溶瘤病毒介导碱基编辑靶向杀伤宫颈癌细胞

Oncolytic virus-mediated base editing for targeted killing of cervical cancer cells

摘要:

传统肿瘤治疗，如放疗和化疗，常损伤正常细胞，可能引发新肿瘤。与之相比，溶瘤病毒(oncolytic virus,OV)则能选择性攻击肿瘤细胞，减少对正常细胞的影响。多数临床试验中的溶瘤病毒经过基因改造，以增强其靶向肿瘤细胞和激活免疫反应的能力。为了开发特异治疗宫颈癌的溶瘤腺病毒，本研究构建了特异性治疗宫颈癌的溶瘤腺病毒，该病毒通过递送靶向原癌基因的碱基编辑器实现肿瘤细胞的高效杀伤，即通过抑制肿瘤生长和直接裂解肿瘤来高效杀伤癌细胞。本研究利用人端粒酶逆转录酶(telomerase reverse transcriptase,TERT)启动子启动病毒早期复制蛋白(early region 1A,E1A)，成功构建了P-hTERT-E1A-GFP载体，并验证了其在宫颈癌细胞中的启动活性。鉴于MYC原癌基因在肿瘤学研究中的重要作用，筛选高效MYC原癌基因编辑位点是本研究的关键步骤。针对MYC基因设计3个gRNA靶点，将其与ABE8e碱基编辑器质粒共转染至HEK293T细胞，经嘌呤霉素筛选后，Sanger测序分析显示各靶点编辑效率分别为43%(MYC-1)、25%(MYC-2)和35%(MYC-3)，明确MYC-1为最优编辑位点。通过构建P-ABEs-hTERT-E1A-GFP和P-MYC gRNA-hTERT-E1A-GFP载体，成功进行了病毒包装并验证了其特异性和有效性。实验结果表明，这种新型溶瘤腺病毒能有效抑制体外培养的HeLa宫颈癌细胞的生长。本研究基于HeLa细胞模型，为宫颈癌治疗提供了新的实验依据和潜在策略。

Abstract:

Conventional cancer therapies, such as radiotherapy and chemotherapy, often damage normal cells and may induce new tumors. Oncolytic viruses (OVs) selectively target tumor cells while sparing normal cells. Most OVs used in clinical trials have been genetically engineered to enhance their ability to target tumor cells and activate immune responses. To develop a specific OV-based approach for treating cervical cancer, this study constructed an oncolytic adenovirus that delivered a base editor targeting oncogenes to achieve efficient killing of tumor cells through inhibiting tumor growth and directly lysing tumor cells. We utilized the human telomerase reverse transcriptase (TERT) promoter to drive the expression of adenovirus early region 1A (E1A) and successfully constructed the P-hTERT-E1A-GFP vector, which was validated for its activity in cervical cancer cells. Given the critical role of the MYC oncogene in the research of oncology, identifying efficient editing sites for the MYC oncogene is a key step in this study.Three MYC-targeting gRNAs were engineered and co-delivered with ABE8e base editor plasmids into HEK293T cells. Following puromycin selection, Sanger sequencing demonstrated differential editing efficiencies: MYC-1 (43%), MYC-2 (25%), and MYC-3 (35%), identifying MYC-1 as the most efficient editing locus. By constructing the P-ABEs-hTERT-E1A-GFP and P-MYC gRNA-hTERT-E1A-GFP vectors, we successfully packaged the virus and confirmed its specificity and efficacy. The experimental results demonstrate that this novel oncolytic adenovirus effectively inhibits the growth of HeLa cells in vitro, providing new experimental evidence and potential strategies for treating cervical cancer based on the HeLa cell model.