Develop High-Precision and Multiplex Base Editing Approaches for Therapeutic Applications

开发用于治疗应用的高精度和多重碱基编辑方法

• 批准号: 10591575
• 负责人: Xue Gao
• 金额: $ 52.54万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591575
• 关键词: Adenine; Affect; Alleles; Bicarbonates; Biomedical Research; CRISPR/Cas technology; Cell Line; Cells; Characteristics; Chlorides; Chronic lung disease; Complex; Computer Assisted; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytosine; DNA; DNA Sequence Alteration; Deaminase; Deamination; Dependence; Disease; Disease model; Drug Combinations; Endonuclease I; Engineering; Epithelial Cells; Exocrine pancreatic insufficiency; Functional disorder; Genes; Genetic Diseases; Genome; Genomics; Goals; Guide RNA; Hereditary Disease; Heterozygote; Human Engineering; Human Genetics; Human Genome; Ion Channel; Length; Lung; Modeling; Mutation; Nonsense Mutation; Nucleotides; Organ failure; Other Genetics; Outcome; Pancreas; Pathogenicity; Patients; Performance; Pharmaceutical Preparations; Point Mutation; Protein Engineering; Rare Diseases; Regulator Genes; Research; Resolution; Risk; Safety; Site; Somatic Cell; Streptococcus pyogenes; Symptoms; System; Terminator Codon; Therapeutic; Trans-Splicing; Transfer RNA; Transportation; Variant; airway epithelium; autosome; base; base editing; base editor; cystic fibrosis patients; design; drug discovery; efficacy validation; flexibility; gene therapy; genetic approach; improved; individual patient; insertion/deletion mutation; intein; mutation correction; next generation; nucleobase; pharmacologic; precise genome editing; premature; prevent; repaired; respiratory; reverse genetics; risk minimization; small molecule; symptomatic improvement; therapeutic genome editing; tool; transition mutation

Project Summary/Abstract Genetic disorders and genetic diseases are caused by insertions, deletions, and base substitutions of a single gene or multiple genes. Cystic fibrosis (CF), an autosomal recessive hereditary disease, is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In healthy cells, CFTR maintains chloride and bicarbonate transportation as an ion channel. Genetic defects of CFTR result in complicated respiratory and systemic organ failure. Point mutations, or single-nucleotide variations (SNVs), account for ~60% of the pathogenic variants causing CF. CF patients can be partially treated by the administration of small molecule drugs to improve symptoms, including chronic pulmonary disease and pancreatic insufficiency. However, CF mutations leading to the premature termination codon (PTC) affect at least 10% of CF patients, whose symptoms cannot be relieved by any of the modulators. Gene therapy is a promising and permanent alternative approach that confers therapeutic benefits to patients who suffer from genetic diseases. The CRISPR- Cas9 system can efficiently cause double-strand breaks (DSBs) to facilitate homology-directed repair (HDR) for accurate gene-editing outcomes. However, safety concerns arising from the DSBs cause unwanted mutations. To surmount this problem, base editors (BEs) use a nickase Cas9 (nCas9) that nicks only the protospacer adjacent motif (PAM)-containing strand, and thus eliminates the risk of DSBs and random indels. BEs use a natural or engineered DNA deaminase fused with a nCas9 and can introduce a C-to-T or an A-to-G conversion within the activity window by the cytosine or adenine deaminase. Both cytosine BEs (CBEs) and adenine BEs (ABEs) can enable base transitions with high efficiency and have already proven successful for a few genetic diseases in proof-of-concept studies. However, before applying BEs to the treatment of human genetic diseases, including CF, several challenges must be overcome. First, indiscriminate conversion of multiple ‘C’s or ‘A’s within CBE or ABE’s characteristic deamination activity window, usually more than five nucleotides, results in undesired bystander editing. Second, the targeting scope of BEs has been largely constrained by the NGG PAM requirement of nSpCas9, the canonical Cas9 from Streptococcus pyogenes. A large proportion of the base transition pathogenic mutations is thus unavailable for editing. Third, the lack of multiplexity of BEs impedes its practicality in processing multiple mutations simultaneously for the treatment of complex genetic diseases. In this proposed research, we aim to develop precise and multiplex BEs that will make it possible to target the vast majority of human genome sites (Aim 1 & 2). We will apply high-precision BEs to generate and correct homozygous and compound heterozygous CF disease models that mirror individual patients, which will also greatly facilitate pharmacological research and drug discovery for personalized CF treatment (Aim 3). In summary, high-precision BEs will contribute to personalized gene therapy for cystic fibrosis as well as many other genetic diseases.

项目概要/摘要 遗传病和遗传病是由单个基因的插入、缺失和碱基取代引起的。 基因或多个基因。囊性纤维化（CF）是一种常染色体隐性遗传病，由突变引起 囊性纤维化跨膜电导调节因子（CFTR）基因。在健康细胞中，CFTR 维持 氯离子和碳酸氢盐作为离子通道的运输。 CFTR的遗传缺陷导致复杂的 呼吸系统和全身器官衰竭。点突变或单核苷酸变异 (SNV) 约占 60% 引起 CF 的致病变异。 CF患者可以通过服用小剂量药物来部分治疗 改善症状的分子药物，包括慢性肺部疾病和胰腺功能不全。 然而，导致提前终止密码子 (PTC) 的 CF 突变影响至少 10% 的 CF 患者， 任何调节剂都无法缓解其症状。基因治疗是一种有前途且永久的治疗方法 为患有遗传性疾病的患者带来治疗益处的替代方法。 CRISPR- Cas9系统可以有效地引起双链断裂（DSB），以促进同源定向修复（HDR） 准确的基因编辑结果。然而，DSB 引起的安全问题会导致不必要的突变。 为了克服这个问题，碱基编辑器 (BE) 使用切口酶 Cas9 (nCas9)，仅切口原型间隔区 包含相邻基序 (PAM) 的链，从而消除 DSB 和随机插入缺失的风险。 BE 使用 与 nCas9 融合的天然或工程 DNA 脱氨酶，可以引入 C 到 T 或 A 到 G 的转换 在胞嘧啶或腺嘌呤脱氨酶的活性窗口内。胞嘧啶 BE (CBE) 和腺嘌呤 BE （ABE）可以高效地实现碱基转换，并且已经证明对一些遗传基因是成功的 概念验证研究中的疾病。然而，在将BE应用于人类遗传疾病的治疗之前， 包括CF在内，必须克服一些挑战。首先，不加区别地转换多个“C”或“A” CBE 或 ABE 的特征脱氨活性窗口（通常超过 5 个核苷酸）会导致不希望的结果 旁观者编辑。其次，BE 的目标范围在很大程度上受到 NGG PAM 的限制 nSpCas9（化脓性链球菌的经典 Cas9）的要求。基地比例很大 因此，过渡致病突变无法进行编辑。第三，BE 缺乏多重性阻碍了其 同时处理多个突变以治疗复杂遗传疾病的实用性。在 在这项拟议的研究中，我们的目标是开发精确且多重的BE，从而能够针对广泛的 大多数人类基因组位点（目标 1 和 2）。我们将应用高精度BE来生成和纠正 反映个体患者的纯合和复合杂合 CF 疾病模型，这也将 极大地促进个性化 CF 治疗的药理学研究和药物发现（目标 3）。在 总之，高精度 BE 将有助于囊性纤维化以及许多疾病的个性化基因治疗 其他遗传病。