Document text
Principal Investigator: James Pickel
Organization: NATIONAL INSTITUTE OF MENTAL HEALTH
Fiscal Year: 2022
Award: $2,105,007
Funding agency: National Institute of Mental Health
1) Production of rodent transgenics
Mouse transgenic production projects: 22
Rat transgenic production projects: 4
Cryopreservation projects: 26
Rederivation projects: 20
2) SARS-Cov2
a) Fibroblast cultures have been propagated using methods developed in the lab (see below) over several years. These cultures have proven useful as in vitro models of ACE2 expression. The ACE2 protein (which acts as a binding site for the SARS-CoV2 virus spike protein) has been introduced into cultured marmoset fibroblasts. Both the marmoset protein and the human protein have been expressed on these fibroblasts to compare the potential for viral infection. Chimeric, or humanized marmoset ACE2 has also been expressed in fibroblasts. Several cell lines expressing each of these three constructs have been immortalized to create reproducible testbeds to enable other investigators to evaluate the binding of isolated viral spike protein.
b) Marmoset embryos grown in culture have been injected with CRISPR constructs that alter the ACE2 gene. Binding of the viral spike protein requires ACE2 protein domains. Two of these domains have been altered in cultured marmoset embryos. The frequency of targeting of the segments of the exons that encode these domains is being optimized.
3) Techniques for transgenic production in marmoset monkeys
Manipulating embryos to produce a line of transgenic marmosets is no longer a feasible option for most research. We have developed a more effective method of introducing transgenes into brain cells using AAV vector variants. These unique AAVs, which can cross the blood-brain barrier and migrate into the brain, can be infused into the peripheral circulation. They avoid accumulating in the liver like most virus, but instead preferentially home to the brain. These variants were developed in collaboration with the Gradinaru laboratory at Cal Tech; the variants were then selected by screen pools of potential variants in the core facility. We have produced several animals that carry transgenes and express them in the brain. With other collaborators we have characterized both new variants with higher transduction frequency and have designed vector payloads to express the exogenous gene in specific cells as well as control endogenous gene expression using modified CRISPR molecules.
AAV variant development to Old World primates has been an important extension of this work:
Chuapoco M, Flytzanis N, Goeden N, Octeau JC, Roxas KM, Chan KY, Scherrer J, Winchester J, Blackburn RJ, Campos LJ, Arokiaraj CM, Miles TF, Jang MJ, Vendemiatti J, Deverman BE, Pickel J, Fox AS, Gradinaru V. Intravenous gene transfer throughout the brain of infant Old World primate using AAV.
bioRxiv 2022.01.08.475342; doi: https://doi.org/10.1101/2022.01.08.475342
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