Complement component 3 (C3) is an attractive therapeutic target for several inflammatory diseases; however, the strict primate specificity of emerging C3 inhibitors mandates the use of nonhuman primates (NHPs) for preclinical evaluation, creating significant ethical and economic barriers. Although we previously developed a human C3 complementary DNA knockin rat, the model was limited by low expression levels of human C3 and an inability to support gene-targeted therapies requiring genomic architecture. To overcome these limitations, we developed a second-generation humanized rat model by introducing the full-length human C3 gene onto a rat C3-deficient background. These rats exhibited significantly increased levels of human C3 in all tissues examined while maintaining normal renal function. We validated the utility of this new model using AMY-101, a clinical-stage, primate-specific C3 inhibitor, demonstrating its suppression of complement-mediated hemolysis both in vivo and in vitro. Notably, this inhibition primarily targeted the alternative pathway while sparing the classical pathway, consistent with the reported C3-bypass phenomenon. This second-generation C3 humanized rat model represents a robust, cost-effective preclinical platform for evaluating diverse human C3-targeted therapeutics, thereby reducing reliance on NHP models.

The Transgenic Core has used Cas9, ZFNs, and TALENs, to target more 500 genes in mice and rats.In the current era, all projects involve gene knockins, such as floxed alleles, recombinase knockins, reporter knockins, cDNA knockins to the ROSA26 locus. Simple gene knockouts are not in demand as they were when CRISPR/Cas9 technology was reported n 2013. It is essential to protect sensitive RNA components from degradation during zygote microinjection and/or electroporation. All materials mixed with gRNA or mRNA components, such as Cas9 protein (used to prepare ribonucleotide protein complexes), DNA donors (such as oligonucleotides, long single stranded DNA donors, and double stranded circular plasmids), and AAV donors must be resuspended in a buffer that is free from endonucleolytic activity.Fine particulate contaminants can be reduced in microinjection solutions by judicious use of 0.02 µm filters. The suggestions below are based our successful experience in the production of genetically engineered mice, rats, and cultured cells.

CCR Translation for This Article from Cabozantinib Inhibits Prostate Cancer Growth and Prevents Tumor-Induced Bone Lesions

PDF file 88K, Supplementary Figure Legends

Supplementary Figure 4 from p21CIP-1/WAF-1 Induction Is Required to Inhibit Prostate Cancer Growth Elicited by Deficient Expression of the Wnt Inhibitor Dickkopf-1

Supplementary Methods, Figure Legends 1-4 from p21CIP-1/WAF-1 Induction Is Required to Inhibit Prostate Cancer Growth Elicited by Deficient Expression of the Wnt Inhibitor Dickkopf-1

PDF file 183K, Impact of cabozantinib on MET, phospho-MET, VEGFR2 and phospho-VEGFR2 expression of subcutaneous and intratibial PC-3 tumors in mice

PDF file - 66K, Figure S1. Mouse body weights were not impacted by AR79 treatment.

PDF file - 56K, DNA profiling of LNCaP, C4-2B and DU145

PDF file 95K, Cabozantinib does not inhibit progression of PC-3luc PCa cells in bone in vivo

<p>Supplemental Figure Legends. Figure legends accompanying all supplementary figures and table.</p>

PDF file - 290K, Figure S2. AR79 has no impact on p-HIPK2 expression in prostate cancer cell lines.

Supplementary Figure 3 from p21CIP-1/WAF-1 Induction Is Required to Inhibit Prostate Cancer Growth Elicited by Deficient Expression of the Wnt Inhibitor Dickkopf-1

PDF file 173K, Impact of cabozantinib on MET, phospho-MET, VEGFR2 and phospho-VEGFR2 expression of subcutaneous LuCaP 35 and intratibial C4-2B tumors in mice