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，以剧烈的疼痛、末梢器官为特征。 损害和早期死亡率(1，2)。它影响了大约10万美国人和全球数百万人 (3，4)，但SCD的治疗选择仍然非常有限。药物疗法： 羟基脲或慢性输血最多只能调节疾病的严重程度，但不能。 治愈病人(5例)。目前，治疗镰状细胞病(SCD)的唯一有效方法是 有限的临床试验是一种造血干细胞移植(HSCT)，通常来自匹配的 亲属供者，只有~15%的患者(6，7)可以获得。因以下原因引起的发病率和死亡率 当使用匹配的无血缘关系的供者(8)或半相合时，HSCT显著增加 捐赠者(9人)。最近一项对SCD中非血缘关系供者进行HSCT的前瞻性研究得出结论，如果没有 对现有方案的修改，这种疗法广泛采用是不安全的(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-珠蛋白致病的A-T点突变 (Hbb)(11-14)，(Ii)诱导胎儿血红蛋白(Hbf)(15，16)，以及(Iii)β- 珠蛋白、γ-珠蛋白或抗镰状β-珠蛋白盒(17)，其中A-T校正 突变或产生足够高水平的HBF可能是治愈的。我们和其他人最近 证明通过将CRISPR gRNA/Cas9核糖核蛋白(RNPs)与 单链寡核苷酸供体模板导入SCD患者来源的研究 造血干细胞和祖细胞(SCD HSPC)，高达37%的突变HBB等位基因可以 基因矫正(12，14)。免疫缺陷小鼠体内注射基因编辑的SCD-HSPC NOD/SCID/IL-2rgnull(NSG)小鼠表现出临床相关的植入水平， 移植后16-19周可检测到的基因校正水平(14)。 我们已经证明，通过使用高保真的Cas9来保持相同的OnTarget级别 基因修饰可以显著降低脱靶效应(14)。然而， 可能在HBB靶标切割部位大量删除和插入，以及脱靶效应，如 作为基因编辑的SCD中的染色体易位和倒位，HSPC仍然是一个重要的 安全问题，因为即使是极少数的肝星状细胞含有这些有害的 这些事件可能会在体内克隆扩张，并导致癌症等疾病。此前，我们 优化的滴状数字聚合酶链式反应(DdPCR)定量检测大片段缺失和反转 HBB中R-66 SCD gRNA靶点和gRNA/Cas9 WT中已知的非靶点(OT18) RNP处理的SCD HSPC(14例)。对于高吞吐量的发现和量化如此庞大的 经过修改，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 短读型NGS的成本竞争力。第一种是LongAmp-Seq(长距离PCR 基于扩增的测序)，第二种是新的-SEQ(核酸酶活性 通过全基因组测序鉴定)。LongAmp-Seq可以识别和量化 HBB靶切位点的大片段缺失(高达5.2kb)和插入(高达300bp)。这个 新的序列分析可以发现罕见的大规模染色体重排，如倒位和 靶上切割部位和已知或未知的脱靶部位之间的易位。我们的 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，高保真的Cas9在可比的情况下诱导了大量的目标修改 评级为WT Cas9。当两个RNP都发生时，较大的缺失和插入的频率降低 和单链ODN被交付。拟议研究的目标是优化和验证 用LongAmp-Seq和New-Seq方法定量测定 HBB靶切位点的缺失/插入与总的染色体重排 由于SCD HSPC在细胞培养和植入NSG后的非靶点切割 老鼠。我们的工作将揭示一系列不同基因和表型的后果 CRISPR/Cas9编辑的SCD CD34+细胞中的突变对 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.