Multilayered HIV-1 resistance in HSPCs through CCR5 Knockout and B cell secretion of HIV-inhibiting antibodies

Abstract

Allogeneic transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs engraft and reconstitute multiple hematopoietic lineages in vivo and can be engineered to express multiple antibodies simultaneously (in pre-clinical models). Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the foundation for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

Fig. 1. HIV inhibiting antibodies maintain function with a peptide linker.

a Diagram of antibody expression with and without a peptide linker driven by the cytomegalovirus (CMV) promoter. b SDS-PAGE western blot of purified traditional and linker antibodies. c IC₅₀ of traditional and linker antibodies against a panel of HIV-1 pseudoviruses measured in vitro with TZM-bl infection assay (IC₅₀ was calculated from technical duplicate infections across serial dilutions of each antibody). Dotted lines indicate the linker antibody did not inhibit infection within the tested antibody concentrations (≤5 µg/mL) for a given pseudovirus. Pseudoviruses listed in each inset were not inhibited within the tested antibody concentrations (≤5 µg/mL) by both the traditional and linker antibody (NN) or the linker antibody only (NN-Linker). Source data are provided as a Source Data file.

Fig. 2. Efficient targeted integration of antibody expression cassettes at the CCR5 locus in HSPCs.

a Schematic of gene editing strategy to knock-in linker antibody expression cassettes at the CCR5 locus. Created in BioRender. Feist, W. (2025) https://BioRender.com/l43f611. b Allelic integration frequency in CB CD34⁺ HSPCs targeted with AAV6 cassettes for linker antibodies as indicated. Each AAV6 was used at a multiplicity of infection (MOI) of 1250. Values represent independent biologic donors (n = 6 for mock and 10-1074 and n = 3 for Ibalizumab, PGDM1400, CAP256V2LS, and 10-1074+Ibalizumab). c Percent of (knock-in) KI alleles integrated with 10-1074 (blue) or Ibalizumab (purple) within the 10-1074+Ibalizumab targeted cells shown in b (n = 3). d Distribution frequency of WT (gray), INDEL (blue), and KI (green) alleles in CB CD34⁺ HSPCs targeted at CCR5 with AAV6 MOI as indicated with or without 0.5 μM AZD7648 (AZD, n = 3). e Percent of knock-in alleles integrated with 10-1074 (blue) or Ibalizumab (purple) within the “10-1074+Ibalizumab” targeted cells shown in d (n = 3). f Distribution frequency of WT (gray), INDEL (blue), and KI (green) alleles in CB CD34⁺ HSPCs used for the CFU assay described in G, H, and I. Cell were targeted at CCR5 with an AAV6 MOI of 625 for each construct with or without AZD7648 (n = 2). g Number of BFU-E (light gray), CFU-GM (dark gray), and CFU-GEMM (black) colonies formed per 500 cells plated (n = 2, with technical duplicates wells for each donor). h Relative frequency of BFU-E (light gray), CFU-GM (dark gray), and CFU-GEMM (black) colonies formed within the CFU assay (n = 2, with technical duplicate wells for each donor). i Frequency of genotypes from single-cell-derived colonies within the 10-1074+Ibalizumab targeted condition with or without AZD7648 (n represents the total number of colonies genotyped across both donors; KI/KI light blue, INDEL/KI dark blue, INDEL/INDEL purple, WT/KI maroon, WT/INDEL red, WT/WT orange). All replicates represent independent biological donors unless otherwise noted. All bars represent mean and error bars represent standard deviation (SD). Integration frequencies were measured by ddPCR, INDEL and wild-type frequencies were measured by ICE analysis. Source data are provided as a Source Data file.

Fig. 3. Antibody edited HSPCs maintain engraftment capacity and multilineage reconstitution in vivo.

a Diagram for editing and engraftment of CB CD34⁺ HSPCs in NSG mice. Created in BioRender. Feist, W. (2025) https://BioRender.com/l43f611. b Distribution frequency of WT (gray), INDEL (blue), and KI (green) alleles in CB CD34⁺ HSPCs prior to transplantation. Cells were targeted with an AAV6 MOI of 1250 for each construct (n = 1 pooled sample from 5 donors). c Percent of KI alleles integrated with 10-1074 (blue) or Ibalizumab (purple) within the 10-1074+Ibalizumab targeted cells in panel b (n = 1). d Percent human chimerism in the bone marrow at endpoint (n = 5 mice for mock, 10-1074, and 10-1074+Ibalizumab, n = 4 for Ibalizumab). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant, P = 0.3287; *P = 0.0496; **P = 0.0082). e Percent human chimerism in the spleen at endpoint (n = 4 for mock, 10-1074, and 10-1074+Ibalizumab, n = 3 for Ibalizumab). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant, P = 0.0806 for Mock/10-1074, P = 0.1363 for Mock/Ibalizumab; *P = 0.0216). f Percent of human cells in the bone marrow (n are the same as in d) or g spleen (n are the same as in e) that are CD19⁺, CD33⁺, or within other lineages in mice engrafted with mock (black) or gene edited HSPCs (10-1074, blue; Ibalizumab, purple; 10-1074+Ibalizumab, red). h Percent of KI human alleles from the bone marrow or spleen (n are the same as in (d, e), respectively). i Percent of KI human alleles from the bulk bone marrow (shown in panel h) or from bone marrow CD19⁺ cells (n are the same as in d). Lines connect measurements from the same mice. Two-tailed Mann–Whitney test (*P = 0.0135). j Percent of human alleles with an INDEL at CCR5 (n are the same as in d). This analysis only includes alleles without KI. All bars represent mean. Integration frequencies were measured by ddPCR, INDEL and wild-type frequencies were measured by ICE analysis. Source data are provided as a Source Data file.

Fig. 4. HSPCs with high frequency knock-in maintain edited alleles following engraftment in vivo.

a Distribution frequency of WT (gray), INDEL (blue), and KI (green) alleles in CB CD34⁺ HSPCs prior to transplantation in NBSGW mice. Cells were targeted at CCR5 with an AAV6 MOI of 625 for each linker antibody construct and treated with 0.5 µM AZD7648 (n = 2 independent biologic donors; data also shown in Fig. 2f). b Percent of KI alleles integrated with 10-1074 (blue) or Ibalizumab (purple) within the 10-1074+Ibalizumab targeted cells treated with 0.5 µM AZD7648 and shown in panel a (n = 2). c Percent human cell chimerism in the bone marrow 12 weeks post-transplantation with cells shown in panel a (treated with 0.5 µM AZD7648 and targeted as indicated). Two-tailed Mann–Whitney test (**P = 0.0040, n = 4 mice for mock and n = 8 mice for 10-1074+Ibalizumab). d Percent of human cells in the bone marrow that are CD19⁺ (B cell lineage), CD33⁺ (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited (red) HSPCs (n are the same as in c). e Percent of human alleles with knock-in from the bulk bone marrow or in positively selected bone marrow CD19⁺ cells. Knock-in frequency was measured by ddPCR. Lines connect measurements from the same mice (n are the same as in c). Two-tailed Mann–Whitney test (ns, not significant, P = 0.7178). f Percent of human alleles from the bone marrow with an INDEL at CCR5 (n are the same as in c). This analysis only includes alleles without KI. All bars represent mean and all error bars represent SD. Integration frequencies were measured by ddPCR, INDEL and wild-type frequencies were measured by ICE analysis. Source data are provided as a Source Data file.

Fig. 5. Antibody engineered B cells secrete functional linker antibodies.

a Allelic integration frequency in adult peripheral blood CD19⁺ B cells targeted with AAV6 cassettes for linker antibodies as indicated. Each AAV6 was used at an MOI of 25,000 and integration frequency was measured with ddPCR (dots represent independent biologic donors, n = 8 for mock and 10-1074, n = 5 for Ibalizumab and 10-1074+Ibalizumab, n = 3 for PGDM1400 and CAP256V2LS). Donor 1 (green circle), Donor 2 (blue triangle), and Donor 3 (purple diamond) are specifically noted for their continued use in (b, d, e). Bars represent mean and error bars represent SD. b Concentration of each antibody (10-1074, blue; Ibalizumab, purple; PGDM1400, gold; CAP256V2LS, orange) as determined by antigen specific ELISA from B cell supernatant from cells targeted with AAV6 cassettes as indicated (n = 3 biological donors comprised of Donor 1, Donor 2, and Donor 3 from a). Six days post-editing, B cells were plated at 1 × 10⁶ cells per mL and supernatant was collected after 5 days. Bars represent mean and error bars represent SD. c Measured IC₅₀ for each antibody against TRO11 or CNE55 pseudoviruses as determined by TZM-bl assay in Fig. 1c (NN, not-neutralizing). d Inhibition of infection with TRO11 or e CNE55 HIV-1 pseudovirus by culture supernatant from B cells engineered to express linker antibodies as indicated, as determined by TZM-bl assay. Percent infection for each dose of supernatant from gene targeted B cells is normalized to infection at each dose of supernatant from mock B cells from the same donor (n = 3 biological donors; Donor 1 (green circle), Donor 2 (blue triangle), and Donor 3 (purple diamond) from a). Data points and error bars represent mean with range of technical duplicate infections. Source data are provided as a Source Data file.