Nuclease-free Engineering of B-cells against HIV

B-cell engineering with CRISPR/Cas9 is a promising avenue in the fight against HIV. It has recently been shown that adoptive transfer of B cells engineered to express HIV-broadly neutralizing antibodies (bNAbs) can facilitate the production of HIV-neutralizing antibody titers in immunocompetent mice. Nevertheless, engineering using CRISPR/Cas9 may lead to genotoxicity. Here, we developed site-specific B-cell engineering without nucleases, based only on endogenous class switch recombination (CSR) induced breaks. We use recombinant adeno-associated viral vectors to introduce genes coding for the anti-HIV bNAb 3BNC117 and GFP into CSR induced breaks at the IgH locus of B cells. Our cassette contains a GFP open reading frame, followed by a 2A peptide and a single-chain version of 3BNC117, with only the variable part of the heavy chain provided. The introduced cassette is encoded under a derivative of a murine IgH promoter, which is active only upon on-target integration in proximity to the endogenous enhancers. Antibody expression as a membranal B-cell receptor is facilitated only upon successful integration into the IgH locus. Engineered splenic B cells were GFP-sorted and propagated on feeder cells expressing CD40L and BAFF, for activation and proliferation. Four days post-sorting, 57% of the cells expressed GFP, out of which 73% bound the HIV antigen gp120, demonstrating efficient nuclease-free B cell engineering. We also detected up to 4 ug/ml of 3BNC117 as a secreted IgG in cell culture media. Our method may provide a safe alternative to the rising field of CRISPR-based B-cell editing against the rapidly evolving HIV.