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

项目摘要 CRISPR-Cas9技术通过实现快速， 精确和可编程的基因组编辑，以研究基因功能或纠正细胞和体内的突变。 在CRISPR介导的基因编辑方法中，胞苷和腺嘌呤碱基编辑器（CBE和ABE）使得能够 在分裂和非分裂细胞类型中有效和精确的单碱基转换。基本编辑器包括一个 与胞嘧啶脱氨酶（CBE）或腺嘌呤脱氨酶（ABE）融合的催化受损的切口酶Cas9（nCas 9） 其被“向导RNA”引导至靶位点。有可能使所有四个核苷酸转换在 在碱基对（C：G→T：A或A：T→G：C）的背景下，碱基编辑器具有治愈广泛的遗传缺陷的潜力。 紊乱实现这一希望需要有效和安全的体内递送方法。 碱基编辑器的体内递送依赖于腺相关病毒（AAV）载体。由于包装有限 AAV的容量和nCas 9直系同源物的大尺寸（例如，SpyCas 9来自化脓性链球菌），递送 需要两个编码内含肽分裂的nCas 9-BE的AAV，所述nCas 9-BE在共表达时融合成功能复合物 在细胞中。编码内含肽分裂的SpyCas 9-BE的双腺相关病毒实现了治疗相关水平的碱基编辑 临床前疾病模型，包括中枢神经系统（CNS）。尽管如此，双AAV SpyCas 9- BE递送受到几个限制，包括：病毒载量增加的毒性;高载体产量; 由nCas 9-BE组分的持续表达引起的免疫应答和脱靶编辑;以及 指导RNA指定多重编辑的能力有限。 在Erik Sontheimer（CRISPR）、Miguel Sena Esteves（AAV递送）、Anastasia Khvorova博士的指导下， （寡核苷酸化学）和Athma派（测序，生物信息学），该提案旨在开发灵活的 用于体内有效和安全的碱基编辑的递送方法。该项目将利用现有的 基因治疗模式，包括编码来自N.脑膜 （Nme 2Cas 9）和化学修饰的寡核苷酸。目标1将优化和验证一体化AAV 编码紧凑型Nme 2Cas 9-ABE及其指导RNA用于小鼠中的有效体内碱基编辑。的使用 用于AAV递送的紧凑ABE将降低病毒载量、生产成本并可提高递送效率。 目标2将开发用于共递送的化学修饰的Nme 2Cas 9 crRNA（指导RNA的靶向特异性部分） 与编码Nme 2Cas 9-ABE和tracrRNA（指导RNA的不变部分）的AAV分开。这种方法 将提供一种控制nCas 9-BE表达并通过递送多个DNA片段来简化多重碱基编辑的方法。 crRNA。尽管这些递送方法适用于各种组织，但本研究将集中于CNS， 其中基于AAV和利培酮的疗法已经显示出一些成功，但迫切需要变革性的治疗。 治疗仍然存在。这项研究的完成将建立新的交付方法，以促进 用于体内应用的CRISPR，包括功能基因组研究和碱基编辑疗法。

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.