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Development and application of Rabies Virus-based approaches for genomic editing of neural circuits in healthy and diseased brain

基于狂犬病病毒的健康和患病大脑神经回路基因组编辑方法的开发和应用

基本信息

批准号：
EP/W03672X/1
负责人：
Ernesto Ciabatti
金额：
$ 36.63万
依托单位：
MRC Laboratory of Molecular Biology
依托单位国家：
英国
项目类别：
Fellowship
财政年份：
2023
资助国家：
英国
起止时间：
2023 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FW03672X%2F1
关键词：
Development application Rabies Virus based

项目摘要

Genetic manipulation is at the basis of modern life sciences and has contributed to the emergence of several novel fields, such as biotechnology, bioengineering and synthetic biology. CRISPR technologies provide the most powerful, precise and effective means to rewrite genomic DNA and have been successfully used in many different organisms, tissues and cell types. The recent developments of base and prime editors have further expanded CRISPR potential, which is now capable, in principle, of targeting and reverting any known human pathological genomic variant. However, groundbreaking advances in CRISPR efficiency and specificity have not yet been followed by the development of a sufficiently wide repertoire of delivery methods capable of targeting any tissues and cell types in the human body, limiting the impact of genome editing technologies on human life. As a result, most clinical trials focus on the ex-vivo genome-editing of patients' cells outside the body and only a minority aims to treat accessible tissues in vivo. As a consequence, despite neurological pathologies represent the major worldwide healthcare burden, the lack of efficient strategies to target the nervous system in vivo largely precludes CRISPR use in the brain. Current delivery strategies can be divided in viral and non-viral methods. Engineered viral vectors have been employed for gene therapy for decades and have been proven safe, can offer specific tropism, and induce high transcriptional levels. On the other hand, they often have limited packaging capacity, produce indefinite long-term expression, and can integrate into the genome of infected cells. Conversely, non-viral methods can prevent genomic integration and induce transient CRISPR activity, thus minimising off-target effects. However, they often display low expression levels and non-specific tropism. Here, I propose to develop a novel viral vector for CRISPR delivery in vivo that combines the best properties of current technologies and could open the door to genome editing in the nervous system. Our recently developed Self-inactivating Rabies virus (SiR) is a non-toxic RNA virus that can transduce neural circuits and disappear from infected cells shortly after their permanent genetic labelling. SiR is an ideal neuronal CRISPR-delivering vector: it induces transient expression, resides only in the cytosol (thus precluding genomic integration and genotoxicity), has a large packaging capacity fitting even large base and prime editors, and has an exceptional neuronal tropism. Moreover, SiR ability to spread between synaptically connected neurons could be exploited to target and treat entire neuronal circuitry. Current research points to distributed neural networks, rather than single areas of the brain or the entire nervous system, to be the primary affected substrate in many neurological and neurodegenerative diseases (e.g. Alzheimer Disease, Parkinson Disease, Amyotrophic Lateral Sclerosis and Depression). Thus, SiR-CRISPR represents not only an innovative viral vector to deliver CRISPR elements in vivo, but could pave the way to the development of neural network therapies for neuronal pathologies with a genetic component and a dysfunctional neural network.

遗传操作是现代生命科学的基础，并促进了几个新领域的出现，如生物技术、生物工程和合成生物学。CRISPR技术提供了最强大、精确和有效的重写基因组DNA的手段，并已成功地应用于许多不同的生物体、组织和细胞类型。碱基和引物编辑器的最新发展进一步扩大了CRISPR的潜力，原则上，它现在能够靶向和恢复任何已知的人类病理基因组变异。然而，在CRISPR效率和特异性方面取得突破性进展之后，还没有开发出足够广泛的能够靶向人体任何组织和细胞类型的递送方法，这限制了基因组编辑技术对人类生命的影响。因此，大多数临床试验都侧重于在体外对患者细胞进行离体基因组编辑，只有少数旨在治疗体内可获得的组织。因此，尽管神经系统疾病是全球主要的医疗负担，但缺乏针对体内神经系统的有效策略在很大程度上阻碍了CRISPR在大脑中的应用。目前的递送策略可分为病毒式和非病毒式两种。工程病毒载体已被用于基因治疗几十年，并已被证明是安全的，可以提供特异性趋向性，并诱导高转录水平。另一方面，它们往往具有有限的包装能力，产生不确定的长期表达，并且可以整合到感染细胞的基因组中。相反，非病毒方法可以阻止基因组整合并诱导短暂的CRISPR活性，从而最大限度地减少脱靶效应。然而，它们通常表现出低表达水平和非特异性倾向。在这里，我建议开发一种新的病毒载体，用于CRISPR在体内的传递，它结合了当前技术的最佳特性，并可能打开神经系统基因组编辑的大门。我们最近开发的自我灭活狂犬病毒（SiR）是一种无毒的RNA病毒，可以转导神经回路，并在感染细胞永久遗传标记后不久从感染细胞中消失。SiR是一种理想的神经元crispr传递载体：它诱导瞬时表达，仅存在于细胞质中（因此排除了基因组整合和遗传毒性），具有大的包装容量，适合大型碱基和引物编辑器，并且具有特殊的神经元向性。此外，SiR在突触连接的神经元之间传播的能力可以用于靶向和治疗整个神经元回路。目前的研究指出，分布式神经网络，而不是大脑的单一区域或整个神经系统，是许多神经和神经退行性疾病（如阿尔茨海默病、帕金森病、肌萎缩侧索硬化症和抑郁症）的主要受影响的基质。因此，SiR-CRISPR不仅代表了一种在体内传递CRISPR元件的创新病毒载体，而且可以为开发具有遗传成分和功能失调神经网络的神经病理学的神经网络疗法铺平道路。