Senior Scientist Revvity Cambridgeshire, England, United Kingdom
Abstract: Pluripotent stem cells (PSCs) hold great promise for manufacturing advanced cell therapies. Off-the-shelf allogeneic products derived from PSCs, engineered to be compatible with large patient cohorts, can broaden access to these therapies. However, their sensitivity to DNA damage presents challenges for complex genome editing. Base editors offer a solution due to their reduced genotoxicity compared to nuclease-based technologies. We developed the Pin-point platform, which enables the modular assembly of base editors composed of DNA binding Cas and DNA modifying deaminase components associated via an aptamer encoded in the sequence-targeting guide RNA (gRNA). Owing to the aptamer-dependent recruitment of the deaminase component to target DNA sequences, the Pin-point platform uniquely allows multi-purposing of a single Cas nickase component for simultaneous multiplexed base editing and targeted transgene knock-in. To demonstrate the platform's utility for engineering allogeneic PSCs, transient delivery of mRNAs encoding a Pin-point base editor (Rat APOBEC1 and SpCas9 nickase) with synthetic aptamer-encoding gRNAs were used to generated clonal hypoimmunogenic iPSC lines with various genotypes, including large cargo transgene integration, using an automated clone tracking and picking workflow. These hypoimmunogenic iPSC lines, created via multiplexed base editing and simultaneous base editing with targeted transgene integration, retained pluripotency, exhibited expected human leukocyte antigen (HLA) phenotypes, and evaded both innate and adaptive immune responses when differentiated into therapeutic cell products. The Pin-point platform thus represents a safe and efficient solution for multiple genome engineering operations via a novel single-step process compatible with downstream automation, dramatically streamlining the development of allogeneic iPSC-derived cell therapies. Pin-point™ Technology Platform: The Pin-point™ base editing platform technology is available for clinical or diagnostic study and commercialization under a commercial license from Revvity.
