The Construction Of Bicistronic Adenovirus Delivering Neurturin And Th Providing Combinatorial Gene Therapy To Macaca Rhesus Model Of Parkinson's Disease

Parkinson's disease (PD) is a neurodegenerative disorder which is characteristic by loss of dopaminergic neurons and reduced dopamine synthesis. The main clinical symptoms were resting tremor, bradykinesia, myotonia, abnormalities of posture and gait. The final results of this progressive diseases is death which is usually caused by lung infection. Parkinson's disease is mainly caused by the aging of central nervous system and drug abuse. The pathogen of the disease is the loss of the dopaminergic neurons in substantia nigra and subsequent reduced dopamine synthesis, which would inhibit the excited glutaminergic neural projection from thalamus to cerebral cortex indirectly. There are over 4 million of world population who are disturbed by PD, and half of them are Chinese. Therefore, as the development of our country, the aging of the population structure would bring about more and more patients with Parkinson's disease. However, there is no effective treatment, apart from L-dopa, thereby imposing an increasing burden on social healthcare systems.Currently, the treatments for PD are mainly focusing on pharmaceutics administration and neural surgeries, however, neither of them could give a satisfied recovery. As the pathogens of the disease are located in striatum and substantial nigra, thus gene therapy would be suite for PD treatment. Neurturin is a member of glial cell line derived neurotrophic factor, and could provide neuroprotection and promote the survival to dopaminergic neurons. Tyrosine hydroxylase (TH) is a rate-limiting enzyme of dopamine biosynthesis which catalyzes the conversion of L-dopa from L-tyrosine. Abnormalities of tyrosine hydroxylase function in dopaminergic neurons, which may be caused by genetic or metabolic factors, can give rise to severe PD symptoms. Thus, correction of the function of tyrosine hydroxylase in vivo by molecular methods is another way to alleviate the symptom of PD.To date, different viral delivery systems are broadly employed in gene therapy of PD. Recombinant viruses delivering GDNF have obtained successful results in different experimental stages. Adeno-associated virus carrying hybrid NTN named CERE 120 has shown good neuroprotective effect in Macaca Rhesus Model of Parkinson's Disease. Another study also using chimeric NTN delivered by lentivirus has indicated that efficient protection of dopaminergic neurons were observed in rat model of PD. A satisfied PD therapy strategy is not only to stop the progression of the disease and also to relieve the symptoms of PD caused by the loss of dopaminergic neurons.In this study, we have synthesized a chimeric Neurturin by overlapping PCR consisting of the prepro part of beta NGF and mature peptide part of wtNTN. Firstly, we integrated the hybrid NTN and HTH cDNA into the genome of adenovirus. By transfecting the recombinant plasmid into Ad-293 cells respectively, we have obtained the recombinant adenovirus Ad-NGFpreproNTN and Ad-HTH. Based on the two virus could express NTN and HTH in Vero cells efficiently, we construct a bicistronic adenovirus named HJ326 which can express modified Neurturin and tyrosine hydroxylase simultaneously in biologically active form. Through the in vitro activity assays, the results show that the NTN expressed by HJ326 could promote the dorsal root ganglia of 9 days chick embryo to grow neural firbers outside the root and could promote the survival of dopaminergic neurons in ventral mesencephalon rat embryo.Concerning on the animal model test, we have established semi-Parkinsonian monkey model, and carried out behavior change observation for about four months. Then histochemistry and HE staining are made to ensure dopaminergic neurons in MPTP lesioned side were lost. Subsequently, were prepared the purified recombinant adenovirus HJ326 and Ad-NGFreproNTN in large scale and inject the therapeutic virus into the target nucleus of monkey model of PD through stereotaxic injection guided by MR imaging. After the behavior and physiological evaluation, the results indicate that the delivery of HJ326 to the MPTP treated monkeys can bring about obviously faster behavior recovery compared to Ad-NTN injected group, which indicated that TH functioned as an accelerator in the antiparkinsonian therapy. The addition of TH has assisted in relieving the parkinsonian state of monkeys during the competition on protection or destroy of dopaminergic neurons between Neurturin and MPTP.