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
Authors: Denise A. Carbonaro-Sarracino, Michael L. Kaufman, Xiangyang Jin, Sabine Geiger, Danielle Clark, Donald B. Kohn
Journal: Molecular Therapy (2015)

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.