527. Improvement of Pre-Clinical Non-Human Primate Model for Pluripotent Stem Cell Based Therapies by Introducing Marker Genes in Safe Harbor Locus
Authors: Ravi Chandra Yada, So Gun Hong, Jizhong Zou, Kyujoo Choi, Arnaud Carpentier, T. Jake Liang, Randall Merling, Colin Sweeney, Harry Malech, Moonjung Jung, Marcus Corat, Youngshun Lin, Ilker Tunc, Xujing Wang, Maryknoll Palisoc, Stefania Pittaluga, Thomas Winkler, Cynthia Dunbar
Journal: Molecular Therapy (2016)

Induced pluripotent stem cells (iPSCs) are being developed as sources for clinical cellular regenerative therapies, as well as valuable in vitro human disease models. Derivation of iPSCs from non-human primates (NHP) affords the opportunity to test the safety, feasibility and efficacy of proposed iPSC-derived cellular delivery in species with physiology, immunology and scale similar to humans. However, there is a need for stable and safe labeling methods for iPSCs and their differentiated progeny allowing analysis of survival, proliferation, tissue integration and biodistribution, in vitro and in vivo. Typically, marker genes have been inserted into target cells by transduction with randomly-integrating viral vectors. However, these methods raise concerns regarding genotoxicity and transgene silencing, particularly in pluripotent stem cells, limiting their utility for tracking and eventual clinical applications. Targeted integration into genomic "safe harbors" offer a promising alternative approach to mark target cells, potentially circumventing these issues. The adeno-associated virus integration site 1 (AAVS1) has been proposed as a suitable safe harbor for human cells, and we now investigate its utility in our rhesus macaque NHP iPSC model. We have efficiently knocked-in both a truncated CD19 (hΔCD19) marker gene a non-immunogenic and clinical relevant marker, or green fluorescent protein (GFP) at the homologous AAVS1 site in rhesus iPSCs (RhiPSCs) using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) system. PCR and Southern blot analyses demonstrated highly efficient knock-in into the AAVS1 locus, with over one third of clones screened containing only targeted but not random integrations. (Table 1). Edited RhiPSC-GFP/hΔCD19 clones retained a normal karyotype and pluripotency - as shown by teratoma formation. Directed differentiation of these clones to neutrophils, hepatocytes or cardiomyocytes was not hindered by the knock-in of marker genes into the AAVS1 sites. Notably, transgene expression was stable in undifferentiated RhiPSCs and differentiated cell types derived from the RhiPSC (Figure 1), in contrast to prior experience with viral vector delivery. We have established a computational platform to assess off-target effects of guide RNAs in the rhesus genome. Genetically marked RhiPSCs afford a unique opportunity to develop clinically relevant models for iPSC-based cell therapies.Table 1Summary of CRISPR-mediated gene editing in rhesus iPSCsOriginal iPSC cloneReporter geneClones with TI/Clones screened1based on by PCR analysisClones without RI/Clones with TI2based on Southern blot analysisZG15-M11-10hΔCD194/42/8GFP14/145/9ZG32-3-4hΔCD19ND1/4GFPND2/4ZH26-HS41hΔ CD19ND1/4Total18/18(100%)11/29 (37.9%)RI: random integration, TI; targeted integration, ND: not determined1 based on by PCR analysis2 based on Southern blot analysis Open table in a new tab RI: random integration, TI; targeted integration, ND: not determined