Scholarly article on topic '587. The Suicide Fusion Gene YCD-YUPRT Induces High and Specific Cytotoxicity in Small Cell Lung Cancer Cell Lines and Tumor Growth Delay in Xenografts When Regulated from a Cancer-Specific Promoter'

587. The Suicide Fusion Gene YCD-YUPRT Induces High and Specific Cytotoxicity in Small Cell Lung Cancer Cell Lines and Tumor Growth Delay in Xenografts When Regulated from a Cancer-Specific Promoter Academic research paper on "Biological sciences"

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Academic research paper on topic "587. The Suicide Fusion Gene YCD-YUPRT Induces High and Specific Cytotoxicity in Small Cell Lung Cancer Cell Lines and Tumor Growth Delay in Xenografts When Regulated from a Cancer-Specific Promoter"

Cancer - Apoptosis and Suici

metabolism assays. However, given the currently low in vivo transfection efficiencies for gene therapy, a necessary component of this system is the bystander effect, or the transfer of cytotoxic metabolites from transfected cells to untransfected neighbor cells. While our variant bCD enzyme displays strong bystander-cell killing, our variant-fusion enzymes exhibit a reduced bystander effect. We hypothesize the decreased bystander effect arises from lower levels of the diffusible metabolite 5FU present in cells expressing fusion enzymes due to increased 5FU metabolism. To measure 5FU levels as well as to investigate any potential shift in the bystander-cell killing mechanisms in the presence of fusion enzymes, we utilized highperformance liquid chromatography, immunoblot assays, diffusion assays, fluorescence microscopy, and flow cytometry. Additionally, we examined the possibility that 5FU catabolism by the endogenous enzyme dihydropyrimidine dehydrogenase (DPD) decreases in the presence of fusion enzymes due to the lower 5FU concentrations. As high levels of DPD are associated with decreased 5FU efficacy, a reduction of 5FU availability to DPD enzymes may increase the overall effectiveness of this system, even in the absence of a robust bystander effect. Understanding bCD-mediated metabolism of 5FC aids in designing and improving engineered variants for more effective suicide gene therapy.

585. Engineering Cytosine Deaminase/Uracil Phosphoribosyltransferase Fusions for Improved Cancer Gene Therapy

Andressa Ardiani,1 Margaret E. Black.12

'School of Molecular Biosciences, Washington State University, Pullman, WA; 2Department of Pharmacology and Toxicology, Washington State University, Pullman, WA.

Suicide gene therapy is a particularly attractive cancer therapy because of its ability to localize toxicity to tumor cells. We are interested in the bacterial uracil phosphoribosyltransferase (UPRT) and its application in conjunction with the cytosine deaminase (CD)/5-fluorocytosine (5FC) system to enhance tumor ablation. Cytosine deaminase (CD) is an enzyme responsible for deaminating cytosine to form uracil. It also recognizes 5FC, an anti-fungal drug, and is able to deaminate 5FC to 5-fluorouracil (5FU), a highly toxic anti-cancer drug. UPRT is an important enzyme involved in the pyrimidine salvage pathway, catalyzing the transfer of a ribosyl-phosphate group to uracil to form uridine-monophosphate. The enzyme also converts 5FU to 5FUMP which is then further catalyzed to antimetabolites by endogenous enzymes. Because the conversion of 5FC to 5FU by bCD is rate limiting, we sought to shift the substrate preferences from its natural substrate, cytosine, to 5FC by performing regio-specific random mutagenesis of the substrate binding site of bCD. From these series of experiments one bCD variant (1525) was shown to have significant increases in tumor cytotoxicity and bystander killing activity in vitro and in vivo. Earlier reports demonstrated that the fusion of CD with UPRT (CD/ UPRT) along with 5FC imparts a greater tumor killing activity than CD alone. Therefore, to further enhance the production of cytotoxic compounds, we sought to incorporate improvements previously identified in bCD 1525 into new fusion constructs containing both bCD and UPRT activities. The fusion constructs were evaluated for their cell killing and bystander effects in vitro. Because mutant fusion enzyme offers only a modest increase in sensitivity toward 5FC and shows no improvement in regards to bystander killing effect, we hypothesize that the conversion of 5FU to 5FUMP by UPRT is rate limiting in this drug activation pathway. To overcome this limitation, we performed regio-specific random mutagenesis to generate UPRT mutants and coupled with genetic complementation in E. coli, identified variants with increased activity toward 5FU. Superior UPRT mutants with improved activity towards 5FU may increase tumor killing efficacy directly by generating higher levels

of active antimetabolites and indirectly through a broader bystander effect. Ultimately, the combination of fusions containing both bCD and UPRT mutants with exceptional prodrug converting properties will be clinically beneficial because they will allow administration of lower doses of 5FC, thereby minimizing side effects without the loss of potency. The use of such novel mutant fusion constructs will advance suicide gene therapy treatment for cancer and improve the likelihood of complete tumor ablation in cancer patients.

586. A Novel, Codon-Optimised HSVtk(A168H) Mutant for Suicide Gene Therapy

Ellen Preuß,2 Alexandra Treschow,3 Jürgen Otte,2 Daniela Brücher,2 Ulrika Felldin,3 Evren Alici,3 Sirac Dilber,3 Boris Fehse.12 'Frankfurter Stiftung für Krebskranke Kinder, Frankfurt am Main, Germany; 2Karolinska Institute, Frankfurt am Main, Huddinge, Sweden; 3University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.

Suicide genes are broadly used in gene therapy, e.g. for the conditional elimination of infused cells or for directly targeting tumours. The Herpex simplex virus thymidine kinase (HSVtk) gene together with its prodrug Ganciclovir (GCV) is the currently most widely used suicide gene/prodrug combination. We developed a codon-optimised HSVtk (coHSVtk) and modified this by introducing the A168H (Karlsson) mutation [coHSVtk(A168H)]. Performance of these two new suicide genes was assessed and compared to that of splice-corrected wild-type scHSVtk in different preclinical models. After transduction of different human haematopoietic cell lines and primary T cells with retroviral sort-suicide vectors containing combinations of positive selection markers (tCD34, OuaSelect) and the different HSVtk variants we found strongly improved killing kinetics for coHSVtk(A168H) as compared to both other HSVtk's. Long-term expression and functionality data also showed an advantage of coHSVtk(A168H) over the other TK genes in different blood cells tested indicating reduced unspecific toxicity. To assess the potential of coHSVtk(A168H) in killing cancer cells, various established tumour cell lines were transduced with LeGO vectors expressing this or one of the control genes in conjunction with sort-selection markers. Again, highest killing rates were found for coHSVtk(A168H) at significantly lower GCV doses as compared to the controls. Moreover, in killing experiments with mixtures of transduced and non-transduced cells we found a strongly improved (app. one order of magnitude) bystander effect for coHSVtk(A168H) as compared to scHSVtk. In summary, we show that the novel codon-optimised HSVtk(A168H) gene combines improved killing performance (with regard to absolute efficacy, kinetics and bystander effect) with low unspecific cell-toxicity. It therefore represents a promising alternative to currently used HSVtk's for suicide gene-based therapeutic strategies.

587. The Suicide Fusion Gene YCD-YUPRT Induces High and Specific Cytotoxicity in Small Cell Lung Cancer Cell Lines and Tumor Growth Delay in Xenografts When Regulated from a Cancer-Specific Promoter

Camilla L. Christensen,1 Torben Gjetting,1 Thomas T. Poulsen,1 Hans S. Poulsen.1

'Department of Radiationbiology, Section 632', Copenhagen University Hospital, The Finsen Center, Copenhagen, Denmark.

Small cell lung cancer (SCLC) is a highly metastatic disease with poor prognosis and novel treament strategies are therefore in great demand. Treatment must, due the disseminated nature of the cancer, be performed systemically requiring a high level of targeting. Utilizing cancer-specific promoters therapeutic gene expression can be selectively targeted to the cancer cells. For that purpose we have identified several promoter regions, which are highly promising

Molecular Therapy Volume 17, Supplement 1, My 2009 Copyright © The American Society of Gene Therapy

Cancer - Apoptosis

candidates for transcriptionally targeted gene therapy for small cell lung cancer (SCLC). The fusion suicide gene consisting of the yeast cytosine deaminase and the yeast uracil phosphoribosyl transferase (YCD-YUPRT) was cloned for regulated expression from the SCLC specific promoter Insulinoma-associated 1 (INSM1) generating the plasmid vector INSM1-YCD-YUPRT. YCD converts the prodrug 5-fluorocytosin (5-FC) into the known chemotherapeutic agent 5-fluorouracil (5-FU) while YUPRT augments the conversion of 5-FU into active cytotoxins. SCLC cell lines transiently transfected with INSM1-YCD-YUPRT plasmid vector were greatly sensitised to 5-FC exposure resulting in extensive cell death as compared to both mock and INSM1-YCD transfected cells. In contrast cancer cell lines of other origins and non-immortalized cell lines were unaffected to YCD-YUPRT/5-FC treatment, manifesting the high specificity of the strategy. To investigate the bystander cytotoxicity, YCD-YUPRT transfected SCLC cells were mixed with untransfected SCLC cells in different ratios and it was established that significant cell death of the total cell population was achieved, when as few as 5 % SCLC cells expressed the YCD-YUPRT transgene. YCD-YUPRT/5-FC therapy was additionally compared to the effect of Herpes simplex virus thymidine kinase (HSVTK) and ganciclovir (GCV). In all tested SCLC cell lines YCD-YUPRT/5-FC therapy was superior to HSVTK/GCV treatment. To study the YCD-YUPRT/5-FC strategy in vivo SCLC xenografts were established on nude mice. Mice were treated for 3 consecutive days with i.t. injections of INSM1-YCD-YUPRT vector encapsulated in a liposome delivery vehicle, DOTAP:Cholesterol in combination with daily i.p. administration of 500 mg/kg 5-FC for 10 days. Preliminary results demonstrate tumor growth delay compared to control treated mice receiving i.t. injection of mock vector and same 5-FC treatment protocol. An update will be presented.

588. Adenovirus Mediated Herpes Simplex Virus-Thymidine Kinase/Ganciclovir Gene Therapy Enhances the Efficacy of TMZ Treatment in Malignant Glioma

Ann-Marie Maatta,12 Haritha Samaranayake,12 Jere Pikkarainen,12 Taina Vuorio,12 Minna Kaikkonen,12 Jaana Siponen,2 Svetlana Laidinen,2 Seppo Yla-Herttuala.13 'Department of Biotechnology and Molecular Medicine, A.I.Virtanen Institute for Molecular Sciences, Kuopio, Finland; 2Ark Therapeutics Oy, Kuopio, Finland; 3Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland.

Current therapies (surgery, chemo- and radiotherapy) used for malignant gliomas do not provide sufficient efficacy and the overall survival remains still poor (< 8-10 months). Temozolomide (TMZ) is a derivative of the alkylating agent dacarbazine and is one of the newest compounds that has been approved as an anticancer agent against recurrent and primary gliomas. However, the efficacy of TMZ has been shown to be affected strongly by three different DNA repair activities. This leads to TMZ resistant population in tumors that needs to be eliminated with some other therapeutic approach such as gene therapy. Adenovirus encoding Herpes simplex type-1 virus thymidine kinase gene therapy with pro-drug ganciclovir (Ad. HSV-tk/GCV) is a well characterised cytotoxic gene therapy based drug and it has been proven to be safe and and efficient in malignant gliomas already in phase I, II and III clinical trials. In this study, the synergistic effect of Ad.HSV-tk/GCV (Cerepro ®, Ark Therapeutics Group plc London, UK and Kuopio, Finland) and TMZ was studied in syngenic BT4C rat glioma model. The efficacy of the treatment was evaluated as an effect on both tumour volume and survival. Other potential effects of the therapy were evaluated by analysis of blood values (leucocytes, red blood cells, haemoglobin, platelets, CD3-, CD4. and CD8- cell counts) and serum values (bilirubin, alkaline phosphatase, alanine aminotransferase and creatinine). When TMZ

was used alone it did not have any effect on survival or tumor volume when compared to untreated controls. However, Ad.HSV-tk /GCV in combination with TMZ improved the survival significantly when compared to the controls (p < 0.01), the TMZ group (p < 0.01) and even to the Ad.HSV-tk/GCV group (p<0.05), suggesting a possible synergistic effect of the combination. This trend was also seen in tumor volumes. Furthermore, the study revealed that leucocyte, T-cell and platelet counts were depleted following TMZ therapy, a side-effect not observed with Ad.HSV-tk /GCV. This study proves that Ad.HSV-tk /GCV gene therapy can be combined safely with the alkylating chemotherapy agent TMZ. The study further suggests a possible synergism between AdHSV-tk/GCV and TMZ in terms of initial cell killing and survival. The mechanism behind the synergism needs to be studied further to understand more the effect on molecular level.

589. Targeted Delivery of FCU1 Suicide Gene to Tumor Cells by Oncolytic Adenovirus

Joao D. Dias,1 Kilian Guse,1 Johann Foloppe,2 Ilkka Liikanen,1 Marta Sloniecka,1 Minna Eriksson,1 Tanja Hakkarainen,1 Monika Lusky,2 Philippe Erbs,2 Vincenzo Cerullo,1 Akseli Hemminki.1 'Cancer Gene Therapy Group, Molecular Cancer Biology Program & Transplantation Laboratory & Finnish Institute for Molecular Medicine & Helsinki Biomedical Graduate School, University of Helsinki & Helsinki University Central Hos; 2Transgene SA, Strasbourg Cedex, France.

Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for selective elimination of tumor cells. However, most current suicide gene therapy strategies are still hindered by the poor efficiency of in vivo gene transfer, limited bystander cell killing effect and need of large prodrug doses. We hypothesized that by combining virus replication to prodrug activation system we could overcome these limitations. Therefore, we generated a transductionally and transcomplementationally targeted oncolytic adenovirus (Ad) and a transductionally targeted replication-deficient Ad expressing the fusion suicide gene FCU1 derived from the yeast cytosine deaminase (FCY1) and uracil phosphoribosyltransferase (FUR1) genes. This fusion suicide gene (FCU1) encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine-5'monophosphate(5-FUMP), thus bypassing the natural resistance of certain human tumor cells to 5-FU. Transductional targeting of the viruses was achieved by replacing the normal Ad5 serotype knob fiber with the Ad3 serotype knob fiber, targeting these viruses to the Ad3 receptors. We examined the feasibility of Ad5/3-D24FCU1 and Ad5/3-D24FCU1 alone as well as of combining with 5-FC in human pancreatic (Panc) cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer and gastric cancer cells. Gene expression was confirmed by Western blot and enzymatic conversion assay was assessed by HPLC separation. In vitro, Ad5/3-D24FCU1 demonstrated increased cell killing by combining with 5-FC in Panc and HNSCC cells as measured by MTS assay. The highest cell killing increase was obtained at low doses of Ad5/3-D24FCU1 and 5-FC. In vivo studies on an s.c. tumor model of HNSCC in nude mice are presently on going and the results should be presented at the meeting.

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy