Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer

Abstract

Gene-based therapeutic strategies for cancer mainly packaging cell clone that produced a reasonable titer of include augmentation of immunotherapeutic and chemorecombinant virus and expressed high levels of IL-2 and tk therapeutic approaches. In this study we report the design transcripts. Although transduction efficiency was reduced and functional assay of a novel bicistronic Moloney-based in glioma cells as compared with murine NIH 3T3 cells, retroviral vector expressing human interleukin-2 (IL-2) and transgene expression was effectively achieved. Transherpesvirus thymidine kinase (tk) through a cap-dependent duced glioma cells were sensitive to ganciclovir and translation and an internal ribosome entry site (IRES)-regusecreted around 1000 U/ml IL-2 in the culture superlated translation, respectively. This construct has the natants. Simultaneous production of IL-2 and tk in vivo by potential for allowing combination of cytokine and suicide genetically treated tumor cells would hopefully potentiate gene therapy, especially in areas such as the brain, comthe effect of gangiclovir-induced metabolic suicide, posed of post-mitotic cells refractory to transduction by possibly by boosting the immune response associated type C retroviral vectors. Accordingly, human glioma cells with tumor debulking or by amplifying the bystander were used as targets for gene transfer after selecting a response.

Gene therapy of glioblastoma multiforme via combined expression of suicide and cytokine genes: a pilot study in humans

“…Our attempt was guided by the use of a single vector simultaneously expressing HSVtk and IL-2 as therapeutic genes. 8 The study was purposely designed to ascertain the efficacy and biological effects of tumour cell transduction. This evaluation was accomplished by taking stereotactic biopsies at a predetermined distance from the site of the RVPC injection, before GCV administration and tumourinduced suicide.…”

“…pLIL-2-TK construction and pre-clinical application of a high titre clone (2.5 × 10 5 transducing units/ml of culture supernatant) have been previously reported. 8 Three patients were stereotactically injected into the tumour mass with freshly cultured 1.5-3 × 10 8 viable RVPC in 6-9 ml suspension (the maximum injectable volume at which cell viability was preserved). Releasing a small fragment of a titanium clip, which is MRI compatible and clearly identified in CT and radiographic films, marked the site of injection.…”

“…Seven to 14 days after implantation the patients were treated by GCV intravenously at a dose of 5 mg/kg body weight twice daily for 14 days. [1][2][3]5,8,23,24 Detection of RCR and vector sequences in clinical samples Blood, urine and stool specimens were tested weekly for the presence of RCR by a conventional reverse transcriptase (RT) assay. 25 Peripheral blood mononuclear cells and cerebral biopsy tissues were analysed with direct and reverse PCR for the presence of tk, neo and gag genes and specific transcripts thereof.…”

“…Figure 1 shows a schematic representation of the pLIL-2-TK vector used in the study. 8 Clinical features of GBM patients and response to combined TK-IL-2 treatment in vivo The salient clinical features of the four treated patients suffering from relapsing GBM are presented in Table 1. All patients underwent standard treatment including microsurgical bulk removal and conventional-fractionated or stereotactic-interstitial radiotherapy.…”

Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons

“…14,45 In tumor gene therapy, combinations of immunostimulatory, antiangiogenic and suicide genes may be expressed simultaneously from a single vector to maximize antitumor effects. [49][50][51] Moreover, effective vaccination against two or more tumor or viral antigens expressed at high levels from one vector may be feasible. Other applications may include the coordinate synthesis of IL-12 subunits that must be expressed at similar levels in order to achieve maximal cytokine activity.…”