Adeno-Associated Viral Vectors for Gene Delivery to the Non-Human Primate Brain

Author: Chuapoco, Miguel Roberto Estella

Year: 2023

Degree: Dissertation (Ph.D.)

Advisor: Gradinaru, Viviana

Committee Members: Bjorkman, Pamela J.; Hay, Bruce A.; Van Valen, David A.; Gradinaru, Viviana

Option: Bioengineering

DOI: 10.7907/gngs-q727

Abstract

Viral vectors are efficient gene-delivery carriers for somatic cell gene therapy, and replication-deficient vectors are actively being used in the clinic to replace and correct disease-causing genes and mutations. Mirroring their therapeutic effectiveness, viral vectors are also powerful in vivo gene-delivery tools in basic research. In the field of neuroscience, adeno-associated viruses (AAVs) in particular have emerged as a major workhorse that enable efficient in vivo expression of opsins for optogenetics, designer GPCRs for chemogenetics, and GCaMP for calcium imaging. Recently, AAV engineering efforts by our group and others have expanded the toolbox of AAV vectors to include capsid variants that traverse the blood-brain-barrier (BBB) in rodents. However, not all engineered AAV capsids translate from mice to non-human primates (NHPs). This is especially true for translation to the rhesus macaque (Macaca mulatta), an Old World primate that is the predominant NHP model and shares a more recent common ancestor with humans (~25 million years ago) compared to rodents (~75 million years ago) and New World primates, such as the common marmoset (Callithrix jacchus; ~35 million years ago). The primary contents of this dissertation will focus on the development of AAV.CAP-Mac as a vector for non-invasive gene-transfer to the NHP brain and demonstrations of its utility to interrogate neuronal morphology and physiology in the macaque central nervous system. We identified and selected CAP-Mac using a multi-species selection strategy in adult marmosets and infant macaques, where it demonstrated improved delivery efficiency compared to AAV9 and other engineered variants. In individual characterization, CAP-Mac was biased towards neurons in two infant Old World primate species, the rhesus macaque and the green monkey (Chlorocebus sabaeus). Given this neuronal tropism in Old World primates, we demonstrated how CAP-Mac can be readily used for non-invasive, Brainbow-like labeling of macaque neurons and calcium imaging of GCaMP ex vivo. In closing, we describe CAP-Mac tropism across multiple developmental states, species, and routes of administration. Additionally, we present preliminary data on “orphan capsids,” capsid variants that were engineered to be non-infective, but can be readily re-functionalized using known receptor-ligand pairs. Collectively, the work covered in this dissertation disseminates non-invasive, gene-delivery tools for NHP researchers, and lays the groundwork for further development of more specific and efficacious AAVs that access the NHP brain.

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