[Show abstract][Hide abstract] ABSTRACT: Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.
The Journal of clinical investigation 07/2009; 119(7):1952-63. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic granulomatous disease (CGD) is characterized by recurrent infections and granuloma formation. In addition, we have observed a number of diverse autoimmune conditions in our CGD population, suggesting that patients with CGD are at an elevated risk for development of autoimmune disorders. In this report, we describe antiphospholipid syndrome, recurrent pericardial effusion, juvenile idiopathic arthritis, IgA nephropathy, cutaneous lupus erythematosus, and autoimmune pulmonary disease in the setting of CGD. The presence and type of autoimmune disease have important treatment implications for patients with CGD.
The Journal of allergy and clinical immunology 10/2008; 122(6):1097-103. · 12.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the phagocyte nicotinamide dinucleotide phosphate oxidase catalytic subunit gp91(phox). Gene therapy targeting hematopoietic stem cells (HSCs) can correct CGD, but permanent correction remains a challenge. Lentiviral vectors have become attractive tools for gene transfer, and they may have the potential to transduce very primitive HSCs. We used a self-inactivating RD114/TR-pseudotyped simian immunodeficiency virus (SIVmac)-based vector encoding human gp91(phox) for ex vivo transduction of peripheral blood-mobilized stem cells (PBSCs) from patients with X-CGD. In PBSCs from two patients, ex vivo transduction efficiencies of 40.5 and 46% were achieved, and correction of oxidase activity was observed in myeloid cells differentiating in culture. When transduced PBSCs from these patients were transplanted into nonobese diabetic/severe combined immunodeficient mice and compared to normal control, 10.5 and 7.3% of the human myeloid cells in bone marrow developing at 6 weeks from the human xenografts expressed the gp91(phox) transgene. Sustained functional correction of oxidase activity was documented in myeloid cells differentiated from engrafted transduced PBSCs. Transgene marking was polyclonal as assessed by vector integration site analysis. These data suggest that RD114/TR SIVmac-based vectors might be suitable for gene therapy of CGD and other hereditary hematologic diseases.
[Show abstract][Hide abstract] ABSTRACT: A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the DeltadblGATA mouse. We demonstrate here that bone marrow progenitors isolated from DeltadblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured DeltadblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured DeltadblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1E(B)), located approximately 3700 bp downstream to the previously described first exon found in hemopoietic cells (1E(A)) and approximately 42 bp upstream to another variant first exon, 1E(C). These data suggest that cultured progenitors are able to circumvent the effects of the DeltadblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.
The Journal of Immunology 09/2007; 179(3):1693-9. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retroviral gene therapy can restore immunity to infants with X-linked severe combined immunodeficiency (XSCID) caused by mutations in the IL2RG gene encoding the common gamma chain (gammac) of receptors for interleukins 2 (IL-2), -4, -7, -9, -15, and -21. We investigated the safety and efficacy of gene therapy as salvage treatment for older XSCID children with inadequate immune reconstitution despite prior bone marrow transplant from a parent. Subjects received retrovirus-transduced autologous peripherally mobilized CD34(+) hematopoietic cells. T-cell function significantly improved in the youngest subject (age 10 years), and multilineage retroviral marking occurred in all 3 children.
[Show abstract][Hide abstract] ABSTRACT: WHIM(warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis) syndrome is a rare immunodeficiency caused in many cases by autosomal dominant C-terminal truncation mutations in the chemokine receptor CXCR4. A prominent and unexplained feature of WHIM is myelokathexis (hypercellularity with apoptosis of mature myeloid cells in bone marrow and neutropenia). We transduced healthy human CD34(+) peripheral blood-mobilized stem cells (PBSCs) with retrovirus vector encoding wild-type (wt) CXCR4 or WHIM-type mutated CXCR4 and studied these cells ex vivo in culture and after engraftment in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model. Neither wt CXCR4 nor mutated CXCR4 transgene expression itself enhanced apoptosis of neutrophils arising in transduced PBSC cultures even with stimulation by a CXCR4 agonist, stromal cell-derived factor-1 (SDF-1 [CXCL12]). Excess wt CXCR4 expression by transduced human PBSCs enhanced marrow engraftment, but did not affect bone marrow (BM) apoptosis or the release of transduced leukocytes into PB. However, mutated CXCR4 transgene expression further enhanced BM engraftment, but was associated with a significant increase in apoptosis of transduced cells in BM and reduced release of transduced leukocytes into PB. We conclude that increased apoptosis of mature myeloid cells in WHIM is secondary to a failure of marrow release and progression to normal myeloid cell senescence, and not a direct effect of activation of mutated CXCR4.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-5 (IL-5) promotes signal transduction and expansion of eosinophil colonies in bone marrow via interactions with its heterodimeric receptor (IL-5R). Two variants encoding soluble forms of the alpha subunit (sIL-5R alpha) have been described, although the signals promoting and/or limiting differential transcription remain to be clarified.
Our intent was to explore the role of IL-5 in regulating differential transcription of these splice variants in vivo.
We have designed a quantitative reverse transcriptase-polymerase chain reaction assay to detect transcripts encoding the transmembrane, soluble 1 and 2 forms of IL-5R alpha in two strains of wild-type (BALB/c and C57BL/6) and corresponding IL-5 gene-deleted mice. Wild-type mice respond to S. mansoni infection with a gradual increase in serum IL-5 and eosinophilia, which is not observed in IL-5 gene-deleted mice.
We find that IL-5 is not necessary for differential splicing to occur in vivo, as all three forms of the IL-5R alpha are detected in both strains of IL-5 gene-deleted mice, with ratios of transcript expression (transmembrane : soluble 1 : soluble 2) that were indistinguishable from their wild-type counterparts. Differential splicing does vary markedly between strains, potentially because of local effects of strain-specific polymorphisms.
European Journal Of Haematology 10/2006; 77(3):181-90. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lentiviral vectors are capable of transducing non-dividing/slow proliferating cells. Thus, they are suggested to more efficiently transfer genes into long-term repopulating hematopoietic stem cells (HSCs). Human immunodeficiency virus (HIV)-1-based vectors have so far dominated the field of lentiviral vector development for transduction of HSCs. Because HIV is a known human pathogen, alternative lentiviral vectors e.g. derived from simian immunodeficiency virus (SIV) might be preferred for application in humans if similar efficiency could be achieved. We investigated different variables influencing the titer of transiently produced green fluorescent protein (GFP) encoding SIV-derived lentiviral vectors pseudotyped with different envelopes. 293T HEK cells were co-transfected with transfer vector (v), gag-pol encoding (gp), rev-tat encoding (rt), and envelope encoding (e) plasmid DNA at different concentrations. The virus titer produced was dependent on the ratio of the components with the best results at 5 (v) : 3 (gp) : 1.2 (rt) : 1 (e) for VSV-G and a MLV-amphotropic envelope and 4 (v) : 2.4 (gp) : 1 (rt) : 2 (e) for a modified RD114 envelope (RD114+). DNA quality is very critical and if transfection efficiency is high but virus production poor endotoxin contamination or other problems with the purity of the plasmid DNA should be considered. The application of chloroquine or sodium butyrate did not result in a benefit. In our studies the use of IMDM supplemented with 10% FBS was the best medium to use compared to DMEM + 10% FBS and IMDM + 5% or + 0% FBS. FuGENE6-based transfection did not outperform our calcium-phosphate-precipitation protocol. We also found that different individuals following the same protocol had significantly different production efficiencies, and that individuals over time showed greatly increased ability to produce much higher titers presumably through a |[ldquo]|learning|[rdquo]| process, but could not always pinpoint why their results improved. Concentration of large volumes of virus production medium containing virus particles could be achieved by high-speed (non-ultra-) centrifugation (18,600 g for 4 h) for both VSV-G and modified RD114 pseudotyped vectors and by lower speed centrifugation (8,000 g for 3 h) for MLV-amphotropic vectors with excellent recovery. Thus, under best conditions and after concentration (150 to 200-fold) vector titers of approximately 2|[times]|10E7 IU/ml could be achieved as determined using the human erythroleukemia cells K562 as a target. We demonstrated excellent performance of the SIV vectors in human, rhesus, and canine CD34+. It was possible to achieve ex vivo transduction rates in human CD34+ cells of greater than 90% after three overnight transductions. It is of note that mouse bone marrow cells could not be transduced with VSV-G pseudotyped SIV-vectors in contrast to a similar HIV-1-based vector demonstrating profound host cell difference in these vectors. In conclusion, these studies might be helpful to optimize SIV-derived lentiviral vector production and performance with a special focus on their use for human and large animal (rhesus, dog) HSCs.
[Show abstract][Hide abstract] ABSTRACT: X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency and early mortality, the only potential cure being hematopoietic stem cell (HSC) transplantation or gene therapy. Current clinical gene therapy protocols targeting HSCs are based upon ex vivo gene transfer, potentially limited by the adequacy of HSC harvest, transduction efficiencies of repopulating HSCs, and the potential loss of their engraftment potential during ex vivo culture. We demonstrate an important proof of principle by showing achievement of durable immune reconstitution in XSCID dogs following intravenous injection of concentrated RD114-pseudotyped retrovirus vector encoding the corrective gene, the interleukin-2 receptor gamma chain (gamma c). In 3 of 4 dogs treated, normalization of numbers and function of T cells were observed. Two long-term-surviving animals (16 and 18 months) showed significant marking of B lymphocytes and myeloid cells, normalization of IgG levels, and protective humoral immune response to immunization. There were no adverse effects from in vivo gene therapy, and in one dog that reached sexual maturity, sparing of gonadal tissue from gene transfer was demonstrated. This is the first demonstration that in vivo gene therapy targeting HSCs can restore both cellular and humoral immunity in a large-animal model of a fatal immunodeficiency.
[Show abstract][Hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S256|[ndash]|S256; doi: 10.1016/j.ymthe.2006.08.741
664. WHIM Syndrome Type Myelokathexis In Vivo in the NOD/SCID Mouse Model Following Transplant of Healthy Human Stem Cells Transduced with C-Terminus Truncated CXCR4
Toshinao Kawai1, Uimook Choi1, Lanise Cardwell1, Suk See Deravin1, Nora Naumann1, Narda L. Whiting-Theobald1, Gilda F. Linton1, Sebastian Brenner1,2, Philip M. Murphy1 and Harry L. Malech11Laboratory of Host Defenses, NIAID NIH, Bethesda, MD2Department of Pediatrics, University Clinic Carl Gustav Carus, TU-Dresden, Germany
[Show abstract][Hide abstract] ABSTRACT: In addition to increased susceptibility to infections in patients with chronic granulomatous disease (CGD), a higher incidence of sterile inflammatory disorders in these patients has been noted. However, sarcoidosis has not been reported previously in CGD. In this report, we describe two patients who have CGD and a disorder consistent with sarcoidosis on the basis of unequivocal clinical-radiographic presentations, their responses to treatment, and serum angiotensin-converting enzyme levels. Serum angiotensin-converting enzyme levels were measured in 26 other patients with CGD to establish an appropriate reference range. A possible relationship between CGD and sarcoidosis is discussed.
[Show abstract][Hide abstract] ABSTRACT: Lentiviral vectors have become an attractive tool for gene transfer into hematopoietic stem cells (HSCs) because they may more efficiently transduce non-dividing multipotent HSCs. Most lentiviral gene transfer studies use human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with VSV-G envelope. We investigated the ability of vectors based on simian immunodeficiency virus (SIVmac) and pseudotyped with modified RD114 envelope to efficiently transduce human HSCs using both marker constructs and a construct containing the therapeutic gene for X-linked chronic granulomatous disease (CGD) gp91phox. RD114-pseudotyped SIV vector particles transiently produced following transfection of 293T HEK cells were concentrated by high-speed centrifugation. The marker gene construct encoded a green fluorescent protein-mutant methylguanine methyltransferase fusion protein (GFP-MGMT*) which confers resistance to O6-benzylguanine (BG) and a DNA alkylating agent such as 1,3-bis (2-chloroethyl)-1-nitrosurea (BCNU) and retains green fluorescence. Mobilized healthy human CD34+ peripheral blood stem cells (PBSCs) were efficiently transduced (>90% GFP+ on day 4 of culture) and could be selected with BG/ BCNU ex vivo on day 4 of culture (1 hour 5 μM BG followed by 2 hours 7 μM BCNU) to increase stable ex vivo marking to 97%. When non-selected transduced PBSCs were transplanted into NOD/ SCID mice, marking of human cells at 6 weeks post transplant was consistently greater than 50%. In vivo selection of these animals is ongoing. We next created an IRES bicistronic SIV modified RD114 pseudotyped vector encoding both gp91phox and MGMT*, and demonstrated that we could transduce gp91phox-deficient X-linked CGD patient CD34+ PBSCs to a level of 30% ex vivo and this could be increased in culture to 55% following BG/BCNU selection at day 4. Transplant of the unselected cells into NOD/SCID mice resulted in 2.2% marking of the engrafted CGD human myeloid cells and using the ex vivo selected cells marking was 5.1%. Thus, while the therapeutic selective vector did perform, we find that it is more difficult to obtain high pre-concentration titers of this larger bicistronic construct. However, preliminary experiments using significantly higher concentrations of centrifuged vector indicate that low starting titers can be augmented by concentration to achieve much higher transduction rates similar to those seen with the marker vectors and these experiments are ongoing.