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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.

项目总结/文摘