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.

项目摘要/摘要 遗传疾病和遗传疾病是由插入，缺失和基本取代引起的 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，是由突变引起的 囊性纤维化跨膜电导调节剂（CFTR）基因的。在健康细胞中，CFTR保持 氯化物和碳酸氢盐作为离子通道。 CFTR的遗传缺陷导致复杂 呼吸和系统性器官衰竭。点突变或单核苷酸变化（SNV）约为60％ 引起CF的致病变异。 CF患者可以通过小型给药来部分治疗 分子药物可以改善症状，包括慢性肺部疾病和胰腺功能不全。 但是，导致过早终止密码子（PTC）的CF突变影响至少10％的CF患者， 任何调节器都无法解释其符号。基因疗法是一种承诺和永久性的 为患有遗传疾病的患者承认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），以促进同源指导的维修（HDR） 准确的基因编辑结果。但是，DSB引起的安全问题会导致不良突变。 为了克服这个问题，基础编辑器（BES）使用nickase cas9（NCAS9），该nick cas9（NCAS9）仅刻痕 相邻基序（PAM）含有链，从而消除了DSB和随机indels的风险。 bes使用 天然或工程的DNA脱氨酶与NCAS9融合，可以引入C-TO-T或A-TO-G转换 在活动窗口内通过胞嘧啶或腺嘌呤脱氨酶。胞嘧啶BES（CBE）和腺嘌呤BES （ABES）可以以高效率实现基本过渡，并且已经证明了一些通用的成功 概念验证研究中的疾病。但是，在将BES应用于治疗人类遗传疾病之前， 包括CF在内，必须克服几个挑战。首先，多个'C或'a内的偶联转换 CBE或ABE的特征脱氨酸活动窗口，通常超过五个核动肽，导致不希望的 旁观者编辑。其次，BES的靶向范围在很大程度上受到NGG PAM的限制 NSPCAS9的需求，来自链球菌的典型Cas9。大部分基础 因此，过渡致病突变无法进行编辑。第三，BES缺乏多重性阻碍了它的 仅用于处理复杂遗传疾病的多个突变处理多个突变的实用性。在 这项拟议的研究，我们旨在发展精度和多重效果，这将使靶向疫苗成为可能 大多数人类基因组站点（AIM 1和2）。我们将应用高精度BES以生成和纠正 纯合子和复合杂合的CF疾病模型，这些模型反映了个别患者，这也将 为个性化CF治疗的大量准备的药物研究和药物发现（AIM 3）。 摘要，高精度BES将有助于囊性纤维化的个性化基因疗法以及许多 其他遗传疾病。