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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1084: CRISPR-Activated Biomedical Reporter Pigs for In Vivo Genome Editing Pre-Clinical Studies

Type: Poster Session

Poster Board Number: 1084
Presentation Details
Session Title: Thursday Poster Session
Start Time: 5/18/2023 12:00
End Time: 5/18/2023 14:00

Translating promising gene editing technologies such as CRISPR-Cas and base editor from basic research tools to therapeutics that cure genetic diseases require relevant preclinical testing to ensure the safety and efficacy. Preclinical studies in large animal models will provide critical data on dosing and safety of both the editor and the delivery vehicle prior to human use. Genetically engineered swine are beneficial as a preclinical model due to their similar size, anatomy, and physiology to humans as well as their <4 months gestation time and high fecundity (>6 per litter). Technology to produce gene edited swine has rapidly advanced, and custom-engineered swine that harbor the targeted therapeutic site from humans in their genome can now be made and delivered in less than 7 months. These animals are particularly useful for late-stage IND-enabling studies like dose-escalation, pharmacokinetics and pharmacodynamics analyses to streamline preclinical evaluation for gene editing-based therapies. In addition, these engineered animals can elucidate which organs, tissues, and cells will be targeted by the delivery vehicle in a large animal and allow for toxicity studies to be performed. One way of measuring the specificity of in vivo gene editing in specific organs, tissues, or cells is by using reporter lines, like those pioneered by the rodent community, including the Ai9 reporter. We have developed a biomedical research pig line with a modified Ai9 reporter at the ROSA26 safe-harbor locus in pigs as part of the NIH Somatic Cell Genome Editing consortium. The modified Ai9 line includes a human Ubiquitin C or CAG promoter driving a tdTomato fluorescent reporter that is activated after DSB-induced removal of a series of transcriptional stop signals (Figure 1). After activation, analysis can be performed in organs, tissues, and cells using immunohistochemistry, single cells using fluorescence activated cell sorting, molecular analysis, or direct sequencing (Figure 2-4). In addition to the fluorescent protein, the reporter will express a sodium iodide symporter (NIS) that will allow reporter activation to be detected non-invasively by PET or SPECT imaging for longitudinal studies. This model is available with a ~4 month lead time, and pre-clinical experiments can be performed at Recombinetics or through a contract research organization.

Jarryd M. Campbell, Rebecca A. Milliken, Derek M. Korpela, Dennis A. Webster, Hinsoukpo V. Dagan, Adrienne Watson, Daniel F. Carlson

Recombinetics, Inc., Eagan, MN
 J.M. Campbell: None.

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