A5 - AAV Vectors - Non-Human Primates and Large Animal Models
517: Swine Reporter Models for Somatic Cell Genome Editing
Type: Poster Session
Poster Board Number: 517
Presentation Details
Session Title: Wednesday Posters: AAV Vectors - Non-Human Primates and Large Animal Models
Delivery of gene editing reagents to the appropriate tissues and cells at an effective dose is a significant challenge for in vivo gene editing therapies. Rodent reporter models activated by recombinases or gene editing reagents have been indispensable for development of new delivery modalities. However, their small size and anatomy do not accurately mimic the effectiveness of delivery to large mammals. As part of the NIH Somatic Cell Genome Editing (SCGE) consortium, the Recombinetics Swine Reporter Models are swine models designed to track gene editing in any tissue or cell in the body. It does this by harboring a genetic reporter in every cell that can only be activated by gene editors like CRISPR or Base Editor or by Cre recombinase. Once activated, the reporter will permanently make fluorescent markers that can be used to track gene delivery in any tissue or cell in as soon as one week or in months to years even with non-viral vectors. The first Swine Reporter Model (SRM-1) harbors a genetic reporter that is based on the rodent Ai9 allele. The Ai9 allele has been modified to allow for activation by a SpCas9, SaCas9, AsCas12a, or Cre. The reporter is driven by a highly active and ubiquitous CAG promoter and was positioned at a safe harbor site by gene editing to avoid genotoxicity caused by the integration. After removal of a 3xStop cassette by CRISPR or Cre the cell will begin producing a fluorescent gene (tdTomato) and a sodium iodide symporter gene (NIS) that can be tracked by fluorescent activated cell sorting (FACS) or immunohistochemistry (IHC). The NIS gene also allows for non-invasive imaging of cell activation after injection with a radioactive iodide tracer using sensitive imagine modalities like single-proton emission computed tomography (SPECT) or positron emission tomography (PET). We intravenously injected AAV9-Cre (7.3x1012 GC/kg) into 3-week old SRM-1 piglet and collected 41 tissues after three weeks for analysis of AAV9 viral distribution and reporter activation measured by DNA recombination and tissue immunohistochemistry. AA9 injected in a male piglet was found to have widespread tropism with most accumulation in liver, muscle, adrenal glands, and testicular tissue. A subset of tissues was analyzed by immunohistochemistry, co-staining for tdTomato and NIS, to visualize the reporter activation. The resulting images showed clear and robust reporter activation can be visualized at the tissue and cellular level. The level of activation was highest in skeletal muscle, liver, and testicular tissue, followed by heart muscle, spleen, and no activation in the brain tissue. Overall, this experiment demonstrates the utility of the reporter for tissue- and cell-specific delivery of AAV. We next plan to deliver CRISPR editing tools by packaging mRNA with LNPs and delivering to the brain and liver to the reporter to further validate model utility. The second reporter animal, SRM-2, can be activated by adenine base editor and will also undergo in vivo validation studies. We anticipate these animal lines will be useful for both discovery studies of new delivery modalities and late-stage IND-enabling studies such as dose-escalation, pharmacokinetics, and pharmacodynamics analyses to streamline preclinical evaluation for gene editing-based therapies.
Plain Language Summary
Delivering gene editing tools to specific body tissues poses a challenge in treating diseases. While rodents are commonly used for testing, their small size doesn't accurately reflect larger animals or humans. The Recombinetics Swine Reporter Models, developed as part of the NIH Somatic Cell Genome Editing consortium, are pig models designed to track the delivery of gene editing reagents throughout the body. These pigs carry a genetic reporter that permanently expresses a new marker where gene editors are active. This marker aids scientists in tracing the effectiveness of gene editing tools in target and non-target tissues. The first model, SRM-1, functions like a rodent Ai9 reporter allele and was validated in vivo through an AAV9 i.v. injection. Future studies will explore delivering nonviral gene editing tools to the brain and liver using these models, proving valuable for both early and advanced gene editing therapy testing.
Jarryd M. Campbell1, Rebecca Milliken1, Sierra Long1, Hinsoukpo Dagan1, Dennis Webster1, Derek Korpela1, Daniel F. Carlson2
1Recombinetics, Inc., Eagan, MN,2Recombinetics, Eagan, MN"
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