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

基本信息

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
项目类别：
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

项目摘要

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。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) 在健康细胞中保持不变。 CFTR 的遗传缺陷导致氯离子和碳酸氢盐的离子通道变得复杂。呼吸系统和全身器官衰竭约占 60%。引起 CF 的致病变异可以通过给予小剂量来部分治疗。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的基因疗法是一种有前途且永久的疗法。 CRISPR-为患有遗传性疾病的患者提供有益的治疗的替代方法。 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）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，从而消除了 DSB 和随机插入缺失使用 a 的风险。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。（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，必须克服几个挑战，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其次，BE 的目标范围在很大程度上受到 NGG PAM 的限制。 nSpCas9 的要求，来自化脓性链球菌的经典 Cas9 碱基的很大一部分。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编辑。同时处理多个突变以治疗复杂遗传疾病的实用性。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子和复合杂合子 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）。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。