously described.11,13
Results and discussion
Following transduction, 2.8 ? 106and 35 ? 106total cells/kg were
infused into P1 (body weight ? 53 kg) and P2 (body weight ? 34
kg) respectively, 30% and 13% of which coexpressed CD34 and ?c
(Figure 1). This transduction rate is comparable to that obtained for
patients younger than 1 year of age as previously reported.9,10No
adverse events were observed. Both patients were followed up for
at least 180 days (Table 1).
For P1, despite persisting PCR signals indicating the presence
of transgene at low levels, there was no significant change in
immunological parameters or clinical status up to 2 years after gene
therapy. Specifically, there was no evidence for production of naive
CD45RA?or CD27hiCD45RO?T cells, and the numbers of
TRECs remained very low (? 20/105peripheral blood lympho-
cytes [PBLs]). Molecular analysis of integration sites in sorted
blood lineages performed 6 months after gene therapy revealed
polyclonal marking in B, NK, and myeloid cells comparable to that
observed in other patients treated by the same protocol. This
indicated successful transduction and engraftment of CD34?cells.
However, the pattern of T-cell marking showed few clones (Figure
1, right panel). These observations are consistent with levels of
marking in purified T, B, and myeloid lineages less than 1%
determined by quantitative PCR up to 2 years after engraftment,
and are suggestive of a host-dependent restriction to T-cell
Figure 1. CD34?cell transduction and LAM-PCR analysis of peripheral leuko-
cyte subsets. (A) ?c expression on P1 CD34?cells before and after transduction. (B)
LAM-PCR analysis of the vector insertion site restriction length polymorphism
present in peripheral blood leukocytes of P1. DNA samples of approximately 1 to 50
ng isolated directly from sorted peripheral blood leukocytes were analyzed at 6
months after gene therapy. Prominent bands in all lanes at the bottom of the gel are
internal PCR controls. Left lane shows 100 bps DNA marker; other lanes: T, CD3?
cells; B, CD19?cells; NK, CD16/56?cells; and M, CD33?myeloid cells.
Table 1. Immunologic and molecular investigations of the patient’s peripheral blood leukocytes
? 15 d
? 15 d
? 60 d
? 120 d
? 180 d
valuesP1P2P1 P2P1P2P1P2P1 P2
ALC, ? 109/L
?c integration study
ND indicates not done;ALC absolute lymphocyte counts; — indicates no control value.
*Positive signal studied by quantitative PCR shows ? 1% level of gene marking.
4256THRASHER et alBLOOD, 1 JUNE 2005?VOLUME 105, NUMBER 11
For P2, PCR analysis of PBLs or CD3?sorted cells also Download full-text
revealed a very low ?c transgene signal, which disappeared 6
months after gene therapy (Table 1). CD4?lymphopenia persisted,
as did the absence of T-cell proliferative responses. There was no
evidence for the emergence of naive T cells and the numbers of
TRECs remained very low (? 20/105PBLs). No improvement of
his clinical condition was observed. Despite intensive therapy for
recurrent skin and pulmonary infections, his condition deteriorated
and he died of respiratory failure 1.5 years after gene therapy. To
verify successful gene transfer, cells preserved at completion of
gene transfer were thawed and cocultured on a MS-5 feeder cell
line.After 5 weeks of culture, both myeloid (CD33?) and lymphoid
(CD19?) precursors were present. Immunoselected CD33?cells
were strongly positive for the ?c transgene by PCR, indicating that
early hematopoietic precursors had been transduced and were
viable at the time of engraftment (data not shown).
The failure of gene therapy to produce therapeutic effects in
these 2 patients raises important issues for the extension of this type
of therapy, as CD34?progenitor cells were successfully transduced
in vitro.The capacity to initiate (or reinitiate) thymopoiesis is likely
to be time dependent and influenced by chronic infection, previous
GVHD, and physiological aging. The absence of ongoing thymo-
cyte and thymic epithelial cell (TEC) interaction results in disorga-
nization of thymic architecture and hypoplasia, which may become
irreversible.14-18This likely explains why allogeneic HSCT per-
formed for SCID patients in the neonatal period results in superior
thymic output compared with those performed later.6,7,19Similar
arguments may well apply to patients with attenuated disease in
whom thymopoiesis is severely compromised. The results of
gene therapy in these 2 patients with SCID-X1 indicate that there
may well be age-related restrictions to efficacy. Therefore, we
suggest that intervention should be considered at the earliest
We are indebted to families of the patients for their continuous
support, and to the medical and nursing staff of the Pediatric
Immunology Unit at the Necker Hospital and the Immunology Unit
at Great Ormond Street Hospital. We would also like to acknowl-
edge Fabian Gross, Chantal Martinache, and Jean-Marc Luby for
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FAILURE OF SCID-X1 GENE THERAPY IN OLDER PATIENTS 4257BLOOD, 1 JUNE 2005?VOLUME 105, NUMBER 11