234. Polyclonal Insertion Sites in Two Rhesus Macaques after Non-Myeloablative Transplantation with MFGS-gp91phox Transduced Autologous CD34+PBPC

Mutagenesis activation of the LMO-2 gene was observed to be associated with the development of lymphoid leukemia several years after two infants with X-SCID had their immune systems restored by gene therapy with a murine onco-retrovirus vector encoding the human IL2 receptor common gamma chain. Just recently, a third infant treated with the same vector was reported to also have developed T cell leukemia, however, insertion site analysis is pending in this case. These observations highlight the importance of the conductance of large animal marking studies which follow the insertional pattern of vectors planned for human treatments. We have recently reported that a high titer RD114 pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the oxidase defect in CD34+ cells from patients with X-linked chronic granulomatous disease in the xenotransplant NOD/SCID mouse model. In anticipation of considering clinical use of this vector, we transduced G-CSF-FLT3L fusion protein mobilized peripheral blood CD34+ cells (CD34+ PBPC) of two healthy rhesus macaques with the RD114-MFGS-gp91phox. Transduced CD34+PBPC were transplanted on culture day 4 into two non-myeloablative (2|[times]|300 Rads) irradiated animals. The in vivo marking decreased over the first months and stabilized at about 8 months post transplant. Marking for animal J976 and D406 at 15 and 19 months, respectively is 1.3% and 0.3% in B lymphocytes, 0.3% and 0.3% in neutrophils and 0.7% and 0.4% in T lymphocytes at which time the animals remained healthy. Vector insertion analysis was performed using LAM-PCR and gel electrophoresis. Analyses of cloned and sequenced integration sites revealed 44 integrations sites in monkey J976, and 33 integration sites in monkey D406 with no predominant clone seen to emerge over time. 27% (J976) and 12% (D406) of the insertion sites were identified as coding regions (intron or exon). Nested PCR with insertion site specific primers enabled recovery of identified sites from different cell lineages at different time points. In summary, we detected a high number of integration sites in two rhesus macaques transplanted with gene-modified CD34+ cells despite low marking due to non-myeloablative conditioning.