Antitumor efficacy of VP22-CD/5-FC suicide gene system mediated by lentivirus in a murine uveal melanoma model.

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, which has high frequency of metastasis to the liver, typically causing a fatal outcome. Chemo-resistance remains a major obstacle in the therapeutic approach to UM, leaving limited choice for treating UM. Other possible treatments have been explored but the results are yet to be evident. To improve therapy for UM, transcriptional suicide genes were transfected into the OCM-1 cell line. In the current study, OCM-1 cells transfected with lentiviral-meditated EGFP, cytosine deaminase (CD)/EGFP, and VP22-CD/EGFP were established. Of the three groups, we examined the cell growth in vitro and in vivo by using the MTT method with cell culture media and MRI in murine UM models. According to our results, the cell proliferation in the transfected CD/EGFP group was slower than the non-suicide gene group. The VP22-CD/EGFP group manifested superior cell cytotoxicity than the CD/EGFP group. Further analysis of MRI and fluorescent imaging of the murine UM model identified significant differences in tumor volume among the three groups. Collectively, our study demonstrated that CD/5-FC is a potent therapeutic approach for UM. With the efficacy of VP22, suicide gene-induced cytotoxicity was superior to applying CD alone. Taken together, we concluded that novel therapy with the VP22-CD suicide gene may further contribute to treatment of UM.

Chimeric antigen receptor-modified T cells for the treatment of solid tumors: Defining the challenges and next steps.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in CD19 expressing hematologic malignancies, but how to translate this success to solid malignancies remains elusive. Effective translation of CAR T cells to solid tumors will require an understanding of potential therapeutic barriers, including factors that regulate CAR T cells expansion, persistence, trafficking, and fate within tumors. Herein, we describe the current state of CAR T cells in solid tumors; define key barriers to CAR T cell efficacy and mechanisms underlying these barriers, outline potential avenues for overcoming these therapeutic obstacles, and discuss the future of translating CAR T cells for the treatment of patients with solid malignancies.

Double suicide gene therapy using human neural stem cells against glioblastoma: double safety measures.

With recent advancements in stem cell-based gene therapy, concerns about safety have grown. Stem cell-based gene therapies may pose the risk of immunological problems and oncogenesis. We investigated the feasibility of treating glioblastomas with neural stem cells [(NSCs), HB1.F3 cells] expressing double prodrug enzymes [cytosine deaminase (CD) and tyrosine kinase (TK)] to eliminate the NSCs following treatment for safety purposes. First, the in vitro and in vivo therapeutic efficacies of NSCs engineered with double prodrug enzymes (HB1.F3-CD.TK cells) were compared to cells expressing a single prodrug enzyme (HB1.F3-CD). Second, the degree of safety achieved by NSC elimination was compared with an in vitro viability assay of the NSCs after treatment with the double prodrugs. We further compared the differences in in vivo proliferation of control, single prodrug enzyme and double prodrug enzyme expressing NSCs. HB1.F3-CD.TK cells showed a better or comparable treatment outcome than HB1.F3-CD cells in vitro and in vivo. For safety, HB1.F3-CD.TK cells showed the least viability in vitro after treatment with prodrugs compared to HB1.F3 and HB1.F3-CD cells. Additionally, the in vivo proliferation among the injected NSCs found in the tumor was the smallest for HB1.F3-CD.TK cells. Double-prodrug enzyme-directed gene therapy shows good therapeutic efficacy as well as efficient eradication of the NSCs to ensure safety for clinical applications of stem cell-based gene therapies.

[Genetic, immunological, and pharmacological strategies to generate improved oncolytic viruses]. / Stratégies génétiques, immunologiques et pharmacologiques au service d'une nouvelle génération de virus anticancéreux.

Since over a century, medical literature has reported cases of viral infections leading to tumour regression. This phenomenon, now understood, can be exploited for cancer therapy. It involves viruses defined as "oncolytic". These viruses, either wild-type or genetically engineered, replicate preferentially in malignant cells. They induce tumour regression through various mechanisms including direct cell lysis and stimulation of an anti-tumour immune response. Several oncolytic viruses have reached late-stage clinical investigation and could be approved soon for treating certain neoplasms. While already promising, there is still room for improvement and various genetic, immunological, and pharmacological strategies are currently under development to increase their therapeutic efficacy.

New gene therapy strategies for cancer treatment: a review of recent patents.

Cancer is the second leading cause of death in the Western world. The limited successes of available treatments for cancer mean that new strategies need to be developed. The possibility of modifying the cancer cell with the introduction of genetic material opens the way to a new approach based on gene therapy. There are still many technical difficulties to be overcome, but recent advances in the molecular and cellular biology of gene transfer have made it likely that gene therapy will soon start to play an increasing role in clinical practice, particularly in the treatment of cancer. Gene therapy will probably be the therapeutic option in cases in which conventional treatments such as surgery, radiotherapy and chemotherapy have failed. The development of modified vectors, and an improved understanding of interactions between the vector and the human host, are generating inventions that are being protected by patents due to the considerable interest of industry for their possible commercialization. We review the latest strategies, patented and/or under clinical trial, in cancer gene therapy. These include patents that cover the use of modified vectors to increase the security and specificity, recombining adenovirus that leads to loss or gain of gene function, activation of the patient's own immune cells to eliminate cancer cells by expression of molecules that enhance immune responses, silencing genes related to the development of drug resistance in patients, inhibition of angiogenesis of solid tumors by targeting the tumor vasculature, and the development of enzymes that destroy viral or cancerous genetic material.

The adeno-associated virus-mediated HSV-TK/GCV suicide system: a potential strategy for the treatment of bladder carcinoma.

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. The emergence of cancer gene therapy potentially offers a number of exciting treatments. The majority of approaches involve strategies to suppress the function of activated oncogenes to restore the expression of functional tumour suppressor genes or to initiate tumour self-destruction. One gene therapy approach against tumours that holds great promise is suicide gene therapy. Herpes simplex virus thymidine kinase (HSV-TK) phosphorylates ganciclovir (GCV), which in turn interacts with cellular DNA polymerase and interferes with DNA synthesis to cause death of rapidly dividing cells. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, we investigated the suppression effect of AAV-mediated HSV-TK/GCV system on the bladder cancer cells and in mice xenograft models of bladder cancer. Our data demonstrate that rAAV-HSV-TK system controlled tumour cell growth and achieves strong antitumour efficacy in vivo. These findings provide a foundation for the development of potential targeted clinical therapies for bladder cancer in humans.

A reconstituted telomerase-immortalized human corneal epithelium in vivo: a pilot study.

PURPOSE: Telomerase-immortalized human corneal epithelial cells have been reported to stratify and differentiate in vitro similar to native tissue. The purpose of this study was to assess the ability of a telomerase-immortalized human corneal epithelial cell line to generate a full thickness epithelium in vivo in athymic mice. METHODS: Telomerized corneal epithelial cells were transduced with a retroviral vector encoding the herpes simplex thymidine kinase gene. Efficacy of the thymidine kinase suicide gene was confirmed using a live/dead assay. The epithelium was mechanically removed from athymic nude mice and remaining cells were treated with mitomycin C to prevent re-epithelialization. Telomerized corneal epithelial cells were seeded onto the denuded cornea and allowed to adhere for 4 and 24 hours. Cellular attachment was assessed using a fluorescent cell tracker. Stratification and differentiation were assessed after 7 days using phalloidin and a mouse monoclonal antibody to K3. RESULTS: Telomerized corneal epithelial cells were visualized across the denuded stromal surface at 4 and 24 hours, with multi-layering evident at the latter time point. No epithelium was present in the non-treated eye. After 7 days post-transplantation cells stratified into a multilayered epithelium, with positive K3 expression in basal and suprabasal cells. Treatment with ganciclovir induced significant loss of viability in vitro. CONCLUSIONS: The findings in this pilot study demonstrate that telomerized corneal epithelial cells possess the capacity to reconstitute a stratified corneal epithelium in vivo. The introduction of thymidine kinase allowed for the successful induction of cell death in proliferating cells in vitro. Collectively, these data suggest that a telomerase-immortalized corneal epithelial cell line transduced with thymidine kinase represents a potential model for studying differentiation and epithelial-niche interactions in vivo with potential applications in tissue engineering.

A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells.

An unmet need in cell engineering is the availability of a single transgene encoded, functionally inert, human polypeptide that can serve multiple purposes, including ex vivo cell selection, in vivo cell tracking, and as a target for in vivo cell ablation. Here we describe a truncated human EGFR polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux). After lentiviral transduction of human T cells with vectors that coordinately express tumor-specific chimeric antigen receptors and huEGFRt, we show that huEGFRt serves as a highly efficient selection epitope for chimeric antigen receptor(+) T cells using biotinylated cetuximab in conjunction with current good manufacturing practices (cGMP)-grade anti-biotin immunomagnetic microbeads. Moreover, huEGFRt provides a cell surface marker for in vivo tracking of adoptively transferred T cells using both flow cytometry and immunohistochemistry, and a target for cetuximab-mediated antibody-dependent cellular cytotoxicity and in vivo elimination. The versatility of huEGFRt and the availability of pharmaceutical-grade reagents for its clinical application denote huEGFRt as a significant new tool for cellular engineering.

Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo.

Herpes simplex virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We described earlier two strategies to overcome this limitation: (1) generation of HSVTK mutants with improved GCV activation potential and (2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants, mutant 30 and SR39, were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, shows significant reduction in IC(50) values of approximately 12 500-fold, 100-fold, and 125-fold compared with HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses show that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1 mg kg(-1)) that has no effect on wild-type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2500-fold or 25-fold compared with HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC(50) values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 seems to elicit stronger bystander killing effects in which 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1 mg kg(-1) GCV. However, at a lower dose of GCV (0.1 mg kg(-1)), MGMK/SR39 seems to have slightly greater tumor growth inhibition properties compared with SR39 (P< or =0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1 mg kg(-1) GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.

Different degrees of somatotroph ablation compromise pituitary growth hormone cell network structure and other pituitary endocrine cell types.

We have generated transgenic mice with somatotroph-specific expression of a modified influenza virus ion channel, (H37A)M2, leading to ablation of GH cells with three levels of severity, dependent on transgene copy number. GH-M2(low) mice grow normally and have normal-size pituitaries but 40-50% reduction in pituitary GH content in adult animals. GH-M2(med) mice have male-specific transient growth retardation and a reduction in pituitary GH content by 75% at 42 d and 97% by 100 d. GH-M2(high) mice are severely dwarfed with undetectable pituitary GH. The GH secretory response of GH-M2(low) and GH-M2(med) mice to GH-releasing peptide-6 and GHRH was markedly attenuated. The content of other pituitary hormones was affected depending on transgene copy number: no effect in GH-M2(low) mice, prolactin and TSH reduced in GH-M2(med) mice, and all hormones reduced in GH-M2(high) mice. The effect on non-GH hormone content was associated with increased macrophage invasion of the pituitary. Somatotroph ablation affected GH cell network organization with limited disruption in GH-M2(low) mice but more severe disruption in GH-M2(med) mice. The remaining somatotrophs formed tight clusters after puberty, which contrasts with GHRH-M2 mice with a secondary reduction in somatotrophs that do not form clusters. A reduction in pituitary beta-catenin staining was correlated with GH-M2 transgene copy number, suggesting M2 expression has an effect on cell-cell communication in somatotrophs and other pituitary cell types. GH-M2 transgenic mice demonstrate that differing degrees of somatotroph ablation lead to correlated secondary effects on cell populations and cellular network organization.

Antiglioma immunological memory in response to conditional cytotoxic/immune-stimulatory gene therapy: humoral and cellular immunity lead to tumor regression.

PURPOSE: Glioblastoma multiforme is a deadly primary brain cancer. Because the tumor kills due to recurrences, we tested the hypothesis that a new treatment would lead to immunological memory in a rat model of recurrent glioblastoma multiforme. EXPERIMENTAL DESIGN: We developed a combined treatment using an adenovirus (Ad) expressing fms-like tyrosine kinase-3 ligand (Flt3L), which induces the infiltration of immune cells into the tumor microenvironment, and an Ad expressing herpes simplex virus-1-thymidine kinase (TK), which kills proliferating tumor cells in the presence of ganciclovir. RESULTS: This treatment induced immunological memory that led to rejection of a second glioblastoma multiforme implanted in the contralateral hemisphere and of an extracranial glioblastoma multiforme implanted intradermally. Rechallenged long-term survivors exhibited anti-glioblastoma multiforme-specific T cells and displayed specific delayed-type hypersensitivity. Using depleting antibodies, we showed that rejection of the second tumor was dependent on CD8(+) T cells. Circulating anti-glioma antibodies were observed when glioblastoma multiforme cells were implanted intradermally in naïve rats or in long-term survivors. However, rats bearing intracranial glioblastoma multiforme only exhibited circulating antitumoral antibodies upon treatment with Ad-Flt3L + Ad-TK. This combined treatment induced tumor regression and release of the chromatin-binding protein high mobility group box 1 in two further intracranial glioblastoma multiforme models, that is, Fisher rats bearing intracranial 9L and F98 glioblastoma multiforme cells. CONCLUSIONS: Treatment with Ad-Flt3L + Ad-TK triggered systemic anti-glioblastoma multiforme cellular and humoral immune responses, and anti-glioblastoma multiforme immunological memory. Release of the chromatin-binding protein high mobility group box 1 could be used as a noninvasive biomarker of therapeutic efficacy for glioblastoma multiforme. The robust treatment efficacy lends further support to its implementation in a phase I clinical trial.

Combinatorial control of suicide gene expression by tissue-specific promoter and microRNA regulation for cancer therapy.

Transcriptional targeting using a tissue-specific cellular promoter is proving to be a powerful means for restricting transgene expression in targeted tissues. In the context of cancer suicide gene therapy, this approach may lead to cytotoxic effects in both cancer and nontarget normal cells. Considering microRNA (miRNA) function in post-transcriptional regulation of gene expression, we have developed a viral vector platform combining cellular promoter-based transcriptional targeting with miRNA regulation for a glioma suicide gene therapy in the mouse brain. The therapy employed, in a single baculoviral vector, a glial fibrillary acidic protein (GFAP) gene promoter and the repeated target sequences of three miRNAs that are enriched in astrocytes but downregulated in glioblastoma cells to control the expression of the herpes simplex virus thymidine kinase (HSVtk) gene. This resulted in significantly improved in vivo selectivity over the use of a control vector without miRNA regulation, enabling effective elimination of human glioma xenografts while producing negligible toxic effects on normal astrocytes. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of gene delivery vectors with high targeting specificity for cancer therapy.

The effect of herpes simplex virus virion host shutoff gene- a new suicide gene- on tumor cells.

BACKGROUND: The herpes simplex virus (HSV) UL41 gene product, virion host shutoff (Vhs) protein, mediates the rapid degradation of both viral and cellular mRNA. This ability suggests that Vhs protein can be used as a suicide gene in cancer gene therapy applications. The recent reports have shown that the degradation of cellular mRNA during herpes simplex infection is selective. RNA containing AU-rich elements (ARE) in their 3' untranslated ends are the targets for the Vhs protein. RNA that are not subject to Vhs protein-dependent degradation are up-regulated during HSV infection. ARE are frequently found in mRNA that encode proto-oncogenes, nuclear transcription factors, and cytokines. In many human cancers, the AU-rich stretch of proto-oncogenes and regulatory genes has impaired. METHODS: To investigate whether Vhs protein might be useful for inhibition of tumor cell proliferation, a eukaryotic expression vector containing Vhs protein gene was constructed. Cell degradation and RNA content of HeLa and MRC-5 tumor cells after transfection with the constructed vector were studied. RESULTS: The results showed a strong inhibitory activity in proliferation of transfected tumor cells and a sharp decrease in their RNA content. CONCLUSION: These data suggest that Vhs protein can be considered as a candidate for suicide cancer gene therapy.

Abrogation of microsatellite-instable tumors using a highly selective suicide gene/prodrug combination.

A substantial fraction of sporadic and inherited colorectal and endometrial cancers in humans is deficient in DNA mismatch repair (MMR). These cancers are characterized by length alterations in ubiquitous simple sequence repeats, a phenotype called microsatellite instability. Here we have exploited this phenotype by developing a novel approach for the highly selective gene therapy of MMR-deficient tumors. To achieve this selectivity, we mutated the VP22FCU1 suicide gene by inserting an out-of-frame microsatellite within its coding region. We show that in a significant fraction of microsatellite-instable (MSI) cells carrying the mutated suicide gene, full-length protein becomes expressed within a few cell doublings, presumably resulting from a reverting frameshift within the inserted microsatellite. Treatment of these cells with the innocuous prodrug 5-fluorocytosine (5-FC) induces strong cytotoxicity and we demonstrate that this owes to multiple bystander effects conferred by the suicide gene/prodrug combination. In a mouse model, MMR-deficient tumors that contained the out-of-frame VP22FCU1 gene displayed strong remission after treatment with 5-FC, without any obvious adverse systemic effects to the mouse. By virtue of its high selectivity and potency, this conditional enzyme/prodrug combination may hold promise for the treatment or prevention of MMR-deficient cancer in humans.

Genetic control of wayward pluripotent stem cells and their progeny after transplantation.

The proliferative capacity of pluripotent stem cells and their progeny brings a unique aspect to therapeutics, in that once a transplant is initiated the therapist no longer has control of the therapy. In the context of the recent FDA approval of a human ESC trial and report of a neuronal-stem-cell-derived tumor in a human trial, strategies need to be developed to control wayward pluripotent stem cells. Here, we focus on one approach: direct genetic modification of the cells prior to transplantation with genes that can prevent the adverse events and/or eliminate the transplanted cells and their progeny.

High mobility group box2 promoter-controlled suicide gene expression enables targeted glioblastoma treatment.

Achievement of specific tumor cell targeting remains a challenge for glioma gene therapy. We observed that the human high mobility group box2 (HMGB2) gene had a low level of expression in normal human brain tissues, but was significantly upregulated in glioblastoma tissues. With progressive truncation of a 5'-upstream sequence of the HMGB2 gene, we identified a 0.5-kb fragment displaying a high transcriptional activity in glioblastoma cells, but a low activity in normal brain cells. To test the feasibility of using the HMGB2 promoter sequence in targeted cancer therapy, we constructed a baculoviral vector expressing the herpes simplex virus thymidine kinase (HSVtk) gene driven by the HMGB2 promoter. Transduction with the viral vector induced cell death in glioblastoma cell lines in the presence of ganciclovir (GCV), but did not affect the survival of human astrocytes and neurons. In a mouse xenograft model, intratumor injection of the baculoviral vector suppressed the growth of human glioblastoma cells and prolonged the survival of tumor-bearing mice. Our results suggest that the novel 5' sequence of HMGB2 gene has a potential to be used as an efficient, tumor-selective promoter in targeted vectors for glioblastoma gene therapy.

Understanding apoptotic signaling pathways in cytosine deaminase-uracil phosphoribosyl transferase-mediated suicide gene therapy in vitro.

Cytosine deaminase-uracil phosphoribosyl transferase (CD-UPRT) fusion gene is known to exhibit therapeutic effect by inducing apoptosis in vitro. However, bystander effects of 5-flurocytosine (5-FC)/CD-UPRT and the molecular mechanism for apoptosis are yet to be established. In the present study, we have generated BHK21 cell line expressing both CD-UPRT and green fluorescent protein (GFP) from two separate transcripts, where GFP was used as a noninvasive probe to monitor the therapeutic effect of CD-UPRT. Enzyme activity of CD-UPRT in the stable cell line was measured by the reverse phase high-performance liquid chromatography analysis. Inhibition of cell growth and strong bystander effects of 5-FC/CD-UPRT were established, whereas characteristic surface morphology of apoptotic cell death was identified by AFM analysis. Involvement of various apoptotic signaling genes using semi-quantitative RT-PCR has been explored to substantiate the potential application of 5-FC/CD-UPRT suicide gene in therapy.

Bacterial deoxyribonucleoside kinases are poor suicide genes in mammalian cells.

Transfer of deoxyribonucleoside kinases (dNKs) into cancer cells increases the activity of cytotoxic nucleoside analogues. It has been shown that bacterial dNKs, when introduced into Escherichia coli, sensitize this bacterium toward nucleoside analogues. We studied the possibility of using bacterial dNKs, for example deoxyadenosine kinases (dAKs), to sensitize human cancer cells to gemcitabine. Stable and transient transfections of bacterial dNKs into human cells showed that these were much less active than human and fruitfly dNKs. The fusion of dAK from Bacillus cereus to the green fluorescent protein induced a modest sensitization. Apparently, bacterial dNKs did not get properly expressed or are unstable in the mammalian cell.