Davis Alicia, Morris Kevin V, Shevchenko Galina
Center for Gene Therapy, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA.
Irell & Manella Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010, USA.
Mol Ther Methods Clin Dev. 2022 Mar 16;25:158-169. doi: 10.1016/j.omtm.2022.03.008. eCollection 2022 Jun 9.
Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia-responsive element (HRE) sequences within the promoter of target genes. HRE-driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the cytomegalovirus (CMV) and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid-packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions while maintaining tight regulation with no significant changes to cell viability in normoxia.
缺氧是实体瘤的一个特征，它有助于肿瘤的侵袭性，并与癌症治疗耐药性相关。缺氧诱导因子-1（HIF-1）转录因子复合物通过与靶基因启动子内的缺氧反应元件（HRE）序列结合来介导缺氧特异性基因表达。作为实现癌症特异性基因表达的一种策略，HRE驱动的治疗性载体表达已得到广泛探索。通过利用该系统，我们实现了两种治疗相关载体元件的缺氧特异性表达：单纯疱疹病毒胸苷激酶（HSV-tk）自杀基因和CRISPR-Cas9核酸酶。使用一个包含五个源自血管内皮生长因子基因的HRE拷贝的表达载体，我们能够在缺氧环境中的细胞中显示出高转基因表达，类似于使用巨细胞病毒（CMV）和CBh启动子所达到的水平。此外，我们能够使用质粒包装的脂质纳米颗粒（LNP）将我们的治疗性载体高效递送至肿瘤细胞，以在缺氧条件下实现肿瘤细胞的特异性杀伤，同时在常氧条件下保持严格调控，细胞活力无显著变化。
