Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer

Abstract

Background: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system.

Methods: We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models.

Results: We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan.

Conclusions: We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier - a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

Figure 1

Schematic representation of the expression constructs used. On the left – construct names. The SV40 polyA signal is omitted from the schemes. CMV – major immediate-early promoter of human cytomegalovirus; hGM-CSF and mGM-CSF – human and mouse genes of granulocyte macrophage colony stimulating factor, respectively; HSVtk –herpes simplex virus thymidine kinase gene, IRES - internal ribosome entry site of encephalomyocarditis virus.

Figure 2

Effect of ex vivo transformation of LLC cells with the TK and TKmGM constructs combined with administration of GCV on A) tumor growth rate, and B) animal lifespan after transplantation of the transfected cells into C57Bl/6 mice. The data represent mean values for treatment groups of ten animals and control groups of six animals. We studied 6 groups of mice: two control groups (K/GCV and K/PBS) inoculated with non-transfected cells; two experimental groups (TK/GCV and TK/PBS) inoculated with LLC cells transfected with TK; and two experimental groups (TKmGM/GCV and TKmGM/PBS) inoculated with LLC cells transfected with TKmGM using LFA. The animals of groups К/GCV, ТK/GCV and TKmGM/GCV received intraperitoneal injections of ganciclovir in a dose of 75 mg/kg twice a day for 10 days. The animals of groups К/PBS, ТK/PBS and TKmGM/PBS received phosphate buffered saline (PBS) as a placebo instead of GCV. Starting from day 6 after transplantation, we measured the volume of developed tumors. The euthanasia criterion was the tumor volume that exceeded 2000 mm³. A) Tumor volume (in mm³, Y-axis) versus time since cell transplantation (X-axis). Mean ± SEM values are shown. B) Survival period of mice after transplantation of the transfected and non-transfected LLC cells.

Figure 3

Survival of S37-bearing mice after injection of TK-PPT, mGM-PPT and TKmGM-PPT with or without ganciclovir. TKmGM (CMV-HSVtk-mGM-CSF-pGL3 construct), TK (CMV-HSVtk-pGL3), mGM (CMVmGM-CSF-pGL3); PPT - polyethylenimine-polyethylene glycol-TAT peptide copolymer; PBS – phosphate buffered saline (placebo); GCV – ganciclovir.

Figure 4

Histological images of tumors and lymph nodes. Subcutaneously transplanted mouse sarcoma 37 on day 15 of tumor growth (a,d). Metastatic ipsilateral inguinal lymph nodes on the day 30 of tumor growth in control mice (b,c,f,g). Images show tumor cells infiltrating lymph node parenchyma (f) and totally replacing lymph node tissue (g). A lymph node taken on day 30 after the beginning of the treatment from a mouse treated with TKmGM/GCV (h); note that its parenchyma is free of tumor cells. The sections are made through the largest cross dimension of the tissue samples. Low-power field images (a-d, 40×) demonstrate differences in size between positive (metastatic) and negative (metastasis free) lymph nodes. High-power field images (e-h, 400×) represent detailed histological features of the specimens. Formalin fixed and paraffin embedded tissues (H&E staining).

Figure 5

Survival period of mice inoculated with A) adenocarcinoma C26; B) cervical squamous carcinoma CSC5 after injection of TKmGM-PPT. TKmGM (CMV-HSVtk-mGM-CSF-pGL3 construct); PPT - polyethylenimine-polyethylene glycol-TAT peptide copolymer; PBS – phosphate buffered saline (placebo); GCV – ganciclovir.