In-vivo engineering of B cells elicits memory retention and allows for secretion of broadly neutralizing antibodies in mice

Abstract

Abstract A potential single-shot HIV therapy may be transplanted engineered B cells allowing strong secretion of broadly neutralizing antibodies (bNAbs). However, extensive, and expensive ex-vivo manipulations performed in specialized facilities hinders clinical potential of this approach. Furthermore, allogeneic B cell therapy necessitates MHC-II compatibility to receive mandatory T-cell help. To overcome these limitations, we engineer B cells in-vivo. In particular, we demonstrate that a single, systemic dose of dual AAV, one coding for CRISPR/Cas9 and another coding for a bNAb donor cassette, allows for site specific integration in B cells. Following immunizations, we show memory retention and bNAb secretion at high titers. Antibodies secreted by the engineered B cells were found to be of multiple isotypes and IgGs could neutralize autologous and heterologous pseudoviruses. We found engineered B cell subsets in the spleen and blood. We detected homing of in-vivo engineered cells to germinal centers and bone marrow. Biodistribution of the donor AAV over time and as compared to a CRISPR- group, indicated expansion of engineered B cells in lymphatic tissues. We determined minimal CRISPR/Cas9 off-target cleavage, using unbiased, highly sensitive, CHANGE-Seq analysis. Finally, we diminished on-target, non-productive double-strand breaks at undesired tissues by expressing Cas9 from a B cell specific promoter. Eliciting a specific, neutralizing serological response to hypervariable viruses is a long-standing challenge in medicine. B cell engineering provides an opportunity to express therapeutic antibodies to generate an adaptive and evolving immunity.