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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 × 107 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. [ABSTRACT FROM AUTHOR]

Using a mouse model of adenosine deaminase–deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1–based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.Molecular Therapy (2006) 13, 1110–1120; doi: 10.1016/j.ymthe.2006.02.013 [ABSTRACT FROM AUTHOR]

Abstract: Mucopolysaccharidosis type I (MPS I) is a lysosomal glycosaminoglycan (GAG) storage disorder caused by deficiency of α-l-iduronidase (IDUA). In this study, we evaluated the potential to perform gene therapy for MPS I by direct in vivo injection of a lentiviral vector, using an IDUA gene knockout murine model. We compared the efficacy in newborn versus young adult MPS I mice of a single intravenous injection of the lentiviral vector. The extent of transduction was dose-dependent, with the liver receiving the highest level of vector, but other somatic organs reaching almost the same level. The phenotypic manifestations of disease were partially improved in the mice treated as young adults, but were nearly normalized at every end-point measured in the mice treated as neonates. In the neonatally treated mice, the expressed IDUA activity resulted in decreased GAG storage, prevention of skeletal abnormalities, a more normal gross appearance, and improved survival. Most strikingly, significant levels of IDUA enzyme were produced in the brain of mice treated as neonates, with transduction of neurons at high levels. The sustained expression of enzymatically active IDUA in multiple organs had a significant beneficial effect on the phenotypic abnormalities of MPS I, which may be translated to clinical gene therapy of patients with Hurler disease. [Copyright &y& Elsevier]

An abstract of the article "A Novel Form of Enzyme Replacement Therapy for ADA-Deficiency: In Vivo Transduction by Neonatal Injection of Lentivirus Expressing ADA," by Denise A. Carbonaro, Xiangyang Jin, Denise Petersen and Donald B. Kohn is presented.

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]

Abstract: Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a “humanized” dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1–3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load. [Copyright &y& Elsevier]

Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells. [Copyright &y& Elsevier]

An abstract of the article "Effective Suicide Gene Therapy of Leukemia in a Novel Model of Retroviral Insertion," by Martina Blumenthal, Dianne Skelton, Denise A. Carbonaro, Karen A. Pepper, Donald B. Kohn is presented.