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GENE THERAPY
From the National Human Genome Research Institute, the
Howard Hughes Medical Institute/National Institutes of Health Research
Scholar Program, and the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD; and the
Department of Medicine, University of Nevada School of Medicine, Reno.
X-linked severe combined immunodeficiency (XSCID) is caused by
mutations of the common gamma chain of cytokine receptors, X-linked severe combined immunodeficiency (XSCID)
is caused by mutations in the IL2RG gene leading to absent
or defective common gamma chain protein ( Gene transfer into autologous hematopoietic stem cells may become an
alternative to current standard treatment with BMT or an adjunctive
post-BMT therapy. The biology of Several in vitro studies have demonstrated retroviral transduction of
normal IL2RG complementary DNA (cDNA) into B-cell lines from
XSCID patients with restoration of cell-surface In recent work by our group, IL2RG was transduced into
The choice of retrovirus envelope also affects transduction efficiency
of retroviral vectors into human hematopoietic progenitors. Most
vectors used to date for gene transfer into human cells have had the
amphotropic envelope.24 However, transduction efficiency is poor, consistent with the very low levels of amphotropic envelope receptor expressed by highly enriched human hematopoietic cell populations.25,26 Therefore alternative envelopes have
been considered for transduction of human hematopoietic stem cells. The
gibbon ape leukemia virus (GALV) and the feline endogenous virus RD114
permit transduction of human hematopoietic cells,27-35 and
their receptors are expressed at higher levels than the amphotropic receptor on CD34+CD38 In the present study, we compared amphotropic, GALV-pseudotyped, and
RD114-pseudotyped MFGS-gc vectors by transducing autologous cells from
post-BMT XSCID patients who had persistent defects in B-cell immunity
or combined B- and T-cell immunity. Both XSCID B-cell lines and
mobilized peripheral blood CD34+ hematopoietic progenitors
demonstrated provirus integration and new cell-surface Human subjects, cell lines, and primary cells
Volunteers and XSCID patients were given subcutaneous injections of
granuloctyle colony-stimulating factor (G-CSF) (Filgrastim) (Amgen,
Thousand Oaks, CA) 10 µg/kg/d for 5 days.42 On day 5, leukapheresis was performed by means of a Fenwal CS3000 continuous-flow blood cell separator (Baxter Healthcare, Deerfield, IL).
CD34+ cells were purified by means of the Isolex 300I
paramagnetic bead immunoaffinity-positive selection system (Newell,
Irvine, CA). CD34+ cells were either used fresh or
cryopreserved and thawed for transduction experiments. Human cord blood
cells were purchased from National Disease Research Interchange
(Philadelphia, PA).
DNA fingerprinting of posttransplantation XSCID patients.
MFGS-gc-pseudotyped retrovirus producer lines. PG13 and FLYRD18 packaging cell lines were cultured in Dulbecco modified Eagle minimum essential medium supplemented with 10% newborn calf serum, 2 mM L-glutamine, and penicillin/streptomycin (Life Technologies). The 293SPA packaging cell line was cultured in improved modified Eagle medium zinc option medium with 10% FBS and 2 mM L-glutamine.The MFGS-gc vector was generated by cloning the human IL2RG
cDNA into the NcoI and BamHI sites of MFGS
(Figure 1).20 An amphotropic
MFGS-gc producer line was made by transfection of 293SPA cells
with the MFGS-gc plasmid by calcium-phosphate coprecipitation followed
by colony selection. GALV-pseudotyped MFGS-gc was produced by
transducing PG13 packaging cells44 with supernatant from the ecotropic GP+E86 MFGS-gc.20
RD114-pseudotyped MFGS-gc was prepared by transducing the FLYRD18
packaging cell line with supernatant from PG13 MFGS-gc. Transduced
cells were sorted after immunofluorescent staining of
Retroviral transduction EBV-transformed B cells were cultured overnight without serum and then placed in RPMI 1640 medium supplemented with 20% FBS, 2 mM L-glutamine, and penicillin/streptomycin for 4-hour recovery at 37°C, 5% CO2. The cells were then cultured at 5 × 105/mL in 24-well polystyrene plates coated with fibronectin (Retronectin CH-296; Takara Shuzo, Otsu, Japan), in 50% filtered supernatant from amphotropic, GALV- or RD114-enveloped retrovirus producer cells supplemented with 6 µg/mL polybrene (Sigma, St Louis, MO). The B cells were placed at 32°C, 5% CO2 for 2 hours15,21 followed by 7 hours at 37°C. Transductions of B-cell lines were performed on 2 consecutive days. Following the second transduction, cells were cultured in complete medium supplemented with IL-2 (Hoffman-LaRoche, Nutley, NJ). After 2 to 3 days of growth, a third and fourth transduction were performed as above.CD34+ cells were transduced following previously described protocols.35,45 Briefly, purified mobilized peripheral CD34+ cells were prestimulated by culturing overnight in X-Vivo 10 medium (BioWhittaker, Walkersville, MD) containing 1% human serum albumin and cytokines at 37°C in 5% CO2. Cytokines used for transductions with amphotropic and GALV-pseudotyped viruses were Pixykine (IL-3/granulocyte-macrophage-CSF fusion protein; 50 ng/mL) (Immunex, Seattle, WA); Flt-3 ligand (100 ng/mL); and stem cell factor (SCF) (50 ng/mL). In some experiments, IL-6 (50 ng/mL), IL-3 (1 ng/mL), and G-CSF (10 ng/mL) were substituted for Pixykine. For transduction with the RD114-pseudotyped vector, the cytokine cocktail consisted of Flt-3 ligand (300 ng/mL), SCF (300 ng/mL), and IL-6 (50 ng/mL). Transductions were performed in fibronectin-coated wells with 1 to 1.5 × 106 cells per well and 1 to 2 × 105 cells per milliliter. Cytokines other than Pixykine were from R&D Systems (Minneapolis, MN). Following prestimulation, transductions with amphotropic and GALV-pseudotyped retroviruses were performed as daily 6- to 7-hour incubations on 4 consecutive days with 90% fresh, filtered virus supernatant supplemented with 6 µg/mL polybrene. RD114 transductions were performed by adding the stimulated cells to fibronectin-coated wells that had been preloaded with retroviral particles on day 2 of cytokine activation as described by Kelly et al.35 In utero transplantation to generate human-sheep chimeras Following transduction, CD34+ cells were resuspended in medium containing 10% FBS and sent by overnight mail for injection into preimmune fetal sheep at 55 to 60 days of gestation by means of the amniotic bubble procedure described previously.46 An average of 5 × 105 untransduced, mobilized peripheral blood CD34+ cells from a healthy adult donor were injected into 3 sheep fetuses. Another sheep fetus received 1 × 105 untransduced human cord blood CD34+ cells. Two fetuses received an average of 4 × 105 untransduced XSCID CD34+ cells from patient 1; 5 fetuses each received 1.8 × 105 RD114-transduced XSCID CD34+ cells, also from patient 1. Single sheep fetuses each received either 2 × 106 untransduced or 5 × 106 RD114-transduced XSCID CD34+ cells from patient 3.Detection of provirus DNA Semiquantitative PCR and Southern blot analysis were performed on DNA isolated from untransduced and transduced B-cell lines and CD34+ cells of patients and healthy controls. Human lymphoid and myeloid cells were recovered from peripheral blood of human-sheep chimeras by means of serial immunomagnetic bead selection of CD3+, CD19+, CD14+, and CD45+ cells (Dynabeads M-450; Dynal, Lake Success, NY).For PCR, the 5' primer (5'-CAGTGTTTTGTGTTCAATGTCG) and 3' primer (5'-TGCAGTTTTAGCA TCTGTGTGG) were located in exons 2 and 3, respectively, of human IL2RG, allowing coamplification of both a genomic DNA segment spanning intron 2 (473 base pairs [bp]) and a provirus cDNA segment (265 bp). Each PCR reaction contained 32P-deoxycytidine 5'-triphosphate (dCTP) and was carried out with 35 cycles of 94°C for 20 seconds, 60°C for 20 seconds, and 72°C for 1 minute. The genomic and cDNA IL2RG signals were quantitated with a Molecular Dynamics PhosphorImager and ImageQuant software (both from Molecular Dynamics, Sunnyvale, CA), with the genomic IL2RG band used as an internal control (modified from Aviles Mendoza et al20). Southern blotting of SstI-digested DNA was performed by means of 32P-dCTP-labeled IL2RG cDNA as a probe. Analysis of transduced cells Expression of Restoration of Analysis of Statistical analysis We performed 2 tests using the
population comparison platform in the FlowJo software package. The
platform allows direct comparison between different populations through
the Probability Binning (Chi(T) or PB) comparison algorithm, related to
the Cox 2 approach. The 2 is converted
into a metric, T(), that can be used to estimate the
probability that a test population is different from a control population. When T() = 0, the 2 populations are
indistinguishable (P = .5). When T() = 1, the
populations differ by 1 SD, giving the probability that the 2 populations differ as P < .17. A T() that exceeds 4 implies that the 2 distributions are different, with
P < .01 (99% confidence). All reported nonzero
percentages for cell populations are statistically different from
control populations.
Donor cell engraftment in posttransplantation XSCID patients Table 1 summarizes the mutations in IL2RG, BMT treatment and outcome, and latest lymphocyte functional status of the 4 XSCID patients. Patient 1 carried a nonsense IL2RG mutation in exon 5, causing undetectable levels of messenger RNA (mRNA) and no cell-surface c in
EBV-transformed B-cell lines. Patient 2 had a defect in the
poly(adenylic acid) addition signal of IL2RG, with
absent to trace amounts of c. Patients 3 and 4 had
mutations truncating the intracellular domain, but resulting in normal
c expression at the cell surface. All XSCID patients
included in this study had received haploidentical, parental T
cell-depleted transplants without cytoreductive conditioning within
the first year of life, but had not achieved full reconstitution of
their immune systems (Table 1). They continued to require
immunoglobulin replacement and additional treatments as noted. Patients
2 and 3 were lymphopenic, with poor to absent T-cell responses to
mitogens and antigens as compared with healthy controls.
Chimerism analysis by PCR was performed to delineate the post-BMT donor and host contributions to hematopoietic lineages in our patients (Table 1). No patient had any detectable donor-derived alleles in DNA from myeloid cells. Patient 2 had no detectable donor cell alleles in either T- or B-cell compartments. Patients 1, 3, and 4 had 5% or fewer donor B cells. The same assay was used to analyze CD34+ progenitor cells purified after G-CSF mobilization and apheresis. In no patient were donor-derived hematopoietic progenitor cells detectable. Retroviral transduction of patient cells Titers of the producer lines were estimated by Southern blotting and RNA slot-blot analysis to be equivalent and high (see "Patients, materials, and methods"). XSCID B-cell lines were transduced as described with amphotropic, GALV-, and RD114-pseudotyped virus supernatants. With the use of coding sequence primers flanking IL2RG intron 2, genomic and provirus DNA were amplified in the same PCR reaction (Figure 2A). Provirus equivalents per genome (relative to genomic signal) were calculated, and within each set of transductions, a relative ratio of provirus integration was obtained by normalizing to the amphotropic virus transduction rate. For all XSCID patients and controls, transduction with GALV- and RD114-pseudotyped viruses produced higher rates of provirus integration than did the amphotropic virus, with RD114 more than 10-fold higher than either of the other 2. The relative amounts of provirus integration were further analyzed by Southern blot, confirming that the RD114 envelope afforded the most provirus integration, followed by GALV and then amphotropic (Figure 2B).
Transduced B-cell lines from patient 1, patient 2 (both
Restoration of c signaling pathway, we
took advantage of the specificity of the interaction of
c with JAK3 and the subsequent activation of STAT5,
using IL-2-induced pSTAT5 as an indicator of c signal
transduction. We found no pSTAT5 by Western analysis in untransduced
B-cell lines from XSCID patients, but detectable pSTAT5 in all
transduced cell lines following IL-2 stimulation (not shown). In
contrast to normal control cells assayed by flow cytometry (Figure
4A), untransduced XSCID cells had no pSTAT5 following IL-2 exposure. The proportion of pSTAT5 in transduced cells matched that of cells to which c expression was
newly restored (Figure 4B). Comparison of IL-2-stimulated cells with
their unstimulated controls showed that untransduced XSCID B-cell lines
did not phosphorylate STAT5. Trace amounts of pSTAT5 induced by IL-2
exposure were detected in cells transduced with amphotropic-enveloped
MFGS-gc. Higher amounts of pSTAT5 production on average were achieved
with GALV MFGS-gc, but the highest proportion of pSTAT5+
cells was seen following transduction with RD114 MFGS-gc. In the latter
cells, the levels of pSTAT5 were comparable to those seen in cells from
normal controls (Figure 4A). For the XSCID B-cell line of patient 3 with truncated c, detection of pSTAT5 was more
informative than cell-surface staining for c.
Engraftment and lineage development of human cells in sheep To evaluate whether transducedc-deficient
CD34+ cells could differentiate into T and B lymphocytes,
thus demonstrating correction of the XSCID defect, we transplanted the
human cells into fetal sheep to create stable chimeras. In a
preliminary experiment (Table 3), we
compared CD34+ cells from normal cord blood, known to have
high numbers of hematopoietic stem cells, to peripheral blood
CD34+ cells obtained from a healthy donor using our G-CSF
mobilization protocol. Immunomagnetic bead-selected CD34+
cells from freshly harvested cord blood were cultured for 4 days in
cytokine-supplemented medium and then injected into a preimmune sheep
fetus. Another sheep fetus received cytokine-cultured mobilized peripheral blood CD34+ cells from a healthy adult. At 1 month after birth of the sheep, their peripheral blood was analyzed for
human cell engraftment. Comparable levels of chimerism were found in
the recipient of cord blood and of peripheral blood, 3.0% and 2.7%,
respectively (Table 3). Similar percentages of human-derived cells were
also found in the T-cell, B-cell, and myeloid lineages. The
c-expression profiles of human lymphoid and myeloid
cells were similar in the sheep engrafted with cord blood and
peripheral blood CD34+ cells (not shown).
Untransduced XSCID peripheral CD34+ cells from patient 1 and patient 3 and the same cells transduced with RD114 MFGS-gc were transplanted into preimmune fetal sheep to generate human-sheep hematopoietic chimeras. Flow cytometric analyses of peripheral blood
obtained postnatally from the sheep are summarized in Table 4. The mononuclear cells bearing
human-specific CD45 ranged from 1.4% to 5.1%. The 5 sheep that
received identical aliquots of 1.8 × 105 transduced
cells from patient 1 had engraftment of CD45+ human
leukocytes varying from 2.0% to 5.1%, and all developed roughly
similar proportions of mature human lineages. As expected, untransduced
and transduced XSCID CD34+ cells were equally capable of
development into human myeloid cells, demonstrated with CD11b and/or
CD15, accounting for 47% to 95% of the human leukocytes. As
illustrated in Figure 5, the myeloid
cells were
Post-BMT XSCID patients with varying degrees of persistent immune
compromise were studied. One patient had no donor cell engraftment despite multiple attempted BMTs. The other 3 patients had T cells derived from their bone marrow donor, but B cells and myeloid cells of
host origin. No patient had detectable CD34+ cells
from the bone marrow transplant donor, suggesting that the
stem cells or progenitors giving rise to T cells in these individuals
were rare, and underlining the positive selection for T cells with
intact Since the first successful BMT for XSCID was reported in
1968,47 BMT has become the standard treatment. Techniques
such as T-cell depletion have improved BMT outcome for patients without an HLA-matched sibling donor, making possible survival of up to 70% to
90% or more of infants with XSCID.8,48,49 However, immune
reconstitution, especially of specific antibody production by B cells,
remains incomplete in many post-BMT XSCID patients,8-10 a
finding possibly related to their ability to make
Successful retroviral gene transfer depends on several factors
involving the retrovirus vector and the target cell. The impact of
retrovirus vector backbone on transgene expression was recently highlighted in a murine transplantation model by Aviles Mendoza et
al.20 In a comparison of 5 retrovirus vectors containing IL2RG cDNA, the highest transgene expression level, achieved
by MFGS-gc, was required to overcome competition with endogenous B-lymphoid progenitors to produce B cells in unirradiated
W/Wv hosts. We have now extended these findings
by testing the hypothesis that MFGS-gc viruses of equivalent titer, but
with different pseudotypes, would transduce human hematopoietic stem
cells with varying efficiency and thus lead to varying degrees of
functional restoration. DNA analysis of provirus in transduced patient
cells illustrated the significant differences between amphotropic,
GALV-pseudotyped, and RD114-pseudotyped retrovirus transduction
efficiencies. As reflected in the relative ratios of provirus
equivalents per genome, RD114 facilitated the transduction of the
highest proportion of target cells.35 The differences
observed in provirus integration paralleled the relative levels of
retrovirus receptor mRNA expressed in successive enrichments for
hematopoietic stem cells (D.M.B., unpublished data, December
2001). The most striking finding on DNA analysis of cells
transduced with RD114-enveloped virus was that the provirus per copy
number was greater than 1 (the signal from provirus cDNA exceeded that
from genomic DNA). The improvement in transduction efficiency over
amphotropic-enveloped virus that was achieved with the GALV and RD114
envelopes was further confirmed by analyses of new The human-sheep hematopoietic chimera, an excellent model for studying
human hematopoietic stem cell engraftment and maturation, has become an
important tool for our preclinical evaluation of gene therapy
strategies for XSCID. Despite reported phenotypic and functional
differences among progenitors from mobilized peripheral blood, cord
blood, and bone marrow,50-53 we achieved equivalent human
lymphoid and myeloid cell development in sheep transplanted with cord
blood or mobilized peripheral CD34+ cells from
healthy human donors, and we showed durable human chimerism
lasting for 10 months. These findings suggest that peripheral mobilized
CD34+ cells are appropriate target cells for a human XSCID
gene transfer trial. The sheep model also allowed us to demonstrate
directly the successful development of
We thank Stacie Anderson, Gilda Linton, and Roxanne Fischer for technical assistance; Joie Davis for coordinating patient visits and samples; Charlie Carter for CD34+ cell isolation; and the XSCID patients and their families for their ongoing participation and encouragement.
Submitted November 6, 2001; accepted February 25, 2002.
Supported by the Howard Hughes Medical Institute (E.J.T.); the Veterans Administration (E.D.Z.); the Mathers Charitable Foundation (E.D.Z.); and National Institutes of Health grants HL40722, HL46566, and HL39875 (E.D.Z.).
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: Jennifer M. Puck, Genetics and Molecular Biology Branch, NHGRI/NIH, Bldg 49, Rm 4A14, 49 Convent Dr, Bethesda, MD 20892-4442; e-mail: jpuck{at}nhgri.nih.gov.
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M. L. Lucas, N. E. Seidel, C. D. Porada, J. G. Quigley, S. M. Anderson, H. L. Malech, J. L. Abkowitz, E. D. Zanjani, and D. M. Bodine Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes Blood, July 1, 2005; 106(1): 51 - 58. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
B+
phenotype, has readily given rise to donor T cells, but often failed to
give rise to donor B cells, whether or not cytoreductive conditioning
was given prior to BMT. A long-term study of chimerism and B-cell
function in post-BMT T
. Cells stimulated with IL-2
were compared with unstimulated cells. For flow cytometric analysis,
the percentage of p-STAT5+ cells was estimated by an
isotype gate of fewer than 1.0%.
cells in cord blood and bone marrow.
Blood.
1995;86:3745-3753