In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice

Abstract

As a potential single-shot HIV therapy, transplanted engineered B cells allow robust secretion of broadly neutralizing antibodies (bNAbs). However, ex vivo engineering of autologous B cells is expensive and requires specialized facilities, while allogeneic B cell therapy necessitates MHC compatibility. Here, we develop in vivo B cell engineering, by injecting two adeno associated viral vectors, one coding for saCas9 and another coding for a bNAb. Following immunizations, we demonstrate memory retention and bNAb secretion at neutralizing titers. We observed minimal CRISPR/Cas9 off-target cleavage, using unbiased CHANGE-Seq analysis, while on-target cleavage at undesired tissues is reduced by expressing saCas9 from a B cell specific promoter. In vivo B cell engineering is thus a safe, potent and scalable method for expressing desired antibodies against HIV and beyond.

A CRISPR-Cas Cure for HIV/AIDS

“…Alternatively, one could use CRISPR-Cas to mediate the integration of antiviral genes of interest. Nahmad et al described a dual adeno-associated virus (AAV) CRISPR–saCas9-mediated integration of antibody genes into the Ig loci of primary human B cells to express anti-HIV broadly neutralizing antibodies (bNAbs) in vivo [ 62 , 116 ]. Using this method, the authors achieved high titers of anti-HIV bNAbs that neutralize infection-competent pseudoviruses in mice [ 62 , 116 ].…”

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin

“… 12 , 20 , 21 , 22 , 23 Directed B cell immunoglobulin gene engineering to introduce novel anti-HIV paratropes has the distinct advantage that class switching and affinity maturation can occur, thereby potentially improving anti-HIV-1 breadth and neutralization potency. 24 , 25 , 26 , 27 , 28 However, an ongoing concern in vaccine development and the use of a single bNAb for therapy remains the potential for HIV-1 resistance. 24 , 25 To limit HIV-1 escape, there are ongoing discussions to deliver bNAb cocktails with bNAbs of differing specificity with the intent to limit resistance development.…”

Heavy-chain CDR3-engineered B cells facilitatein vivoevaluation of HIV-1 vaccine candidates

“…However, current wild-type rodent, rabbit, or macaque models are not optimal in large part because the diversity (D) gene segments, key contributors to the HCDR3, are highly species-specific (Lefranc, 2014;Lefranc et al, 2015).Transgenic mice can be engineered to express mature or progenitor apex bnAbs (Crooks et al, 2021;Melzi et al, 2022), but these mice are slow to generate or modify, and their antigen reactive repertoires are essentially monoclonal, biasing antigen comparisons. Strategies to engineer mature murine B cells to express human bnAbs and adoptively transfer these cells into wildtype mice have been developed for novel cell-based therapies (Voss et al, 2017;Hartweger et al, 2019;Moffett et al, 2019;Voss et al, 2019;Huang et al, 2020;Nahmad et al, 2020;Nahmad et al, 2022). These engineered B cells proliferate in response to antigen and generate neutralizing sera, but this response has impaired somatic hypermutation, and is monoclonal and therefore unrepresentative of a human repertoire.…”