CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease

Abstract

Background: Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.

Methods: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923.

Results: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period.

Conclusions: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).

Figure 1.. OTQ923 molecular approach and preclinical characterization.

Panel A shows the targeting site of the ribonucleic acid–protein complex (RNP) consisting of Cas9 complexed with guide RNA-68 (gRNA-68). Genes in the β-like globin cluster on chromosome 11 are shown as boxes, with RNP targeting sites in HBG1 and HBG2 promoters indicated. The protospacer sequence of gRNA-68 is shown below in blue, and the protospacer adjacent motif (PAM) sequence is shown in green. The sequence is numbered in accordance with the start of transcription being at +1. The predicted cutting site is indicated by the blue arrow in the protospacer sequence. Panel B shows the editing outcomes as determined by qPCR, targeted NGS, and UnIT (Supplemental Methods). Most edits generated a deletion of ~5 kb caused by simultaneous double-stranded DNA breaks at the two target sites, thereby generating a single functional HBG2–HBG1 fusion gene with the HBG2 promoter sequence fused to the downstream HBG1 (48.8–87.4% alleles). Editing also resulted in small indels at either targeting site that accounted for 7.1–36.9% of alleles. No inversion of the ~5-kb fragment was detected. Panel C shows the distribution of editing outcomes in OTQ923 or control cells generated from healthy-donor CD34+ cells (n=4). Panel D shows the percentages of F-cells in erythroblasts generated by in vitro differentiation of OTQ293 or control cells described in Panel C (n=4 healthy donors). Panel E shows the amount of HbF protein estimated by HPLC in erythroblasts generated by in vitro differentiation of OTQ293 or control cells derived from healthy donors (n=2 healthy donors). Panel F shows the distribution of editing outcomes in OTQ923 cells or control cells generated from individuals with sickle cell disease (n=4). Panel G shows the percentages of F-cells in erythroblasts generated by in vitro differentiation of OTQ293 or control cells described in Panel E (n=3 sickle cell disease donors). Panel H shows the amount of HbF protein estimated by HPLC in erythroblasts generated by in vitro differentiation of OTQ293 or control cells derived from sickle cell disease patients (n=3). All graphs show data as the mean ± SD.

Figure 2.. Total hemoglobin and its fractions, F-cell percentage, and tracking of edited alleles in the first two participants.

Panel A shows the total hemoglobin and hemoglobin fractionation over time for Participant 1. Participant 1 experienced a transient decline in hemoglobin from 11.0 to 8.0 g/dL between months 8 and 9, coincident with an intercurrent viral infection and associated bone marrow suppression, which recovered spontaneously at the subsequent timepoint. Panel B shows the number of blood transfusions received by Participant 1 over time. Panel C shows the percentage of allelic editing in OTQ923 product (2 batches), nucleated peripheral blood cells (up to 18 months after OTQ923 infusion), or bone marrow (at 12 months after OTQ923 infusion) from Participant 1. Panel D shows the total hemoglobin and hemoglobin fractionation over time for Participant 2. Panel E shows the number of transfusions received by Participant 2 over time. Panel F shows the percentage of allelic editing in OTQ923 product (3 batches), nucleated peripheral blood cells (up to 12 months after OTQ923 infusion), or bone marrow (at 12 months after OTQ923 infusion) from Participant 2. Panel G shows the total hemoglobin and hemoglobin fractionation over time for Participant 3. Panel H shows the number of transfusions received by Participant 3 over time. Panel I shows the percentage of allelic editing in OTQ923 product (2 batches), or nucleated peripheral blood cells (up to 6 months after OTQ923 infusion) from Participant 3. In panels A, D and G, green or gray labels indicate the proportions of fetal hemoglobin (HbF) or sickle hemoglobin (HbS), respectively, as percentages of the total hemoglobin at those respective time points. Total hemoglobin values are reported above each bar. The line graph (black line) represents the percentage of F-cells over time after OTQ923 infusion and indicates near-pancellular HbF expression. In panels B, E and H each black dot represents a transfusion. The time periods prior to enrollment, after enrollment on the study but prior to OTQ923 infusion, and after OTQ923 infusion are shown in different colors. In panels C, F and I orange bars represent the percentages of indels detected by next-generation sequencing (bright orange indicates indels at the HBG1 locus, pale orange indicates indels at the HBG2 locus). Blue bars represent the ~5-kb deletion that was detected in the infused product by quantitative PCR or by droplet digital PCR (ddPCR; also see Figure S5).