915. Insertional Analyses in Rhesus Monkey Blood Cells after Non-Myeloablative Hematopoietic Stem Cell Marking with a Therapeutic Onco-Retroviral Vector for X-Linked Chronic Granulomatous Disease

Abstract

Wild-type murine onco-retroviruses cause cancer in part by insertional mutagenesis, but until recently, cancers had not been observed in humans or animals subjected to marking or treatment with the highly modified murine retrovirus vectors in current use. Recently, 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 therapeutic onco-retrovirus vector. Insertional mutagenesis activation of other oncogenes associated with development of cancers has been reported in vector-marked mice. These observations highlight the importance of encouraging many investigators to conduct large animal marking studies which follow the insertional pattern of vectors planned for human treatments. We have recently reported that a very 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 NOD/SCID mouse model. In anticipation of considering clinical use of this vector, we transduced mobilized peripheral blood CD34+ cells (PBSC CD34+) of two healthy rhesus macaques with the RD114-MFGS-gp91phox. Transduced PBSC CD34+ with an average two vector copies per cell were transplanted on culture day 4 into two non-myeloablative (2× 300 Rads) irradiated animals. The moderately high levels of in vivo marking (>6%) seen in the first months following gene marking decreased 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 by LAM-PCR, using gel electrophoresis and GeneScan analysis to demonstrate that despite the relative low marking at 10 months and later animal D406 had more than 25 and animal J976 had more than 40 separate insertional events. 34% of integration sites are located in coding regions. No predominant clone was seen to emerge over time and no patterns of insertion site preferences were detected up to this point.