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C6 - Gene Targeting and Gene Correction New Technologies

14: Retron Mediated Exon-Sized Genome Insertion Using an All- RNA System

Type: Oral Abstract Session

Presentation Details
Session Title: New Technologies for Gene Targeting and Gene Correction

While exogenous DNA has been instrumental in advancing precise genome editing technology, effective delivery of donor DNA remains a substantial hurdle in therapeutic applications. DNA is frequently packaged in viral vectors, which presents challenges such as non-specificity, liver toxicity, immunogenicity, and high manufacturing costs. There is a need for a non-viral “all-RNA” gene editing modality that enables in vivo targeted insertions and potential re-dosing. Retrons, which hail from bacterial retroelements involved in bacteriophage defense, are minimally two-component RNA systems comprising a non-coding RNA (ncRNA) and an associated reverse transcriptase (RT) which together can be harnessed to create ssDNA templates required for targeted insertion when coupled with a CRISPR nuclease or nickase. We have screened and optimized retrons that show vastly improved editing efficiency of exon-sized gene insertions in cell lines and primary cells. Starting with an in silico retron prediction algorithm we identified >7000 novel retrons. After screening a phylogenetically diverse subset of hundreds of these for precise insertion activity we identified >50 retrons with up to a 45-fold better performance than our Eco1 benchmark. We have utilized a combination of retron RT engineering, ncRNA minimization, chemical modification, and homology-dependent repair manipulation to improve retron-mediated target insertion. Retron system optimization using in silico modeling-based RT engineering achieved 2-fold higher 305 base pair insertion. These combinations of ncRNA engineering and retron system optimization has led to ~40% editing efficiency in hematopoietic stem cells and T cells. Together, our initial data demonstrates retron capabilities to produce repair templates for exon-sized insertions using an all-RNA system in mammalian cells.

Kyong-Rim Kieffer-Kwon, Jacob Layer, Zhongxia Yi, Socheata Ly, Elise Keston-Smith, Shailesh Gurung, Elizabeth Narayanan, Sierra Harken, Abril Fleitas, Aisha A. Aljanahi, Dawn Ciulla, Matthew Pandelakis, Hari Jayaram, Vlad Presnyak, Inna Shcherbakova

ReNAgade Therapeutics, Cambridge, MA"

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