37. A Novel Form of Enzyme Replacement Therapy for ADA-Deficiency: In Vivo Transduction by Neonatal Injection of Lentivirus Expressing ADA
Authors: Denise A. Carbonaro, Xiangyang Jin, Denise Petersen, Donald B. Kohn
Journal: Molecular Therapy (2005)

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.