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A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34[SUP+] cells was started in 1993.ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking[SUP1,2]. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants[SUP3,4]. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single prethymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID)[SUP5,6]. [ABSTRACT FROM AUTHOR]

ADA deficiency in humans results in a SCID phenotype, characterized by extremely low levels of T, B and NK cells, and other organ systems also may be affected (skeletal, hepatic and CNS). In mice, however, knockout of the ADA gene is lethal because ADA is required for gestation. A viable murine model of ADA- deficient SCID was developed in which the ADA-deficient embryos are rescued with an ADA mini-gene expressed only during gestation. Post-natally, these mice die from a non-infectious pulmonary insufficiency by day 19-20, which can be rescued using enzyme replacement therapy (ERT) with PEG-ADA. In these studies, newborn ADA-deficient pups (whole litters) were transplanted with normal congenic bone marrow (5.0 × 10^6 cells in 100 ul PBS via the superficial temporal vein). These non-ablated neonatal congenic BMT resulted in long-term survival (up to 360d) and multi-system improvement, including restored fertility and immune function. To characterize the extent and nature of the graft, we have analyzed these mice at 16, 60 and 180 days after neonatal BMT, compared to normal mice and to ADA-deficient mice maintained on ERT. By day 16, untreated ADA deficient pups (n=4) were profoundly lymphopenic with dramatically decreased levels of thymic T-cells, splenic T-cells and B-cells, and no lymphocyte proliferation in response to stimulation with ConA. Thymocyte and splenocyte cell population numbers and function were improved at 16, 60 and 180 days in neonatal recipients of normal congenic bone marrow, and while sub-normal in several aspects, were consistently better than in mice maintained on ERT. The levels of donor cell chimerism in recipient tissues (thymus, spleen, bone marrow, liver and lung) were similar, in the range of 0.3-3.0% (determined by Q-PCR and Southern blot analysis for the normal ADA gene allele). We obtained increases in the chimerism of donor cells in each of the cell compartments of recipients by administering low dosages of total body irradiation to the neonatal pups prior to BMT. ADA deficient pups born from dams receiving busulfan on d18 of gestation (n=7) had higher levels of chimerism than those receiving no ablation. In similarly-treated neonatal common γc gene knockout mice (X-linked SCID) receiving normal congenic bone marrow, we observed mostly donor-derived cells in the lymphoid compartment, in contrast to myeloid engraftment that was at low levels, similar to those seen in all lineages in the ADA gene knock-out mice. In summary, despite low engraftment, ADA-deficient neonates can be rescued with normal bone without further treatment. The ADA-deficient SCID mouse model does not show a selective proliferation of donor-derived, genetically normal T lymphoid cells after non-ablated BMT, suggesting that other mechanisms are responsible for the immune reconstitution.Molecular Therapy (2006) 13, S418–S418; doi: 10.1016/j.ymthe.2006.08.1191 [ABSTRACT FROM AUTHOR]

In humans, the genetic deficiency of adenosine deaminase (ADA) is responsible for approximately 20% of Severe Combined Immune Deficiency (SCID) and results in severe lymphopenia. The current standard is either an allogeneic bone marrow transplant from an MHC-matched donor or enzyme replacement therapy (ERT) with PEG-ADA; BMT from a donor other than a matched sibling has poor outcome. Because of a postulated selected advantage for corrected T cells, ADA-deficient SCID has been an early candidate disease for gene therapy. Recent studies by Aiuti and co-workers using oncoretroviral vectors to transduce CD34+ cells from the bone marrow of ADA-deficient SCID patients, together with cessation of ERT and moderate cytoreduction with Busulfan show clinical benefit, however, the relative roles of the cytoreduction and the cessation of PEG-ADA have yet to be elucidated. A murine model of ADA-deficient SCID was produced by Rodney Kellems and co-workers (U.Texas, Houston) by ADA gene knock-out. These mice die from non-infectious pulmonary insufficiency between days 19-20 post-natal, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. In these studies, we evaluated the role of ERT as a single variable on the ability of gene-corrected cells to repopulate 10-week old ADA-deficient mice that received a cytoablative dosage of total-body irradiation (900 cGy) prior to receiving ADA-deficient marrow transduced with the retroviral vector MND-MFG-huADA. These mice, previously maintained on ERT, were either continued on the same low dose ERT (n=8) or not (n=8). ADA -/- mice receiving ERT had 89% survival and those not receiving ERT had 75% survival after radiation and BMT. Mice were euthanized between 4-5 months post transplant and were perfused with PBS prior to tissue removal. Proviral marking was assessed by qPCR in the peripheral blood leukocytes, thymus, spleen, bone marrow, lung and liver, as well as in sorted cell populations from the thymus (CD4/8), spleen (CD19) and bone marrow (CD11b). Gene marking was seen in all of the tissues assessed with little difference between those animals receiving ERT and those that did not. Although not significant, marking was slightly higher in the hematopoietic tissues analyzed from mice receiving ERT. Proviral marking (copies/ cell) was highest in the thymus and spleen and lowest in the bone marrow. Cell populations enriched from these organs had marking similar to the organ from which they were isolated. Marking in the lung and liver was similar to that seen in the hematopoietic tissues and may be relevant due to the severe lung and hepatocellular pathology seen in ADA-deficient mice. With or without ERT, ADA- deficient mice receiving gene therapy had improved immune cellularity and function compared to ADA-deficient mice receiving mock transductions and maintained on ERT. In these studies, we have observed no selective lymphoid expansion of the gene-corrected T cells, whether ERT was given or withheld, but nevertheless there was immunologic improvement from host (ADA-deficient) cells.Molecular Therapy (2006) 13, S419–S419; doi: 10.1016/j.ymthe.2006.08.1192 [ABSTRACT FROM AUTHOR]

Background: Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study.Methods: Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution.Results: With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant.Conclusion: These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile.Trial Registration: ClinicalTrials.gov NCT00794508.Funding: Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124. [ABSTRACT FROM AUTHOR]