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

项目摘要 CRISPR-Cas9技术通过使快速、 精确、可编程的基因组编辑，用于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程序(CBE和ABES)使 在分割和非分割细胞类型中进行高效和精确的单碱基转换。基本编辑器由一个 催化受损镍酶Cas9(NCas9)与胞嘧啶脱氨酶(CBE)或腺嘌呤脱氨酶(ABE)融合 由一个“引导RNA”引导到目标位置。有可能实现所有四个核苷酸的转变 在碱基对(C：G→T：A或A：T→G：C)的背景下，碱基编辑有可能治愈广泛的遗传 精神错乱。要实现这一希望，需要高效、安全的体内给药方法。 碱基编辑的体内传递有赖于腺相关病毒(AAV)载体。由于包装有限 AAVs的容量和nCas9的大尺寸同源物(例如，来自化脓链球菌的SpyCas9)，递送 需要两个编码内含子的AAV-Split nCas9-BE，当共表达时，该内含子融合成功能复合体 在牢房里。编码内含子分裂的SpyCas9-BES的双AAV实现了与治疗相关的碱基编辑水平 临床前疾病模型，包括中枢神经系统(CNS)。尽管如此，Dual AAV SpyCas9- BE的传递受到几个限制，包括：病毒载量增加带来的毒性；高载体产量 成本；持续表达nCas9-BE组分导致的免疫反应和非靶点编辑；以及 向导RNA指定多路复用编辑的能力有限。 在Erik Sonthemer博士(CRISPR)、Miguel Sena Esteves(AAV Delivery)、Anastasia Khvorova的指导下 (寡核苷酸化学)和Athma PAI(测序，生物信息学)，这项提议旨在开发灵活的 有效和安全的体内碱基编辑的传递方法。该项目将利用已建立的 基因治疗方式，包括编码脑膜炎奈瑟氏菌致密Cas9的单个AAV载体 (Nme2Cas9)和化学修饰的寡核苷酸。AIM 1将优化和验证一体式AAV 编码紧凑的Nme2Cas9-ABE及其引导RNA，用于在小鼠体内进行有效的碱基编辑。的用法 用于AAV递送的紧凑型ABE将降低病毒载量，降低生产成本，并可能提高递送效率。 AIM 2将开发化学修饰的Nme2Cas9 crRNA(导向RNA的靶标特定部分)，用于联合传递 与编码Nme2Cas9-ABE和trrRNA(引导RNA的不变部分)的AAV分开。这种方法 将提供一种控制nCas9-BE表达的方法，并通过提供多个 CrRNA。虽然这些给药途径适用于各种组织，但本研究将重点放在中枢神经系统， 基于AAV和寡核苷酸的疗法已经显示出一些成功，但迫切需要变革性的 治疗公司仍然存在。这项研究的完成将建立新的交付方法，以提高 CRISPR用于活体应用，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.