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Engineering Synthetic Guide RNAs and Compact Base Editors for Enhanced In Vivo Delivery

用于增强体内递送的工程合成指南 RNA 和紧凑碱基编辑器

基本信息

批准号：
10541817
负责人：
Nathan Bamidele
金额：
$ 3.39万
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：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10541817
关键词：
Adenine Affect Animal Model Base Pairing Benchmarking Biodistribution Bioinformatics C57BL/6 Mouse Capsid Cell model Cells Chemicals Chemistry Complex Cultured Cells Cytidine Cytosine deaminase DNA Deaminase Dependovirus Disease model Endonuclease I Engineering Exhibits GTP-Binding Protein alpha Subunits, Gs Genes Genetic Diseases Genetic Research Genome Goals Guide RNA Human Immune response Impairment Injections Interphase Cell Liver Mediating Methods Modality Modification Mus Muscle Mutation Neisseria meningitidis Neuraxis Neurons Nucleotides Oligonucleotides Organ Orthologous Gene Pathway interactions Pattern Plant Roots Production RNA Ribose Site Specific qualifier value Speed Streptococcus pyogenes System Technology Therapeutic Therapeutic Studies Tissues To specify Toxic effect Trans-Splicing Viral Load result adeno-associated viral vector base base editing base editor cell type cost design flexibility functional genomics gene function gene therapy genome editing genomic locus immune activation in vivo intein mouse model novel novel strategies nuclease phosphodiester pre-clinical programs repaired success tool vector

项目摘要

Project Summary CRISPR-Cas9 technology has profoundly advanced genetic research and gene therapy by enabling rapid, precise, and programmable genome editing to study gene function or correct mutations in cells and in vivo. Among CRISPR-mediated gene editing approaches, cytidine and adenine base editors (CBEs and ABEs) enable efficient and precise single-base transitions in dividing and non-dividing cell types. Base editors comprise a catalytically-impaired nickase Cas9 (nCas9) fused to a cytosine deaminase (CBE) or adenine deaminase (ABE) that is guided to a target site by a “guide RNA”. With the potential to enable all four nucleotide transitions in the context of a base-pair (C:G→T:A or A:T→G:C), base editors have the potential to cure a wide range of genetic disorders. Realizing this hope requires efficient and safe in vivo delivery methods. In vivo delivery of base editors relies on adeno-associated virus (AAV) vectors. Due to the limited packaging capacity of AAVs and the large size of nCas9 orthologs (e.g., SpyCas9 from Streptococcus pyogenes), delivery requires two AAVs encoding an intein-split nCas9-BE that fuses into a functional complex when co-expressed in cells. Dual AAVs encoding intein-split SpyCas9-BEs achieve therapeutically-relevant levels of base editing in pre-clinical disease models, including in the central nervous system (CNS). Nonetheless, dual AAV SpyCas9- BE delivery suffers from several limitations, including: toxicity from increased viral load; high vector production costs; immune response and off-target editing caused by sustained expression of nCas9-BE components; and limited ability of guide RNA to specify multiplexed edits. Under guidance from Drs. Erik Sontheimer (CRISPR), Miguel Sena Esteves (AAV delivery), Anastasia Khvorova (oligonucleotide chemistry), and Athma Pai (sequencing, bioinformatics), this proposal aims to develop flexible delivery approaches for efficient and safe base editing in vivo. This project will take advantage of established gene therapy modalities, including a single AAV vector encoding a compact Cas9 from N. meningitidis (Nme2Cas9), and chemically-modified oligonucleotides. Aim 1 will optimize and validate an all-in-one AAV encoding a compact Nme2Cas9-ABE and its guide RNA for efficient in vivo base editing in mice. The use of a compact ABE for AAV delivery will decrease viral load, production costs and may increase delivery efficiency. Aim 2 will develop chemically-modified Nme2Cas9 crRNA (target-specify portion of guide RNA) for co-delivery separate from an AAV encoding Nme2Cas9-ABE and tracrRNA (invariant portion of guide RNA). This approach will provide a way to control nCas9-BE expression and streamline multiplexed base editing via delivery of multiple crRNA. Although these delivery approaches are applicable to variety of tissues, this study will focus on the CNS, where AAV- and oligonucleotide-based therapies have shown some success, but a dire need for transformative therapeutics remains. Completion of this study will establish novel delivery approaches to advance the utility of CRISPR for in vivo applications, including functional genomic studies and base editing therapies.

项目总结