In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods

Abstract

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34⁺ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12-14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ^-/- Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.

Keywords: CD34; Crispr/Cas9; NBSGW41 mice; NHEJ versus HDR; gene editing; hematopoietic stem cells; hemoglobin disorders; homology-directed repair; in vivo engraftment; rAAV6; sickle cell disease; ssODN; stem cell cures.

Figure 1

Screening of Nucleases to Create DSBs in Exon 1 of the HBB Gene (A) Schematic representation of the genomic HBB gene showing the location of sgRNA-binding sites. A nucleotide substitution from GAG (codon 6 in red) to GTC or GTG changes the amino acid from glutamate to valine and causes SCD. (B) Optimizing the Cas9:sgRNA ratio to maximize editing efficiency in mPBSCs. NHEJ rates were analyzed by TIDE/ICE sequencing (Cas9:sgRNA ratio of 1:1 [40 pmol each], donor n = 2 or ratio of 1:2.5 [20 pmol of Cas9 and 50 pmol of sgRNA], donor n = 15). (C) Evaluating on-target disruption at HBB and possible off-target disruption at HBD by MiSeq analysis in mPBSCs using sgRNA-g1 delivered as RNP (donor n = 7). All bar graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p value was calculated by comparing each sample mean with the respective control sample mean by two-way ANOVA with Dunnett’s multiple comparison. See also Figure S1 and Table S2.

Figure 2

Homology-Directed Repair at the HBB Nuclease Target Site Using rAAV6 Donor Template (A) Schematic representation of rAAV6 cassettes designed to drive either a GTC (E6V) introducing a sickle mutation or a GAA (E6optE) introducing a codon-optimized change at codon 6 by HDR. (B) Experimental timeline for testing gene editing with RNP and rAAV6 delivery followed by erythroid differentiation in mPBSCs. (C) WT (%) and HDR (%) measured by ddPCR and NHEJ (%) measured by ICE sequencing, respectively, following electroporation with RNP alone, transduction with rAAV6 donor template alone, or co-delivery of RNP and GTC (E6V) rAAV6 donor template, at the indicated concentrations (donor n = 4). (D) RP-HPLC analysis of erythroid cells to measure β-globin expression in cells treated with RNP only, rAAV6 only, or RNP plus GTC (E6V) rAAV6 (donor n = 7). (E) WT (%) and HDR (%) measured by ddPCR and NHEJ (%) measured by ICE sequencing, respectively, following electroporation with RNP alone, transduction with rAAV6 donor template alone, or co-delivery of RNP and GAA (E6optE) rAAV6 donor template, at the indicated concentrations (1% rAAV6; donor n = 3). (F) RP-HPLC analysis of erythroid cells to measure β-globin expression in cells treated with RNP only, rAAV6 only, or RNP plus GAA (E6optE) rAAV6 (donor n = 3). β^A, adult globin; β^S, sickle globin; γ^G, gamma 2; γ^A, gamma 1. All bar graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p value was calculated by comparing each sample mean of NHEJ (%), HDR (%), WT (%), or globin sub-type (%) with the respective NHEJ (%), HDR (%), WT (%), or globin sub-type (%) of the mock sample by two-way ANOVA with Dunnett’s multiple comparison. Asterisks are color matched to the respective mock sample. See also Figures S2–S4.

Figure 3

Homology-Directed Repair at the HBB Nuclease Target Site Using ssODN Donor Template (A) Schematic representation of ssODN cassette designed to drive either a GTC (E6V) introducing a sickle mutation or a GAA (E6optE) introducing a codon-optimized change at codon 6 by HDR. (B) Experimental timeline for testing gene editing with RNP and ssODN delivery followed by erythroid differentiation in mPBSCs. (C) WT (%) and HDR (%) measured by ddPCR and NHEJ (%) measured by ICE sequencing, respectively, following electroporation with RNP alone or co-delivery of RNP and GTC (E6V) ssODN donor template, at the indicated concentrations (50 pmol ssODNs, donor n = 5). (D) RP-HPLC analysis of erythroid cells to measure β-globin expression in cells treated with RNP only or RNP plus GTC (E6V) ssODNs (50 pmol ssODNs, donor n = 5). (E) WT (%) and HDR (%) measured by ddPCR and NHEJ (%) measured by ICE sequencing, respectively, following electroporation with RNP alone or co-delivery of RNP and GAA (E6optE) ssODNs, at the indicated concentrations (50 pmol ssODNs, donor n = 8). (F) RP-HPLC analysis of erythroid cells to measure β-globin expression in cells treated with RNP only or RNP plus GAA (E6optE) ssODNs (50 pmol ssODNs, donor n = 6). α, alpha; β^A, adult; β^S, sickle; γ^G, gamma 2; γ^A, gamma 1. All bar graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p value was calculated by comparing each sample mean of NHEJ (%), HDR (%), WT (%), or globin sub-type (%) with the respective NHEJ (%), HDR (%), WT (%), or % globin sub-type (%) of the mock sample by two-way ANOVA with Dunnett’s multiple comparison. Asterisks are color matched to the respective mock sample. See also Figures S5–S8.

Figure 4

Comparison of Outcomes of ssODN and rAAV6 Donor Template Delivery Methods by MiSeq Analysis (A) Quantification of HDR versus NHEJ edits by MiSeq analysis in cells treated with GTC (E6V) rAAV6 (n = 6) versus ssODNs (using GTC [E6V, n = 8], GTG [E6V, n = 3], or GAA [E6optE, n = 2] ssODNs). (B) Indel spectrum analysis by MiSeq comparing RNP-mediated editing alone to residual indels present in cells after the promotion of HDR with either rAAV6 or ssODN delivery (donor n = 6). (C) The various gene-editing outcomes, WT, NHEJ (insertion, substitution, deletion), and HDR, measured in the following samples: mock, RNP alone, and co-delivery of RNP with rAAV6 and RNP with ssODNs. The samples analyzed were the pre-transplant input and in vitro-edited samples analyzed on day 14 post-editing (n = 6). All bar graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p value was calculated by comparing each sample mean of NHEJ (%) and HDR (%) with the respective NHEJ (%) and HDR (%) of the mock sample by two-way ANOVA with Dunnett’s multiple comparison. Asterisks are color matched to the respective mock sample. See also Figure S9.

Figure 5

Engraftment Potential of rAAV6- versus ssODN-Edited HSCs (A) Experimental timeline for testing gene editing with GTC (E6V) rAAV6- or ssODN-treated cells in vitro in mPBSCs and in vivo in the NBSGW mouse model. Red lines indicate placement of cells in erythroid differentiation conditions. (B) Human cell (hCD45⁺) chimerism in the BM and spleen at days 84–96, with gating based on forward scatter (FSC), side scatter (SSC), and single cells. (C) Human CD19⁺ and CD33⁺ subsets within the BM hCD45⁺ population. (D) Human CD235⁺ cells in the BM gated on the mCD45⁻ population. The BM cells were cultured ex vivo for 14 days in erythroid differentiation media, and CD235⁺ (ex vivo) was measured by flow cytometry. (E) Proportion of human CD34⁺ and CD34⁺CD38^lo cells within the BM hCD45⁺ population. (F) HDR rates determined by ddPCR within the GTC (E6V) rAAV6- or ssODN-treated input cells (day 14, n = 4 transplants, single donor), at 3 weeks post-transplant (day 21, n = 2), and at 12–14 weeks post-transplantation (days 84–96; mock n = 8, RNP + rAAV6 n = 17, RNP + ssODN n = 18). (G) NHEJ rates determined by ICE sequencing for GTC (E6V) rAAV6- or ssODN-treated input cells (day 14), at 3 weeks (day 21) post-transplant, and at 12–14 weeks (days 84–96) post-transplant. All bar graphs show mean ± SD. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p value was calculated by comparing each sample mean of NHEJ (%), HDR (%), and WT (%) with the respective NHEJ (%), HDR (%), and WT (%) of the mock sample by two-way ANOVA with Dunnett’s multiple comparison. Asterisks are color matched to the respective mock sample. See also Figures S10–S15.