In vivo B cell engineering

As cost and complexity of autologous B cell engineering ex vivo may be prohibitive, we developed the first ever in vivo B cell engineering protocol, based on a single systemic injection of adeno associated viral (AAV) vectors coding for CRISPR/Cas9 and for the desired bNAb cassette, which is targeted for integration at the IgH locus. Following immunizations, we demonstrated memory retention and bNAb secretion at neutralizing titers. We showed that CRISPR/Cas9 off-target cleavage is minimal, using unbiased CHANGE-Seq analysis. We further reduced the on-target cleavage at undesired tissues by expressing saCas9 from a B cell specific promoter. Our in vivo B cell engineering is thus a safe, potent, and scalable method for expressing desired antibodies against HIV and beyond.