The Construction Of General Tetracycline-regulatable AAV Vectors And The Study Of Its Application In Genetic Therapy Of PD

Parkinson-disease is a neurodegenrative disease characterised by resting tremor, rigidity, bradykinesia. Traditionally, PD is treated by oral administration of L-dopa. With time, the therapeutic effect of L-dopa decrease dramatically and PD patients become intolerable to its adverse effcets. Now, gene therapy is a potentially powerfUl approach to the treatment of Parkinson抯 disease. For this, based on the tetracycline regulatory system, we have constructed two general tetracycline-regulatable adeno-associated virus vectors which different each other. The fUnction of this vectors, in which carried a reporter gene, GFP or EGFP, were evaluated following transfer into cell line, and the conditional expression of TH gene was investigated in vitro by administration of Doxycycline. The main results are as following: 1. Using a tetracycline-repressible construct, two different general tetracycline-regulatable Adeno-associated rims vector plasmids were constructed to at least provide as operatively linked components in the direction of transcription, named pSVtTA and pSVbitTA respectively. Plasmid pSVtTA contains the following DNA sequences operatively linked from 5?to 3? polyadenylation signal, multiple cloning site, the human cytomegalovirus promoter IE combined with tet operator sequences, and the tetracycline-responsive transcriptional activator (tTA) expression codon, all this element are placeed in between two ITR. Another plasmid pSVbitTA is a autoregulated bi-directional expression vector in which the condition expression of two genes, the interested gene and tTA, can be tightly regulated, it is also a positive feed-back regulatory system. The vector comprising neomycin and kanamycin resistance cassette, and can be used to transfer gene in mammalian cell. -2- 2. The GFP was cloned into multiple cloning site of pSVtTA, and enhance version GFP was inserted into the corresponding sites of pSVbitTA driven by bidirectional tetracycline-response promoter, then transfected BHX-21 cell line respectively. Depending on the antibiotic-resistance cassette present in the plasmids, the cell lines obtained by supplemented with G418. Doxycycline, an tetrycycline analog, was present at the concentrations and time indicated in the experiments. In the absence of Dox, GFP and EGFP was observed, and in the presence of Dox, no expression of reporter gene was detected in cells, because Dox prevents tTA from binds to the tetracycline- responsive element. Results demonstrated that expression quantitative of reporter gene was tightly controlled by Dox, and induced in a dose-dependent manner in cells. The pSVbitTA exhibit low basal expression on repression. 3. BHK-2 1 cell line was stably transfect with pSVtTA-GFP and pSVbitTA-EGFP respectively, gained the multiclonal packaging cell lines which selected with G418. Upon subsequent culture of the producer cells, recombinant AAV virions harboring the DNA of interest, are produced. Further concentrate and purify the crude products to get purified pseudovirus, the titer is about 1O11-1O13 particles/mi. BHK-21 and 293 cell lines can be infected with rAAV, and expression of reporter gene was observed in cells which can be regulated by Dox in a tight-controlled manner. These results demonstrate that we have successfully generated rAAV-GFP and rAAV- EGFP vector, and rAAV vector can be used for reliably inserting interest gene into target cells, such as into neurons. 4. pSVbitTA-TH expression vec...