Addressing safety issues by quantify large deletions and chromosomal rearrangements in HBB gene editing

镰状细胞病（SCD）是一种严重的慢性疾病，其特征是严重的疼痛，终末器官

Sickle cell disease (SCD) is a devastating chronic illness marked by severe pain, end organ damage and early mortality (1, 2). It affects ~100,000 Americans and millions more worldwide (3, 4), but treatment options for SCD remain very limited. Pharmacological therapy with hydroxyurea or chronic blood transfusions at best modulates the disease severity but does not cure patients (5). Currently, the only curative therapy for sickle cell disease (SCD) outside of a limited clinical trial is a hematopoietic stem cell transplant (HSCT), typically from a matched related donor, which is available to only ~15% of patients (6, 7). Morbidity and mortality from HSCT increases significantly when using matched unrelated donors (8), or haploidentical donors (9). A recent prospective study of unrelated donor HSCT in SCD concluded that, without modifications to existing regimens, this therapy is not safe for widespread adoption (10). With the advancement of CRISPR/Cas9 technology, there are several possible gene editing strategies to ameliorate SCD: (i) correction of the causative A-T point mutation in β-globin (HBB)(11-14), (ii) induction of fetal hemoglobin (HbF)(15, 16), and (iii) gene addition of a β- globin, γ-globin, or anti-sickling β-globin cassette (17), among which correction of the A-T mutation or producing high enough levels of HbF could be curative. We and others recently demonstrated that, by delivering CRISPR gRNA/Cas9 ribonucleoproteins (RNPs) together with single-stranded oligonucleotide (ssODN) donor templates into SCD patient-derived hematopoietic stem and progenitor cells (SCD HSPCs), up to ~37% of mutant HBB alleles can be gene corrected (12, 14). Injection of gene-edited SCD HSPCs into immunodeficient NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment, with detectable levels of gene correction 16-19 weeks post-transplantation (14). We have shown that by using a high-fidelity Cas9 that maintained the same level of ontarget gene modification, the off-target effects could be significantly reduced (14). However, potential large deletions and insertions at the HBB on-target cut-site, and off-target effects such as chromosomal translocation and inversion in gene-edited SCD HSPCs remain a significant safety concern, since even a very small number of HSCs harboring these detrimental events could clonally expand in vivo and cause a disease such as cancer. Previously, we optimized droplet digital PCR (ddPCR) assay to quantify large deletions and inversions between the R-66 SCD gRNA target site in HBB and a known off-target site (OT18) in gRNA/Cas9 WT RNP-treated SCD HSPCs (14). For high throughput discovery and quantification of such large modifications, we recently developed two next-generation sequencing (NGS) based methods based on short-read high-throughput illumina NGS platform leveraging the high sensitivity and cost-competitiveness of short-read NGS. The first is the LongAmp-Seq (Long-range PCR Amplification based Sequencing) assay, and the second is the NEW-Seq (Nuclease-activity identified by gEnome-Wide Sequencing) assay. The LongAmp-Seq can identify and quantify large deletions (up to 5.2 kb) and insertions (up to 300 bp) at the HBB on-target cut site. The NEW-Seq assay can discover rare gross chromosomal rearrangements such as inversions and translocations between the on-target cut-site and known or unknown off-target site. Our preliminary study using a SCD model cell-line and SCD HSPCs has shown that despite the enhanced specificity, the high-fidelity Cas9 induced large on-target modifications at comparable rate as WT Cas9. The frequency of large deletions and insertions decreased when both RNP and ssODN are delivered. The goal of the proposed research is to optimize and validate the LongAmp-Seq and NEW-Seq assays to quantitatively determine the degree of large deletions/insertions at the HBB on-target cut site and the gross chromosomal rearrangements due to off-target cutting in SCD HSPCs, both in cell culture and after engraftment into NSG mice. Our work will uncover genotypic and phenotypic consequences of a diverse array of mutations in the CRISPR/Cas9 edited SCD CD34+ cells which have important implications for clinical applications.