Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease

Abstract

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (HBB). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34⁺ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

Fig. 1.. Therapeutic gene correction protocol optimization in healthy donor derived HSPCs.

(A) Schematics of cell manufacturing protocol, in vitro readouts and experimental design of in vivo NSG mouse studies. (B) Percentage of in vitro gene corrected alleles (gcHBB) (left), viability and CD34 content (right) in up to 13 cell donors; black lines indicate mean values. (C) Human chimerism in bone marrow of NSG mice at week-16 post-injection. Control cells were i. electroporated only (Mock), ii. coupled with either RNP (Cas9 only) or iii. AAV6 (AAV6 only). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test; ns, not significant. (D) Percent distribution of human hematopoietic lineages within the human cell population. (E) Percent distribution of gcHBB alleles in the human cell population (bulk) and in the respective hematopoietic lineages. (F) Percent gcHBB alleles distribution in bulk population and in a mouse-by-mouse analysis of human lineages in bone marrow samples, which show either good representation of all lineages (left) or mostly myeloid cells composition (right). (G) Quantification of HBB alleles distribution in single CFU colonies. WT: wild type, INDEL: insertion/deletions, HR: homologous recombination. Dot colors indicate HBB cellular outcome: white “neutral”, red “deficient”, green “corrective”. Brackets group genotypes per cellular outcomes; black bars indicate mean values. (H) Percent of gcHBB alleles in vivo (bone marrow) linked to genotype of single sorted HSPC derived CFU. Pearson r test, p<0.001. Black bars and lines indicate median values if not differently specified.

Fig. 2.. Effective gene correction, recovery of normal hemoglobin output, and engraftment capability in SCD patient derived HSPCs.

(A) Percent allele modification in six gcHBB-SCD biological replicates (Exp 1–6). (B) Viability and CD34 content in vitro. (C) Percent of in vitro differentiated erythroid cells displaying CD71+ CD235a+ markers (see Fig. S9A). (D) Quantification of hemoglobin tetramers (HPLC) on erythroid differentiated population (n=4). (E) Human chimerism in the bone marrow of NSG mice analyzed at week-16 post-injection. Mann-Whitney test; **, p<0.01. Black bars indicate median values. (F) Percent distribution of human hematopoietic lineages in the NSG bone marrow. (G) Percent of gcHBB alleles in the human cell population (bulk) in vitro and in vivo mouse-by-mouse, along with the respective human hematopoietic lineages collected from NSG bone marrow. Mann-Whitney test; *, p<0.05. Black bars and lines indicate mean (± SD) if not differently specified.

Fig. 3.. Efficient scale-up cell manufacturing in clinically relevant CD34+ cells with lack of tumorigenicity in a long-term toxicology study.

(A) Percent of allele modification in six medium-scale cell manufacturing runs (Run 1–6). (B) CFU frequency for gcHBB-SCD cell product and untreated cell counterpart. Paired t test. *, p<0.05. (C) Scatter plot linking gene correction of HBB alleles in bulk cell population to single genotyped colonies. Pearson r test *, p<0.05. (D) Quantification of HBB alleles distribution in single CFU colonies (genotype). WT: wild type, INDEL: insertion/deletions, HR: homologous recombination. Dot colors indicate HBB cellular outcome: white “neutral’, red ‘deficient’, green ‘corrective’. Brackets group genotypes per cellular outcomes. (E) Human chimerism in hematopoietic tissues (peripheral blood, bone marrow and spleen) of NSG mice in long-term toxicology study. Untreated HSPCs (in blue) and gcHBB-SCD (in green) groups were segregated by sex. One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test; ns, not significant; *, p<0.05; **, p<0.001; ***, p<0.0001. (F) Percent gcHBB alleles in hematopoietic tissues collected at study end from gcHBB-SCD NSG cohort (n=39). (G) Percent human engraftment and (H) percent gcHBB alleles in bone marrow of NSG mice injected with cell manufacturing run 5 at Stanford lab (n=10). Black bars and lines indicate median values.

Fig. 4.. Next generation sequencing (NGS) based-techniques assess very minimal genotoxicity in gcHBB-SCD cell product.

(A) NGS results of the 67 off-target rank list assessed in the six medium-scale manufacturing runs for toxicology studies (Run 1–6). On-target activity=percent INDELs+HR. Dotted line indicates the assay detection threshold. (B) Circos plots of genome-wide prey junctions binned into 5-Mb regions (black bars) are plotted on a log scale with indicated ticks; frequency ranges are colored from light orange (10 – 100) to increasingly darker orange colors by factors of 10. Red arrow connects the HBB bait site on chromosome 11 to the off-target hotspot on chromosome 9. (C) IGV plot of HBB off-target on chromosome 9 (coordinates bottom left); junctions are shown on a logarithmic scale. Red and blue numbers indicate the number of junctions from the specified region that translocated in the plus and minus orientation, respectively. The bar (bottom right) indicates 50 base pairs length. (D) Junction frequency at the main HBB-OT (OT1, Chr9) for WT and HiFi Cas9 (mean ± SD). Paired t-test; ***, p<0.001.