Comparative Analysis of Gene Therapy Systems Expressing Two Oncotherapeutic Genes under Control of a Single Promotor

To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus ( HSVtk ) and mouse OX40L gene ( m0X40L ) involved in regulation of immune response was constructed. Therapeut...

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Published in:Molecular genetics, microbiology and virology microbiology and virology, 2018, Vol.33 (1), p.15-20
Main Authors: Alekseenko, I. V., Kostina, M. B., Serebrovskaya, E. O., Potapov, V. K., Sverdlov, E. D.
Format: Article
Language:English
Subjects:
Biomedical and Life Sciences
Colon
Comparative analysis
Cytomegalovirus
Cytotoxicity
Experimental Works
Expression vectors
Flow cytometry
Gene therapy
Herpes simplex
Immune response
Immunofluorescence
Internal ribosome entry site
Life Sciences
Microbiology
Molecular Medicine
Ox40L protein
Peptides
Ribonucleic acid
RNA
Suicide
Suicide genes
Thymidine
Thymidine kinase
Transfection
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Summary:To improve the safety profile and specificity in cancer gene therapy applications, а set of singleplasmid vectors for expression of suicide thymidine kinase gene of the herpes simplex virus ( HSVtk ) and mouse OX40L gene ( m0X40L ) involved in regulation of immune response was constructed. Therapeutic transgene expression was directed by a strong constitutive promoter of human cytomegalovirus (CMV), while translation occurred on an RNA template due to an encephalomyocarditis virus internal ribosome entry site (construct CMV-HSVtk-IRES-mOX40L) or 2A-peptide of Porcine Teschovirus-1 (construct CMV-HSVtk-2AmOX40L) inserted between them. The constructs obtained were functionally validated by transient transfection of C26 cells (mouse colon carcinoma). Both bicystronic vectors demonstrated strong cytotoxic activity and produced cytotoxic protein HSVtk at the similar level. To analyze efficiency of mOX40L expression, C26 transfected cells were immunofluorescence-labelled with anti-OX40L-antibodies conjugated with phycoerythrin following flow cytometric analysis. Production of mOX40L was higher in the case of CMV-HSVtk- 2A-mOX40L. Better performance of this expression vector was correlated with our data. Previously, we have demonstrated that a higher level of transgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence. Thus, transfection with the vector based on the 2A peptide sequence is optimal for achieving high efficiency of expression of both oncotherapeutic genes.
ISSN:0891-4168
1934-841X
DOI:10.3103/S0891416818010020