671. SIV-Derived Lentiviral Vectors Pseudotyped with Modified RD114 Envelope and Containing Mutant MGMT Efficiently Transduce Human CD34+ PBSCs and Allow Selection

Abstract

Lentiviral vectors have become an attractive tool for gene transfer into hematopoietic stem cells (HSCs) because they may more efficiently transduce non-dividing multipotent HSCs. Most lentiviral gene transfer studies use human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with VSV-G envelope. We investigated the ability of vectors based on simian immunodeficiency virus (SIVmac) and pseudotyped with modified RD114 envelope to efficiently transduce human HSCs using both marker constructs and a construct containing the therapeutic gene for X-linked chronic granulomatous disease (CGD) gp91phox. RD114-pseudotyped SIV vector particles transiently produced following transfection of 293T HEK cells were concentrated by high-speed centrifugation. The marker gene construct encoded a green fluorescent protein-mutant methylguanine methyltransferase fusion protein (GFP-MGMT|[ast]|) which confers resistance to O6-benzylguanine (BG) and a DNA alkylating agent such as 1,3-bis (2-chloroethyl)-1-nitrosurea (BCNU) and retains green fluorescence. Mobilized healthy human CD34+ peripheral blood stem cells (PBSCs) were efficiently transduced (>90% GFP+ on day 4 of culture) and could be selected with BG/ BCNU ex vivo on day 4 of culture (1 hour 5 |[mu]|M BG followed by 2 hours 7 |[mu]|M BCNU) to increase stable ex vivo marking to 97%. When non-selected transduced PBSCs were transplanted into NOD/ SCID mice, marking of human cells at 6 weeks post transplant was consistently greater than 50%. In vivo selection of these animals is ongoing. We next created an IRES bicistronic SIV modified RD114 pseudotyped vector encoding both gp91phox and MGMT|[ast]|, and demonstrated that we could transduce gp91phox-deficient X-linked CGD patient CD34+ PBSCs to a level of 30% ex vivo and this could be increased in culture to 55% following BG/BCNU selection at day 4. Transplant of the unselected cells into NOD/SCID mice resulted in 2.2% marking of the engrafted CGD human myeloid cells and using the ex vivo selected cells marking was 5.1%. Thus, while the therapeutic selective vector did perform, we find that it is more difficult to obtain high pre-concentration titers of this larger bicistronic construct. However, preliminary experiments using significantly higher concentrations of centrifuged vector indicate that low starting titers can be augmented by concentration to achieve much higher transduction rates similar to those seen with the marker vectors and these experiments are ongoing.