Fig. 4: 5A2-DOT-X LNPs simplify generation of complex mouse models.
a To create an in situ liver-specific cancer model, 5A2-DOT-5 LNPs encapsulating Cas9/sgP53/sgPTEN/sgRB1 RNPs were injected into adult C57BL/6 mice weekly (three injections, 2.5 mg kg−1 total sgRNA, IV, n = 4). After 12, 15, and 20 weeks, mice were sacrificed and livers were collected to analyze tumor generation. b T7EI cleavage results from genomic DNA extracted from livers confirmed gene editing occurred at all three loci. Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. c Representative photograph of a mouse liver containing tumors excised 20 weeks after injection. d H&E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in tumor lesions. Scale bar = 100 μm. e To create an in situ lung-specific cancer model, 5A2-DOT-50 LNPs encapsulating Cas9/sgEml4/sgAlk RNPs were injected into adult C57BL/6 mice once (2 mg kg−1) or twice (1.5 mg kg−1 weekly for 2 weeks) (IV, n = 5). After 10, 16, and 24 weeks, mice were sacrificed and lungs were collected to analyze tumor generation. f T7EI cleavage results from genomic DNA extracted from lungs confirmed gene editing occurred at loci of Eml4 and Alk. Red arrows indicate cleavage bands. Indel percentages shown under gel images were measured by Sanger sequencing and TIDE analysis. g PCR amplicons of Eml4-Alk rearrangements were also detected in all lungs treated with 5A2-DOT-50 LNPs. h Eml4-Alk rearrangements were further confirmed by sub-cloning and DNA sequencing. i H&E and Ki67 staining further confirmed progressive tumor formation. Higher tumor proliferation biomarker Ki67 expression was detected in lung tumor lesions. Scale bar = 100 μm. Data of b, d, f, g, and i were repeated three times independently with similar results.
