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

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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.