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

Alekseenko IV, Snezhkov EV, Chernov IP, Pleshkan VV, Potapov VK, Sass AV, Monastyrskaya GS, Kopantzev EP, Vinogradova TV, Khramtsov YV, Ulasov AV, Rosenkranz AA, Sobolev AS, Bezborodova OA, Plyutinskaya AD, Nemtsova ER, Yakubovskaya RI, Sverdlov ED - J Transl Med (2015)

Bottom Line: We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy.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.Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

View Article: PubMed Central - PubMed

Affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia. irina.alekseenko@mail.ru.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig1: 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.

Mentions: The cDNA of the hGM-CSF gene was amplified from plasmid hGM-CSF-pBK, (kindly provided by S. Larin, IGB RAS, Moscow) using primers 5′-TTATCGATATGTGGCTGCAGAGC and 5′-TTGGATCCTCACTCCTGGACTGG that had at their 5′-ends the restriction sites of ClaI and BamHI, respectively. The amplificate was ligated into pAL-TA vector (Evrogen, Moscow, Russia) containing an SV40 polyA fragment. The hGM-CSF-polyA sequence was excised with ClaI and SphI and cloned into retroviral vector pFB-neo (Stratagene, La Jolla, USA) that contained a picornavirus internal ribosome entry site (IRES) and was hydrolyzed by these restriction endonucleases. The IRES-hGM-CSF-polyA cassette was excised from this vector with NotI and BamHI, and its ends were filled in with Klenow fragment. After this, the cassette was blunt-end ligated into CMV-HSVtk-pGL3 vector [58], split at a unique site by XbaI and treated with Klenow fragment. This gave the construct CMV-HSVtk-hGM-CSF-pGL3 (designated as TKhGM, Figure 1) harboring the HSVtk and hGM-CSF genes under the control of the CMV promoter.Figure 1


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

Alekseenko IV, Snezhkov EV, Chernov IP, Pleshkan VV, Potapov VK, Sass AV, Monastyrskaya GS, Kopantzev EP, Vinogradova TV, Khramtsov YV, Ulasov AV, Rosenkranz AA, Sobolev AS, Bezborodova OA, Plyutinskaya AD, Nemtsova ER, Yakubovskaya RI, Sverdlov ED - J Transl Med (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359447&req=5

Fig1: 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.
Mentions: The cDNA of the hGM-CSF gene was amplified from plasmid hGM-CSF-pBK, (kindly provided by S. Larin, IGB RAS, Moscow) using primers 5′-TTATCGATATGTGGCTGCAGAGC and 5′-TTGGATCCTCACTCCTGGACTGG that had at their 5′-ends the restriction sites of ClaI and BamHI, respectively. The amplificate was ligated into pAL-TA vector (Evrogen, Moscow, Russia) containing an SV40 polyA fragment. The hGM-CSF-polyA sequence was excised with ClaI and SphI and cloned into retroviral vector pFB-neo (Stratagene, La Jolla, USA) that contained a picornavirus internal ribosome entry site (IRES) and was hydrolyzed by these restriction endonucleases. The IRES-hGM-CSF-polyA cassette was excised from this vector with NotI and BamHI, and its ends were filled in with Klenow fragment. After this, the cassette was blunt-end ligated into CMV-HSVtk-pGL3 vector [58], split at a unique site by XbaI and treated with Klenow fragment. This gave the construct CMV-HSVtk-hGM-CSF-pGL3 (designated as TKhGM, Figure 1) harboring the HSVtk and hGM-CSF genes under the control of the CMV promoter.Figure 1

Bottom Line: We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy.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.Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

View Article: PubMed Central - PubMed

Affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia. irina.alekseenko@mail.ru.

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.

No MeSH data available.


Related in: MedlinePlus