Cytosine Deaminase Gene Research Articles

Therapeutic activity of retroviral replicating vector-mediated gene therapy in combination with anti-PD-1 antibody in a murine pancreatic cancer model.

Toca 511, a tumor-selective retroviral replicating vector encoding the yeast cytosine deaminase (yCD) gene, exerts direct antitumor effects through intratumoral prodrug 5-fluorocytosine (5-FC) conversion to active drug 5-fluorouracil by yCD, and has demonstrated therapeutic efficacy in preclinical and clinical trials of various cancers. Toca 511/5-FC treatment may also induce antitumor immunity. Here, we first examined antitumor immune responses activated by Toca 511/5-FC treatment in an immunocompetent murine pancreatic cancer model. We then evaluated the therapeutic effects achieved in combination with anti-programmed cell death protein 1 antibody. In the bilateral subcutaneous tumor model, as compared with the control group, enhanced CD8+ T-cell-mediated cytotoxicity and increased T-cell infiltration in Toca 511-untransduced contralateral tumors were observed. Furthermore, the expression levels of T-cell co-inhibitory receptors on CD8+ T-cells increased during treatment. In the bilateral subcutaneous tumor model, combination therapy showed significantly stronger tumor growth inhibition than that achieved with either monotherapy. In an orthotopic tumor and peritoneal dissemination model, the combination therapy resulted in complete regression in both transduced orthotopic tumors and untransduced peritoneal dissemination. Thus, Toca 511/5-FC treatment induced a systemic antitumor immune response, and the combination therapy could be a promising clinical strategy for treating metastatic pancreatic cancer.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-Vivo Therapy.

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

Engineering of mesenchymal stem cells to express cytosine deaminase gene with a selecable marker gene.

e14050 Background: Suicide genes induce apoptosis not only in the cell itself but also in surrounding cells. The former is called the suicide effect and the latter is called the bystander effect. Two major suicide gene strategies currently pursued, cytosine deaminase (CD)/5-fluorocytosine (5-FC) and the herpes simplex virus-thymidine kinase/ganciclovir. Both genes have similar potential to induce suicide effects, but bystander effect is higher in CD/5-FC. Here we show that the level of CD expression/activity influence the anti-cancer activity of CD-expressing cells and further demonstrate the need to use selectable markers during manufacturing of CD-expressing cells. Finally we test whether live microbes harboring the shed selectable marker genes are controllable by commonly used antibiotics. Methods: We introduced a microbial CD gene and puromycin-N-acetyltransferase ( pac) gene to bone-marrow derived mesenchymal stem cells MSCs) using a retroviral vector and selected the transduced cells with 4 ug/mL puromycin for one week. To determine the effect of purity, we varied the ratio of CD-expressing MSCs with naïve MSCs and 5-FC concentration in the culture and on co-cultured glioma cells. To mimic the situation in which the shed pac gene is transmitted to live microbes in vivo, we introduced the pac gene to E. coli and tested the growth of the pac-expressing E. coli using VITEK following the standard protocol M100-S32 in the presence of diverse antibiotics. Results: We found that selection of CD-expressing cells with puromycin during manufacturing process enhanced the bystander (anti-cancer effect). Lowering the purity of CD-expressing MSCs required more 5-FC with higher IC50. Growth of pac-expressing E. coli was completely inhibited by commonly used antibiotics in the hospital. Conclusions: We proved that antibiotic selection is necessary during manufacturing process. We also showed that the risk of transmission of shed pac gene can be manageable with commonly used antibiotics. We propose that using pac selectable marker gene can be beneficial to maintain the purity of engineered cells.

Enhanced tumor inhibiting effect of 131I-BDI-1-based radioimmunotherapy and cytosine deaminase gene therapy modulated by a radio-sensitive promoter in nude mice bearing bladder cancer.

Radioimmunotherapy (RIT) has great potential in cancer therapy. However, its efficacy in numerous tumors is restricted due to myelotoxicity, thereby limiting the dose of radionuclide. To increase tumor radiosensitivity, we incorporated the recombinant lentivirus into the EJ cells (bladder cancer [BC] cells), and examined the combined anti-tumor effects of RIT with 131I-BDI-1(131I-monoclonal antibody against human BC-1) and gene therapy (GT). The recombinant lentivirus was constructed and packed. The animal xenograft model was built and when the tumor reached about 0.5cm in diameter, the mice were randomly separated into four groups: (1) RIT + GT: the xenografts were continuously incorporated with the recombinant lentivirus for two days. And 7.4MBq 131I-BDI-1 was IV-injected, and 10mg prodrug 5-fluorocytosine (FC) was IV-injected for 7days, (2) RIT: same dose of 131I-BDI-1 as the previous group mice, (3) GT: same as the first group, except no 131I-BDI-1, and (4) Untreated. Compute tumor volumes in all groups. After 28days the mice were euthanized and the tumors were extracted and weighed, and the inhibition rate was computed. The RIT + GT mice, followed by the RIT mice, exhibited markedly slower tumor growth, compared to the control mice. The tumor size was comparable between the GT and control mice. The tumor inhibition rates after 28days of incubation were 42.85 ± 0.23%, 27.92 ± 0.21% and 0.57 ± 0.11% for the four groups, respectively. In conclusion, RIT, combined with GT, suppressed tumor development more effectively than RIT or GT alone. This data highlights the potent additive effect of radioimmune and gene therapeutic interventions against cancer.

A phase II, single-arm, open label, Simon two-stage study of pembrolizumab in patients with metastatic HER2-negative breast cancer: Evaluation of impact of germline variants in APOBEC3B (AUROR).

1026 Background: A germline deletion in the APOBEC3B cytosine deaminase gene [A3Bdel] occurs more frequently in Asian women (45% heterozygous deletion (hetD) and 15% homozygous deletion (homD)) compared to in Caucasian women (15% hetD and 4% homD). Carriers are more likely to develop breast cancer, and cancers in carriers are more likely to have a hypermutator phenotype (with C > T transitions) and to be immune-enriched. In this clinical trial, we aim to evaluate whether the immune activation increases response to checkpoint immunotherapy. Methods: In this open label, single arm Phase II study of single agent pembrolizumab in metastatic HER2-receptor negative breast cancer patients with germline deletion in A3B, 40 evaluable subjects who have received > = 1 but < = 3 lines of therapy in a metastatic setting will be enrolled and given pembrolizumab 200 mg intravenously (IV) every 3 weeks (Q3W) for up to 35 administrations (2 years), with Objective Response Rate (ORR) using RECIST 1.1 as the primary study endpoint. The study applies a Simon two-stage design, where if at least 3 out of 22 evaluable patients achieve CR/PR in stage I, the study will proceed to stage II. Results: To date, 84 breast cancer patients were screened for germline A3Bdel, of whom 46 (54.8%) were heterozygous carriers and 12 (14.3%) were homozygous carriers. The study enrolled 22 female A3Bdel carriers with a median age of 59.4 years (range: 32.1, 82.9 years) between September 2020 and September 2021 for stage I analysis. On average, patients received 2 prior lines of chemotherapy in a metastatic setting [6 with 1, 8 with 2 and 8 with 3 lines of prior chemotherapy]. Complete response (CR) was observed in one patient, while partial response (PR) was observed in 4 patients, with an ORR of 22.7% (5 over 22 subjects) in stage I, meeting the pre-defined criteria to proceed to stage II. Notably, the patient with complete response had received 2 prior lines of chemotherapy, whereas of the patients with partial response, 1 had received 1 prior line and 3 had received 3 prior lines of chemotherapy in a metastatic setting. As the observed ORR was greater than the value of r1 (13.6%), the study has met the statistical criteria to proceed to the stage II enrolment with an additional 18 patients required to complete the entire study. Conclusions: Single agent pembrolizumab demonstrates promising efficacy in germline A3Bdel carriers, who constitute almost two-thirds of Asian patients. Clinical trial information: NCT03989089.

Abstract PR014: Design and construction of evolutionary-guided "selection gene drive" therapy

Abstract Targeted therapies that are rationally designed to inhibit driver oncogenes generally offer more durable responses for cancer patients compared to standard chemotherapies. However, in virtually all cases, the development of drug resistance inevitably subverts eradication attempts. In tyrosine kinases inhibitors (TKIs), resistance is often addressed with next-generation TKIs or combination therapies. Appropriate treatment strategies are selected based on the nature of resistance outgrowth. However, intra- and intertumoral heterogeneity makes it impossible to predict a priori which resistance variants will drive relapse in a given patient, and thus what therapeutic strategy will be optimal. Here, we propose a resistance-agnostic treatment approach that seeks to leverage evolutionary principles, rather than combat them. We designed and demonstrated the capability of a gene therapy, which we term “dual-switch selection gene drives”, that can be used to engineer cancer cells in order to guide the tumor’s evolutionary trajectory towards eradication. The dual-switch gene drive system is a genetic circuit composed of two genes that can be delivered to tumor cells on a single vector in situ. The “switch one” gene is a drug target analog that acts as an inducible, transient resistance marker, enabling rapid selection of modified cells. The “switch two” gene is the therapeutic payload of the system. Its role is to kill tumor cells by activating an inert small molecule. Importantly, the activity of the therapeutic gene is diffuse, and this bystander effect is maximized by hitchhiking off the inducible resistance gene. We developed stochastic dynamic models of tumor evolution to predict parameter regimes where eradication is possible. With these design criteria, we constructed and evaluated our system. First, we generated a panel of optimized “switch one” genes co-opting the kinase domains for a range of TKI targets, including EGFR, ALK, and RET. We demonstrated that these synthetic genes could induce a conditional resistance phenotype and be used to select for modified cells in a mixed population. Next, we evaluated the suicide gene cytosine deaminase (CD), which converts the inert prodrug 5-FC into the nucleoside analog 5-FU. Cell viability assays for mixed populations of wild-type and CD-expressing cells confirmed a strong bystander effect in this system. Finally, we assembled a single vector harboring both switches. In a pooled experiment, gene drive cells were able to drive the mixed population to eradication, even when pre-existing resistance to the TKI was introduced. Our dual-switch design demonstrates the potential to target pre-existing resistant subpopulations, without foreknowledge of the exact nature of resistance. Given that drug resistance, and thus treatment failure, is inevitable in most cancers, our treatment strategy enables us to decide the terms of relapse. Rather than combat drug resistance, we can leverage it to amplify a therapeutic gene’s effect and ultimately collapse a complex tumor population. Citation Format: Scott M. Leighow, Haider Inam, Justin R. Pritchard. Design and construction of evolutionary-guided "selection gene drive" therapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR014.

CTNI-03. EXTENDED BENEFITS IN PATIENTS WITH RECURRENT HIGH GRADE GLIOMA THAT CONTINUOUSLY RECEIVE TOCA FC AFTER TOCA 511 TREATMEN

Abstract Toca 511 (vocimagene amiretrorepvec) is an investigational nonlytic, retroviral replicating vector (RRV) constructed with a codon-optimized yeast cytosine deaminase (CD) gene. Toca 511 infects cancer cells, and stably delivers CD gene whose protein product converts courses of the prodrug TocaFC (5-fluorocytosine) into 5-fluorouracil (5-FU). Several phase 1 studies and a phase 2/ 3 clinical study tested Toca511 in combination with TocaFC in patients undergoing planned resection for recurrent glioblastoma (rGBM) or anaplastic astrocytoma (rAA). The last trial was completed in 2019, and although the Phase 2/3 study did not meet the primary objective, patients who appeared to benefit from the treatment elected to continue TocaFC through an expanded access or compassionate use pathway. Seven patients continued on treatment: 3 men and 4 women; 5 with rGBM, 2 with rAA. Duration of treatment with TocaFC ranges from 29 months to 7 years and 5 months (average 49.2 months). Six patients who are still on active treatment with TocaFC have either stable disease or complete response. TocaFC is well tolerated in these patients, typical side effects include diarrhea. Some patients with rGBM or rAA appear to benefit from extended TocaFC treatment after Toca511. Individual cases will be discussed.

Non-viral Suicide Gene Therapy: Cytosine Deaminase Gene Directed by VEGF Promoter and 5-fluorocytosine as a Gene Directed Enzyme/prodrug System in Breast Cancer Model.

The present study investigated the potential of vascular endothelial growth factor (VEGF) promoter to derive cytosine deaminase (CD) transfected by polyamidoamine (G4-PAMAM) dendrimers to 4T1 murine breast cancer cell line as gene-directed enzyme/prodrug therapy. The VEGF promoter and cytosine deaminase gene were cloned into the pEGFP-N1vector from the genomic DNA of 4T1 and E. coli, respectively. The frequency of transfection for VEGF-CD-pEGFP-N1 and pEGFP-N1- CD treated groups was 35±3 and 36±4, respectively. MTT assay was perform to evaluate the cytotoxic effects of converted 5-flurocytosine on 4T1 cells. Also, the optimal concentration of 5-FC in 4T1 cells transfected by VEGF-CD-pEGFP-N1 plasmid was evaluated. The GFP expression of transfected 4T1 cells by VEGF-CD-pEGFP-N1were observed by fluorescent microscopy and flowcytometry. Results demonstrated that the suicide CD gene was successfully expressed in 4T1 cells determined by RT-PCR and GFP expression. A concentration of 200 μg/ml 5-FC was identified as optimal dose of prodrug. Furthermore, the CD/5-FC enzyme/prodrug system not only demonstrated toxicity on transformed 4T1 cells but also exerted a 'bystander effect' determined by MTT assay. The results showed that by 35% transfection with VEGF-CD-pEGFP-N1and CD-pEGFP-N1 plasmids, 80% and 90% inhibition of the cells growth occurred, respectively.

Enhanced gene transfection of macrophages by photochemical internalization: Potential for gene-directed enzyme prodrug therapy of gliomas.

Drawn by tumor synthesis of chemo-attractive factors, macrophages are frequently found in and around glioblastomas and play an important role both in augmenting as well as inhibiting tumor growth. Patient-derived macrophages have the potential, therefore, to act as targeted delivery vectors for a variety of anti-cancer treatments. Among these is ex vivo gene transfection and re-injection back into the patient of macrophages to target residual tumors. In this study, photochemical internalization (PCI) is investigated as a technique for the non-viral transfection of the cytosine deaminase (CD) prodrug activating gene into macrophages. The CD gene encodes an enzyme that converts the nontoxic antifungal agent, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU) - a potent chemotherapeutic agent. PCI (photosensitizer + light treatment) mediated CD gene transfection of rat alveolar Ma cells was carried out in vitro. CD gene transfected NR8383 macrophages were co-cultured with F98 rat glioma cells in the presence or absence of 5-FC. Cell viability was assayed using the MTS colorimetric assay. Compared to the glioma cells, NR8383 demonstrated enhanced resistance to the toxic effects of 5-FU. PCI greatly increased the transfection efficiency of the CD gene in NR8383 cells. The viability of F98 cells was significantly inhibited by coculture with CD transfected NR8383 macrophages and 5-FC. Although gene insertion into macrophages has proven difficult, the results presented here show that non-viral transfection of the CD gene into these immune cells can be enhanced via PCI. CD transfected NR8383 cells could efficiently convert 5-FC to 5-FU and export the drug, producing a pronounced bystander toxic effect on adjacent non-transfected glioma cells. Compared to single treatment, repetitive PCI-induced transfection was more efficient at low CD plasmid concentration.

Antitumor efficacy of cytosine deaminase-armed vaccinia virus plus 5-fluorocytosine in colorectal cancers

BackgroundVaccinia viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability of tumor tropism and oncolytic property. Cytosine deaminase (CD), which is derived from bacteria or yeast, can convert a relatively nontoxic prodrug 5-fluorocytosine (5-FC) into the active anticancer drug 5-Fluorouracil (5-FU). Vaccinia virus armed with the prodrug-activator CD gene would result in augmented antitumor effects that combined the effect of vaccinia virus and 5-FU together, and particularly limited the anticancer drug to tumor regions.MethodsThe attenuated vaccinia Tian Tan strain Guang 9 (VG9), with active yeast CD expression and thymidine kinase (TK) deficiency, was successfully constructed. Then, in vitro and in vivo antitumor efficacy of vaccinia VG9-CD plus 5-FC administration was evaluated in colorectal cancer cells.ResultsVaccinia viruses displayed different oncolytic potency in vitro cells, no relationship with whether they were cancer cells or normal cells. In colorectal tumor models, mice treated with vaccinia VG9-TK− showed better tumor remission ability and prolonged survival. Moreover, vaccinia VG9-CD in combination with gavage administration of 5-FC displayed the best antitumor efficacy, especially for the prolongation of survival.ConclusionsVaccinia VG9-CD in combination with 5-FC plays combined effect of vaccinia virus and chemotherapy, and becomes a promising virotherapy for cancer.

The enhanced efficacy of herpes simplex virus by lentivirus mediated VP22 and cytosine deaminase gene therapy against glioma

Despite the comprehensive treatment of surgery following by concurrent radiochemotherapy, the prognosis of malignant glioma remains unsatisfactory with an awful median survival time of only 12–18 months. This might be improved by the development of oncolytic herpes simplex virus. However, the biggest challenge of recombinant herpes simplex virus (rHSV) lies in their limited therapeutic efficiency in clinical trials. This study aims to enhance the efficacy of rHSV against glioma by a HSV-1 tegument protein VP22 modified cytosine deaminase/5-flurocytosine (CD/5-FC) suicide gene system. Stable glioma cell lines carrying CD or VP22-CD fusion gene were successfully obtained by lentiviral infection following Fluorescence-activated cell sorting. The lethal effect of the prodrug 5-FC was significantly increased in the transduced VP22-CD glioma cells compared to the transduced CD glioma cells. Interestingly, VP22 was found to elicit the enhanced efficacy of rHSV-1 against glioma. Furthermore, we detected a synergistic efficacy of rHSV-1 combined with lentivirus mediated VP22 and CD suicide gene therapy in the orthotopic glioma xenograft models. In conclusion, we successfully established the stable cell lines carrying VP22-CD fusion genes. The incorporation of VP22 further induced an enhanced efficacy of rHSV-1 as well as CD suicide gene therapy respectively and synthetically in vitro. Also, we demonstrated an increased therapeutic benefit of rHAV-1 by VP22 modified CD gene therapy against glioma in vivo.

Gene Therapy with Cytosine Deaminase and Endostatin Fusion Gene Mediated by Endothelial Progenitor Cells in Hepatomas.

PurposeGene-targeting therapy provides a novel therapeutic approach for tumor treatment using genetically modified endothelial progenitor cells (EPCs) as cellular carriers. This study applied EPCs armed with cytosine deaminase (CD) and endostatin (ES) fusion gene in liver cancer to explore its therapeutic effect.Materials and MethodsEPCs from heart blood of male BALB/c nude mice were cultured and transfected with CD and ES fusion gene. Subsequently, these genetically modified cells were injected into mice bearing hepatoma through their tail veins. The tumor volumes and cell apoptosis were followed up.ResultsTumor volume in the group injected CD/ES-EPCs greatly decreased. The positive rate of VEGF and CD31 in the tumor tissue was lowest in the CD/ES-EPC group. Furthermore, the number of apoptotic cells was highest in the CD/ES-EPC group.ConclusionThe EPCs transfected with CD/ES inhibited tumor growth and preferentially induced tumor cell apoptosis, providing a novel methodology for cancer-targeting therapy.

Inhibitory effect of therapeutic genes, cytosine deaminase and interferon‑β, delivered by genetically engineered stem cells against renal cell carcinoma.

Although the effects of stem cells expressing anticancer genes on tumor growth have been demonstrated by many researchers in various types of cancer, relatively few studies have investigated their inhibitory effects on cancer metastasis. In the present study, we examined the inhibitory effects of cytosine deaminase (CD)/5‑fluorocytosine (5‑FC) and interferon‑β (IFN‑β) using genetically engineered neural stem cells (hNSCs) in a cellular and metastasis model of renal cell carcinoma (RCC). The CD/5‑FC method has the advantage of minimizing damage to normal tissues since it selectively targets cancer cells by the CD gene, which converts prodrug 5‑FC to the drug 5‑fluorouracil. Moreover, we used hNSCs as a tool to effectively deliver the anticancer genes to the tumor site. These stem cells are known to possess tumor‑tropism because of chemoattractant factors expressed in cancer cells. Therefore, we ascertained the expression of these factors in A498 cells, a cell line of RCC, and identified the A498‑specific migration ability of hNSCs. We also confirmed that the proliferation of A498 cells was significantly reduced by therapeutic hNSCs in the presence of 5‑FC. Furthermore, we established an A498 metastasis model. In the animal experiment, the weight of the lungs increased in response to cancer metastasis, but was normalized by hNSCs expressing CD and/or IFN‑β genes, while the incidence of liver metastasis was suppressed by the hNSCs. Overall, the results of this study demonstrate that stem cells expressing anticancer genes have the potential for use as an alternative to conventional therapy for metastatic cancer.

Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

Gene Therapy with Cytosine Deaminase and Endostatin Fusion Gene Mediated by Endothelial Progenitor Cell in Hepatomas

Background: Gene-targeting therapy provides a novel therapeutic approach for tumor treatment using genetically-modified endothelial progenitor cells (EPCs) as cellular carriers. To study the therapeutic effect of EPCs armed with cytosine deaminase (CD) and endostatin (ES) fusion gene in liver cancer. Methods: EPCs derived from heart blood of mice were cultured and transfected with CD and ES fusion gene. EPCs, CD/ES, and CD/ES-EPCs were injected through their tail veins into mice with hepatoma, respectively. Subsequently, the in vivo tumor volumes were observed by MRI. Findings: Tumor volumes in the group injected CD/ES-EPCs were found to be smaller than other groups receiving other treatments. Also, VEGF and apoptotic tumor cells in the tumor tissues were detected after infusion of the cells into the mice. The results showed that the VEGF-positive rate was lowest and the number of apoptotic cells was highest in the CD/ES-EPCs group. Interpretation: The EPCs transfected with CD/ES inhibited tumor growth and preferentially induced tumor cell apoptosis. Thus, this treatment strategy might open a novel avenue for cancer-targeting therapy. Funding Statement: The present work was funded by Zhejiang Provincial Natural Science Foundation of China (Grant No.LZ18H180001), National S&T Major Project of China (NO.2018ZX10301201)，National Natural Science Foundation of China (Grant No.81371658), The Key Research Development Program of Zhejiang province (Grant No.2018C03018) and Key Science and Technology Program of Zhejiang province(No.WKJ-ZJ-1923). Declaration of Interests: The authors declare no potential conflict of interest. Ethics Approval Statement: All experimental procedures of the study were approved by our Institutional Animal Use and Care Committee. Mouse hepatocarcinoma model, aged 6 weeks, was provided by the Experimental Animal Center, College of Medicine, Zhejiang University. All experiments in this study were performed in agreement with the Guidelines for the Welfare of Animals in Experimental Neoplasia and approved our Institutional Animal Use and Care Committee.

Associations of cytosine deaminase gene polymorphisms with effectiveness of gemcitabine/cisplatin chemotherapy in patients of Xinjiang Uyghur and Han nationality with non-small cell lung cancer.

Cytidine deaminase (CDA) polymorphisms may affect the response to gemcitabine/cisplatin chemotherapy in patients with non-small cell lung cancer (NSCLC). This study is designed to investigate the associations of CDA-79A>C and 208G>A polymorphisms and gemcitabine/cisplatin chemotherapy effectiveness in Xinjiang Uyghur and Han patients. This prospective cohort study enrolled consecutive patients with stage IIIb/IV NSCLC administered gemcitabine/cisplatin chemotherapy at the First Affiliated Hospital, Medical College of Shihezi University and the First People's Hospital, Kashgar Region. CDA-A79C and CDA-G208A polymorphisms were detected by direct sequencing. Progression-free survival was analyzed by the Kaplan-Meier method. Associations of A79C and G208A polymorphisms with treatment effectiveness and progression-free survival were analyzed using logistic regression and multivariate Cox regression analyses. Subgroup analyses based on ethnicity were performed. The study enrolled 120 patients. A79C and G208A polymorphisms followed the Hardy-Weinberg equilibrium. The frequencies of the AA, AC, and CC genotypes and the A and C alleles of A79C were 52.2%, 29.9%, 17.9%, 67.2%, and 32.8%, respectively, in Han patients and 75.4%, 18.9%, 5.7%, 84.9%, and 5.1%, respectively, in Uyghur patients. Uyghur patients had lower frequencies of A79C-AC/CC genotypes, A79C-C allele, G208A-GA genotype, and G208A-A allele (P<0.05). Compared with A79C-AA, the odds of ineffective chemotherapy were increased for A79C-AC (odds ratio [OR] 2.818; 95% confidence interval [95% CI] 1.031, 7.705; P=0.043) and A79C-CC (OR 9.864; 95% CI 1.232, 78.966; P=0.031). G208A polymorphisms did not influence chemotherapy effectiveness. Chemotherapy was more effective in Han patients than in Uyghur patients for A79C-AC and G208A-GG. Progression-free survival was longer for A79C-AA versus A79C-AC/CC (10 vs. 7 months, P=0.004) and G208A-GA/AA vs. G208A-AA (12 vs. 8 months, P=0.010). Polymorphisms of A79C (hazard ratio [HR] 1.617; 95% CI 1.009, 2.592; P=0.046) and G208A (HR 2.193; 95% CI 1.055, 4.557; P=0.035) were associated with progression-free survival. For Uyghur and Han ethnic groups, A79C and G208A polymorphisms can be used as a promising biomarker for the chemotherapy efficacy and prognosis of NSCLC.

Efficient exogenous DNA-free reprogramming with suicide gene vectors

Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications.

Abstract 4771: The addition of toca 511 and 5-FC to temozolomide improves response in a temozolomide-resistant murine glioblastoma model and correlates with toca 511 dose

Abstract Background: Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized yeast cytosine deaminase (CD) gene to tumors. Within infected cells, CD converts 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU). The combination of Toca 511 with oral extended release 5-FC (Toca FC), is currently being evaluated in a randomized phase III clinical trial for recurrent high grade glioma (glioblastoma (GBM) and anaplastic astrocytoma) (NCT02414165, Toca 5). Temozolomide (TMZ), in combination with radiation therapy, is the standard of care used for first-line chemotherapy treatment of patients with GBM, the most common and aggressive form of primary brain cancer. Previously, we have shown that: (1) Toca 511/5-FC treatment provides durable response in a syngeneic murine glioma model and supports anti-tumor immune memory; (2) The combination of TMZ and Toca 511/5-FC had synergistic efficacy in a TMZ-sensitive human glioma nude mouse model; (3) TMZ did not inhibit the efficacy of Toca 511/5-FC in a TMZ resistant murine glioma syngeneic model; (4) Toca 511/5-FC caused significant radiosensitization in a radioresistant murine glioma model. Results: To assess the interaction of TMZ with escalating doses of Toca 511 (as defined by percent of tumor transduction by RRV), an orthotopic TMZ-resistant murine glioma model, Tu-2449, was utilized. These results show that moderate levels of tumor transduction of Toca 511 (30% - 50%) with 5-FC treatment prolongs survival in the presence of TMZ compared with lower transduction rates (10%). Additionally, mice treated with Toca 511/5-FC and TMZ are being tested for the establishment of anti-tumor immune memory. Conclusion: These results demonstrate that (1) survival correlates with the transduction levels of Toca 511 when combined with temozolomide in the Tu-2449 orthotopic glioma model and (2) that this combination may support anti-tumor immune memory. These studies along with prior work support evaluation of the combination of Toca 511/5-FC with temozolomide in patients with newly diagnosed GBM (NRG-BN006). Citation Format: Maria E. Rodriguez-Aguirre, Sophie Viaud, Daniel Mendoza, Tiffany Montellano, Derek G. Ostertag, Harry Gruber, Douglas Jolly, Cornelia Bentley. The addition of toca 511 and 5-FC to temozolomide improves response in a temozolomide-resistant murine glioblastoma model and correlates with toca 511 dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4771.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy

BackgroundThe cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool.MethodsVcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice.Results5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 μM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals.ConclusionsWe have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.