BACKGROUND: Recent clinical trials conducted in people living with HIV (PLWH) have shown that the administration of anti-HIV broadly neutralizing antibodies (bNAbs) is safe, significantly decreases the plasma viremia and potentiates HIV-specific host immune responses. However, waning levels of injected bNAbs are frequently associated with viral rebound and bNAb resistance during analytical treatment interruption (ATI). These challenges significantly hamper bNAbs potential. To address this issue, we are interested in applying CRISPR-based gene editing methods to permanently reprogram B cells to express one or more bNAbs from the endogenous immunoglobulin heavy chain (IGH) locus. To facilitate rapid translation to PLWH, we have focused on studies in our well-established nonhuman primate (NHP) model of acquired HIV-1 and anti-HIV gene therapy. Here we present foundational NHP B cell manufacturing data that will be applied in upcoming NHP experiments.
METHODS: Rhesus macaque B cells were isolated and activated in culture before ex vivo editing with IGH-directed CRISPR-Cas9 ribonucleoprotein and adeno-associated virus (AAV). CRISPR targeting promotes the insertion of AAV-delivered homology donor template encoding an engineered VRC01 bNAb into an intronic region of the IGH locus, replacing the endogenous heavy chain expression. IGH editing efficiency and specific integration of VRC01 in the targeted locus were assessed by Inference of CRISPR Edits (ICE) and in-out PCR, respectively. Cell surface VRC01 expression was quantified by flow cytometry.
RESULTS: CRISPR RNP edited the IGH locus in 80-90% of NHP B cells. These edits facilitated the insertion of our VRC01 donor sequence, which was confirmed by in-out PCR. Five to eight days post-editing, flow cytometry assays identified up to 15% of NHP B cells expressing VRC01 bNAb at the cell surface. Our editing approach did not impact NHP B cell proliferation, consistent with successful expression and trafficking of the engineered VRC01 bNAb.
CONCLUSIONS: Our data demonstrate that NHP B cells can be efficiently engineered to express anti-HIV bNAbs. This ongoing study paves the way for further in vivo evaluation in our immunocompetent NHP model. In addition to adoptive transfer of ex vivo­-manufactured B-cells, we are developing in vivo editing approaches to accelerate efficacious, scalable and feasible B cell-based therapies for PLWH.