Human Induced Pluripotent Stem Cells Incorporating Safe Harbor Loci Integrated Inducible Suicide Systems for Use in the Application of Cellular Therapeutics

As the field of human induced pluripotent stem cell (hiPSC) research continues to advance, and as the clinical investigation of genetically-engineered hiPSC-derived cellular therapeutics begins to emerge, safety concerns relating to the administration of genetically-altered cells must be addressed and mitigated. A number of strategies including recombinant peptides, monoclonal antibodies, small molecule-modulated enzyme activity and gene-specific modifications have been explored to facilitate the selective elimination of aberrant cells. Of these, the application of genetically-encoded inducible "suicide" systems that can be rapidly activated by a specific non-toxic chemical inducer represents a very attractive, targeted approach for eliminating administered cells without damaging surrounding cells and tissues. Most previous studies have employed viral vectors and short promoters to stably introduce suicide-genes, including herpes complex virus thymidine kinase or inducible caspase-9 (iCasp9), into human cells. The use of viral vectors can lead to random integration events which can disrupt or activate disease-related genes, potentially causing deleterious effects. In addition, many artificial promoters and genome regions are prone to epigenetic silencing in both pluripotent and differentiated states, resulting in cells becoming unresponsive to suicide gene induction. Thus, it is of great importance to identify optimal integration sites and promoters in order to maintain functional suicide gene responses and facilitate the development of genetically-engineered cellular therapeutics.

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.