Human induced pluripotent stem cells (hiPSCs) are a unique population of cells that can serve as an unlimited source for "off-the-shelf" cellular immunotherapeutics. Similar to master cell lines used in the manufacture of monoclonal antibodies, engineered hiPSC lines have the potential to serve as a renewable cell source for the consistent manufacture of homogeneous populations of effector cells for the treatment of thousands of patients. However, the creation of an effective master line is largely dependent on the ability to genetically edit hiPSCs in a precise, efficient and clonal manner. Furthermore, the genetically edited hiPSCs must maintain their inherent ability to continuously self-renew while retaining ability to express engineered modalities upon directed differentiation to the cell type of choice.We have previously reported the use of stage-specific media compositions to enable the footprint-free generation and long-term maintenance of single cell naïve hiPSCs with enhanced clonogenicity, an attribute critical for the derivation of engineered single cell-derived lines. Here we demonstrate the use of our naïve hiPSC platform to precisely introduce, in a site-specific manner, multiple genes into multiple safe harbor loci. By combining our single-cell naïve hiPSC platform with different nuclease-independent and -dependent strategies, we are able to generate large numbers of precisely engineered iPSC clones. The single cell-derived hiPSC clones were subsequently screened in a multiplexed fashion for successful multi-parameter engineering, maintained pluripotency and propensity for differentiation with lack of undesired phenotypes and genomic alterations. Using this approach, we derived individual clones containing a uniform population (>99%) of multi-engineered modalities consisting of tumor targeting, a controllable safety switch and a tracking marker. Moreover, we show that engineered modalities are expressed in undifferentiated and differentiated hiPSCs, including being expressed in >95% of both CD34 positive hematopoietic progenitor cells and CD56 positive natural killer cells. Furthermore, we have generated hiPSC clones with dual suicide genes (inducible Caspase 9 (iCasp9) and Herpes simplex virus thymidine kinase (HSV-TK)) targeted into two independent safe harbor loci, in both mono- and bi-allelic manner. The dual-targeted hiPSC clones were confirmed to have specific insertions into the predicted sites and were screened to exclude random insertions. Concurrent activation of both suicide genes led to complete elimination of engineered hiPSCs and no treatment-refractory cells were observed unlike the case when only one suicide gene was activated. In addition to robust targeted insertions, we were able to generate small insertions and deletions in up to 70% of naïve hiPSCs without selection and homozygous knockout of gene of interest in 100% of cells after selection. Finally, we will discuss efforts to temporally synchronize ectopic gene expression through endogenously regulated promoters by simultaneous endogenous gene disruption and transgene insertion. Overall, we show our naïve hiPSC platform is an ideal renewable source to efficiently generate, genetically engineer, isolate and bank clonally-derived homogenous population of pluripotent cells. These highly-stable pluripotent clonal banks can be repeatedly tapped to facilitate the consistent production of homogenous populations of potent, persistent, scalable and safer off-the-shelf cellular immunotherapeutics. DisclosuresAbujarour:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Meza:Fate Therapeutics: Employment, Equity Ownership. Robinson:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.
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