Neuroprotective Effect Of Recombinant Adeno-associated Virus Encoding Cdc2-siRNA On Niemann-Pick Disease Type C Mice

Part One Packaging and functional identification of recombinant adeno-associated virus encoding cdc2-siRNA by Helper Free systemObjectiveCyclin dependent kinases(cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. As the first step toward exploring the possibility of cdks related gene therapy for Niemann-pick disease type C, we packed recombinant adeno- associated virus (rAAV) encoding cdc2-siRNA.MethodsThe expressing plasmid pAAV-EGFP-U6-cdc2-siRNA was constructed by molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated cotransfection of pAAV-EGFP-U6-cdc2- siRNA, p-RC and p-Helper into AAV-293 cells.ResultsDNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV- MCS-EGFP. After 72 hours'packaging, the expression of EGFP could be detected in AAV-293 cells. Cdc2 gene expression in AAV-293 cells assessed by Western Blot was down-regulated markedly after infection of rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. ConclusionOur data indicate that the packaging of rAAV encoding cdc2-siRNA is successful. It can silence cdc2 gene effectively, which might offer a novel means for treating Niemann- Pick disease type C.Part Two Effect of recombinant adeno-associated virus encoding cdc2-siRNA on the behavior of Niemann-Pick disease type C miceObjectiveTo observe the effect of cerebellar injection of recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA on the behavior of Niemann-Pick disease type C (NPC) mice.MethodsThe rAAV encoding cdc2-siRNA was injected into the cerebellum of 2 weeks old npc-/- mice. The mice were weighed dynamically and coat hanger test and footprint test were performed to assess their motor ability from 4 to 8 weeks age.Results①Weight loss was significantly delayed in cdc2-siRNA group.②Coat hanger test showed that the motor defect was remarkably ameliorated in cdc2-siRNA group. ③Footprint test showed that the relative stride in cdc2-siRNA group (75.1%±8.6%) was markedly longer than that in the controlled viral group and the non-surgical group.ConclusionThe cerebellar injection of rAAV encoding cdc2-siRNA improves the behavior of npc-/- mice, which may offer a novel means for treating NPC.Part Three Effect of recombinant adeno-associated virus encoding cdc2-siRNA on the neuropathology of Niemann-Pick disease type C miceObjectiveTo assess the effect of cerebellar injection of recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA on the neuropathology of Niemann-Pick disease type C (NPC) mice.MethodsRAAV encoding cdc2-siRNA was injected into the cerebellum of 2 weeks old npc-/- mice. The neuropathology in the treated mice was evaluated by immunohistology, HE staining and immunoblotting technology.Results①rAAV encoding cdc2-siRNA was widely expressed in npc-/- mice brain.②rAAV encoding cdc2-siRNA remarkably reduced the number of axonal spheroids, and ameliorated the deletion of purkinje cells in npc-/- mice. ③rAAV encoding cdc2-siRNA markedly inhibited the expressions of cdc2, phospho- neurofilament (recognized by SMI31), phospho-mitotic associated proteins (recognized by TG-3 and MPM-2), and phospho-Tau (recognized by PHF-1).ConclusionThe rAAV encoding cdc2-siRNA injected into the cerebellum of npc-/- mice can protect the neuronal cytoskeletal pathology, which implys that cdc2 might be a novel therapeutic target in NPC.