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

基因操纵是现代生命科学的基础，并促成了若干新领域的出现，如生物技术、生物工程和合成生物学。CRISPR技术提供了重写基因组DNA的最强大、精确和有效的手段，并已成功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，它现在能够靶向和逆转任何已知的人类病理基因组变异。然而，CRISPR效率和特异性方面的突破性进展尚未被开发出能够靶向人体内任何组织和细胞类型的足够广泛的递送方法，限制了基因组编辑技术对人类生活的影响。因此，大多数临床试验都集中在体外对患者细胞进行体外基因组编辑，只有少数旨在治疗体内可接近的组织。因此，尽管神经系统疾病是全球主要的医疗负担，但缺乏有效的体内靶向神经系统的策略在很大程度上阻碍了CRISPR在大脑中的应用。目前的递送策略可以分为病毒和非病毒方法。基因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，可以提供特定的向性，并诱导高转录水平。另一方面，它们通常具有有限的包装能力，产生不确定的长期表达，并且可以整合到受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瞬时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向性。在这里，我建议开发一种用于体内CRISPR递送的新型病毒载体，该载体结合了当前技术的最佳特性，可以为神经系统中的基因组编辑打开大门。我们最近开发的自失活狂犬病病毒（SiR）是一种无毒的RNA病毒，可以破坏神经回路，并在永久遗传标记后不久从受感染的细胞中消失。SiR是一种理想的神经元CRISPR递送载体：它诱导瞬时表达，仅存在于胞质溶胶中（因此排除了基因组整合和遗传毒性），具有适合甚至大碱基和引物编辑器的大包装容量，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来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，而不是大脑的单个区域或整个神经系统，是许多神经系统和神经退行性疾病（例如阿尔茨海默病，帕金森病，肌萎缩性侧索硬化症和抑郁症）的主要受影响基质。因此，SiR-CRISPR不仅代表了一种在体内递送CRISPR元件的创新病毒载体，而且可以为具有遗传成分和功能失调的神经网络的神经元病理的神经网络疗法的开发铺平道路。

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.