Studys On The Recombinant Baculovirus As A Novel Vector For Gene Therapy

The baculovirus Autographa californica multiple Nucleopolyhedrovirus (AcMNPV) has been traditionally used as an efficient vehicle to over-express recombinant proteins in insect cells. Its host specificity was originally thought to be restricted to cells derived from arthropods. Recently, however, accumulating evidence has revealed that baculovirus, carrying mammalian cell-active promoters, is capable of transferring and effective expressing foreign genes in a variety of mammalian cells. Thanks to its highly efficient gene delivery mechanism, increasing interest has been shown in baculovirus as a novel vector for gene therapy. In addition, other potential advantages, including easy manipulation, high recombinant viral titers, simple scale-up, a large DNA insertion capacity, lack of replication in mammalian cells, and lack of toxicity, make this vector highly promising for gene therapy.Apoptin, a protein derived from the chicken anemia virus, can induce programmed cell death in a large panel of human transformed and malignant cells, but not in normal, diploid cells. The intrinsic tumor specificity of Apoptin makes it a promising new tool for cancer gene therapy.Duchenne musclar dystrophy (DMD) is an X-linked recessive disease with characteristic of progressive degeneration and necrosis in skeletal muscle. There is no effective treatment for DMD at present, gene therapy is a promising therapeutical approaches for the treatment of DMD. Howeve, DMD gene therapy is challenged by the viral vectors for their small insert capacity, replication-deficient and require helper virus functions for propagation and raising biosafety concerns. These all impede their clinical application. Enhancement the experssed efficiency in vivo and selection ideal vector are the keys to success to treat DMD.Based on the above mentioned research background, the present study investigate the anti-tumor effects in vitro and in vivo of a recombinant baculovirus encoding Apoptin protein, evaluated the microdystrophin protein expression of the recombinant baculovirus containing microdystrophin gene, at last, compared the gene therapy ability of the recombinant baculovirus and recombinant adenovirus in mdx mice. The main projects are:1. Construction of recombinant baculovirus expressing the Apoptin proteinFull-length cDNA for Apoptin was amplified by polymerase chain reaction from plasmid pVAX-Apoptin, and subcloned into pcDNA-HisB vector to generate pcDNA3.1-His-Apoptin. The DNA fragment containing CMV-IE enhancer/promoter-Apoptin was obtained from pc-Apoptin and inserted into pFast-VSV-G to generate pFB-G-Apoptin. The recombinant baculoviruses were subsequently generated by using the Bac-to-Bac(?) system (Invitrogen) following the manufacturer's instructions. The virus was further amplified by propagation in Sf-9 cells.2. The research on effect of transduction with recombinant baculovirus BV-ApoptinWe investigate the correct subcellular localization of BV-Apoptin in cells. We found that Apoptin was mainly located in the nucleus of HepG2 cells, whereas in normal HEK293 cells BV-Apoptin was localized in the cytoplasm. The results indicated that BV-Apoptin could mediate efficient gene delivery and the greater gene transduction efficiency of the BV-Apoptin was confirmed by comparing the Apoptin expression level in a variety of multiplity of infection.3. The antitumor effect of BV-Apoptin in vitro and in vivoTo determine whether BV-Apoptin can induce apoptosis in transduced HepG2 cells, the DNA ladder and TUNEL assays were performed. An obvious DNA ladder pattern could be observed in BV-Apoptin-transduced HepG2 cells. The results indicated that BV-Apoptin induced apoptosis in a dose-dependent manner, specifically in transduced tumor cells but not in transduced normal cells. C57BL/6 mice model bearing H22 hepatoma was constructed by transplanting H22 cells into the right hind limb of the mice and the anti-tumor effect on H22 hepatoma of BV-Apoptin was observed. The results indicated that the treatment with BV-Apoptin can confer significant survival benefits and reduction of tumor size in vivo. Taken altogether, BV-Apoptin shows the good anti-tumor effect may be used as new and powerful strategy for cancer bio-therapy in future.4. Construction of recombinant baculovirus expressing the microdystrophin proteinThe DNA fragment containing CMV-IE enhancer/promoter was released from pcDNA3.1 (+) and inserted into pFast-VSV-G, resulting in pFB-G-CMV. Full-length cDNA for human microdystrophin gene was acqured from the plasmid pBSK-MICRO and subcloned into pFB-G-CMV to generate pFB-G-MICDYS. The recombinant baculoviruses were subsequently generated by using the Bac-to-Bac(?) system (Invitrogen) following the manufacturer's instructions. The virus was further amplified by propagation in Sf-9 cells.5. Research on the effect of transduction with BV-MICDYS in C2C12 cellsThe C2C12 cells were transduced with BV-Apoptin at different MOIs (50, 100 and 200). At 48 h after infection, the expression of microdystrophin was detected. Obvious differences in number of positive fluorescence cells were observed between wells infected with 50,100, 200 MOI of BV-MICDYS as the progression of the transduction. The microdystrophin protein expression by BV-MICDYS is in a dose-dependent manner in C2C12 cells. The results also indicated that in the same dosage, the microdystrophin positive of BV-MICDYS is higher than the microdystrophin expression with recombinant adenovirus Ad-MICDYS. These results indicated that BV-MICDYS could mediate efficient gene delivery and expression of microdystrophin protein in a dose-dependent manner in mammalian cells, and the transduction efficiency was higher than adenovirus.6. Research on the effect of transduction with recombinant baculovirus mediated microdystrophin in mdx miceTA muscles of mdx mice were injected with 10¹⁰pfu/mL, 10⁹pfu/mL, 10⁸pfu/mL of recombinant baculovirus BV-MICDYS. Cryostat sections were prepared from the snap frozen muscle samples after 2 weeks and 8 weeks injection. HE evaluated pathological change of TA and immunocytochemistry evaluated the expression of microdystrophin; at the same time, the isometric contractile properties of TA were tested in situ. The results indicated in 2 weeks that transduction of the microdystrophin BV-MICDYS could retard significantly the degeneration and distribution in TA of mdx mice. The gene therapy ability of BV-MICDYS is in a dose-dependent pattern. But the BV-MICDYS lost its ability in gene therapy ability 8 weeks after injection. These results indicated that BV-MICDYS are suit for DMD short-term gene therapy.