CBMS-3 Potent bystander effect in suicide gene therapy using genetically engineered stem cells from human exfoliated deciduous teeth

HSV thymidine kinase (TK)/ganciclovir (GCV) has a long history of application in malignant glioma and we have previously demonstrated its bystander effect on gliomas using several stem cell types as a vehicle. The main reason for applying stem cells is that they have a unique tumor-trophic activity that allows them to deliver TK genes efficiently to nearby the tumor. Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells easily harvested from dental pulp and no studies have reported suicide gene therapy using SHED as a carrier for malignant gliomas. For transduction of SHED with the HSVTK gene (SHEDTK), we used HSVTK retrovirus-producing cells.In vitro experiments showed a significant migration ability of SHEDTK toward tumor-conditioned medium and representative tumor growth factors. We also detected a significant bystander effect of SHEDTK on gliomas in the presence of GCV. In vitro time-lapse imaging showed that both SHEDTK and glioma cells underwent gradual morphological apoptosis and activation of caspase 3/7 was observed in both cell types. In intracranial tumor models using nude mice, SHEDTK migrated around the U87 cell mass implanted in the contralateral hemisphere. Additionally, coculture suspensions of SHEDTK and U87-luciferase cells were xeno-transplanted followed by intraperitoneal administration of GCV for 10 days. All mice of treatment group survived for more than 100 days, whereas those treated without GCV died of tumor growth with median survival of 42 days after tumor implantation. Furthermore, pre-existing intracranial U87 model mice were injected intratumorally with SHEDTK followed by GCV administration as described above. The tumor volume was significantly reduced during the treatment period, and over-all surivial in treatment group is prolonged significantly to that of control groups. These results indicate that SHEDTK-based suicide gene therapy might offer a new promising therapeutic modality for human malignant gliomas.

Effective Oncoleaking Treatment of Pancreatic Cancer by Claudin-Targeted Suicide Gene Therapy with Clostridium perfringens Enterotoxin (CPE)

Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

Suicide gene therapy may be effective in the treatment of malignant glioma

Suicide gene therapy has a long history, and its effectiveness is now under investigation. The bystander death effect therapeutic strategy proved successful in treating malignant glioma; nevertheless, non-replicating viral vectors may not be able to cover a large invading region of glioma cells. Tumor-trophic stem cell migration and viral replication capacities have the potential to overcome the refractory mechanisms of malignant glioma. Surprisingly, the time to response (partial or total response) was delayed for more than 6 months after the initial Toca 511 treatment, reflecting a stronger antitumor immune response. Immunosuppressive cells such myeloid-derived suppressor cells and tumor-associated macrophages were found to be lower in human glioblastoma tissues after treatment. These mechanisms may have a long-term impact on people with malignant glioma. NSCs and MSCs, for example, might be employed to cover glioma cells' more broad invading areas. However, we must investigate which brain stem cells are the most suited. More study is needed to confirm the migratory potential and survival ratio of the implanted stem cells in the brain utilizing comparative analysis. Suicide gene therapy using stem cell migratory capability and/or viral replicating capability for malignant glioma may recapture the spotlight.

Development of iRGD-Modified Peptide Carriers for Suicide Gene Therapy of Uterine Leiomyoma

Uterine leiomyoma (UL) is one of the most common benign tumors in women that often leads to many reproductive complications. Suicide genetherapy was suggested as a promising approach for UL treatment. In the present study, we describe iRGD ligand-conjugated cysteine-rich peptide carrier RGD1-R6 for targeted DNA delivery to αvβ3 integrin-expressing primary UL cells. The physico-chemical properties, cytotoxicity, transfection efficiency and specificity of DNA/RGD1-R6 polyplexes were investigated. TheHSV-1thymidine kinase encoding plasmid delivery to PANC-1pancreatic carcinoma cells and primary UL cells resulted in significant suicide gene therapy effects. Subsequent ganciclovir treatment decreased cells proliferative activity, induced of apoptosis and promoted cells death.The obtained results allow us to concludethatthe developed RGD1-R6 carrier can be considered a promising candidate for suicide gene therapy of uterine leiomyoma.

Molecular Basis of NDT-Mediated Activation of Nucleoside-Based Prodrugs and Application in Suicide Gene Therapy

Herein we report the first proof for the application of type II 2′-deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of LdNDT over the nucleoside-based prodrugs 2′-deoxy-5-fluorouridine (dFUrd), 2′-deoxy-2-fluoroadenosine (dFAdo), and 2′-deoxy-6-methylpurine riboside (d6MetPRib). Such activity was significantly increased (up to 30-fold) in the presence of an acceptor nucleobase. To shed light on the strong nucleobase dependence for enzymatic activity, different molecular dynamics simulations were carried out. Finally, as a proof of concept, we tested the LdNDT/dFAdo system in human cervical cancer (HeLa) cells. Interestingly, LdNDT/dFAdo showed a pronounced reduction in cellular viability with inhibitory concentrations in the low micromolar range. These results open up future opportunities for the clinical implementation of nucleoside 2′-deoxyribosyltransferases (NDTs) in cancer treatment.