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JOURNAL ARTICLE
RESEARCH SUPPORT, NON-U.S. GOV'T
RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S.
RESEARCH SUPPORT, U.S. GOV'T, P.H.S.
Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus.
Gene Therapy 2003 August
The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5'/3' ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems - exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5'/3' ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.
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