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

镰状细胞病（SCD）是一种毁灭性的慢性疾病，其特征是严重的疼痛，终末器官功能障碍， 损伤和早期死亡（1，2）。它影响了大约10万美国人和全世界数百万人 （3，4），但SCD的治疗选择仍然非常有限。药物治疗， 羟基脲或慢性输血充其量只能调节疾病的严重程度， 治愈病人（5）。目前，镰状细胞病（SCD）的唯一治愈性疗法， 有限的临床试验是造血干细胞移植（HSCT），通常来自匹配的 相关供体，仅约15%的患者可用（6，7）。的患病率和死亡率 当使用匹配的无关供体（8）或单倍体相合供体时，HSCT显著增加 捐助者（9）。最近一项对SCD患者进行的非亲缘供者HSCT的前瞻性研究得出结论， 尽管对现有方案进行了修改，但这种疗法对于广泛采用来说并不安全（10）。 随着CRISPR/Cas9技术的进步，有几种可能的基因编辑 改善SCD的策略：（i）纠正β-珠蛋白中的A-T点突变 （ii）胎儿血红蛋白（HbF）的诱导（15，16），和（iii）β-血红蛋白（HBB）的基因添加（11-14）， 珠蛋白、γ-珠蛋白或抗镰状化β-珠蛋白盒（17），其中A-T 突变或产生足够高水平的HbF可能是治愈性的。我们和其他人最近 证明，通过将CRISPR gRNA/Cas9核糖核蛋白（RNP）与 单链寡核苷酸（ssODN）供体模板转化为SCD患者来源的 在造血干细胞和祖细胞（SCD HSPC）中，高达~37%的突变HBB等位基因可 基因校正（12，14）。将基因编辑的SCD HSPC注射到免疫缺陷的 N 0 D/SCID/IL-2 rgnull（NSG）小鼠显示出临床相关的植入水平， 移植后16-19周可检测到基因校正水平（14）。 我们已经证明，通过使用高保真Cas9，保持相同的靶向水平， 通过基因修饰，脱靶效应可以显著降低（14）。然而，在这方面， HBB靶向切割位点的潜在大缺失和插入，以及脱靶效应， 由于基因编辑的SCD HSPC中的染色体易位和倒位仍然是一个重要的问题， 安全问题，因为即使是极少数的HSC携带这些有害的 事件可以在体内克隆扩增并引起疾病如癌症。此前我们 优化的液滴数字PCR（ddPCR）测定，以定量 HBB中R-66 SCD gRNA靶位点和gRNA/Cas9 WT中已知脱靶位点（OT 18 RNP处理的SCD HSPC（14）。对于高通量发现和定量这种大的 修改，我们最近开发了两种基于下一代测序（NGS）的方法 基于短读高通量Illumina NGS平台，利用高灵敏度和 短读NGS的成本竞争力。第一种是LongAmp-Seq（长距离PCR 第二种是NEW-Seq（核酸酶活性测定）， 通过全基因组测序鉴定）测定。LongAmp-Seq可以识别和定量 在HBB靶切割位点处的大缺失（高达5.2 kb）和插入（高达300 bp）。的 NEW-Seq检测可以发现罕见的总染色体重排，如倒位和 靶向切割位点和已知或未知的脱靶位点之间的易位。我们 使用SCD模型细胞系和SCD HSPC的初步研究表明，尽管 增强的特异性，高保真度Cas9在相当的水平上诱导了大的靶向修饰。 以WT Cas9计。当两种RNP基因同时表达时， 和ssODN被递送。所提出的研究的目标是优化和验证 LongAmp-Seq和NEW-Seq测定以定量确定大的 HBB靶切割位点的缺失/插入和总染色体重排 由于SCD HSPC中的脱靶切割，无论是在细胞培养中还是在植入NSG后 小鼠我们的工作将揭示一系列不同的基因型和表型的后果， CRISPR/Cas9编辑的SCD CD 34+细胞中的突变，这些突变对 临床应用。

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.