Non-Invasive Functional Gene Delivery to the Central and Peripheral Nervous System Across Species

Author: Chen, Xinhong

Year: 2023

Degree: Dissertation (Ph.D.)

Advisor: Gradinaru, Viviana

Committee Members: Mazmanian, Sarkis K.; Oka, Yuki; Lois, Carlos; Gradinaru, Viviana

Option: Neurobiology

DOI: 10.7907/48a2-0y07

Abstract

The normal function of the central nervous system (CNS) and peripheral nervous system (PNS) relies on precise regulation. When this regulation breaks down in diseases, genetic access to the nervous system is critical for therapeutic intervention. However, access to the nervous system remains difficult, reflecting the critical need for development of effective and non-invasive gene delivery vectors across species. By applying directed evolution approach, we identified 2 capsids, AAV-MaCPNS1 and AAV-MaCPNS2, which efficiently transduced the PNS in rodents following intravenous administration. Combining with rational optimization, we also identified AAV-X1 capsid family, which transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. Some previously-engineered AAVs that target the nervous system fail to translate across non-human primate (NHP). We thus also further tested our novel vectors across species and showed that AAV-MaCPNS1/2 efficiently transduced both the PNS and CNS in NHPs. AAV-X1.1 also exhibit superior transduction of the CNS in rhesus macaques and ex vivo human brain slices although the endothelial tropism is not conserved across species.

With these enhanced systemic AAVs, we wanted to explore whether they could enable neuronal recording and modulation which has been challenging with the nature AAV serotypes. We used AAV-MaCPNS1 to systemically deliver the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia. We observed specific nodose neuronal response to physiological modulation in the gut. Furthermore, we showed that the MaCPNS1-delivered neuronal actuator DREADD to dorsal root ganglia could enable non-invasive neuronal modulation and create a model of pain. The functional utility of the novel systemic vectors demonstrated here provide a non-invasive approach to better explore the nervous system, which would lead to better therapeutic intervention. To this end, we also demonstrated that the X1 capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory for production of therapeutic agents for CNS. We showed that vasculature-secreted Hevin (a synaptogenic protein), whose coding sequence is delivered by X1 vectors, rescued synaptic deficits in a mouse model.

AAV repeated dosing could be favorable for certain therapeutic applications, however, neutralizing antibody generated following the first injection creates major obstacle for second injection. We explored whether serotype switching with 2 AAV capsids that have a distinguished neutralizing antibody profile could be a potential solution. To this end, we firstly showed that the X1 capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ. We then combined the different engineered serotype to enable serotype switching for sequential AAV administration in mice, showing the first AAV-delivered receptor for the second AAV could boost its CNS targeting.

In general, we developed strategies to enable non-invasive functional gene delivery to the central and peripheral nervous system across species, which would be incremental for both basic neuroscience research and gene therapies for neurological disorders.

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