Program: Oral and Poster Abstracts
Session: 801. Gene Therapy and Transfer: Poster II
To effectively select and test suicide systems under the control of various promoters in combination with different safe harbor loci integration strategies, we took advantage of our proprietary hiPSC platform, which enables single cell passaging and high-throughput, 96-well plate-based flow cytometry sorting. In a single hiPSC per well manner, we utilized both nuclease-independent and nuclease-dependent strategies to efficiently and precisely integrate various suicide gene expression cassettes in AAVS1 or ROSA26 safe harbor loci. Several integration vectors, each containing a suicide gene expression cassette downstream of various exogenous and endogenous promoters, including endogenous AAVS1 or ROSA26, cytomegalovirus, elongation factor 1α, phosphoglycerate kinase, hybrid CMV enhancer/chicken β-actin and ubiquitin C promoters, were tested to systematically analyze and compare the activity of different suicide systems in both hiPSCs and hiPSC-derived differentiated cells. To conduct high-throughput analyses of these integration and expression strategies, we selected an iCasp9 suicide gene platform where rapid caspase-9 mediated cell death can be induced by small molecule chemical inducers of dimerization such as AP1903. Several endogenous promoters were found to drive persistent expression of iCasp9 during clonal expansion of hiPSCs, but the expression level was determined to be too low to effectively respond to AP1903 treatment. Expression of iCasp9 under the control of various exogenous promoters was lost during prolonged clonal expansion of hiPSCs, and failed to drive AP1903-induced cell death. However, one promoter maintained high levels of iCasp9 expression during the long-term clonal expansion of hiPSCs. Furthermore, these iCasp9-integrated clonal lines underwent rapid cell death in the presence of AP1903, and no residual cell survival was observed when cultures were allowed to recover in the absence of the dimerizing molecule. To test whether epigenetic landscape alterations would abrogate suicide gene-mediated response, hiPSC clones were differentiated into three somatic lineages in vitro and were found to be completely subject to AP1903-induced cell death. Clones were also specifically differentiated towards hematopoietic cells to demonstrate complete induction of cell death by AP1903 treatment. When injected into NSG mice to form teratomas, similar cell death-mediated response was observed in vivo. Notably, one hiPSC clone contained certain rare cells and did escape induced cell death, and this clone and these rare cells were characterized to assess the molecular mechanisms of escape. Our study describes novel findings toward designing optimal safety systems for integration into hiPSC-derived cellular therapies.
Disclosures: Huang: Fate Therapeutics Inc: Employment . Lan: Fate Therapeutics Inc: Employment . Parone: Fate Therapeutics Inc: Employment . Clarke: Fate Therapeutics Inc: Employment . Abujarour: Fate Therapeutics Inc: Employment . Robinson: Fate Therapeutics Inc: Employment . Meza: Fate Therapeutics Inc: Employment . Lee: Fate Therapeutics Inc: Employment . Shoemaker: Fate Therapeutics Inc: Employment . Valamehr: Fate Therapeutics Inc: Employment .
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