一种通过mRNA-脂质纳米颗粒递送的新型病毒衍生表观遗传基因疗法，用于抑制HPV相关恶性肿瘤

Human Papillomavirus (HPV) is associated with a range of malignancies, including oropharyngeal, cervical, and anal carcinomas. Approximately 47,000 carcinoma cases annually in USA are attributed to HPV causing significant morbidity and mortality. Integration of the episomal HPV genome into the host's DNA is a critical event in oncogenesis. HPV's long control region (LCR) promoter overexpresses the viral E6 and E7 proteins, which inhibit the tumor suppressors p53 and Rb1 respectively, driving the oncogenic transformation. E6 and E7 are also critical for tumor maintenance by creating a state of 'oncogene addiction', making them ideal targets for therapeutic intervention. They are considered undruggable, but they present ideal targets for gene therapy interventions. However, gene therapies face significant challenges such as targeting multiple HPV oncogenes within sequence-diverse high-risk HPV subtypes. To this end, we have exploited a viral self-regulatory genetic circuit to develop a compact, single component epigenetic repressor (BE2K). BE2K targets highly conserved sites in the LCR which can silence multiple oncogenes simultaneously in virtually all HPV subtypes. BE2K inhibited the proliferation of high-risk HPV16 and HPV18-associated oropharyngeal, cervical, and anal cancer cell lines. BE2K had no effect on non-HPV associated cell lines. BE2K suppressed E6 and E7 expression resulting in reactivation of the p53 tumor suppressor and caspase-mediated apoptosis. Global mRNA profiling confirmed E6 and E7 reduction, an increase in p53 signaling and apoptosis pathways, as well as a reduction in the E2F transcription factor targets, downstream of Rb1. Of note, an upregulation in interferon pathways and inflammatory response was observed, which may additionally contribute to the anti-proliferative effects in HPV-associated cancers in an immune-competent context. To deliver BE2K intratumorally, it was formulated as mRNA in MC3 lipid nanoparticles (LNPs). An HPV-associated head and neck SCC154 xenograft model in NSG mice was used and LNP-mRNA BE2K reduced tumor volume and improved clinical scores in the mice compared to controls. Immunohistochemistry showed an elevated p53 with a reduction in the Ki-67. Safety profiling showed no effect on serum ALT/AST levels, or liver and spleen histology. To validate our findings, a patient-derived xenograft (PDX) model was established from a primary tonsil HPV-associated head and neck squamous cell cancer. Preliminary experiments in the PDX showed that BE2K treatment resulted in a correlation between tumor growth inhibition and p53 and caspase-3 activation. Overall, BE2K, as a scalable LNP-mRNA formulation, represents a promising new epigenetic mRNA treatment towards a more generalizable gene therapy for HPV-associated malignancies.