Construction, Expression And Characterization Of A Chimeric Multi-domain Protein Mediating Specific DNA Transfer

The basic objective of gene therapy is to treat human diseases by the transfer of therapeutically relevant genes, and the specific delivery and expression of these genes to defined target cells is a major goal of gene therapeutic strategies. In the past few decades, these systems can generally be divided into two categories, viral and non-viral. Viral-mediated gene transduction generally facilitates highly efficient delivery and expression of foreign genes in a host cell. However, several types of viruses that have been explored for gene therapy, including retrovirus, adenovirus, adenoassociated virus (AAV), and herpes simplex virus, have their limitations. Retroviral vectors can permanently integrate into the genome of the infected cell, but require dividing cells during mitosis for transduction. Adenoviral vectors can efficiently infect both dividing and non-dividing cell types, but transient expression and immune responses often limit gene delivery in vivo. Similar to adenovirus, AAV can deliver genes to dividing cells, but has a limited DNA capacity. Herpes simplex virus can deliver large amounts of exogenous DNA; however, latent infection remains an obstacle.Therefore, non-viral systems for gene transfer are desirable and safe due to the absence of viral genomic sequences and are highly flexible in the size of the DNA incorporated. Examples of non-viral gene transfer approaches currently being used include:introduction of naked DNA, electroporation, gene gun, cationic liposomes, cationic polymers, cationic proteins (polylysine and protamine), and cationic peptides. Each of these methods has advantages as well as limitations for in vivo gene delivery.A novel approach for the delivery of genes is through the use of fusion protein-based vehicles. The functions of these fusion proteins include:specific binding to the cell surface, entry, endosomal escape, translocation to the nucleus, and stable integration into the target cell genome. The amino terminal 147 amino acids of GAL4 contain a DNA-binding domain were shown to have high affinity binding to a 17 bp oligonucleotide recognition sequence (17 mer). Thus, GAL4 can be used as part of a gene transfer vehicle due to its ability to bind to a specific DNA sequence that may be incorporated into a variety of gene therapy vectors. Endocytosis is a major cellular mechanism for the uptake of macromolecules from the environment. Cell-penetrating peptides (CPPs) are a group of short peptides with the ability to enter the plasma membrane of cells, and CPPs derived from proteins are often referred to as protein transduction domains (PTDs). CPPs such as the well-studied HIV-1 Tat have already been successfully exploited for the delivery of various cargoes from peptides, proteins, nanoparticles, liposomes, to oligonucleotides.In this study, we generated a novel genetically engineered chimeric protein, TG and TNG, in which one domain (GAL4) acts as a carrier for plasmid DNA uptake, while another (Tat) enables the compounds (TG/plasmid or TNG/plasmid) to be endocytosed. To evaluate the TG- or TNG- mediated cell transfection, the HepG-2 human hepatoma cell line was analyzed by fluorescence. HepG-2 cells were seeded the day before the assay and incubated with the complexes of TG/pUAS-EGFP, TNG/pUAS-EGFP, G/pUAS-EGFP or NG/pUAS-EGFP, respectively, for 48 h at 37℃. The HepG-2 cells incubated with TG/pUAS-EGFP or TNG/pUAS-EGFP complex exhibited strong green fluorescence. In contrast, no fluorescence was visualized in the cells incubated with G/pUAS-EGFP or NG/pUAS-EGFP complex under the same conditions. These results demonstrated that the Tat peptide fusion in the TG or TNG protein mediated efficient gene transfer and resulted in significant expression of the exogenous gene.Anti-tumor effects of TG and TNG in HepG-2 cells were identified by MTT. The results showed that TG/pUAS-Apoptin or TNG/pUAS-Apoptin kill HepG-2 cells in varying degrees. While, there was no significant difference between the anti-tumor effects of TG/pUAS-Apoptin and the anti-tumor effects of TNG/pUAS-Apoptin. In the certain treatment doses, the anti-tumor effects of TG/pUAS-Apoptin or TNG/pUAS-Apoptin were heightened with the extending of treatment time, and resulted in some time-effect relationship.By using AO/EB assay, DAPI assay, Caspases assay.Δφassay and ROS assay, we analyzed the mechanism of the anti-tumor effects mediated by TG/pUAS-Apoptin or TNG/pUAS-Apoptin. The results showed that the TG/pUAS-Apoptin or TNG/pUAS-Apoptin complex can induce apoptosis of HepG-2 cells. While, there was no significant difference between the apoptosis induce function of TG/pUAS-Apoptin and the poptosis induce function of TNG/pUAS-Apoptin. We also constrcted the C57BL/6 mice model bearing H22 by transplanting H22 cells into the right hind limb of the mice. And then, the in vivo anti-tumor effects of TG/pUAS-Apoptin or TNG/pUAS-Apoptin were observed through the mice model. The results showed that TG/pUAS-Apoptin and TNG/pUAS-Apoptin treatment groups demonstrate varying degree anti-tumor effects. While, there was no significant difference between the suppression rate of TG/pUAS-Apoptin and the suppression rate of TNG/pUAS-Apoptin. We, then, observed the mean survival of the mice model. The results showed that the mean survival rates of both TG/pUAS-Apoptin and TNG/pUAS-Apoptin treatment groups were 66.7% and 83.3%, respectively, and much higher that Saline, pUAS and pUAS-Apoptin treatment groups. To approach the effects of TG/pUAS-Apoptin and TNG/pUAS-Apoptin on immune system, the contents of IL-2 level, IFN-γlevel, IL-4 level, IL-10 level, NK activity and CTL activity were identified. The results showed that TG/pUAS-Apoptin and TNG/pUAS-Apoptin did not affect the concentration of IL-2, IFN-γ, IL-4 and IL-10 and the activity of NK and CTL.In summary, the recombinant protein TG and TNG basing on GAL4/147 and Tat peptid in this study, which has both UAS specific recognition and membrane penetration abilities, can transduct plasmid containing UAS sequences, and suppress HepG-2 cells and inhibit solid tumor in animal model by transducting Apoptin containing plasmid, effectively. The study provid a novel subsystem for the expanding use of nacked plasmid DNA.