Xiangyang Jin

β-globin gene transfer to human bone marrow for sickle cell disease

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Preclinical Demonstration of Lentiviral Vector-mediated Correction of Immunological and Metabolic Abnormalities in Models of Adenosine Deaminase Deficiency

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

Gene therapy/bone marrow transplantation in ADA-deficient mice: Roles of enzyme-replacement therapy and cytoreduction

Gene therapy (GT) for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada(-/-)). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion.

Adenosine deaminase (ADA)-deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose-dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: Studies in infant ADA-Deficient mice and rhesus monkeys

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

442. Decreased Survival and Engraftment in Adult ADA-Deficient Mice Co-Transplanted With Uncorrected Lineage-Positive Bone Marrow Cells Combined With Gene-Corrected Lineage-Negative Bone Marrow Cells

ADA-deficiency primarily results in a severe combined immunodeficiency (SCID) but because it is a disorder of the purine salvage pathway the accumulation adenosine and deoxy-adenosine also causes skeletal, neurologic, hepatic and pulmonary abnormalities. Autologous transplantation of retroviral vector-mediated gene-corrected hematopoietic cells after non-myeloablative conditioning for the treatment of ADA-deficient SCID has been shown to be safe and efficacious, especially in very young children, with more variable results in older children. Previously, we have shown that ADA-deficient (ADA-/-) mice transplanted with gene-corrected whole marrow or ADA+/+ marrow had significantly higher engraftment of gene-corrected cells in the bone marrow, liver, and lung compared to transplantation with gene-corrected lineage negative (Lin-) bone marrow cells. The improved engraftment with whole marrow might be due the presence of specific cell populations and/or factors that could facilitate engraftment, including the additional ADA expressed from terminally differentiated cells in the niche during the engraftment period. In new experiments (n=3) we compared outcomes in ADA-/- mice transplanted with gene-corrected Lin- cells (5×10^5) alone (n=10) to these end-points in ADA-/- mice transplanted with gene-corrected Lin- cells combined with either gene-corrected lineage positive (Lin+) cells (5×10^6)(n=8), or with uncorrected Lin+ cells (5×10^6)(n= 10). Marrow cells were fractionated by immunomagnetic sorting and transduced with the lentiviral vector, EFS-ADA at 3 × 10^7 TU/ml (Lin- MOI 60, Lin+ MOI 6) for 24 hours and transplanted into adult (16 weeks) ADA-/- recipients conditioned with 500 cGy and maintained on enzyme replacement for one month after transplant. Unexpectedly, survival to day 100 was significantly reduced in ADA-/- mice transplanted with gene-corrected Lin- cells combined with uncorrected Lin+ cells (37%) compared to mice transplanted with either gene-corrected Lin- cells alone (90%) or with gene corrected Lin- cells co-transplanted with gene-corrected Lin+ (88%)(p<0.0001). Overall engraftment and VCN were measured in the thymus, spleen, bone marrow, lung and liver and neither was significantly improved with the addition of gene-corrected Lin+ cells when compared to transplant of gene-corrected Lin- cells alone. Even with so few ADA-/- mice transplanted with Lin- and uncorrected Lin+ cells surviving (n=3), overall engraftment in spleen was significantly lower (8%) when compared to ADA-/- co-transplanted with gene-corrected Lin+ cells (66%)(p<0.001). Likewise, these mice also had fewer splenic T and B cells numbers when compared to mice co-transplanted with gene-corrected Lin+ cells (p<0.05). Taken together with the very poor survival, we hypothesize the uncorrected Lin+ cells may place an extra burden systemically or within the bone marrow niche to decrease engraftment and survival.

37. A Novel Form of Enzyme Replacement Therapy for ADA-Deficiency: In Vivo Transduction by Neonatal Injection of Lentivirus Expressing ADA

Genetic deficiency of adenosine deaminase (ADA) is responsible for approximately 20% of human Severe Combined Immune Deficiency (SCID). A murine model of ADA-deficient SCID was produced by Blackburn and Kellems (U. of Texas) by ADA gene knock-out. These mice die from non-infectious pulmonary insufficiency between days 19|[ndash]|20 post-natal, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. We previously reported that neonatal mice injected with a lentiviral vector expressing hu ADA have increased survival (ASGT 7th annual meeting, June 2004). We have continued our efforts to characterize the effects of systemic ADA enzyme expression in this model of ADA-deficient SCID, using a SIN HIV-1-based lentivirus, SMPU-R-MND-ADA, pseudotyped with VSV-G and given via the temporal vein on day 2 of life. Viral supernatant was produced at high titer (1|[times]| 10^10 TU/ml). When mice received a dosage of 1.0 |[times]| 10^7 TU/kg surivial was not prolonged, but when mice received a dosage of 1.6 |[times]| 10^8 TU/kg, survival was significantly prolonged. An initial cohort of mice received PEG-ADA ERT until day 45 after neonatal injection of vector (n=4). A second cohort of mice was injected with the lentiviral vector as neonates and never given ERT (n=5). Both cohorts had survival greater than 180 days without further treatment. Mice were either sacrificed at 2 or 6 months of age. Immunophenotype analysis revealed the absence of a SCID phenotype with normal numbers of T and B lymphocytes and adequate lymphocyte proliferation in response to stimulation with ConA. Vector proviral copy number was determined by Q-PCR in peripheral blood, lung, liver, thymus, spleen and bone marrow. Proviral marking was highest in the liver (1.2+/|[minus]|0.18 copies/cell) and lung (0.12+/|[minus]|0.013 copies/cell), as has been reported for multiple bio-distribution studies of lentivirus (and AAV) given by tail vein to mice. Proviral marking in the thymus (0.0024+/|[minus]|0.0001 copies per cell), spleen (0.0037+/|[minus]|0.0001 copies/cell) and bone marrow (0.0023+/|[minus]|0.0001 copies/cell) were at least 10|[ndash]|100 fold lower than the level of marking observed in the liver and lung tissues analyzed. T-cells (CD4/8) sorted from thymus or B-cells (CD19) sorted from spleen had proviral marking similar to the marking observed in the organs from which the cells were isolated. Peripheral blood marking was very low at 2 mo (0.00007+/|[minus]|.0006 copies/cell) and undetectable by 6 mo. To determine if there was transduction of hematopoietic stem cells in surviving mice, bone marrow was transplanted into lethally irradiated, secondary recipients (n=6). There was no marking in the peripheral blood, bone marrow or colony forming units (CFUs) from bone marrow. Thus, the mice had restoration of immune function, despite very low levels of gene correction of T lymphocytes and hematopoietic cells. The findings suggest that ADA enzyme made in highly transduced organs (e.g. liver, lung) may be rescuing ADA-deficient T lymphocytes in trans; this approach of intravenous lentiviral vector delivery of the ADA gene may provide a novel form of in vivo enzyme replacement therapy.