Targeted DNA methylation of the JSRV LTR suppresses Env-driven pulmonary adenocarcinoma: Epigenetic silencing as a potential veterinary therapeutic strategy.

Abstract

Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic disease caused by jaagsiekte sheep retrovirus (JSRV), leading to significant economic losses due to the absence of effective treatments. Based on the discovery that endogenous JSRV LTR is highly methylated in healthy tissues while exogenous JSRV LTR is demethylated in OPA lung tissues, this study explored targeted DNA methylation as an epigenetic therapeutic strategy. We constructed dCas9-DNMT3A and dCas9-KRAB epigenome editing systems targeting key CpG hotspots in the U3 region of exogenous JSRV LTR and established an exJSRV-LTR-driven Env overexpression tumor model in mouse lungs. Results showed that epigenome editing therapy significantly reduced tumor burden by 42.4&#x202f;% (P&#x202f;<&#x202f;0.001), prolonged survival as determined by Kaplan-Meier analysis and Cox proportional hazards modeling (hazard ratio 0.34-0.41, P&#x202f;<&#x202f;0.005), while Env protein expression was downregulated by 72.4&#x202f;% (immunohistochemistry) and 70.5&#x202f;% (Western blot quantification). Safety assessment indicated no abnormal changes in histology, biochemical parameters, or inflammatory responses in treated mice. Mechanistic studies confirmed that targeted methylation significantly reduced transcription factor occupancy in the U3 region. This was achieved by recruiting heterochromatin marks, including upregulated H3K9me3 and H3K27me3, and downregulated H3K4me3, along with methylation reader proteins. These epigenetic changes collectively inhibited LTR transcriptional activity. Translational application analysis suggested good feasibility of AAV vector-based pulmonary delivery systems, with treatment costs significantly lower than traditional culling strategies. LTR methylation detection could serve as an early diagnostic biomarker and breeding selection tool. This study provides innovative strategies for OPA prevention and control, advancing veterinary precision medicine development and offering important reference for other retroviral diseases.