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

项目摘要/摘要 遗传性疾病和遗传性疾病是由单个基因的插入、缺失和碱基替换引起的 基因或多个基因。囊性纤维化是一种常染色体隐性遗传性疾病，由突变引起 囊性纤维化跨膜传导调节因子(CFTR)基因。在健康细胞中，CFTR坚持认为 氯化物和碳酸氢盐作为离子通道运输。CFTR的遗传缺陷导致复杂 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的致病变异体。Cf患者可以通过使用小剂量的 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，导致过早终止密码子(PTC)的CF突变至少影响10%的CF患者， 其症状不能被任何调节剂缓解。基因治疗是一种有希望的、永久的 为患有遗传病的患者提供治疗益处的另一种方法。CRISPR- Cas9系统可有效地导致双链断裂(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，基本编辑(BE)使用了一个昵称Cas9(NCas9)，它只删除Protspacer 含有邻近基序(PAM)的链，从而消除了DSB和随机INDELs的风险。BES使用 天然或工程DNA脱氨酶与nCas9融合，可实现C-T或A-G转换 活性窗口内的胞嘧啶或腺嘌呤脱氨酶。胞嘧啶BES(CBEs)和腺嘌呤BES (ABES)可以高效地实现碱基转换，并已被证明对一些基因成功 概念验证研究中的疾病。然而，在将BES应用于治疗人类遗传病之前， 包括合作伙伴关系在内，必须克服几个挑战。首先，不分青红皂白地转换内部的多个C或A CBE或ABE特有的脱氨活性窗口，通常超过5个核苷酸，导致不受欢迎的结果 旁观者编辑。其次，BES的目标范围在很大程度上受到了NGG PAM的限制 化脓性链球菌对典型Cas9基因nSpCas9的要求基地的很大比例 因此，无法编辑过渡致病突变。第三，缺乏BES的多元性阻碍了其 同时处理多个突变以治疗复杂遗传病的实用性。在……里面 这项拟议的研究，我们的目标是开发精确的和多路BES，使其有可能针对广大 大多数人类基因组位置(目标1和2)。我们将应用高精度的BES来生成和校正 反映单个患者的纯合子和复合杂合性CF疾病模型，这也将 极大地促进个性化治疗的药理研究和药物发现(目标3)。在……里面 总结：高精度的BES将有助于囊性纤维化的个性化基因治疗，以及许多 其他遗传性疾病。

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.