Preparation And Gene Transfection Properties Of Gene Vectors Based On Functional Peptides

After the first human gene therapy trial succeeded in 1989, thousands clinical trials have been completed for treating various forms of diseases and its potential in cancer therapy is widely recognized. Gene therapy holds potential for diseases such as cancer, monogenic disorders, cardiovascular diseases, infectious diseases, neurological diseases and ocular diseases. In addition to target genes, a gene delivery system which would be nontoxic and efficient is essential for gene therapy. Although viral vectors are efficient DNA delivery systems, there also exist some disadvantages, such as the risk of an immune response and the size limitations of the gene that can be used. Hence, non-viral vectors have acquired more attention due to low immune response and simplicity for preparation. Among thousands of non-viral vectors, they still have defects, including lacking tumor-targeted ability, low transfection efficiency, high cytotoxicity and poor serum stability, which directly limited their clinical applications. Hence, the aim of this study is to prepare a series of non-viral vectors for gene therapy. They will be multifunctional and biocompatible systems to incorporate functional peptides to overcome low gene transfer capacity or lead to specific gene delivery.In chapter 1, the current definition and barriers of gene therapy were introduced. The functional peptides used in gene therapy were reviewed.Cellular uptake and nuclear localization are two barriers to gene delivery, in chapter 2, we designed new gene delivery carriers with an N-terminal stearylated (STR) nuclear localization signal (NLS), PKKKRKV, present in the Simian Virus 40 large T antigen with the aim to overcome limitations, such as cell membrane and nuclear pores, offering attractive possibilities to enhance gene delivery. Four vectors with different structures of N-stearylated nuclear localization signal-octaarginine peptide (STR-PKKKRKV-R8 or STR-NLS-R8, STR-VKRKKKP-R8 or STR-reverse NLS-R8, PKKKRKV-R8 or NLS-R8, and VKRKKKP-R8 or reverse NLS-R8) were compared. The gene expression mediated by these vectors in dividing and non-dividing cells (both in 293 T and HeLa cell lines) was investigated. The most efficient N/P ratio was 4 for STR-PKKKRKV-R8, STR-VKRKKKP-R8, and 0.25 for PKKKRKV-R8, VKRKKKP-R8. The maximum transfection activity of these vehicles (VKRKKKP-R8) was up to 80% as effective as jetPEITM and the vehicles did not exhibit cytotoxicity. Interestingly, N-stearylated peptides presented lower transfection activity compared to peptides without N-stearylation at lower N/P ratios (0.25 to 1). Confocal study showed that the vectors could effectively promote the nuclear translocation."Human cancer targeting" is also a key requirement in gene therapy, in chapter 3, a non-viral vector with iodine-nuclear localization sequence (namely NLS-I) targeting to breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to NLS. In addition, compared to that of PEI 1800/DNA complex, PEI 1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct non-viral delivery system for targeted gene transfer into breast cancer cells.To construct a novel gene delivery vector used in vivo, polyethylenimine (PEI) 5 kDa was used as backbone modified with the novel small molecule anti-tumor peptide tyrosyl-seryl-leucine(YSL) and polyethyleneglycol (PEG) 550 Da, named YSL-PEI-PEG. YSL has been documented to inhibit tumor growth both in vitro and in vivo, in chapter 4, the co-administration of YSL and therapeutic gene p53 has been tested. Firstly, the new synthesized vector YSL-PEI-PEG could efficiently condense plasmid DNA and transfect foreign DNA in the presence of FBS. Then to fully characterize the anticancer properties of YSL-PEI-PEG/p53, it is necessary to perform large-scale experiments both in vitro and in vivo. As well, the anti-tumor cells effect was enhanced compared to PEI 25 kDa/p53, the tumor growth inhibition rate were also greater, by contrast, the cytotoxicity in normal cells was not shown. All the results indicated that YSL-PEI-PEG/p53 system could be potentially useful for further clinical application.At last, in chapter 5, an optimal method of coating gene delivery nanoparticles with functional peptide was selected. In the study, in order to evaluate the targeting ability of three methods, we synthesized disulfide-containing polyethyleneimine-GRGDSF (SS-PEI-GRGDSF), GRGDSF, EEEEEEEEGRGDSF (E8GRGDSF) and compared three approachs for ligand-specific delivery. SS-PEI-GRGDS conjugate was used to represent a covalently conjugated to SS-PEI; the terminology "inclusion complex" of GRGDS and SS-PEI was used to depict dendrimer containing a free peptide bound by non-covalent interaction; and the addition of free EGRGDS into SS-PEI/DNA binary complexes was used as electrostatic forces. We evaluate the effect on transfection and differentiation capacity of all the three incorporation methods. All the experiments were assessed by employing HeLa cells that express avβ3 intcgrins and COS-7 cells that lack the expression of the integrin. In HeLa cells, SS-PEI-GRGDSF/DNA and SS-PEI/DNA/E8GRGDSF increased transfection ability compared to SS-PEI and SS-PEI/GRGDSF. In addition, compared to that of COS-7 cells, all the three methods exhibited much decreasing luciferase reporter gene expression. The introducing of RGD could target gene transfer into cancer cells, and as the two methods, covalently conjugate and electrostatic forces, have high efficacy, ligand-based specificity, biodegradability, and low cytotoxicity.