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

项目概要/摘要 遗传障碍和遗传疾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性疾病，由基因突变引起 囊性纤维化跨膜传导调节因子（CFTR）基因。在健康细胞中，CFTR维持 氯化物和碳酸氢盐运输作为离子通道。CFTR的遗传缺陷导致复杂的 呼吸和全身器官衰竭。点突变或单核苷酸变异（SNV）占约60%。 导致CF的致病性变异。CF患者可以通过施用小剂量的 分子药物，以改善症状，包括慢性肺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， 其症状不能被任何调节剂缓解。基因治疗是一种有前途的和永久的 这是一种为遗传病患者提供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），以促进同源性定向修复（HDR），从而促进细胞凋亡。 准确的基因编辑结果。然而，DSB引起的安全性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（nCas 9），其仅切口原型间隔区（protospacer 在一些实施方案中，所述方法包括将含有邻近基序（PAM）的链连接到含有邻近基序（PAM）的链，并且因此消除了DSB和随机插入缺失的风险。BE使用 与nCas 9融合的天然或工程化的DNA脱氨酶，并且可以引入C至T或A至G的转化 在活性窗口内通过胞嘧啶或腺嘌呤脱氨酶。胞嘧啶BE（CBE）和腺嘌呤BE （ABE）可以高效地实现碱基转换，并且已经证明对于一些遗传修饰是成功的。 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，必须克服若干挑战。第一，不分青红皂白地转换多个“C”或“A”， CBE或ABE的特征性脱氨活性窗口，通常超过五个核苷酸，导致不期望的脱氨活性窗口。 旁观者编辑其次，生物工程的目标范围在很大程度上受到NGG PAM的限制 需要nSpCas 9，即来自化脓性链球菌的典型Cas9。很大比例的基数 因此，转换致病突变不可用于编辑。第三，BE缺乏多样性阻碍了其 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和多重BE，使其能够针对广大的 大多数人类基因组位点（目标1和2）。我们将应用高精度的BE来生成和校正 反映个体患者的纯合和复合杂合CF疾病模型， 极大地促进了用于个性化CF治疗的药理学研究和药物发现（目的3）。在 总之，高精度BE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.