Ovarian Cancer Gene Therapy Research Articles

Applications of CRISPR Cas-9 in Ovarian Cancer Research.

Ovarian cancer is a highly prevalent malignancy among women and affects a significant population worldwide. Different forms of hormonal treatments or chemotherapies are used to treat ovarian cancer, but the possible side effects, including menopausal symptoms, can be severe, forcing some patients to prematurely stop the treatment. The emerging genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9), has the potential to treat ovarian cancer via gene editing strategies. Studies have reported CRISPR knockouts of several oncogenes that are involved in the pathogenesis of ovarian cancer, such as BMI1, CXCR2, MTF1, miR-21, and BIRC5, and demonstrate the potential of the CRISPR-Cas9 genome editing technique to effectively treat ovarian cancer. However, there are limitations that restrict the biomedical applications of CRISPR-Cas9 and limit the implementation of Gene therapy for ovarian cancer. These include off-target DNA cleavage and the effects of CRISPR-Cas9 in non-target, normal cells. This article aims to review the current state of ovarian cancer research, highlight the significance of CRISPR-Cas9 in ovarian cancer treatment, and establish the groundwork for potential clinical research.

Effect of WWOX on epithelial-mesenchymal transition in ovarian cancer cells in vivo, and its association with Elf5/Snail-1 signal pathway

Purpose: To investigate how WW domain-containing oxidoreductase (WWOX) gene affects epithelial-mesenchymal transition (EMT) of ovarian cancer (OC) cells, and the implication of Elf5/Snail-1 pathway in the process.
 Methods: This study employed female BALB/c nude mice aged 6 weeks. The expression levels of E-cadherin, β-Catenin, N-cadherin, Vimentin, Snail1 and Elf5 were assayed using qRT-PCR. The protein levels of WW1, WW2, E-cad, β-Cat, N-cad, vimentin, Snail-1 and Elf5 were determined using western blotting (WB). Invasion changes in cells in each group were analyzed by Transwell invasion test while WWOX and Elf5 were evaluated by GST pull-down test; furthermore, WWOX and ELF5 were evaluated by co-immunoprecipitation (COIP).
 Results: Relative to control group, tumor volume and weight of WWOX high-expression mice were significantly decreased, while those of WWOX low-expression group were increased (p < 0.05). The levels of E-cad, Elf5 and β-catenin increased, but those of N-cad, Snail-1 and vimentin decreased in WWOX high expression group. In the WWOX low-expression group, the levels of E-cad, Elf5 and β-cat were down-regulated, while those of N-cad, Snail-1 and vimentin were up-regulated (p < 0.05). The expression of E-cad, Elf5 and β-cat declined; on the other hand, those of N-Cad, Snail-1 and vimentin were elevated after mutation of WW1 domain in WWOX gene (p < 0.05).
 Conclusion: Based on the in vivo results, the role of WWOX/Elf5/Snail1 signal route in the epithelial-mesenchymal transition of ovarian cancer, and its related mechanisms have been clarified. This provides a new target for gene therapy of ovarian cancer.

Emerging targeted drug delivery strategies toward ovarian cancer.

Ovarian cancer is a high-mortality malignancy in women. The contemporary clinical chemotherapy with classic cytotoxic drugs, targeted molecular inhibitors would mostly fail when ovarian cancer cells become drug-resistant or metastasize through the body or when patients bare no more toleration because of strong adverse effects. The past decade has spotted varying targeted delivery systems including antibody-drug conjugates (ADCs), peptide/folate/aptamer-drug conjugates, polymer-drug conjugates, ligand-functionalized nanomedicines, and dual-targeted nanomedicines that upgrade ovarian cancer chemo- and molecular therapy effectively in preclinical/clinical settings via endowing therapeutic agents selectivity and bypassing drug resistance as well as lessening systemic toxicity. The targeted delivery approaches further provide means to potentiate emergent treatment modalities such as molecular therapy, gene therapy, protein therapy, photodynamic therapy, dual-targeting therapy and combination therapy for ovarian cancer. This review highlights up-to-date development of targeted drug delivery strategies toward advanced, metastatic, relapsed, and drug resistant ovarian cancers.

Liposomal Delivery of MicroRNA-7 Targeting EGFR to Inhibit the Growth, Invasion, and Migration of Ovarian Cancer.

Ovarian cancer is highly aggressive and has high rates of recurrence and metastasis. Due to the limited effects of current treatments, it is necessary to conduct research and develop new treatment options. The application of gene therapy in tumor therapy is gradually increasing and has exciting prospects. MicroRNA-7 (miR-7) has been reported to inhibit the growth, invasion, and metastasis of a variety of solid tumors. Cationic liposomes are safe and effective gene delivery systems for transfection in vivo and in vitro. To realize the application of miR-7 in the treatment of ovarian cancer, cationic liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol. The miR-7 liposomes had a suitable particle size, potential, and a high cellular uptake rate. MiR-7 encapsulated by liposomes could be effectively delivered to ovarian cancer cells and successfully targeted to the tumor site in a mouse xenograft model of ovarian cancer. In vitro and in vivo experiments revealed that the miR-7 liposomes had a significant ability to inhibit the growth, invasion, and migration of ovarian cancer, probably by inhibiting the expression of the epidermal growth factor receptor. Our studies of miR-7 liposomes demonstrated a safe and efficient microRNA delivery system for the gene therapy of ovarian cancer.

Arf6-mediated macropinocytosis-enhanced suicide gene therapy of C16TAB-condensed Tat/pDNA nanoparticles in ovarian cancer.

The use of cell-penetrating peptides (CPPs), typically HIV-Tat, to deliver therapeutic genes for cancer treatment is hampered by the inefficient delivery and complicated uptake route of plasmid DNA (pDNA). On the one hand, surface charges, particle size and shape essentially contribute to the endocytosis pathway of Tat/pDNA nanocomplexes, and on the other hand, endogenous cellular factors dominantly determine their intracellular trafficking fate and biological outcome. Recent advances in surfactant-modified nanomaterial and dual molecular imaging technology have offered new opportunities for suicide gene therapy. In this study, we employed the cationic surfactant C16TAB to further condense Tat/pDNA nanocomplexes for improving their delivery efficiency and tested the therapeutic effect of Tat/pDNA/C16TAB (T-P-C) nanoparticles carrying the GCV-converted HSV-ttk suicide gene for ovarian cancer. The cellular endocytosis pathway and underlying signal mechanism of T-P-C nanoparticles were further determined. The obtained T-P-C nanoparticles exhibited a small size, positive surface charge, irregular granular shape and high pDNA encapsulation efficiency. The in vitro experiments showed that T-P-C nanoparticles mainly used the macropinocytosis pathway for uptake in ovarian cancer cells. Their internalization and payload gene expression were controlled by the Arf6 GTPase-dependent, Rab GTPase-activated signal axis. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that T-P-C nanoparticles significantly increased the targeted delivery and suicide gene therapy in a mouse model xenografted with human ovarian cancer. More importantly, Arf6-mediated macropinocytosis remarkably enhanced the delivery efficiency and suicide gene therapy effect of T-P-C nanoparticles. Therefore, these C16TAB-condensed Tat/pDNA nanoparticles combined with the dual molecular imaging strategy provides a novel intracellular delivery platform for high-efficient, precise suicide gene therapy of ovarian cancer.

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 131I, which is important for radionuclide therapy and nuclear medicine imaging in recent years. However, the rapid iodine efflux has become a bottleneck for NIS-mediated radionuclide gene therapy. Our previous studies found that the early growth response-1 (Egr1) promoter containing CC(A/T)6GG (CArG) elements had an 131I radiation-positive feedback effect on the NIS gene. Other research showed the synthesized Egr1 promoter containing four CArG elements, E4, was nearly three times as sensitive as the Egr1 promoter. In our study, BMSC-E4-NIS was engineered to express NIS under the control of E4 promoter using lentivirial vectors. After BMSC-E4-NIS implantation, no tumors were seen in BALB/c nude mice and BMSC-E4-NIS did not promote the growth of SKOV3 tumor. BMSCs migrated toward ovarian cancer samples in chemotaxis assays and to ovarian tumors in mice. Using micro-single-photon emission computed tomography/computed tomography (SPECT/CT) imaging, we found that E4 promoter produced a notable increase in 125I uptake after 131I irradiation, the radionuclide uptake is almost three and six times more than Egr1 and cytomegalovirus (CMV) promoters. These studies confirmed the feasibility of using BMSCs as carriers for lentivirus-mediated E4-NIS gene therapy for ovarian cancer. Further research on BMSC-E4-NIS gene therapy for ovarian cancer in vivo will also be carried on, and if successful, this might provide a new adjuvant therapeutical option for platinum-resistant ovarian cancer patients and provide a new method for dynamic evaluation of curative effect.

Molecular imaging-monitored radiofrequency hyperthermia-enhanced intratumoral herpes simplex virus-thymidine kinase gene therapy for rat orthotopic ovarian cancer

Objective To establish the technique of intratumoral combination therapy of radiofrequency hyperthermia (RFH) with herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy for rat ovarian cancers. Material and methods This study consisted of three parts: (1) in vitro experiments to establish the ‘proof of principal’ that combination of RFH and HSV-TK gene therapy has the synergistic effect on human ovarian cancer cells; (2) creation of bioluminescence imaging-detectable rat ovarian cancer model; and (3) in vivo experiments using this rat model to validate the technical feasibility of the combination therapy. Cells and nude rats were divided into four groups: (i) combination therapy (HSV-TK/GCV + RFH); (ii) RFH; (iii) HSV-TK/GCV; and (iv) phosphate-buffered saline (PBS). Data were analyzed using Dunnett t-test or Kruskal–Wallis test. Results Cell proliferation assay demonstrated significantly greater reduction in viable cells with the combination therapy [0.52 (0.43, 0.61)] compared to other treatments [RFH 0.90 (0.84, 0.96), HSV-TK/GCV 0.71 (0.53, 0.88), PBS 1 (1, 1); p < .05]. For 24 rat models with bioluminescence imaging-detectable orthotopic ovarian cancer (n = 6 per group), optical imaging demonstrated significantly decreased relative bioluminescence signal with the combination therapy [0.81 (0.52, 1.08)] compared to other treatments [RFH 3.60 (2.34, 4.86), HSV-TK/GCV 2.21 (1.71, 2.71), PBS 3.74 (3.19, 4.29); p < .001]. Ultrasound imaging demonstrated the smallest relative tumor volume with the combination therapy [0.78 (0.45, 1.11) versus 3.50 (2.67, 4.33), 2.10 (0.83, 3.37), 3.70 (1.79, 5.61); p < .05]. Conclusion The feasibility of intratumoral RFH-enhanced HSV-TK/GCV gene therapy was established on a unique rat model with molecular imaging-detectable orthotopic ovarian cancer.

Increased temperature-related adeno-associated virus vectors transduction of ovarian cancer cells - essential signatures of AAV receptor and heat shock proteins.

Recombinant adeno-associated viruses (rAAVs) are becoming more commonly used in clinical trials involving gene therapy. Additionally AAV-based drugs have already been registered. Gene therapy aims to increase transduction efficiency, increase in vivo selectivity and reduce side effects. One approach to achieve this is the use of physical factors, such as temperature or more specifically, hyperthermia, which is already utilized in oncology. The aim of the present study was to investigate the effect of hyperthermic conditions (40°C and 43°C) on the rAAV transduction efficiency of ovarian cancer cells (Caov-3 and NIH:OVCAR-3) and non-cancerous cells (AAV-293). The present study was designed to identify functional associations between the level of gene transfer and the expression of representative genes for rAAV transmission (AAVR (AAV receptor), heparan sulfate proteoglycan (HSPG) 1 and HSPG2) and heat shock proteins (HSPs). The expressions of selected genes were measured via reverse transcription-quantitative PCR and cell adhesion/invasion chamber tests were also performed. The results revealed that ovarian cancer cell lines were more efficiently transduced with rAAV vectors at an elevated temperature. Additionally, the expression patterns of AAVR, HSPG1 and HSPG2 genes were different between the tested lines. The expression of certain receptors in ascites-derived NIH:OVCAR-3 ovarian cancer cells was higher compared with tumor-derived Caov-3 cells at 37, 40 and 43°C, which indicates a higher transduction efficiency in the formerly mentioned cells. Ascites-derived ovarian cancer cells were characterized by high expressions of HSP40, HSP90 and HSP70 families. Lower levels of HSP expression were demonstrated in less-effectively transduced Caov-3 cells. Furthermore, expressions of the examined genes changed with increasing temperature. The results indicated that temperature-dependent transduction is associated with the expression of the rAAV receptor and HSP genes. The results of the current study may aid the design of effective protocols for ovarian cancer gene therapy.

Bioengineered adipose-derived stem cells for targeted enzyme-prodrug therapy of ovarian cancer intraperitoneal metastasis

The objective of this study was to develop a stem cell-based system for targeted suicide gene therapy of recurrent, metastatic, and unresectable ovarian cancer. Malignant cells were obtained from the ascites of a patient with advanced recurrent epithelial ovarian cancer (named OVASC-1). Cancer cells were characterized to determine the percentages of drug-resistant ALDH+ cells, MDR-1/ABCG2 overexpressing cells, and cancer stem-like cells. The sensitivity and resistance of the OVASC-1 cells and spheroids to the metabolites of three different enzyme/prodrug systems were assessed, and the most effective one was selected. Adipose-derived stem cells (ASCs) were genetically engineered to express recombinant secretory human carboxylesterase-2 and nanoluciferase genes for simultaneous disease therapy and quantitative imaging. Bioluminescent imaging, magnetic resonance imaging and immuno/histochemistry results show that the engineered ASCs actively targeted and localized at both tumor stroma and necrotic regions. This created the unique opportunity to deliver drugs to not only tumor supporting cells in the stroma, but also to cancer stem-like cells in necrotic/hypoxic regions. The statistical analysis of intraperitoneal OVASC-1 tumor burden and survival rates in mice shows that the administration of the bioengineered ASCs in combination with irinotecan prodrug in the designed sequence and timeline eradicated all intraperitoneal tumors and provided survival benefits. In contrast, treatment of the drug-resistant OVASC-1 tumors with cisplatin/paclitaxel (standard-of-care) did not have any statistically significant benefit. The histopathology and hematology results do not show any toxicity to major peritoneal organs. Our toxicity data in combination with efficacy outcomes delineate a nonsurgical and targeted stem cell-based approach to overcoming drug resistance in recurrent metastatic ovarian cancer.

Reducing off target viral delivery in ovarian cancer gene therapy using a protease-activated AAV2 vector platform

Gene therapy is a promising strategy for treating metastatic epithelial ovarian cancer (EOC). However, efficient vector targeting to tumors is difficult and off-target effects can be severely detrimental. Most vector targeting approaches rely on surface receptors overexpressed on some subpopulation of cancer cells. Unfortunately, there is no universally expressed cell surface biomarker for tumor cells. As an alternative, we developed an adeno-associated virus (AAV) based “Provector” whose cellular transduction can be activated by extracellular proteases, such as matrix metalloproteinases (MMP) that are overexpressed in the tumor microenvironments of the most aggressive forms of EOC. In a non-tumor bearing mouse model, the Provector demonstrates efficient de-targeting of healthy tissues, especially the liver, where viral delivery is <1% of AAV2. In an orthotopic HeyA8 tumor model of EOC, the Provector maintains decreased off-target delivery in the liver and other tissues but with no loss in tumor delivery. Notably, approximately 10% of the injected Provector is still detected in the blood at 24 h while >99% of injected AAV2 has been cleared from the blood by 1 h. Furthermore, mouse serum raised against the Provector is 16-fold less able to neutralize Provector transduction compared to AAV2 serum neutralizing AAV2 transduction (1:200 vs 1:3200 serum dilution, respectively). Thus, the Provector appears to generate less neutralizing antibodies than AAV2. Importantly, serum against AAV2 does not neutralize the Provector as well as AAV2, suggesting that pre-existing antibodies against AAV2 would not negate the clinical application of Provectors. Taken together, we present an EOC gene delivery vector platform based on AAV with decreased off-target delivery without loss of on-target specificity, and greater immunological stealth over the traditional AAV2 gene delivery vector.

P53-Bad: A Novel Tumor Suppressor/Proapoptotic Factor Hybrid Directed to the Mitochondria for Ovarian Cancer Gene Therapy.

Clinical trials involving p53 gene therapy for ovarian cancer failed due to the dominant negative inhibition of wild-type p53 and multiple genetic aberrations in ovarian cancer. To overcome this problem, we have designed a more potent chimeric gene fusion, called p53-Bad, that combines p53 with the mitochondrial pro-apoptotic factor Bad. Unlike wild-type p53, which acts as a nuclear transcription factor, this novel p53-Bad construct has multiple unique mechanisms of action including a direct and rapid apoptotic effect at the mitochondria. The mitochondrial localization, transcription activity, and apoptotic activity of the constructs were tested. The results suggest that p53 can be effectively targeted to the mitochondria by controlling the phosphorylation of pro-apoptotic Bad, which can only localize to the mitochondria when Ser-112 and Ser-136 of Bad are unphosphorylated. By introducing S112A and S136A mutations, p53-Bad fusion cannot be phosphorylated at these two sites and always localizes to the mitochondria. p53-Bad constructs also have superior activity over p53 and Bad alone. The apoptotic activity is consistent in many ovarian cancer cell lines regardless of the endogenous p53 status. Both p53 and the BH3 domain of Bad contribute to the superior activity of p53-Bad. Our data suggests that p53-Bad fusions are capable of inducing apoptosis and should be further pursued for gene therapy for ovarian cancer.

Narrowing the field: cancer-specific promoters for mitochondrially-targeted p53-BH3 fusion gene therapy in ovarian cancer

BackgroundDespite years of research, the treatment options and mortality rate for ovarian cancer remain relatively stagnant. Resistance to chemotherapy and high heterogeneity in mutations contribute to ovarian cancer’s lethality, including many mutations in tumor suppressor p53. Though wild type p53 gene therapy clinical trials failed in ovarian cancer, mitochondrially-targeted p53 fusion constructs, including a fusion with pro-apoptotic protein Bad, have shown much higher apoptotic potential than wild type p53 in vitro. Due to the inherent toxicities of mitochondrial apoptosis, cancer-specificity for the p53 fusion constructs must be developed. Cancer-specific promoters such as hTERT, hTC, Brms1, and Ran have shown promise in ovarian cancer.ResultsOf five different lengths of hTERT promoter, the − 279/+ 5 length relative to the transcription start site showed the highest activity across a panel of ovarian cancer cells. In addition to − 279/+ 5, promoters hTC (an hTERT/CMV promoter hybrid), Brms1, and Ran were tested as drivers of mitochondrially-targeted p53-Bad and p53-Bad* fusion gene therapy constructs. p53-Bad* displayed cancer-specific killing in all ovarian cancer cell lines when driven by hTC, − 279/+ 5, or Brms1.ConclusionsCancer-specific promoters hTC, − 279/+ 5, and Brms1 all display promise in driving p53-Bad* gene therapy for treatment of ovarian cancer and should be moved forward into in vivo studies. -279/+ 5 displays lower expression levels in fewer cells, but greater cancer specificity, rendering it most useful for gene therapeutics with high toxicity to normal cells. hTC and Brms1 show higher transfection and expression levels with some cancer specificity, making them ideal for lowering toxicity in order to increase dose without as much of a reduction in the number of cancer cells expressing the gene construct. Having a variety of promoters available means that patient genetic testing can aid in choosing a promoter, thereby increasing cancer-specificity and giving patients with ovarian cancer a greater chance at survival.

The interaction of Lin28A/Rho associated coiled-coil containing protein kinase2 accelerates the malignancy of ovarian cancer

Ovarian cancer (OC) is the leading cause of death among women with gynecologic malignant diseases, however, the molecular mechanism of ovarian cancer is not well defined. Previous studies have found that RNA binding protein Lin28A is a key factor of maintain the pluripotency of stem cells, and it is positively correlated with the degree of several cancers (breast, prostate, liver cancer, etc). Our previous study shows that Lin28A is highly expressed in OC tissues and is involved in the regulation of OC cell biological behavior. In this study, we confirmed that high expression of Lin28A promoted the survival, invasion, metastasis, and inhibited the apoptosis of OC cells. Lin28A interacts with Rho associated coiled-coil containing protein kinase2 (ROCK2) but not ROCK1 and upregulates the expression of ROCK2 in OC cells. The binding sites of each other were identified by truncated mutations and Immuno-precipitaion (IP) assay. After knock down of ROCK2 in cells with high expression of Lin28A, the survival, invasion, metastasis was significantly inhibited and early apoptosis was increased in OC cells and OC xenograft in nude mice. Our experimental data also showed that knock down of ROCK2 but not ROCK1 inhibited the invasion by decreasing the expression of N-cadherin, Slug, β-catenin and increasing ZO-1 expression. Simultaneously, knock down of ROCK2 induced cell apoptosis by increasing cleaved Caspase-9,cleaved Caspase-7, and cleaved Caspase-3. Taken together, Lin28A regulated the biological behaviors in OC cells through ROCK2 and the interaction of Lin28A/ROCK2 may be a new target for diagnosis and gene therapy of OC.

A hierarchical assembly strategy to engineer dextran-enveloped polyurethane nanopolyplexes for robust ovarian cancer gene therapy

A hierarchical assembly strategy is herein investigated to generate bio-responsive, dextran-enveloped, bioreducible polyurethane nanopolyplexes for nonviral gene therapy against ovarian tumor. Initially, a group of poly(urethane amine)s were designed and characterized for in vitro gene transfection. The polyurethane containing 1,4-bis(3-aminopropyl)piperazine residue (PUBAP) could induce the best in vitro transfection efficacy against SKOV-3 or A2780 ovarian cancer cells. Next, dextran-enveloped PUBAP polyplexes (e-polyplexes) were constructed by a hierarchical assembly procedure involving gene neutralization with PUBAP and subsequent gene condensation with a cationic dextran (SSDP800). Such dextran comprised dextran (15 kDa) as the main chain and multiple disulfide-linked branched polyethylenimine (BPEI) oligomers as the side grafts. Additionally, folate-dextran-enveloped PUBAP polyplexes (FA-e-polyplexes) were fabricated by folate-modified SSDP800. These nanoscale-enveloped polyplexes elicited an improved colloidal stability against salt ions and negatively charged heparin, efficient endosomal escaping, and bioreduction-triggered intracellular gene release. In vitro transfection against SKOV-3 cells illustrated that FA-e-polyplexes exerted higher transfection efficiency in the serum than e-polyplexes and 25 kDa BPEI-polyplexes. In vivo, FA-e-polyplexes yielded higher transgene expression level than e-polyplexes in an SKOV-3 tumor-bearing nude mouse model. In the tumor gene therapy with a small hairpin RNA silencing vascular endothelial growth factor, FA-e-polyplexes afforded higher tumor growth inhibition than polyplexes of folate-PEGylated PUBAP and 25 kDa linear polyethylenimine as positive controls. Importantly, such gene therapy had minor toxic effects on the health of the mouse. This work highlights a practical hierarchical assembly method to construct innovative enveloped polyurethane nanopolyplexes enabling robust ovarian cancer gene therapy. Statement of SignificanceIt is indispensable to rationally update binary cationic polyplexes into ternary polyplexes for vigorous tumor gene therapy. In this work, we have confirmed that a hierarchical assembly strategy, by using initial gene neutralization and subsequent gene condensation, is facile and effective to promote cationic polyurethane polyplexes into ternary folate-dextran-enveloped polyurethane polyplexes with a relatively high gene-loading capacity. The enveloped polyplex system enables more efficient gene transfection than the PEGylated polyplex counterpart in ovarian cancer in vitro and in vivo, thereby affording robust ovarian cancer gene therapy. The development of innovative enveloped polyplexes may be a new direction for a non-viral gene delivery system.

Abstract 5928: Suicide gene therapy of ovarian cancer

Abstract Purpose: The objective of this study was to develop a gene directed enzyme prodrug therapy (GDEPT) method that can effectively kill both differentiating and cancer stem-like cells (CSCs) in ovarian cancer. Methods: Ovarian cancer cell lines including A2780, A2780-Cis, SKOV-3, OVCAR-3 and OVASC-1 (malignant cells from ascitic fluid of an ovarian cancer patient) were examined to identify the most drug resistant one. Various chemotherapeutic drugs which are used for suicide gene therapy were tested to identify one that is the most effective against ovarian cancer cells. These include 6-Methylpurine, 5-Fluorouracil, and SN-38. Cisplatin was used as a standard of care control. The anticancer activities of the drugs were studied by a cell toxicity assay and clonogenic assay. Flow cytometry was performed to study surface stem markers, such as CD133, CXCR4, HER2. ALDH1 activity in ovarian cancer cells was studied by ALDH assay kit. The ability of the most effective enzyme/prodrug system to kill drug resistant ovarian cancer cells was studied by a cell viability assay. Results: The results of in vitro dose-response experiments on various ovarian cancer cells showed that SN-38 was the most efficient drug with IC50 in a range of 10 -100 nM, in comparison to 10-500 μM range of all other selected active drugs. OVASC-1 and OVCAR-3 cell lines showed a higher resistance to drug therapy as compared with other cell lines. It was observed that these two cell lines have a higher number of drug resistant cancer cells in their populations. It was also observed that CSCs are strongly associated with resistance to chemotherapy. ALDH assay, clonogenic assay and sphere formation assay showed a direct correlation between the therapy resistance and the percentage of CSCs. OVASC-1 cells in comparison to the other cell lines were found to be a suitable model for therapy resistant studies. After treatment by 100 nM SN-38, no visible colonies of Ascites were observed. Adipose-derived stem cells (ADSCs) were genetically modified to express Carboxylesterase-2 (CE2) and thus could convert CPT-11 to its active form (SN-38). Using this enzyme/prodrug system an effective eradication of highly resistant OVASC-1 ovarian cancer cells was demonstrated. Conclusions: Our findings suggest that Carboxylesterase/CPT-11 enzyme/prodrug system is the most effective GDEPT method against highly drug resistant ovarian cancer cells. Citation Format: Obeid M. Malekshah, Siddik Sarkar, Arash Hatefi. Suicide gene therapy of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5928.

Recent Progress in Gene Therapy for Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy in developed countries. This is due to the lack of specific symptoms that hinder early diagnosis and to the high relapse rate after treatment with radical surgery and chemotherapy. Hence, novel therapeutic modalities to improve clinical outcomes in ovarian malignancy are needed. Progress in gene therapy has allowed the development of several strategies against ovarian cancer. Most are focused on the design of improved vectors to enhance gene delivery on the one hand, and, on the other hand, on the development of new therapeutic tools based on the restoration or destruction of a deregulated gene, the use of suicide genes, genetic immunopotentiation, the inhibition of tumour angiogenesis, the alteration of pharmacological resistance, and oncolytic virotherapy. In the present manuscript, we review the recent advances made in gene therapy for ovarian cancer, highlighting the latest clinical trials experience, the current challenges and future perspectives.

Retro-inverso follicle-stimulating hormone peptide-mediated polyethylenimine complexes for targeted ovarian cancer gene therapy

Background: The development of nanoparticle drug delivery systems with targeted ligands has the potential to increase treatment efficiency in ovarian cancer.Methods: We developed a 21-amino acid peptide, YTRDLVYGDPARPGIQGTGTF (L-FP21) conjugated to polyethylenimine (PEI) and methoxy polyethylene glycol (mPEG) to prepare a nanoparticle drug vehicle to target follicle-stimulating hormone receptor (FSHR) in ovarian cancer. At the same time, we optimized the ligand of the nanoparticle vehicle using D-peptides, which consist of D-amino acids (D-FP21). Nanoparticle vehicles carrying the therapeutic gene plasmid growth-regulated oncogene alpha (pGRO-α) short hairpin RNA (shRNA) (FP21-PEG-PEI/pGRO-α) were prepared for further investigation.Results: Compared with L-FP21, D-FP21 exhibited improved biological stability and higher uptake rate for FSHR-expressing ovarian cancer cells. The cytotoxicity of the L, D-FP21-PEG-PEI/pGRO-α complexes were significantly lower than that of the PEI/pGRO-α complex. The nanoparticle drug with the targeted ligand showed higher transfection efficiencies and improved anti-proliferation effects for ovarian cancer cells than that without the targeted ligand (mPEG-PEI/pGRO-α). Furthermore, an in vivo evaluation of an antitumor assay indicated that D-FP21-PEG-PEI/pGRO-α inhibited the growth of tumor spheroids considerably more than L-FP21-PEG-PEI/pGRO-α; their tumor inhibition rates were 58.5% and 33.3%, respectively.Conclusions: D-FP21-PEG-PEI/plasmid DNA is a safe and efficient gene delivery vehicle for ovarian cancer targeted therapy.

Inhibition of enhancer of zeste homolog 2 increases the expression of p16 and suppresses the proliferation and migration of ovarian carcinoma cells in vitro and in vivo.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase, which targets histone H3 lysine 27. Studies have reported that EZH2 is involved in the development of several types of tumor, including ovarian cancer. p16, a well-known cell cycle regulator, has been demonstrated to be a tumor suppressor gene in a variety of malignant cells. However, the regulatory association between EZH2 and p16 in ovarian cancer remains to be fully elucidated. The present study aimed to determine whether EZH2 is involved in the development of ovarian cancer by regulating the expression of p16. An EZH2 short hairpin RNA (shRNA) lentiviral vector was constructed and used for transducing A2780 and SKOV3 ovarian cancer cell lines. The expression levels of EZH2 and p16 in the ovarian cancer cells were detected using a reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively. The function of the inhibition of EZH2 in cell proliferation and migration were determined using a CCK-8 assay and Transwell assay. In addition, a nude mouse xenograft model was used to determine the function of EZH2 and p16 in the formation of ovarian cancer in vivo. The results revealed that the inhibition of EZH2 increased the expression of p16, and suppressed the proliferation and migration capabilities of ovarian cancer in vitro. The downregulated expression of EZH2 suppressed ovarian tumor formation in vivo. The results of the study revealed that p16 was negatively regulated by EZH2 in ovarian cancer, and that p16 and EZH2 are important in the tumorigenesis of ovarian cancer. EZH2 and p16 represent potential biomarkers for the diagnosis of ovarian cancer and as targets for ovarian cancer gene therapy.

Pseudotyped retroviral replicating vectors for combination prodrug activator gene therapy of ovarian cancer

Pseudotyped retroviral replicating vectors for combination prodrug activator gene therapy of ovarian cancer

Vocimagene amiretrorepvec (Toca 511) for prodrug activator gene therapy in ovarian cancer models

Objective: Ovarian cancer is the leading cause of death in women with gynecologic malignancies in the United Staes, with <50% overall 5-year survival. Our studies to date have demonstrated significant survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for gene therapy in a variety of preclinical cancer models. RRV-mediated prodrug activator gene therapy using yeast cytosine deaminase (RRV-CD, ‘Toca 511’) is now being evaluated in a registered phase IIB/III trial at multiple sites throughout the United States and Canada (NCT02414165) for recurrent glioma. As FDA approved initiation of clinical trials for intravenous delivery of RRV, it is timely to consider applying this approach also to systemic malignancies such as ovarian cancer. Accordingly, we have conducted the first preclinical studies to evaluate the feasibility, safety, and efficacy of RRV-mediated prodrug activator gene therapy for ovarian cancer.