Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons: inhibition of endogenous caspase-3 activity and protection from apoptotic cell death.
Antisense technology, including ribozyme and small interfering RNA, is being developed to mediate the down-regulation of specific intracellular genes. It was observed in this study that both antiluciferase ribozymes and short hairpin RNAs (shRNAs) could significantly reduce the activity of exogenously expressed luciferase in primary hippocampal neurons in a viral titer-dependent manner. shRNAs were more effective gene-silencing agents than ribozymes, although they exhibited some nonspecific gene-silencing effects at high viral titers. We also attempted to increase ribozyme efficacy by using a woodchuck hepatitis posttranscriptional regulatory element (WPRE) in the ribozyme expression cassette. The results showed that adenoviral vectors encoding specific ribozymes could silence the cellular expression of luciferase and endogenous procaspase-3 significantly. Furthermore, the antiprocaspase-3 ribozyme was shown to inhibit staurosporine-mediated cell death. The addition of a WPRE did not, however, increase or decrease ribozyme activity. As far as we are aware, this is the first example of adenovirally mediated delivery of hammerhead ribozymes being used to manipulate gene expression in primary neurons. The results therefore suggest that hammerhead ribozymes may be useful tools for studying neuronal gene function and have potential as therapeutic agents to treat CNS diseases.
Bantounas
Ioannis
I
The Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, United Kingdom.
Glover
Colin P J
CP
Kelly
Stephen
S
Iseki
Sachiko
S
Phylactou
Leonidas A
LA
Uney
James B
JB
D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001562:Benzimidazoles / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004622:Embryo, Mammalian; D005822:Genetic Vectors / Q000502:physiology; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009474:Neurons / Q000378:metabolism* / Q000821:virology; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014161:Transduction, Genetic / Q000379:methods
D001562:Benzimidazoles; D061945:Caspase Inhibitors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; C418481:hairpin ribozyme; C427204:hammerhead ribozyme; D008156:Luciferases; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C017807:bisbenzimide ethoxide trihydrochloride