MicroRNAs are small non-coding RNA molecules that regulate mRNA translation by binding to complementary sequences usually within the 3’ un-translated region (UTR). By inserting four perfectly complementary binding sites for the hepatic specific microRNA mir122 into the 3’ UTR of adenovirus wild type 5 (Ad5 WT) E1A mRNA I show that the acute liver toxicity caused by Ad5 WT in mice can be significantly reduced. This virus, termed Ad5-mir122, is a promising virotherapy candidate and causes no obvious liver pathology whilst maintaining Ad5 WT replication in mir122 negative cells. Data shows that repeat intravenous administration of Ad5-mir122 (2x1010vp) to HepG2 tumour bearing mice mediated significant anti-cancer efficacy. RT-QPCR for E1A mRNA demonstrated a 29-fold reduction when compared to Ad5 WT in murine liver whilst western blot confirmed that all E1A protein variants were knocked down. Viral genomic replication was also reduced in mouse liver by 25-fold compared to Ad5 WT. This control of virus activity reduced alanine and aspartate transaminase release by >15-fold and histological analysis showed little to no pathology in Ad5-mir122 infected livers. Measurement of mature mir122 levels in Ad5-mir122 infected livers by RT-QPCR showed that the quantity of mir122 remained unaffected at therapeutic doses. Complete genome mRNA array profiling of infected livers showed that the transcript levels of >3900 different mRNAs were changed more than 2-fold following Ad5 WT infection whilst less than 600 were changed by Ad5-mir122. A non-replicating control adenovirus vector altered >550 mRNAs. No known mir122 target mRNAs were affected following infection with Ad5-mir122. Western blot analysis of a known mir122 regulated target (Aldolase A) confirmed these results, demonstrating no change in protein level despite infection with Ad5-mir122. These data combined demonstrate that the exploitation of microRNA mir122 regulation to control adenovirus replication is a safe method of control and does not alter the endogenous level or activity of the microRNA or its endogenous mRNA targets. Ad5-mir122 is a potent anti-cancer agent that replicates to wild-type levels in microRNA mir122 negative cells but is specifically and safely attenuated in hepatocytes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:543557 |
| Date | January 2011 |
| Creators | Cawood, Ryan |
| Contributors | Seymour, Leonard W. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:d97d80b5-6272-4aab-b555-d03d0016eeff |
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