Human Gene Therapy

Gene Targeting of HPV18 E6 and E7 Synchronously by Nonviral Transfection of CRISPR/Cas9 System in Cervical Cancer

High-risk human papillomavirus (HPV) E6 and E7 genes display vital oncogenic properties in cervical cancer. Eliminating HPV driver gene or loss of function by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a promising treatment for the HPV-associated cancer. Thus, this study designed a CRISPR/Cas9 system to target the E6 and E7 genes at once, to detect whether it have efficacy in vitro and in vivo . Meanwhile, CRISPR/Cas9 system was measured after transfection with liposomes but virus. Cervical cancer lines (HeLa and SiHa) were used in this study. Sanger sequencing confirmed that the single CRISPR/Cas9 vector [termed E6E7-knockout (KO)] containing guide RNAs could targeting both HPV18 E6 and E7 genes in vitro . In addition, double-targeting E6 and E7 increased p53 protein expression significantly while compared with E6 or E7 targeting, respectively. Mice with xenografts were divided into four groups: three doses of experimental groups (20, 40, and 60 μg) and one control group. The E6E7-KO through liposome delivery was injected into tumors. Tumor growth was measured and protein expression was observed through immunohistochemistry. The toxic side effects in vivo were also evaluated. E6E7-KO induced cell apoptosis and inhibited cell proliferation markedly in vitro . E6E7-KO downregulated the messenger RNA and protein expression of E6 and E7, whereas p53 and p21 protein levels were upregulated accordingly. Notably, E6E7-KO delivery by liposome exhibited an effect in vivo . Tumor growth was inhibited in the E6E7-KO groups, which was accompanied by decreased E6/E7 protein expression and increased p53/p21 protein expression, especially the level of p53 protein expression. Therefore, E6E7-KO could have synergy efficient by p53 pathway. Furthermore, local injection with CRISPR/Cas9 by nonviral delivery may be regarded as a potential therapy for cervical cancer in the future.

Truncated Bid Regulates Cisplatin Response via Activation of Mitochondrial Apoptosis Pathway in Ovarian Cancer

Refractoriness to conventional chemotherapy is a major challenge in the treatment of advanced ovarian cancer (OC). There is increasing evidence that mitochondrial priming correlates with cisplatin response in various cancers. Notably, Bim and Bid, two of the proapoptotic BH3-only proteins, are recognized as the most effective inducers of mitochondrial priming in OC. In this study, we constructed two tumor-specific oncolytic adenoviruses (Ads) coding for Bim (Ad-Bim) or truncated Bid (Ad-tBid), respectively, and performed gain-of-function assays in nine OC cell lines. Ad-tBid exhibited significant antitumor efficacy than the controls. On addition of Ad-tBid pretreatment, mito-primed cells displayed more sensitivity to cisplatin both in vitro and ex vivo . We also found that Ad-tBid induced mitochondrial apoptosis in a Bak-dependent manner. Furthermore, a combined cisplatin plus Ad-tBid therapy markedly inhibited tumor growth in a subcutaneous xenotransplanted tumor model. In mice bearing peritoneal disseminated OC, intraperitoneal administration of Ad-tBid potentiated the antitumor effect of cisplatin. Our findings suggest that Ad-tBid enhances cisplatin response in OC cells, establishing the potential treatment of advanced OC via a combination of cisplatin and Ad-tBid.

Feasibility of Bone Marrow Mesenchymal Stem Cell-Mediated Synthetic Radiosensitive Promoter-Combined Sodium Iodide Symporter for Radiogenetic Ovarian Cancer Therapy

Ovarian cancer is the most lethal gynecological cancer, most patients relapse within 12-24 months, and eventually die, especially platinum-resistant patients. Gene therapy has been one of the most potential methods for tumor treatment. Bone marrow mesenchymal stem cells (BMSCs) have been used for systemic delivery of therapeutic genes to solid tumors. Sodium iodide symporter (NIS) is an intrinsic membrane glycoprotein and can concentrate

M11: A Tropism-Modified Oncolytic Adenovirus Arming with a Tumor-Homing Peptide for Advanced Ovarian Cancer Therapies

Oncolytic adenoviruses (OAds) have shown great promise in cancer therapy, but their efficacy has been greatly limited by poor tumor selectivity and highly off-target liver sequestration. Herein, we generated a novel “stealth” and tumor-targeting OAd vector, M0-TMTP1, by inserting TMTP1 (NVVRQ), a tumor-homing peptide specifically targeting metastasis, into the hypervariable region 5 of hexon. M0-TMTP1 exhibits increased transduction of tumor cells in vitro . In vivo biodistribution of M0-TMTP1 in an intraperitoneal disseminated ovarian cancer model showed significantly reduced virus load in major organs but apparent aggregation in tumors. The tumor-to-liver ratio of M0-TMTP1 was nearly 5,000-fold higher than that of control adenovirus M0. Furthermore, we armed M0-TMTP1 with trunked BID, a mitochondrial apoptosis protein, to obtain M11. Combining M11 with cisplatin (DDP) could induce an intensive antitumor effect in vitro and in vivo . Moreover, this combination therapy showed higher biosafety. Taken together, our results suggest that M11 represents a tumor-targeting, efficacious, and relatively nontoxic virotherapeutic agent, and these findings might offer renewed hope for tumor management.

Clinical Efficacy and Safety of AdV-tk Gene Therapy for Patients with Cervical Squamous Intraepithelial Lesion: A Prospective Study

Cervical cancer is the fourth most common type of cancer for women in 2020, and many more women have cervical precancerous lesion-squamous intraepithelial lesion (SIL). Early treatment of cervical SIL to reverse or delay its progression is an important approach to reduce the incidence of cervical cancer. The efficacy and safety of adenovirus-based vectors expressing the thymidine kinase gene (AdV-tk) in the treatment of multiple types of cancers shows promise for its use in gynecology. We aim to provide relevant clinical efficacy and safety data after introducing AdV-tk for the treatment of cervical SIL for the first time through this prospective study. We conducted a maximum of 6 sessions to administer AdV-tk gene therapy to 23 patients (mean age: 35 years old) with cervical low-/high-grade SIL (LSIL/HSIL) who were enrolled from August 2015 to April 2018 and analyzed the clinical characteristics and follow-up outcomes (mean follow-up period: 7.3 months). The present study consisted of 17 patients (73.9%) with cervical HSIL and 6 patients with LSIL confirmed by colposcopy-directed biopsy. We observed an overall histological remission and regression rate of 87.0% (20/23, 95% confidence interval [95% CI]: 73.2-100,

Aberrantly Expressed Timeless Regulates Cell Proliferation and Cisplatin Efficacy in Cervical Cancer

Timeless is a regulator of molecular clockwork in Drosophila and related to cancer development in mammals. This study aimed to investigate the effect of Timeless on cell proliferation and cisplatin sensitivity in cervical cancer. Timeless expression was determined by bioinformatics analysis, immunohistochemistry, and quantitative polymerase chain reaction (qPCR). Chromatin immunoprecipitation assays and reporter gene assays were applied to determine the transcriptional factor contributing to Timeless upregulation. The effects of Timeless depletion on cell proliferation and cisplatin sensitivity were determined through in vitro and in vivo experiments. Cell apoptosis and senescence were assessed by flow cytometry and β-galactosidase staining. DNA damage and DNA repair pathways were determined by comet assay, immunofluorescent staining, and Western blot analysis. Timeless is aberrantly expressed in ∼52.5% of cervical cancer tissues. E2F1 and E2F4 contribute to the transcriptional activation of Timeless. Timeless depletion inhibits cell proliferation and increases cisplatin sensitivity in vitro and in vivo . Knockdown of Timeless induces cell apoptosis and cell senescence. Mechanically, Timeless silencing leads to DNA damage and impairs the activation of the ATR/CHK1 pathway in response to cisplatin in cervical cancer. Timeless is overexpressed in cervical cancer and regulates cell proliferation and cisplatin sensitivity, presenting an attractive target for cisplatin sensitizer in cervical cancer.

Human Papillomavirus Oncogene Manipulation Using Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 Delivered by pH-Sensitive Cationic Liposomes

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology enables targeted gene editing, but cancer gene therapy with this approach requires improvements to enable safe and efficient delivery of CRISPR/Cas9 to tumors. We developed and evaluated a self-assembled liposome to selectively deliver CRISPR/Cas9 to cancer tissues. Our CRISPR/Cas9 system effectively inhibited proliferation of human papillomavirus (HPV) 16-positive cervical cancer cells and induced apoptosis by inactivating the HR-HPV16E6/E7 oncogene. Based on this system, we prepared a long-circulating pH-sensitive cationic nano-liposome complex with a high cell targeting and gene knockout rate. Intratumoral injection of cationic liposomes targeted to splicing HPV16 E6/E7 in nude mice significantly inhibited tumor growth without significant toxicity in vivo . Liposomes that targeted HPV16 E6/E7 splicing were established as a basis for treatment of HPV16-positive cervical cancer drug candidates. Our study demonstrates that this liposome offers an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine-based cancer therapeutics.