Next Generation IgH-reprogramming for in vivo delivery

Project Summary Genome-modified B cells are promising as functional cure for HIV since they can be used to elicit durable broadly neutralizing antibody (bnAb) memory responses in animal models when they are reprogrammed to express these antibodies as functional B cell antigen receptors. This reprogramming requires insertion of antibody cassettes into the native IgH locus for their expression using native cell regulatory elements and heavy chain constant genes. However, concerns over double strand break (DSB)-mediated genome editing dampens the impact of this therapeutic approach. The field of genome editing is instead focusing on developing technologies for precision genome editing, or genome editing without DSBs. Precision editing is even more necessary when evaluating the potential of in vivo B cell editing for IgH reprogramming, where safety is of top priority. In this project, we describe multiple orthogonal genome engineering approaches towards precision gene insertion of bnAbs into the IgH locus, including prime-editing-assisted insertion genome editing (PASSIGE) and CRISPR- free recombineering. We will discuss using a novel method of retron-mediated donor amplification that can enable an all-RNA system for donor-mediated gene insertion. Finally, individualized delivery systems will be evaluated in vitro alongside each editing approach, weighing features including delivery efficacy, safety of both vector and cargo, ease of production, and clinical precedent, with successful systems progressing to in vivo evaluation. Success of this project will enable an off-the-shelf compound for IgH reprogramming with potential as an in vivo administered treatment for HIV infection.