446. Stable Correction of Alkylator Resistance in Fanconi Anemia Cells By a Mitotically Replicating Non-Integrating Lentiviral Vector

Fanconi anemia (FA) is an inherited multisystem disorder leading to symptomatic hematopoietic defects by early school age in most patients. Observations of lymphoid mosaicism and spontaneous intragenic correction of stem cells leading to clonal hematopoiesis in a small number of patients indicate that phenotypically corrected cells enjoy a survival advantage over FA deficient cells. Accordingly, FA is a leading genetic disorder target for HSPC directed gene therapy. However, reports over the insertional complications of commonly used integrating vectors continue to raise concerns, while available non-integrating lentivectors (NILVs) are generally lost from dividing hematopoietic stem cells. We recently showed that the incorporation of human Scaffold/Matrix Attachment Region (S/MAR) sequences in NILVs results in the cellular retention of NILV episomes in actively dividing cell lines and HSPCs without selection. Southern analysis, Alu PCR and episome specific PCR all confirmed long-term episomal existence of this vector without genomic integration (NAR. 2014; PMID: 24474068). Here, we designed and tested an episome lentivector to phenotypically correct FA cells (aNILV GFP-FANCC). Results showed the vector expressing the human FANCC ORF fused with GFP could be readily packaged, with stable persistence of vector episomes and sustained GFP expression. Taking advantage of the selective expansion of phenotypically corrected cells, we transduced a FANCC -deficient human cell line pD331 by using aNILV-GFP-FANCC vector. Exaggerated sensitivity to DNA alkylating agents (e.g. Mitomycin C, MMC) is a hallmark of FA deficient cells and can be used as a selection agent. At week 2, we observed 15% retention of GFP expressing cells in MMC treated pD331-aNILV-FANCC population versus 3% retention in non-treated pD331-aNILV-FANCC cells. Long-term maintenance of cells up to 6 weeks under MMC selection resulted the enrichment of corrected cells up to 95% that is comparable to the enrichment observed by integrating LV expressingGFP-FANCC in pD331 cells under MMC selection. Molecular studies of aNILV episomes from long-term cultures by episome specific PCR revealed LTR episome persistence. Integration analysis by Alu-PCR from the bulk population with Southern analysis of clones is ongoing. We already reported that episomal S/MAR NILV transduced short-term murine hematopoietic stem and progenitor cells and are currently characterizing this vector in a murine model of Fanconi Anemia. In conclusion, epsiome maintenance under selective pressure and reversal of canonical alkylator sensitivity in FA cells make this novel aNILV-GFP-FANCC vector a consideration for expanded preclinical exploration.