Efficiency of Promoters of Human Genes FAP and CTGF at Organism Level in a Danio rerio Model.

The identification of tissue-specific promoters for gene therapeutic constructs is one of the aims of complex tumor therapy. The genes encoding the fibroblast activation protein (FAP) and the connective tissue growth factor (CTGF) can function in tumor-associated stromal cells but are practically inactive in normal adult cells. Accordingly, the promoters of these genes can be used to develop vectors targeted to the tumor microenvironment. However, the efficiency of these promoters within genetic constructs remains underexplored, particularly, at the organism level. Here, we used the model of Danio rerio embryos to study the efficiency of transient expression of marker genes under the control of promoters of the FAP, CTGF, and immediate early genes of Human cytomegalovirus (CMV). Within 96 h after the injection of vectors, the CTGF and CMV promoters provided similar equal efficiency of reporter protein accumulation. In the case of the FAP promoter, a high level of reporter protein accumulation was observed only in certain zebrafish individuals that were considered developmentally abnormal. Disturbed embryogenesis was the factor of changes in the exogenous FAP promoter function. The data obtained make a significant contribution to understanding the function of the human CTGF and FAP promoters within vectors to assess their potential in gene therapy.

Embryotoxic activity of 3C protease of human hepatitis A virus in developing Danio rerio embryos.

The 3C protease is a key factor in picornavirus-induced pathologies with a comprehensive action on cell targets. However, the effects induced by the enzyme have not been described at the organismic level. Here, the model of developing Danio rerio embryos was used to analyze possible toxic effects of the 3C protease of human hepatitis A virus (3Cpro) at the whole-body level. The transient 3Cpro expression had a notable lethal effect and induced a number of specific abnormalities in Danio rerio embryos within 24 h. These effects are due to the proteolytic activity of the enzyme. At the same time, the 3Cpro variant with reduced catalytic activity (3Cmut) increased the incidence of embryonic abnormalities; however, this effect was smaller compared to the native enzyme form. While the expression of 3Cmut increased the overall rate of abnormalities, no predominance of specific ones was observed. The data obtained point to a presence significant impact of picornavirus 3Cprotease at the whole-organism level and make contribution to the study of the infectious process caused by human hepatitis A virus.

Functional efficiency of PCR vectors in vitro and at the organism level.

The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.