Packaging and functional identification of recombinant adeno-associated virus encoding cdc2-siRNA

Abstract

Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegenerative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

Keywords

small interfering RNA / recombinant adeno-associated virus / gene therapy

References

[1] VincentI., JichaG., RosadoM., et al.. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer’s disease brain. J Neurosci, 1997, 17(10): 3588-3598

[2] McSheaA., LeeH. G., PetersenR. B., et al.. Neuronal cell cycle re-entry mediates Alzheimer disease-type changes. Biochim Biophys Acta, 2007, 1772(4): 467-472

[3] HannonG. J.. RNA interference. Nature, 2002, 418(6894): 244-251

[4] BuéeL., BussièreT., Buée-ScherrerV., et al.. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Rev, 2000, 33: 95-130

[5] LeeM. S., KwonY. T., LiM., et al.. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature, 2000, 405(6748): 360-364

[6] BuB., LiJ., DaviesP., et al.. Deregulation of cdk5, hy-perphosphorylation, and cytoskeletal pathology in the Niemann-Pick type C murine model. J Neurosci, 2002, 22(15): 6515-6525

[7] ZhangM., LiJ., ChakrabartyP., et al.. Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in Nie-mann-Pick Type C mice. Am J Pathol, 2004, 165(3): 843-853

[8] NobleW., OlmV., TakataK., et al.. Cdk5 is a key factor in tau aggregation and tangle formation in vivo. Neuron, 2003, 38(4): 555-565

[9] MinhD. N., MathieuB., JasnaK., et al.. Cell cycle regulators in the neuronal death pathway of amyotrophic lateral sclerosis caused by mutant superoxide dismutase 1. J Neurosci, 2003, 23(6): 2131-2140

[10] XiaH., MaoQ., PaulsonH. L., et al.. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol, 2002, 20(10): 1006-1010

[11] BrummelkampT. R., BernardsR., AgamiR., et al.. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296(5567): 550-553

[12] FlotteT. R.. Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Ther, 2004, 11(10): 805-810

[13] McCartyD. M., YoungS. M., SamulskiR. J.. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet, 2004, 38: 819-845

[14] ShiauA. L., LiuP. S., WuC. L.. Novel strategy for generation and titration of recombinant adeno-associated virus vectors. J Virol, 2005, 79(1): 193-201

[15] Smith-AricaJ. R., BartlettJ. S.. Gene therapy: recombinant adeno-associated virus vectors. Curr Cardiol Rep, 2001, 3(1): 43-49

[16] MachidaY., OkadaT., KurosawaM., et al.. RAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse. Biochem Biophys Res Commun, 2006, 343(1): 190-197

[17] WangT., LiH., ZhaoC., et al.. Recombinant adeno-associated virus-mediated kallikrein gene therapy reduces hypertension and attenuates its cardiovascular injuries. Gene Ther, 2004, 11(17): 1342-1350