Preparation And Transfection Properties Of Peptide-Based Target Gene Vectors

As the development of life science and disease researches, gene therapy has become a promising therapeutic technique from the very beginning of its proposal to the wide application of clinical trials. Gene vectors are always regarded as one of the primary issues for gene therapy. Until now, there have been two kinds of gene vectors:viral vectors and non-viral vectors. Since they are readily available and efficient in transfection, viral vectors attract much attention first. However, the innate immunogenicity, toxicity and limitations with respect to scale-up procedures of viral vectors hinder their clinical applications. And non-viral vectors with controllable structures can overcome the above problems. Therefore, more researches focus on the design of novel non-viral vectors currently, to enhance their transfection efficiency to a level comparable to that of viral vectors. In this study, cationic peptides with good biocompatibility were used as the primary components of non-viral vectors. Furthermore, avidin-biotin interaction was introduced to improve the therapeutic effects of peptide gene vectors both in vitro and in vivo, which were proved to be promising vectors for gene therapy.In chapter1, the definition, categories and development of gene therapy, the categories of therapeutic genes, and physical, biological and chemical techniques of gene therapy were introduced. And the definition and development of avidin-biotin system were also reviewed.In chapter2, in order to develop a potential approach to improve the gene therapy effects of critical limb ischemia, we designed a peptide of TAT-PKKKRKV as the vector for VEGF165plasmid to facilitate in vivo angiogenesis. In in vitro studies, the low cytotoxic TAT-PKKKRKV exhibited efficient transfection ability either with or without serum. Additionally, application of TAT-PKKKRKV/VEGF165complexes in hindlimb ischemia rats obviously promoted the expression of VEGF protein, which further enhanced effective angiogenesis. The results indicated that TAT-PKKKRKV is an efficient gene vector with low toxicity both in vitro and in vivo, which has great potential for clinical gene therapy.In chapter3, based on the study of chapter2, we introduced avidin-biotin system. Through a simple but universal alternative, a novel self-assembled gene delivery system with optimized target ability was developed. TAT-PKKKRKV peptide (P) was synthesized as the primary component of gene carrier. Avidin (A) and biotin-transferrin (T) of different molar ratios (1:1and1:5) were subsequently introduced into the P/DNA complexes to form PAT/DNA complexes (PAT1/DNA and PAT2/DNA) mediated by avidin-biotin interaction. Both PAT1/DNA and PAT2/DNA complexes exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, PAT1/p53complexes showed superior transfection capability in HeLa and HepG2cells rather than COS-7cells, primarily due to the over-expression transferrin receptors on cancer cells. And for PAT2/DNA complexes, the target transfection ability decreased with the excess content of T. This study provides a unique and all-purpose strategy to fabricate functionalized gene vectors, and the results indicate that the PAT1/p53complex system has great potential for targeted cancer therapy.In chapter4, based on the study of chapter3, we designed and prepared a tumor-targeting peptide gene vector, TAC, directly through avidin-biotin system. TAC contained two peptides with different bioactivities to mediate the in vitro and in vivo delivery of p53gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53complexes showed higher transfection efficiency and expression amount of p53protein in MCF-7cells as compared within293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7cells. Additionally, by in situ administration of TAC/p53complexes into tumor-bearing mice, the expression of p53gene was obviously up-regulated in tumor cells, and the tumor growth was significantly suppressed. And the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.In chapter5, based on the study of chapter3and chapter4, we made further use of avidin-biotin system to mediate target gene delivery. By mimicking the biological ligand-receptor principle, the cell membranes were biotinylated for combining avidin as receptors. And the conjugation between avidin "receptors" and biotin "ligands" of biotinylated gene vectors made a new definition of target interaction. Moreover, this design provides an approach with improved flexibility and universality in comparison with existing natural ligand-receptor principle. And then, bio-CHK6HC was adopted to verify the feasibility and efficiency of the strategy. The results revealed that the transfection performed by this avidin-mediated binding strategy was very efficient both in vitro and in vivo. And we believe it opens a window for potential target gene delivery as well as drug delivery.