DEVELOPING THERAPEUTIC BIOIMPLANTS FOR HEMOPHILIA A: BIOSAFETY EVALUATION OF TARGETED TRANSGENE INTEGRATION IN PRIMARY HUMAN UMBILICAL CORD-LINING CELLS USING PHIC31 INTEGRASE AND ZINC FINGER NUCLEASES

Abstract

Gene and cell-based therapies have been shown to successfully treat genetic disorders. However, the inability of current gene therapy vectors to direct transgene integrations precisely into safe genomic sites has been associated with oncogenic mutations and fatal leukemias. A major challenge is devising gene transfer techniques that are efficacious and result in durable transgene expression but are not mutagenic. Using hemophilia A as a disease model, we evaluated the potential of phiC31 integrase and zinc-finger nucleases (ZFNs) to modify primary human umbilical cord-lining epithelial cells (CLECs) to stably integrate and express a factor VIII transgene, with the ultimate aim of developing autologous treatment for pediatric patients with hemophilia A. Transgene integration resulted in durable FVIII secretion. Transgenic CLECs were evaluated by transcriptome and genome copy number profiling, deep-sequencing of predicted off-target sites, spectral karyotyping and in vivo tumorigenicity in immunocompromised mice to gauge their biosafety.