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

通过量化 HBB 基因编辑中的大缺失和染色体重排来解决安全问题

• 批准号: 10087778
• 负责人: Gang Bao
• 金额: $ 117.39万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-25 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087778
• 关键词: Address; Adoption; Affect; Alleles; American; Biological Assay; Blood Transfusion; CD34 gene; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell model; Cells; Chromosomal Rearrangement; Chromosomal translocation; Chromosome inversion; Chronic; Chronic Disease; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Disease; Engraftment; Event; Fetal Hemoglobin; Frequencies; Gene-Modified; Genes; Genotype; Goals; Guide RNA; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Injections; Malignant Neoplasms; Methods; Modification; Morbidity - disease rate; Mus; Mutation; Oligonucleotides; Organ; Pain; Patients; Pharmacology; Phenotype; Point Mutation; Prospective Studies; Regimen; Research; Ribonucleoproteins; Safety; Severity of illness; Sickle Cell Anemia; Site; Site-Directed Mutagenesis; Specificity; Work; base; beta Globin; clinical application; clinically relevant; cost; curative treatments; digital; gamma Globin; gene correction; genome-wide; hydroxyurea; in vivo; insertion/deletion mutation; mortality; mutant; next generation sequencing; nuclease; off-target site; post-transplant; sequencing platform

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.

镰状细胞病（SCD）是一种毁灭性的慢性疾病，以严重的疼痛为特征 损害和早期死亡率（1，2）。它影响了大约100,000美国人，全球范围内有数百万 （3，4），但是SCD的治疗选择仍然非常有限。药理治疗 羟基脲或慢性输血最能调节疾病的严重程度，但不调节 治愈患者（5）。目前，镰状细胞病（SCD）的唯一现代疗法 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）。发病率和死亡率 当使用匹配的无关供体（8）或单倍型时，HSCT会显着增加 捐助者（9）。 SCD中对无关供体HSCT的最新前瞻性研究得出的结论是，没有 对现有方案的修改，这种疗法对于采用宽度并不安全（10）。 随着CRISPR/CAS9技术的发展，有几种可能的基因编辑 改善SCD的策略：（i）校正β-珠蛋白中的病因A-T点突变 （HBB）（11-14），（ii）诱导胎儿血红蛋白（HBF）（15，16）和（iii）添加β-的基因 球蛋白，γ-球蛋白或抗吸收β-珠蛋白盒（17），其中A-T校正 突变或产生足够高的HBF可能是现实的。我们和其他人最近 证明，通过交付CRISPR GRNA/CAS9核糖核蛋白（RNP） 单链寡核苷酸（SSODN）供体模板进入SCD患者来源 造血茎和祖细胞（SCD HSPC），高达37％的突变HBB等位基因可以 纠正基因（12，14）。将基因编辑的SCD HSPC注射到免疫缺陷中 点头/SCID/IL-2RGNULL（NSG）小鼠显示出与临床相关的植入水平，有 转移后16-19周可检测的基因校正水平（14）。 我们已经证明，通过使用维持相同水平的Antarget的高保真性CAS9 基因修饰，脱靶效应可以显着降低（14）。然而， 在HBB的靶向切割站点处的潜在大删除和插入，脱离目标效果此类效果 由于基因编辑的SCD HSPC中的染色体易位和反转仍然是显着的 安全问题，因为即使是携带这些有害的HSC数量很少 事件可能会在体内扩展并引起癌症等疾病。以前，我们 优化的液滴数字PCR（DDPCR）测定法，以量化大删除和反转 HBB中的R-66 SCD GRNA目标位点和GRNA/CAS9 WT中的已知脱离目标（OT18） RNP处理的SCD HSPC（14）。对于如此大的吞吐量发现和量化 修改，我们最近开发了两个基于下一代测序（NGS）的方法 基于短读高通量Illumina NGS平台利用高灵敏度和 短阅读NGS的成本竞争力。第一个是longamp-seq（远程PCR） 基于扩增的测序）测定，第二个是new-seq（核酸酶 - 活性 通过全基因组测序鉴定）测定。 Longamp-seq可以识别和量化 大型删除（高达5.2 kb）和插入（最高300 bp）在HBB靶标切割站点。这 New-Seq分析可以发现罕见的总染色体重排，例如反转和 靶向切割站点与已知或未知脱离目标的易位。我们的 使用SCD模型细胞系和SCD HSPC的初步研究表明，要求 提高特异性，高保真性CAS9在可比的 速率为wt cas9。两个RNP时，大删除和插入的频率下降 和ssodn已交付。拟议研究的目的是优化和验证 longamp-seq和new-seq分析，以定量确定大的程度 HBB内靶切割站点的删除/插入和染色体重排 由于在细胞培养和植入NSG之后，在SCD HSPC中切割了靶标的。 老鼠。我们的工作将发现潜水员阵列的基因型和表型后果 CRISPR/CAS9中的突变编辑了SCD CD34+细胞，对 临床应用。