Advanced Delivery Platforms for Base Editing In Vivo

用于体内碱基编辑的先进交付平台

基本信息

批准号：
10682172
负责人：
ERIK J. SONTHEIMER
金额：
$ 58.37万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10682172
关键词：
Adenine Adverse effects Alleles Amyotrophic Lateral Sclerosis CLN3 gene CRISPR/Cas technology Central Nervous System Central Nervous System Diseases Clinical Clustered Regularly Interspaced Short Palindromic Repeats Cytidine DNA Sequence Alteration Deaminase Dendritic Cells Deoxyribonucleases Dependovirus Development Dinucleoside Phosphates Disease Disease model Effectiveness Endowment Exons Extrahepatic Genes Genetic Diseases Goals Hepatic Tissue Hepatocyte Hepatotoxicity Immune response Immunity Interphase Cell Knowledge Liver Mammalian Cell MicroRNAs Modality Mus Mutagenesis Mutate Mutation Nucleotides Patients Pharmaceutical Preparations RNA RNA Splicing Repression Research Risk Safety Site Specificity Spielmeyer-Vogt Disease Spinal Ganglia System Technology Testing Therapeutic Tissues Toxic effect adverse outcome base editing base editor cell type clinical practice delivery vehicle density disease-causing mutation exon skipping therapy genome editing humanized mouse immunogenicity improved in vivo knockout gene mouse model mutant next generation novel prevent prime editing repaired superoxide dismutase 1 therapeutic genome editing vector

项目摘要

Project Summary The Cas9 platform has enabled genome editing, base editing (BE), and prime editing to induce gene knockouts as well as tailor-made sequence alterations. CRISPR-Cas9 systems have the potential to similarly revolutionize clinical practice through precise editing of disease loci. We have developed Nme2Cas9 as an editing platform with (1) compact size, facilitating single adeno-associated virus (AAV) delivery; (2) a dinucleotide (N4CC) protospacer-adjacent motif (PAM) that affords high target site density; and (3) exceptional accuracy. More recently, we developed Nme2Cas9 adenine base editor (ABE) systems as among the first to be validated in vivo for single-AAV delivery. AAV is a potent editing delivery modality in vivo, especially in extrahepatic tissues such as the central nervous system (CNS). Nonetheless, the therapeutic promise of base editing systems will hinge upon improving editing efficiency, limiting bystander edits (or their consequences), maximizing PAM-dependent targeting scope, and minimizing immunogenicity, toxicity, and prolonged deaminase expression (which can compromise editing efficiency and lead to safety risks such as hepatotoxicity and the accumulation of unwanted edits). Here we propose to capitalize on our establishment of single-vector Nme2-ABE systems to develop novel base editing capabilities with increased effectiveness, targeting scope, utility, and safety, and to validate these systems in the treatment of CNS disease models in mice. The goals of this proposal are (1) to develop next-generation, single-AAV, deaminase-inlaid Nme2-ABEs and guides with increased efficiency, a single-nucleotide PAM, and greater control over bystander editing; (2) to use next- generation, single-AAV Nme2-ABE systems for therapeutic editing of disease genes in the CNS of mouse models of amyotrophic lateral sclerosis and Batten disease; and (3) to develop systems that use repression by endogenous microRNAs and drug-dependent splicing systems to enhance the safety of AAV-delivered Nme2- ABE in vivo. These safety enhancements will reduce anti-Nme2-ABE immune responses, ameliorate potential toxic effects on the liver and on specific CNS cell types, and limit the off-target mutagenesis that can arise from sustained expression of the editing machinery. Successful completion of these aims will provide invaluable enhancements to the delivery, efficacy, and specificity of in vivo genome editing.

项目概要 Cas9平台实现了基因组编辑、碱基编辑（BE）和引物编辑以诱导基因淘汰赛以及定制的序列改变。 CRISPR-Cas9 系统具有类似的潜力通过精确编辑疾病位点彻底改变临床实践。我们开发了 Nme2Cas9 作为编辑平台具有（1）紧凑的尺寸，便于单个腺相关病毒（AAV）的递送； (2) 一个二核苷酸 (N4CC) 原型间隔子相邻基序 (PAM)，提供高靶位点密度； (3) 特殊的准确性。最近，我们开发了 Nme2Cas9 腺嘌呤碱基编辑器 (ABE) 系统，这是第一个已在体内验证单 AAV 递送。 AAV 是一种有效的体内编辑传递方式，尤其是在肝外组织，例如中枢神经系统（CNS）。尽管如此，碱的治疗前景编辑系统将取决于提高编辑效率、限制旁观者编辑（或其后果），最大化 PAM 依赖性靶向范围，并最小化免疫原性、毒性和延长时间脱氨酶表达（可能会影响编辑效率并导致肝毒性等安全风险）以及不需要的编辑的积累）。在这里，我们建议利用我们建立的单向量 Nme2-ABE 系统可开发新颖的碱基编辑功能，提高效率、靶向范围、实用性和安全性，并验证这些系统在小鼠中枢神经系统疾病模型治疗中的作用。的目标该提案是 (1) 开发下一代、单 AAV、脱氨酶镶嵌 Nme2-ABE 和指南提高效率、单核苷酸 PAM 以及对旁观者编辑的更好控制； (2) 使用下一个- 新一代单 AAV Nme2-ABE 系统，用于治疗性编辑小鼠中枢神经系统中的疾病基因肌萎缩侧索硬化症和巴顿病模型； (3) 开发使用镇压的系统内源性 microRNA 和药物依赖性剪接系统可增强 AAV 传递的 Nme2 的安全性体内 ABE。这些安全性增强将减少抗 Nme2-ABE 免疫反应，改善潜力对肝脏和特定中枢神经系统细胞类型的毒性作用，并限制可能产生的脱靶突变编辑机制的持续表达。成功完成这些目标将为增强体内基因组编辑的传递、功效和特异性。