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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0247.
GENE THERAPY
From Genetics and Molecular Biology Branch, National
Human Genome Research Institute (NHGRI), National Institutes of Health
(NIH), Bethesda, MD; Institut de Génétique
Moléculaire de Montpellier, UMR 5535/IFR24, Montpellier, France;
and Etablissement Français du Sang Bourgogne/Franche-Comté,
INSERM EPI0119/UPRES EA 2284, Besançon, France.
Mutations in the ZAP-70 protein tyrosine kinase gene result in a
severe combined immunodeficiency (SCID) characterized by a selective
inability to produce CD8+ T cells and a signal transduction
defect in peripheral CD4+ cells. Transplantation of
genetically modified hematopoietic progenitor cells that express the
wild-type ZAP-70 gene may provide significant benefit to some of these
infants. The feasibility of stem cell gene correction for human
ZAP-70 deficiency was assessed using a ZAP-70 knock-out model.
ZAP-70-deficient murine bone marrow progenitor cells were transduced
with a retroviral vector expressing the human ZAP-70 gene. Engraftment
of these cells in irradiated ZAP-70-deficient animals resulted in the
development of mature CD4+ and CD8+ T cells. In
marked contrast, both populations were absent in ZAP-70 Severe combined immunodeficiency (SCID) is caused
by a variety of mutations that interfere with the differentiation or
function of T and/or B lymphocytes. Patients with ZAP-70 deficiency
have an unusual SCID phenotype characterized by the presence of normal numbers of nonfunctional CD4+ T cells and a selective
absence of CD8 single-positive T cells in the periphery as well
as in the thymus.1-4 ZAP-70 is a 70-kd protein tyrosine
kinase that is recruited to the T-cell receptor (TCR) following its
stimulation.5 It is expressed at approximately equivalent
levels in thymocytes, mature T cells, and natural killer (NK)
cells.6 Extensive studies in murine models as well as in
human T-cell lines have demonstrated a critical role for this protein
in T-cell ontogeny and activation.7
Like patients with other forms of SCID, ZAP-70-deficient patients
present with opportunistic infections and failure to thrive. The
disease is almost universally fatal in infancy unless treated by
allogeneic hematopoietic stem cell (HSC) transplantation. However, for
most patients, histocompatible donors are not available, and they
therefore undergo transplantation with haploidentical T-cell-depleted or HLA-matched unrelated HSC grafts. Unfortunately, transplantation with nonhistocompatible HSCs is associated with a high rate of serious
complications, such as graft-versus-host disease, delayed immune
reconstitution, and abnormal B-cell differentiation.8-10 Thus, the development of gene-based therapies could be beneficial for
patients who do not have histocompatible HSC graft donors. Indeed, the
elegant clinical trial performed by Fischer's group showed the success
of this type of approach for infants with X-linked SCID
(XSCID).11 Retroviral-mediated introduction of
Our previous data, demonstrating that retroviral-mediated transfer of
ZAP-70 into primary ZAP-70-deficient CD4+ T cells restores
T-cell function,12 support the feasibility of gene therapy
for ZAP-70 deficiency. Nevertheless, several important issues remain to
be addressed: (1) Does introduction of the ZAP-70 gene into HSCs lead
to CD4+ and CD8+ development in vivo? (2) Do
the developing lymphoid cells exhibit a polyclonal TCR repertoire and
normal immune responsiveness? (3) Does ectopic ZAP-70 expression in
progenitor cells negatively affect the differentiation and function of
non-T hematopoietic lineages? Importantly, a gene therapy approach for
ZAP-70 deficiency provides challenges not encountered in XSCID
patients: the ZAP-70 protein is normally expressed only in T-cell
lineages, whereas ZAP-70 knock-out mice provide a tool used to address these issues.
However, the phenotype of ZAP-70-deficient mice and humans are not
identical, with ZAP-70-mutant mice exhibiting an earlier block in
T-cell development at the CD4+CD8+ thymocyte
stage.13,14 Although the bases for the differential role
of ZAP-70 in human and murine T-cell development remain unclear, it is
likely that compensatory mechanisms exist in ZAP-70-deficient patients
that allow nonfunctional CD4+ T cells to mature and
emigrate to the periphery. Nevertheless, ZAP-70 knock-out mice
recapitulate many features of ZAP-70-deficient patients and offer an
important model in which the validity of stem cell gene correction for
this disease can be assessed.
Retroviral constructs and producer cell lines
Retroviral transduction of BM cells and transplantation
Western blot analysis Cells (1 × 106) were lysed in a 1% Nonidet P-40 lysis buffer, resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose membranes as previously described.18 Membranes were blotted with an ZAP-70 monoclonal antibody (mAb) specific for
human ZAP-70 (Upstate Biotechnology, Lake Placid, NY) or an ZAP-70
mAb that cross-reacts with both human and mouse ZAP-70 proteins
(Transduction Laboratories, Lexington, KY). Protein loading was
verified with either an -actin polyclonal antibody (Santa Cruz,
Santa Cruz, CA) or an Erk2 mAb (Transduction Laboratories).
Immunoreactive proteins were visualized using enhanced chemiluminescence.
Flow cytometry analysis Phycoerythrin (PE)- and CyChrome (Cy)-conjugatedCD3 ,
CD4, CD8, B220, CD62L, CD25, CD69, IgG1,
IgG3, IgM, CD5, and TCR mouse mAbs were purchased from
Pharmingen (San Diego, CA). Cells were incubated with antibodies for 20 minutes and then washed in phosphate-buffered saline before analysis on
a FACSCalibur (Becton Dickinson, San Jose, CA). Cells transduced with
retroviral vectors harboring EGFP were identified by their FL-1 autofluorescence.
Splenocyte proliferation Splenocytes obtained 18 weeks following bone marrow transplantation (BMT) were seeded in triplicate in 96-well plates (1 × 105 cells per well). Cells were cultured in 100 µL RPMI media supplemented with 10% fetal calf serum and 50 µM 2-mercaptoethanol in the absence or presence of solubleCD3 and
CD28 mAbs (1 and 2 µg/mL, respectively; Pharmingen) or
lipopolysaccharide (LPS) (20 µg/mL; Sigma, St Louis, MO). After 48 hours of culture, cells were pulsed with [3H]thymidine
(0.5 µCi [18.5 KBq] per well) (NEN, Dupont, Boston, MA) for
16 hours, harvested, and incorporated radioactivity was determined
using a scintillation counter.
In vitro immunoglobulin isotype switching assay Splenocytes obtained 18 weeks after BMT were cultured in RPMI medium supplemented with 10% fetal calf serum at a concentration of 1 × 106 cells per milliliter. For the induction of isotype switching, LPS (20 µg/mL) or LPS plus IL-4 (25 ng/mL; Peprotech) was added. After 6 days of culture, cells were collected and stained with a Cy-conjugatedB220 mAb together with either IgG1
or IgG3 mAbs and analyzed by fluorescence-activated cell
sorting (FACS).
T-cell repertoire analysis Total RNA was prepared from splenocytes obtained 18 weeks after BMT using RNAlater (Ambion, Austin, TX). A total of 2 µg RNA was reverse transcribed with random hexanucleotides (Pharmacia Biotech, Orsay, France) using Moloney MLV reverse transcriptase (RT; Gibco, Cergy, France). The cDNAs were amplified (40 cycles) in a 25-µL reaction mixture with 1 of the 24 TCRBV (TCR
chain hypervariable region) subfamily-specific primers and a C
primer recognizing the 2 constant regions C1 and C2 of the chain of the TCR, as previously described.19,20 A total of
2 µL of the 24 TCRBV/C-first-run polymerase chain reaction (PCR)
products was subjected to 2 cycles of elongation using a C
dye-labeled (6-Fam) primer allowing PCR products to be detected on a
337 automated DNA sequencer (Applied Biosystems); 1 µL of the PCR
product was loaded on a 6% acrylamide sequencing gel and analyzed for
size and fluorescence intensity using Immunoscope software. The TCRBV nomenclature proposed by Arden et al was used in this
study.21
Statistical analyses Statistical significance was determined using a Student t test with a 1-tailed distribution and 2-sample equal variance. Data were considered to be statistically different for P < .05. All data are presented as mean ± SD.
Introduction of ZAP-70 into hematopoietic progenitor cells To assess the feasibility of a stem cell-based gene therapy strategy for ZAP-70-deficient patients, the ability of a human ZAP-70-expressing retroviral vector to support T-cell hematopoiesis in a ZAP-70-deficient mouse model was studied. BM from ZAP-70-deficient mice was harvested following treatment with 5-FU, and differentiated hematopoietic cells were removed using a cocktail of lin-specific antibodies. The remaining lin BM progenitor cells
were prestimulated for 48 hours with IL-3, IL-6, and SCF and then
transduced with either a control EGFP or a ZAP-70/EGFP MLV-based
vector packaged in the GP + E86 ecotropic cell line (Figure
1A).
Retroviral transduction using either control EGFP or ZAP-70/EGFP
virions resulted in gene transfer in more than 85% of progenitor cells, as assessed by expression of the EGFP marker (Figure 1B). Moreover, introduction of the ZAP-70/EGFP vector resulted in a high
level of ZAP-70 expression in the lin Thymic differentiation in ZAP-70 / mice with transduced BM
progenitor cells to assess the effects of transgene expression on
long-term differentiation. The block in ZAP-70 deficiency occurs at a
relatively late state of T-cell development as compared with many other
types of genetic immunodeficiencies: at the double-positive (DP)
CD4+CD8+ stage.13 Because DP
cells constitute the majority of thymocytes, it was not surprising that
neither transduction with the control EGFP vector nor with the
ZAP-70/EGFP vector significantly affected thymic cellularity:
2.4 × 107 ± 1.5 × 107 and
3.8 × 107 ± 2.1 × 107, respectively,
vs 5.6 × 107 ± 3.8 × 107 in
ZAP-70-deficient mice undergoing transplantation with WT BM (P > .05). Within the nontransduced ZAP-70-deficient
thymocyte populations, there were only very few single-positive (SP)
thymocytes, and the relative partition of the DP and SP populations was
unchanged in those thymocytes harboring the control EGFP vector (Figure 2A). In marked contrast, within the
population of thymocytes expressing the ZAP-70/EGFP vector, the
percentages of SP thymocytes were significantly elevated
(20.8% ± 9.7% and 8.6% ± 4.0% for CD4+ and
CD8+ thymocytes; P < .05 for both
conditions). Notably, these levels were not statistically different
from those detected in ZAP-70-deficient mice undergoing
transplantation with WT BM (12.9% ± 5.1% and 4.1% ± 1.3% for
CD4+ and CD8+ thymocytes; P > .05
for both conditions) (Figure 2A).
To more closely examine the differentiation of ZAP-70-transduced
thymocytes in these animals, surface expression of the CD5 and TCR Phenotype and function of peripheral T cells in ZAP-70-transduced mice We next assessed the presence of peripheral splenic T cells in ZAP-70-treated mice. As expected, peripheral T cells were not observed in ZAP-70-deficient mice transduced with the control vector. However, introduction of ZAP-70 allowed the development of CD3+ T lymphocytes. Both mature CD4+ and CD8+ T cells were detected in the spleen as well as in the peripheral circulation of all 4 ZAP-70-transduced mice (Figure 3A and data not shown). The activation status of these lymphocytes was determined by assessing the expression of the CD25 and CD69 activation markers. CD4+ T cells from ZAP-70/ mice
undergoing transplantation with ZAP-70-transduced progenitor cells
expressed the CD25 activation marker at levels equivalent to that
observed in WT mice and ZAP-70-deficient mice undergoing transplantation with WT BM (Figure 3B). Expression of the CD69 activation marker was also detected in ZAP-70-transduced
CD4+ T cells, albeit at higher levels than in WT mice
(52.8% ± 5.9% vs 35.7% ± 3.2%; P < .05).
Although both naive and memory T-cell populations were observed in
reconstituted mice 18 weeks after BMT, the percentage of naive
CD4+ T cells (CD62L+) was reduced in the mice
grafted with ZAP-70-transduced progenitors as compared with mice
undergoing transplantation with WT BM (12.7% ± 4.8% vs
49.6% ± 6.6%; P < .005, Figure 3B). Intriguingly,
increases in the percentages of activated (CD69+) and
memory (CD62L) CD4+ T cells were at least
related in part to the pretransplantation irradiation and/or post-BMT
immunoreconstitution because these populations were also augmented in
ZAP-70-deficient mice undergoing transplantation with WT BM as
compared with WT mice not undergoing transplantation (Figure
3B).
ZAP-70 expression in these transduced T cells was assessed using a
ZAP-70-specific mAb that cross-reacts with human and murine ZAP-70.
Importantly, ZAP-70 was expressed in splenocytes from ZAP-70-transduced animals at levels that appear to be approximately 3-fold higher than endogenous ZAP-70 levels (Figure 3C). However, in
these experiments it is not possible to directly compare expression of
endogenous and ectopic ZAP-70 because it is not known whether the
Splenocytes were then analyzed for their ability to respond to TCR
stimulation. Incorporation of [3H]thymidine was measured
3 days following activation with
Repertoire of ZAP-70-corrected T cells Because the absolute number of T cells in ZAP-70-transduced mice was lower than that observed in WT mice, it was important to further characterize the nature of these lymphocytes, especially with regard to their polyclonality. Thus, the relative usage of each TCRBV within the global T-cell population was examined using the Immunoscope method.19,20 This method is based on an RT-PCR of the hypervariable complementarity determining region 3 (CDR3), allowing the lengths of the RNAs encoding the chain of the TCR to be analyzed. A
Gaussian distribution of the CDR3 lengths is indicative of a diverse
and nonbiased T-cell population. A total of 24 PCRs, corresponding to
the 24 different TCRBV subfamilies, were performed and, notably, RT-PCR
signals were detected for each of the 24 TCRBVs in mice undergoing
transplantation with ZAP-70-transduced precursor cells. However, 5 sets of primers, corresponding to TCRBV families 5.3, 10, 17, 18, and
19, did not allow optimal amplification both in WT mice and
ZAP-70-transduced mice and, as such, data corresponding to the
remaining 19 TCRBV families were compared. Analyses of 3 mice in each
group demonstrated that most TCRBV profiles in the ZAP-70-transduced
mice were Gaussian, similar to that observed in both WT mice and
ZAP-70-deficient mice undergoing transplantation with WT BM cells
(Figure 4 and data not shown). Thus,
introduction of ZAP-70 into progenitor cells allows for a polyclonal
T-cell selection in the thymus and, more importantly, the maintenance
of a diverse lymphocyte population in the periphery. Of note, the
profiles from 3 TCRBV subfamilies (9, 11, and 20) were not strictly
Gaussian in the ZAP-70-corrected mice (Figure 4 and data not shown),
but the physiologic significance of this observation remains to be
determined.
Differentiation and function of non-T-cell lineages following ZAP-70 gene transfer in hematopoietic progenitor cells Because ZAP-70 is not expressed in non-T-cell lineages in WT mice, it was important to determine whether there were any transgene-related adverse effects in other hematopoietic cell lineages. Transduction of hematopoietic progenitor cells with the ZAP-70/EGFP vector did not alter the absolute numbers of white blood cells, red blood cells, splenic B cells, or splenic myeloid cells as compared with normal mice or ZAP-70-deficient mice transduced with WT BM (Table 2). Interestingly, ZAP-70-deficient mice transduced with the control EGFP retroviral vector had an increased number of splenic myeloid cells as compared with all other groups of mice, which was associated with a splenomegaly in 2 of 3 animals (Table 2). This phenotype was likely due to the immunodepressed status of the mice, especially in the context of lethal irradiation and BMT. Indeed, this phenotype has also been observed in another SCID mice model: lethally irradiated XSCID mice undergoing transplantation with XSCID progenitor cells (M.O. and F.C., unpublished observations, June 2000).
Following transplantation of progenitor cells transduced with the
ZAP-70/EGFP vector, the presence of the retroviral vector in
differentiated cell lineages was determined by concurrent analyses of
lin marker and EGFP expression. Because ZAP-70 is required for T-cell
differentiation, the finding that most peripheral T cells were
transduced was expected (Figure 5).
Notably, transgene expression was observed in all other assessed
lineages, including short-lived granulocytes. Thus, these data
suggest that at least a subset of the ZAP-70/EGFP-transduced
lin
The percentage of splenic B lymphocytes
(B220+/IgM+) was equivalent in WT mice and
ZAP-70-deficient mice transduced with the control EGFP vector and the
ZAP-70/EGFP vector, ranging from 65% to 77% (Figure
6A). Importantly, introduction of ZAP-70
had no adverse effects on LPS-induced B-cell proliferation because
splenocytes from all groups of treated and control mice proliferated at
comparable levels (Table 1). Additionally, the function of splenic B
lymphocytes was studied by performing in vitro immunoglobulin (Ig)
isotype switching assays. It has previously been shown that LPS
stimulation results in IgG3 production and B cells secrete IgG1 in
response to LPS plus IL-4.23,24 Upon treatment with LPS,
approximately 2.5% to 4.0% of all splenocytes expressed IgG3
regardless of the type of BMT. IgG1 expression was very low in all the
B-cell populations treated with LPS alone (< 1%), whereas the
combination of LPS and IL-4 resulted in the production of IgG1 in
approximately 25% of B cells (Figure 6B). The presence of the ZAP-70
transgene did not alter this response; equivalent responses were
observed in the untransduced (EGFP
The successful retroviral gene transfer trial for XSCID patients11 has provided an enormous boost to gene therapy-based strategies for many other pathologies and especially for other types of SCID. We previously demonstrated that retroviral-mediated introduction of ZAP-70 into CD4+ T cells from ZAP-70-deficient patients results in the reconstitution of their function.12 Nevertheless, because CD8+ T cells are not present in these patients, this type of approach would not reconstitute the cytotoxic arm of the immune system. We therefore used a murine model to determine whether a hematopoietic progenitor cell-based protocol could be beneficial for ZAP-70-deficient patients. Here, we report that T-lymphocyte differentiation and function is reconstituted following gene transfer of ZAP-70 into deficient progenitor cells. ZAP-70 deficiency provides several challenges not encountered in gene therapy strategies for either XSCID or adenosine deaminase deficiency. Specifically, the gene products in both of these latter pathologies are normally expressed in all hematopoietic lineages, whereas ZAP-70 is expressed only in T lin cells.1-3 As expected, ectopic ZAP-70/EGFP was expressed in most T cells in ZAP-70-deficient mice undergoing transplantation with gene-corrected progenitor cells. Importantly, though, all other hematopoietic lineages were transduced, albeit at lower levels than that observed in T cells. Moreover, Ig isotype switching and B-cell proliferation was not modulated by the presence of the ZAP-70 transgene. Altogether, these data suggest that ectopic ZAP-70 expression was associated with neither a selective advantage nor disadvantage in non-T-cell lineages. Although the mice used in these studies were irradiated prior to
transplantation, ZAP-70-deficient patients participating in a clinical
gene transfer protocol would not be conditioned. Thus, upon
introduction of ZAP-70-corrected progenitor cells in an environment of
high numbers of ZAP-70-deficient stem cells, it is likely that the
percentages of transduced non-T lin cells would be very low. This
hypothesis is based on the finding that, following stem cell gene
therapy for adenosine deaminase deficiency, the percentage of
transduced T cells present in the periphery was 2 to 3 logs higher than
the percentage of transduced granulocytes.25 Furthermore,
in gene-corrected XSCID patients, the percentage of transduced T cells
was essentially 100% but the percentage of transduced granulocytes was
only about 0.1%.11 The huge discrepancies are explained
by the observation that adenosine deaminase and One of the primordial questions in hematopoietic gene therapy protocols
concerns the identity of HSCs. Human hematopoietic cells expressing the
CD34 marker are capable of differentiating into T and NK cells in vivo
as well as in multiple experimental systems.11,27
Nevertheless, it is clear that CD34+ cells represent a very
heterogeneous group of cells and, moreover, several researchers have
suggested that CD34 The level of ZAP-70 expressed from this vector was sufficient to restore T lymphopoiesis in ZAP-70-deficient mice but, as noted earlier, the phenotype of ZAP-70-deficient mice and humans are distinct. ZAP-70-mutant mice exhibit an earlier block in T-cell development, at the CD4+CD8+ thymocyte stage,13,14 whereas ZAP-70-deficient patients develop mature, albeit nonfunctional, CD4+ T cells.1-3 This difference in phenotype raises the possibility that ectopic ZAP-70 expression has different consequences in mice and humans. We therefore assessed whether expression of ectopic ZAP-70 modulates human T-cell differentiation. To this end, the ZAP-70/EGFP retroviral vector was introduced into human fetal liver CD34+ cells and injected into a human fetal thymic organ that had already undergone transplantation in a SCID mouse (SCID-hu model). Importantly, the partition of the various thymocyte subsets was not altered by transduction with the ZAP-70 vector (K. Weijer, N.N., N.T., and H. Spits, unpublished observations, September 1999). Further studies will be necessary to determine the etiology of the
decreased numbers of naive T cells relative to memory T cells in the
ZAP-70-reconstituted mice. Intriguingly, the percentages of naive
CD4+ T cells were also relatively reduced in 2 The level of ectopic ZAP-70 in peripheral splenocytes of animals
undergoing transplantation appeared significant (Figure 3), but it may
be important to optimize this vector prior to use in a clinical ZAP-70
gene therapy trial. In this regard, it is notable that the outcome of
gene therapy for murine XSCID has been reported to be dependent on the
MLV-based retroviral vector used to express the This report demonstrates that MLV-based retroviral-mediated gene
correction of lin
We are indebted to Al Singer for providing the ZAP-70 knock-out
mice as well as for his critical insights and assistance in making this
study possible. The precious SCID-hu experiments of K. Weijer and H. Spits are very much appreciated. We are grateful to Remy Bosselut for
his scientific input. We thank Christophe Duperray for his expertise
and assistance with FACS sorting. Dr Ikunoshin Kato and Setsuko
Yoshimura of Takara Shuzo are generously acknowledged for providing the
recombinant fibronectin fragment and for their continuing assistance.
V. DiBartolo generously provided an
Submitted January 28, 2002; accepted March 22, 2002.
Prepublished online as Blood First Edition Paper, April 30, 2002; DOI 10.1182/blood-2002-01-0247.
M.O. and M.S. contributed equally to this work and are listed in alphabetical order.
Supported by the JPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH, Fundacion YPF, Association France-Israel pour la Recherche en Science et Technologie (AFIRST), and March of Dimes (M.O., M.S., P.M., and N.N., respectively). Also supported by funding from the Association Française contre les Myopathies, March of Dimes grant 6-FY99-406, Association France-Israel pour la Recherche en Science et Technologie, Immune Deficiency Foundation, Association pour la Recherche sur le Cancer (ARC), INSERM and Centre National de la Recherche Scientifique (CNRS) (N.T.), and NIH (F.C.).
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.
Reprints: Naomi Taylor, Institut de Génétique Moléculaire de Montpellier, 1919 Route de Mende, 34293 Montpellier, Cedex 5, France; e-mail: taylor{at}igm.cnrs-mop.fr.
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Top
Abstract
Introduction
c, the gene encoding the common 
80°C for further use.
) followed by strepavidin Dynabeads (Dynal, Compiégne, France) to remove differentiated hematopoietic
cells. After lin depletion, progenitor cells were washed and
stimulated for 48 hours with 10 ng/mL murine interleukin-3 (IL-3)
(Peprotech, Rocky Hill, NJ), 100 ng/mL human IL-6 (Amgen, Thousand
Oaks, CA), and 100 ng/mL stem cell factor (rSCF) (Amgen) in Dulbecco
modified Eagle medium supplemented with 15% fetal bovine serum
(Hyclone, Logan, UT). Stimulated progenitor cells from
ZAP-70
), ZAP-70 cDNA, IRES, and EGFP cDNA are indicated.
(B) Stimulated lin
