Fig. 1: Engineering B cells to express an anti-HIV bNAb.

a Targeting scheme. An rAAV-delivered cassette is targeted to the J-C intron of the IgH locus using CRISPR/Cas9. The bicistronic cassette encodes the light and heavy chains of the 3BNC117 anti-HIV bNAb, under the control of an enhancer dependent (ED) promoter. Splicing with endogenous constant segments allows the expression of a BCR and differentiation into memory B cells and Ig secreting plasma cells upon subsequent antigen-induced activation and alternative polyadenylation (Alt. PolyA). Targeting the J-C intron upstream of the intronic enhancer (iEμ) and switch region further facilitates CSR and SHM. See also Supplementary Fig. 1. b Activation and engineering scheme. Human B cells are collected from blood samples, activated using an anti-RP105 (TLR4 homolog) antibody, electroporated by CRISPR/Cas9 RNP and transduced using rAAV-6. Splenic B cells are activated using the TLR4 agonist LPS electroporated by CRISPR/Cas9 RNP and transduced using rAAV-DJ. c Flow cytometry plots measuring binding of the HIV gp120 antigen by the 3BNC117 BCR following activation and engineering of primary cells. Cells transduced with the donor rAAV and without gRNA serve as a negative control, gating on live, singlets. d, e Quantification of C for mouse (n = 5 for –gRNA and n = 8 for +gRNA, each dot represents a biologically independent sample, data represented as mean values +/− SD) d and human (n = 3, each dot represents a biologically independent sample, data represented as mean values +/− SD) e primary cells. ****p < 0.0001, **p = 0.0040; two-tailed t-test. f Flow cytometry plots demonstrating ERK phosphorylation in primary mouse or human B cells engineered with 3BNC117 and in vitro activated with the gp120 antigen of the YU2.DG HIV strain, gating on singlets.