Ribozyme technology as an effective approach to HBV infection

The concept that RNA has catalytic activity is driving the field of ribozyme technology in a number of exciting directions. Exploiting ribozymes as a means of reducing the levels of specific intracellular proteins offers multiple therapeutic opportunities however identifying ways of stabilizing ribozymes must first be identified. Field leaders at Perdue have developed a highly novel strategy for facilitating such stabilization, thereby optimizing gene therapy. This group has in effect mimicked a viral molecular motor that has been shown to play a novel and essential role in packaging bacterial virus phi29 DNA into procapsids. This motor is based on 6 strands of RNA termed packaging RNA or ‘pRNA’.

The pRNA monomer contains two functional domains: the procapsid binding domain and the DNA translocating domain. The procapsid binding domain is located at the central part of the pRNA molecule, while the DNA translocation domain is located at the 5’/3’ paired ends. Available data suggest that phi29 pRNA could be a vector to escort and chaperone small therapeutic RNA molecules. This concept has now been investigated by the Perdue group and reported in the August edition of the journal Gene Therapy. A chimeric pRNA/hammerhead ribozyme was designed using a ribozyme that targets the polyA signal of the mRNA of hepatitis B virus (HBV) as a model system. The ribozyme was designed to cleave a 137-nucleotide HBV-polyA fragment substrate into two fragments of 70 and 67 nucleotides. This was connected to the 5’ and 3’ ends of pRNA. The pRNA was found to be able to chaperone and escort the hammer-head ribozyme to function in the cell, enhancing the cleavage efficiency and inhibition effect of the ribozyme on HBV. The mechanism for such an increase in ribozyme activity is probably due to the fact that the pRNA can prevent the ribozyme from misfolding and protect the ribozyme from degradation by exonucleases present in cells. These are common reasons for the limited therapeutic efficacy of ribozymes developed to date and hence the use of pRNA is this fashion offers a novel way of harnessing the full potential of ribozyme technology. This particular example offers a highly attractive therapy for the treatment of HBV infection, however this technology could be adapted to other ribozymes that are relevant to alternative disease states.

Of particular note is the possibility of developing this technology to improve the targeting of the ribozyme. Since pRNA is a hexamer, multiple subunits can be linked to a separate therapeutic molecule. For example one subunit can be fused to the ribozyme, while another can be fused to a molecules able to bind receptors specific to the target cell. Alternatively therapy for viral infections and cancers therapeutics is characterized by a multimodel approach by using this polyvalent vector. Thus, optimization of ribozyme technology should allow the simultaneous delivery of multiple anti-cancer, anti-virus therapeutics.

Entry date Monday, July 28, 2003

Adapted from Hoeprich, Gene Ther. 2003 Aug;10(15):1258-67.