Gene Therapy of Human Severe Combined
Immunodeficiency (SCID)-X1 Disease
Marina Cavazzana-Calvo,
*123
Salima Hacein-Bey,
*123
Geneviève de Saint Basile,
1
Fabian Gross,
2
Eric Yvon,
3
Patrick Nusbaum,
2
Franĉoise Selz,
1
Christophe Hue,
12
Stéphanie Certain,
1
Jean-Laurent Casanova,
14
Philippe Bousso,
5
Franĉoise Le Deist,
1
Alain Fischer
124
Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked
inherited disorder characterized by an early block in T and natural
killer (NK) lymphocyte differentiation. This block is caused
by mutations of the gene encoding the c
cytokine receptor subunit of interleukin-2, -4, -7, -9, and
-15 receptors, which participates in the delivery of
growth, survival, and differentiation signals to early
lymphoid progenitors. After preclinical studies, a gene therapy
trial for SCID-X1 was initiated, based on the use of complementary
DNA containing a defective c
Moloney retrovirus-derived vector and ex vivo infection
of CD34+ cells. After a 10-month follow-up period, c
transgene-expressing T and NK cells were detected in two
patients. T, B, and NK cell counts and function,
including antigen-specific responses, were comparable to
those of age-matched controls. Thus, gene therapy was
able to provide full correction of disease phenotype and, hence,
clinical benefit.
1 INSERM Unit 429,
2 Gene Therapy Laboratory,
3 Cell Therapy Laboratory,
4 Unité d'Immunologie et d'Hématologie Pédiatriques, Hôpital
Necker, 75743 Paris Cedex 15, France.
5 INSERM Unit 277, Institut Pasteur, 75730 Paris,
France.
* These authors
contributed equally to this work.
To whom correspondence should be addressed at INSERM Unit 429, Hôpital
Necker-Enfants Malades, 149 rue de Sèvres, 75743 Paris
Cedex 15, France. E-mail: fischer@necker.fr
In considering diseases that might be ameliorated by gene therapy, a
setting in which a selective advantage is conferred by transgene
expression, in association with long-lived transduced cells
such as T lymphocytes, may prove critical. SCID-X1 offers a
reliable model for gene therapy because it is a lethal condition
that is, in many cases, curable by allogeneic bone marrow
transplantation (1-4). It is caused by
c
cytokine receptor deficiency that leads to an early block
in T and NK lymphocyte differentiation (1-3).
In vitro experiments of c
gene transfer have shown that c
expression can be restored (5-7), as
well as T and NK cell development (8-9),
while the immunodeficiency of c
mice can be corrected by ex vivo c
gene transfer into hematopoietic precursor cells (10,
11). Long-term expression of human c
has also been achieved by retroviral infection of canine
bone marrow (12). It has been anticipated that
c
gene transfer should confer a selective advantage to transduced
lymphoid progenitor cells because, upon interaction with
interleukin-7 (IL-7) and IL-15, the c
cytokine receptor subunit transmits survival and
proliferative signals to T and NK lymphocyte progenitors, respectively
(2, 3). This hypothesis
received further support from the observation that a
spontaneously occurring c
gene reverse mutation in a T cell precursor in one patient led
to a partial, but sustained, correction of the T cell deficiency,
including at least 1000 distinct T cell clones (13,
14). Spontaneous correction of the
immunodeficiency has otherwise not been observed in
several hundred c-deficient
SCID patients nor in c
mice (2-4).
Two patients, aged 11 months (P1) and 8 months (P2),
with SCID-X1 met the eligibility criteria for an ex vivo c
gene therapy trial. SCID-X1 diagnosis was based on blood
lymphocyte phenotype determination and findings of c
gene mutations resulting either in a tail-less receptor
expressed at the membrane (P1) (R289 X) or in a protein
truncated from the transmembrane domain that was not
expressed at cell surface (P2) (a frameshift causing deletion of
exon 6) (15). After marrow harvesting and CD34+
cell separation, 9.8 × 106 and 4.8 × 106
CD34+ cells per kilogram of body weight from P1 and P2,
respectively, were preactivated, then infected daily for
3 days with the MFG c
vector-containing supernatant (16). CD34+
cells (19 × 106 and 17 × 106/kg,
respectively) were infused without prior chemoablation into P1
and P2, ~20 to 40% and 36% of which expressed the c
transgene as shown by either semiquantitative PCR
analysis (P1) or immunofluorescence (P2). As early as day
+15 after infusion, cells carrying the c
transgene were detectable by PCR analysis (17)
among peripheral blood mononuclear cells. The fraction of
positive peripheral blood mononuclear cells increased
with time (Fig. 1). T lymphocyte counts
increased from day +30 in P1 (who had a low number of
autologous T cells before therapy), whereas c-expressing
T cells became detectable in the blood of P2 at day +60 (Fig.
2). Subsequently, T cell counts, including CD4+
and CD8+ subsets, increased to 1700/µl from day +120 to
+150 and reached values of ~2800/µl after 8 months
(Fig. 2). Transgenic c
protein expression could not be studied on P1 cells given the presence
of the endogenous tail-less protein. However, semi-quantitative
PCR performed at day +150 showed that a high proportion of T
cells carry and express the c
transgene (Fig. 1, A and B). Similar
results were observed at day +275. Southern blot analysis of
provirus integration in peripheral T cells from both patients revealed
a smear indicating that multiple T cell precursors had been
infected by the retroviral vector (18).
Fig. 1. c
transgene integration and expression. Primers used to detect both
PCR and RT PCR products amplify a 904-base pair stretch encompassing
the 3' end of the c
sequence and downstream vector sequence (5). (A)
Semiquantitative PCR analysis of leukocyte subset DNA from P1 and
P2. Blood samples were drawn at day +150. T cells (CD3+),
B cells (CD19+), monocytes (CD14+),
granulocytes (CD15+), and NK cells (CD56+) as
well as CD34+ from a bone marrow sample obtained at day
+150 from P2 were isolated by a FACStar plus cell sorter (Becton
Dickinson) after staining with appropriate mAbs (19).
Purity was >99%. Sorted cells were analyzed for the frequency of
vector-containing cells (17). Actin DNA was
amplified in parallel. Samples from peripheral blood mononuclear
cells (PBMC) obtained before treatment are shown as negative
controls. A standard curve was constructed by diluting cells
containing one copy of the MFG c
vector (5) with noninfected cells. All specimens
were tested at three dilutions: 1:1, 1:20, and 1:200. (B)
Semiquantitative RT-PCR analysis of leukocyte-subset RNA from P1.
The same blood sample as in (A) was used. Actin cDNA was amplified
in parallel as a control of RNA content. The standard curve was
constructed as in (A) (17). No signal was
detected in the absence of reverse transcriptase (not shown). Each
specimen was diluted to 1:1, 1:500, and 1:5000. [View
Larger Version of this Image (33K GIF file)]
Fig. 2. Longitudinal study of lymphocyte subsets
from patient 1 (P1) and patient 2 (P2). Absolute counts of
T cells (CD3+, CD8+, and CD4+), B
cells (CD19+), and NK cells (CD16+, CD56+)
are shown as a function of time. Day 0 is the date of
treatment. The scale for NK cells is on the right- hand side of each
panel. [View
Larger Version of this Image (23K GIF file)]
Immunofluorescence studies showed that c
was expressed on the membrane of T cells in P2. The magnitude of
expression was similar to that of control cells (Fig.
3A), as found in previous in vitro gene transfer
experiments (5, 8, 9).
These results indicate that sufficient transgene expression
had been achieved and that c
membrane expression is likely to be regulated by the
availability of the other cytokine receptor subunits with
which c
associates (3). Both
and
T cell receptor (TCR)-expressing T cells were detected (Fig.
3B). Polyclonality and V
TCR diversity were demonstrated by using antibodies
specific for TCR V
(19) and the immunoscope method (18,
20). In both patients, naïve CD45RA+
T cells were detected, accounting for a majority of the T cell subset
(Fig. 3B). In both patients, T cells proliferated
from day +105 in the presence of phytohemagglutinin (PHA) and
antibodies to CD3 (anti-CD3). The extent of proliferation was
the same as that of age-matched controls (Fig. 4A).
After primary vaccination, in vitro T cell proliferative
responses to tetanus toxoid (P1 and P2: 18,000 and
12,000 cpm, respectively) and polioviruses (P2:
38,000 cpm) were observed within normal range (21).
P1 T cells were also found to proliferate in the presence
of protein pure derivitive (PPD) (12,000 cpm) as a
likely consequence of bacillus Calmette-Guerin (BCG) persistence
after immunization at 2 months of age in this
immunocompromised child. Five months after cessation of
intravenous immunoglobulin (Ig) therapy, antibodies to
tetanus and diphtheria toxoids as well as to polioviruses
were found in the serum of both patients, together with
detectable concentrations of IgG and IgM (Fig. 4B).
A normal level of IgA was also detected in the serum of P1. As
determined by semi-quantitative PCR and reverse
transcriptase-PCR analysis, it was observed that in both
cases, a low fraction of B cells carry and express the c
transgene (Fig. 1). It is therefore
unknown whether antibody responses are provided by
untransduced or the few transduced B cells. Residual persistence
(< 1%) of administered intravenous immunoglobulins
(last given 5 months before measurement of antibody
response) could, in part, also contribute. The c-expressing
NK cells were detected in the blood of P2 by day 30 (Figs.
1, 2, and 3A). These
cells efficiently killed K562 cells in vitro (18).
NK cells became detectable in the blood of P1 only from day
+150.
Fig. 3. c
protein expression and lymphocyte subsets. (A) c
protein detection at the surface of lymphocyte subsets from a
control and from P2 obtained at day +150. c
expression on B cells from P2 after treatment was undetectable (not
shown). The y axis depicts the relative cell number, and the x
axis shows the logarithm of arbitrary immunofluorescence units. Thin
lines are isotype controls; thick lines, staining by the anti-c.
Similar results were observed on blood samples obtained at days 275 (P1)
and 240 (P2). (B) The percentage of CD45RO+
and CD45RA+ among CD4 and CD8 T cells from P1 and P2
obtained at day +275 and 240, respectively, as well as the
percentage of T cells expressing either an
TCR or a
TCR. [View Larger
Version of this Image (32K GIF file)]
Fig. 4. Functional characteristics of transduced
lymphocyte subsets. (A) Longitudinal follow-up of PHA (,
)-
and anti-CD3 (,
)-induced
proliferation of lymphocytes from P1 (open symbols) and P2 (filled
symbols) (8). Background [3H]thymidine
uptake was less than 400 cpm. Positive control values are
>50 × 103 cpm. (B) Serum
immunoglobulin analysis was determined by nephelometry and serum
antibody by enzyme-linked immunosorbent assay after immunization
(see above). Diphtheria toxoid (Dipht. tox.) was also used for
immunization. The last intravenous Ig injections were given at day
+90 in both patients. Tet. Tox., tetanus toxoid. Isohemagglutinins
to blood group A have now been detected in both patients' sera. [View
Larger Version of this Image (13K GIF file)]
As a likely consequence of development and sustained function of
the immune system, clinical improvement was observed in both patients.
In P2, protracted diarrhea as well as extensive graft-versus-host
disease (GVHD)-like skin lesions disappeared. Both patients
left protective isolation at days 90 and 95 and
are now at home 11 and 10 months, respectively,
after gene transfer without any treatment. Both enjoy
normal growth and psychomotor development. No side effects
have been noted. A similar result has since been achieved in
a third patient 4 months after gene transfer (22).
These results demonstrate that in these patients, a selective
advantage was conferred to T and NK lymphocyte progenitors,
enabling full-blown development of mature and functioning
T and NK lymphocytes (23).
These overall positive results contrast with the failure of
previous attempts to perform ex vivo gene therapy in adenosine deaminase
(ADA)-deficient patients (24-27). Concomitant
administration of ADA enzyme to these patients is likely
to have counterbalanced the potential growth advantage of
the transduced cells in this setting (23). Also,
advances in the methodology of gene transfer into CD34+
cells, i.e., the use of a fibronectin fragment (28)
as well as of a cytokine combination enabling potent CD34 cell
proliferation, contributed to the success of c
gene therapy.
Because c
gene transfer was achieved without any additional myeloablative or
immunosuppressive therapy, these results pave the way for
a possible extension of this therapeutic approach to
other genetic diseases characterized by defective cell-subset generation,
such as other forms of SCID (29). The kinetics
of T cell development in c
gene transfer is similar to that observed in SCID patient
recipients of haploidentical stem cell transplantation (4),
suggesting that early progenitor cells have been infected
by the MFG c
virus and effectively transduced. The hypothesis that
transduced autologous T cells in P1 account for the development
of the T cell compartment is unlikely because (i) the infected
CD34+ cell population was contaminated by less than
0.1% CD3+ T cells; (ii) a thymic gland (27 mm by 25 mm
by 25 mm at day +275) became detectable by
ultrasound echography, indicative of thymopoiesis,
whereas most T cells at day +275 exhibit a naïve CD45RA+
phenotype; and (iii) the T cell repertoire was polyclonal and diverse.
In both patients, it was shown that at day +150, a fraction of
bone marrow CD34+ cells harbored and expressed the c
transgene (Fig. 1, P2). It was not
possible to determine whether more primitive cells, i.e.,
CD34+CD38
cells, were transduced because of insufficient bone marrow sample.
In the mouse, a common lymphoid progenitor (CLP) gives rise to
the different lymphocyte populations (30).
If a human counterpart of CLP exists, it would be the
best candidate from among the earliest cells that were
transduced ex vivo from these patients. Identification of
integration sites in the various cell lineages could help
determine the permissive differentiation stage. The
question of the persistence of T and NK cell generation has yet
to be addressed. If infected cells have no self-renewal capacity
and have a short life-span, new generation of T and NK cells
should cease. However, the fact that a thymic gland is
still detectable 9 months after c
gene transfer suggests that thymopoiesis is still
ongoing. Follow-up of the SCID-X1 patient in whom a spontaneous
reversion mutation occurred in a T cell precursor (13,
14) indicates that gene transfer could be
sufficient to provide a functional memory T cell pool for
a number of years. This optimistic view will require
careful sequential appraisal. Kohn et al. have
previously shown that transgenes placed under the control
of the long-terminal repeat (LTR) viral promoter can be
silenced in quiescent T cells (31). Although the
identification of silencing sequences in the MFG LTR makes
this a strong possibility (31),
down-regulation of c
expression has not been observed so far in these two
patients, in c-deficient
mice treated by ex vivo c
gene transfer (11), or in cell lines
maintained in culture over 1 year (5).
Follow-up will be required to assess the long-term effects of ex
vivo c
gene transfer in CD34+ cells of SCID-X1 patients. To
date, this methodology has resulted in the sustained
correction (up to 10 months) of the SCID-X1 phenotype in
two patients, including a patient in whom the mutated protein is
expressed at the cell surface. It is presumed that the effect results
from a strong positive selective pressure provided to the
corrected lymphoid progenitors.
REFERENCES AND NOTES
-
M. Noguchi, et al., Cell 73, 147 (1993)
[ISI]
[Medline]
.
-
K. Sugamura, et al., Annu. Rev. Immunol. 14,
179 (1996)
[ISI]
[Abstract/Full
Text]
.
-
W. J. Leonard. Annu. Rev. Med. 47, 229 (1996).
-
R. H. Buckley, et al., N. Engl. J. Med. 340,
508 (1999)
[ISI]
[Medline]
.
-
S. Hacein-Bey, et al., Blood 87, 3108
(1996)
[ISI]
[Abstract]
.
-
F. Candotti, et al., Blood 87, 3097
(1996)
[ISI]
[Abstract]
.
-
N. Taylor, et al., Blood 87, 3103
(1996)
[ISI]
[Abstract]
.
-
M. Cavazzana-Calvo, et al., Blood 88,
3901 (1996)
[ISI]
[Abstract]
.
-
S. Hacein-Bey, et al., Blood 92, 4090
(1998)
[ISI]
[Abstract/Full
Text]
.
-
M. Lo, et al., Blood 94, 3027 (1999)
[ISI]
[Abstract/Full
Text]
.
-
C. Soudais, et al., Blood 95, 3071
(2000)
.
-
T. Whitwam, et al., Blood 92, 1565
(1998)
[ISI]
[Abstract/Full
Text]
.
-
V. Stephan, et al., N Engl. J. Med. 335,
1563 (1996)
[ISI]
[Medline]
.
-
P. Bousso, et al., Proc. Natl. Acad. Sci. U.S.A.
97, 274 (2000)
[ISI]
[Abstract/Full
Text]
.
-
Patient 1 had pneumocystis carinii pneumonitis and had
received BCG immunization. Patient 2 suffered from
recurrent oral candidiasis, pneumocystis carinii infection,
protracted diarrhea, failure to thrive, and GVHD-like skin
lesions. Neither patient had an HLA (human leukocyte
antigen)-identical sibling. Patients were placed in a sterile
isolation ward and received nonabsorbable oral antibiotics and
intravenous Igs every 3 weeks for 3 months. Parents
gave informed consent for participation in the trial.
-
The defective MFG c
vector has been described previously (5). It was packaged in the
crip
cell line. The MFG c
vector-containing supernatant was manufactured and provided by
Genopoietic (Lyon, France) under GMP guidelines. The vector
supernatant was free of replication-competent retrovirus as
determined by S+L- assay and a -galactosidase
mobilization test [ R. H. Bassin, N. Tuttle, P. J. Fischinger, J.
Cancer 6, 95 (1970)
; M. Printz, et al., Gene Ther. 2, 143
(1995)
[ISI]
[Medline]
]. Concentration of the virus in the supernatant was 5 × 105
infectious virus particles (5). Marrow CD34+ cells
were positively selected by an immunomagnetic procedure (CliniMACS,
Miltenyi Biotec, Bergish Gladbach, Germany). CD34 cells were
cultured in gas-permeable stem cell culture (PL-2417) containers
(Nexell Therapeutics, Irvine, CA), at a concentration of 0.5 × 106
cells/ml in X-vivo 10 medium (Biowhittaker, Walkerville,
MD) containing 4% fetal cell serum (Stem Cell Technologies,
Vancouver, Canada), stem cell factor (300 ng/ml, Amgen),
polyethylene glycol-megabaryocyte differentiation factor (100 ng/ml,
Amgen), IL-3 (60 ng/ml, Novartis), and Flt3-L (300 ng/ml,
R&D Systems, Minneapolis, MN) for 24 hours at 37°C in
5% CO2. Containers were precoated with the CH296
human fragment of fibronectin (50 µg/ml) (TaKaRa, Shiga,
Japan). Retroviral containing supernatant was added every day
for 3 days. Cells were then harvested, washed twice, and
infused back into the patients.
-
For semiquantitative PCR and RT-PCR analysis, DNA was
isolated from the indicated cell populations. A reference
standard curve was constructed by diluting cells from a
SCID-X1-derived Epstein-Barr virus (EBV)-B cell line containing
one copy per cell of the MFG c
provirus (5) in uninfected cells from the same EBV-B cell line
(100, 10, 1, 0.1, 0.01, and 0.001%).
DNA from each sample was also quantified by actin gel
amplification. MFG c
primers sequences and actin primers sequences are available on
request. DNA was amplified in a 50 µl of PCR reaction
mixture by using 30 cycles at an annealing temperature of
60°, for c
primers and 68°C for actin primers. A sample of the amplified
product was separated on a 1% agarose gel and analyzed by
ethidium bromide staining. RNA was prepared with the RNA easy
kit (Qiagen) and was reverse-transcribed with the Superscript
Preamplification System (Gibco-BRL). c
proviral and -actin
cDNA amplification were performed as described above.
Quantification of expression was made by comparison with RNA
isolated from the same standard curve of diluted cells.
-
M. Cavazzana-Calvo et al., data not shown.
-
The following monoclonal antibodies (mAbs) were used in
immunofluorescence studies: anti-c
chain: Tugh 4 (rat IgG2, PharMingen, San Diego, CA);
anti-CD3: Leu 4 (IgG2a, Becton Dickinson, San Diego, CA);
anti-CD4: Leu3a (IgG1, Becton Dickinson); anti-CD8: Leu 2a
(IgG1, Becton Dickinson); anti-CD19: J4 119 (IgG1
Immunotech, Marseille, France); anti-CD14: Leu M3 (Becton
Dickinson); anti-CD16: 3G8 (IgG1, Immunotech); anti-CD56: MY31
(IgG1, Becton Dickinson); anti-CD15 (IgM, PharMingen); anti-TCR :
BMA031 (IgG1, Immunotech); anti-TCR :
IMMU 515 (IgG1, Immunotech); anti-CD45RO: UCHL1 (IgG2a,
Immunotech); anti-CD45RA: 2H4 (IgG1, Coulter Clone, Margency,
France); anti-CD34: HPCA-2 (IgG1, Becton Dickinson); anti-TcR V2:
MPB2D5 (IgG1, Immunotech); anti-TcR V3:
CH92 (IgM, Immunotech); anti-TcR V5.1:
IMMU 157 (IgG2a, Immunotech); anti-TcR V5.2:
36213 (IgG1, Immunotech); anti-TcR V5.3:
3D11 (IgG1, Immunotech); anti-TcR V8:
56C5.2 (IgG2a, Immunotech); anti-TcR V9:
FIN9 (IgG2a, Immunotech); anti-TcR V13.1:
IMMU 222 (IgG2, Immunotech); anti-TcR V13.6:
JU74.3 (IgG1, Immunotech); anti-TcR V14:
CAS1.13 (IgG1, Immunotech); anti-TcR V17:
E17.5F3.15.13 (IgG1, Immunotech); anti-TcR V21.3:
IG125 (IgG2, Immunotech). Fluorescence staining was done with
phycoerythrin- or fluorescein isothiocyanate-conjugated mAbs.
Cells were analyzed on a FACScan flow cytometer (Becton
Dickinson).
-
C. Pannetier, et al., Proc. Natl. Acad. Sci.
U.S.A. 90, 4319 (1993)
[ISI]
[Abstract]
.
-
Unstimulated lymphocyte proliferations were <1000 cpm.
Control positive values of antigen-stimulated proliferations
were >10,000 cpm.
-
This patient was treated at 1 month of age. Within 3 months,
T and NK lymphocyte counts reached age-matched control values.
The c
expression at T and NK cell surfaces was fully restored. The
child is at home without any therapy, 4 months after
treatment.
-
C. Bordignon, Nature Med. 4, 19 (1998)
[ISI]
[Medline]
.
-
C. Bordignon, et al., Science 270, 470
(1995)
[ISI]
[Abstract]
.
-
D. B. Kohn, et al., Nature Med. 1, 1017
(1995)
[ISI]
[Medline]
.
-
D. B. Kohn, et al., Nature Med. 4, 775
(1998)
[ISI]
[Medline]
.
-
V. W. Van Beusechem, et al., Gene Ther. 3,
179 (1996)
[Medline]
.
-
H. Hannenberg, et al., Nature Med. 2,
876 (1996)
[Medline]
.
-
A. Fischer and B. Malissen, Science 280, 237
(1998)
[ISI]
[Abstract/Full
Text]
.
-
M. Kondo, I. L. Weissman, K. Akashi, Cell 9,
661 (1997)
.
-
P. B. Robbins, et al., Proc. Natl. Acad. Sci.
U.S.A. 95, 10182 (1998)
[ISI]
[Abstract/Full
Text]
.
-
We thank the medical and nursing staff of the Unité
d'Immunologie et d'Hématologie pédiatriques, Hôpital des
Enfants-Malades, for patient care. We also thank C. Harré
and C. Jacques for technical help; D. Bresson for
preparation of the manuscript; N. Wulfraat for patient
referral; O. Danos, M. Fougereau, P. Mannoni, C. Eaves,
and L. Coulombel for advice; A. Gennery for assistance
with English translation; B. Bussière, C. Cailliot,
and J. Caraux (Amgen, France) for providing SCF and MGDF;
J. Bender and D. Van Epps (Nexell Therapeutics,
Irvine, CA) for providing containers; and S. Yoshimura and
I. Kato (Takara Shuzo, Shiga, Japan) for providing the
CH-296 fibronectin fragment. Supported by grants from INSERM,
Association Franĉaise des Myopathies, Agence Franĉaise du
Sang, and the Programme Hospitalier de Recherche Clinique
(Health Ministry).
28 December 1999; accepted 10 March 2000
Volume
288, Number 5466, Issue of 28 Apr 2000, pp. 669-672.
Copyright © 2000 by The American Association for the Advancement of
Science.
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