Miniature Oncopig®癌症模型作为临床前药物评估的创新型大型动物平台

Background: Advances in cancer care rely on human-like preclinical animal models to evaluate safety, metabolism, pharmacokinetics, and efficacy of novel therapeutics in a translational manner. We previously developed the transgenic Oncopig ® , a genotypically, anatomically, metabolically, and physiologically relevant large animal model that develops inducible tumors on demand. While the Oncopig ® platform is ideal for short-term device-based therapeutic and diagnostic studies, its rapid growth and large size limits use for pharmaceutical and long-term efficacy studies. To overcome this limitation, we used the Wisconsin Miniature Swine TM (WMS TM ) to develop a miniature version of the Oncopig ® (WMS TM -Oncopig ® ). The WMS TM -Oncopig ® is a valuable model for preclinical drug studies due to its reduced size in addition to predisposition to obesity, metabolic disorders, and other clinically relevant cancer comorbidities. Methods: The WMS TM -Oncopig ® transgene cassette was designed to harbor Cre recombinase-inducible KRAS G12D and TP53 R167H driver mutations. The cassette also includes a mScarlet fluorescence gene flanked by LoxP sites, serving as a negative marker of Cre-induced recombination and driver mutation expression. Using CRISPR/Cas9 technology, the transgene cassette was inserted into the Rosa26 locus of WMS TM fibroblasts. After validation, the modified fibroblasts were used for reproductive cloning of WMS TM -Oncopigs ® by somatic cell nuclear transfer. Tumor induction studies were conducted on WMS TM -Oncopig ® clones (n=2) with established Oncopig ® liver tumor induction protocols, while the remaining clones were used for breeding. Results: WMS TM -Oncopig ® fibroblasts were selected based on fluorescent imaging (mScarlet) and sequencing. Transgene cassette functionality was confirmed based on KRAS G12D and TP53 R167H expression, increased proliferation, and loss of red fluorescence following nucleofection with a Cre recombinase-expressing plasmid. WMS TM -Oncopigs ® formed liver tumors within 2 weeks of induction using established Oncopig ® tumor induction protocols. Finally, cells isolated from 3 of 5 fetuses generated by breeding WMS TM -Oncopig ® clones to wild-type WMS™ displayed red fluorescence, with genomic PCR confirming germline transmission of the transgene cassette. Conclusions: The WMS TM -Oncopig ® model enables on-demand tumor induction in a clinically relevant pig breed with reduced size and growth compared to the current Oncopig ® model. This, combined with predisposition to obesity and other clinically relevant cancer comorbidities supports the translational relevance of the WMS TM -Oncopig ® for preclinical oncologic drug metabolism, pharmacokinetic, safety, and efficacy studies. The WMS TM -Oncopig ® thus fulfills the current unmet need for translationally relevant models for preclinical investigation of novel cancer therapeutics.