INHIBITING HEPATITIS B VIRUS GENE EXPRESSION WITH HAMMERHEAD RIBOZYMES THAT TARGET THE HBx OPEN READING FRAME

Hepatitis B virus (HBV) infection is endemic to several populous regions and is often complicated by cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important medical objective. The X open reading frame (ORF) of HBV, HBx, is a conserved sequence that overlaps with the polymerase ORF and viral c/'s-elements, and is present within all viral transcripts. In addition, the HBx ORF encodes a 17 kDa transactivator protein, HBx, which is required for the establishment of viral infection and has been implicated in HBV-associated hepatocarcinogenesis. The HBx sequence thus represents a compelling target for applying nucleic acid hybridisation-based therapeutic agents for the inhibition of HBV gene expression and replication. Hammerhead ribozymes are RNA enzymes that can be designed to hybridise to short complementary RNA sequences and catalyse the specific endonucleolytic cleavage of the phosphodiester backbone in trans at conserved 5' NUH 3' triplet sequences. Trans-cleaving hammerhead ribozymes have been applied as therapeutic agents to a number of different diseases for the targeted ‘knockdown’ of both viral and cellular gene expression. Although previous studies have shown that HBV RNA is susceptible to hammerhead ribozyme cleavage in vitro, the intracellular inhibitory effects of hammerhead ribozymes targeted to HBV remained unresolved. Liver-derived cells were co-transfected with anti-HBx ribozyme expression vectors (along with catalytically inactive and antisense RNA control vectors) together with plasmids that constitutively express HBx or reconstitute intracellular HBV infection. Hammerhead ribozymes that were catalytically active in vitro were able to inactivate HBx mRNA, and inhibit HBx trans-activation function in cultured cells. Using a replication-competent HBV vector, ribozymes inhibited markers of HBV replication by inhibiting viral gene expression and decreasing the secretion of HBsAg and HBeAg into the culture medium. To confirm ribozyme antireplicative effects, a sensitive in situ measurement of ribozyme action in co-transfected Huh7 cells was assessed using an HBV vector, where the preS2/S region was replaced by DNA encoding enhanced green fluorescent protein (EGFP). Ribozymes inhibited EGFP marker gene expression in situ and provided an accurate measurement of ribozyme antireplicative efficacy in cell culture. Flowever, the data do not exclude a dominant antisense effect. Vectors were developed that include head-to-tail concatamers of different ribozyme-encoding units, each with a respective 3'-flanked cis complementary target cleavage sequence. The aim was to generate expression cassettes encoding single transcripts with many c/s-cleaving hammerhead ribozymes that retain the ability to cleave simultaneously in trans. All multimeric hammerhead ribozyme transcripts generated in vitro, self-cleaved to release 5' and 3' trimmed trans-acting hammerhead ribozymes. Expression vectors that encode multimeric cisand trans-cleaving hammerhead ribozymes decreased HBsAg and FIBeAg secretion when co-transfected along with HBV vectors in Fluh7 cells. These ribozymes demonstrated an improved antireplicative effect when compared to previous single-unit, or catalytically defective ribozyme controls. The most elaborate multimeric hammerhead ribozyme expression vector, a 24-mer construct, that generates eight units of each of the three ribozymes, inhibited EGFP marker gene expression 10-15% more efficiently than single-unit ribozyme counterparts when measured in situ using a modified HBV EGFP vector. In conclusion, the hammerhead ribozyme-encoding vectors generated represent a significant improvement on previously described anti-HBV ribozyme constructs. Moreover, the HBx ORF proved to be a suitable target site for hammerhead ribozyme-mediated inhibition of HBV gene expression and markers of replication in cell culture. For future use in vivo, expression cassettes encoding multimeric hammerhead ribozymes may be incorporated into liposomes or viral delivery vectors. Should the endogenously expressed hammerhead ribozymes presented in this thesis prove to be safe and efficacious in animal models of viral infection they may be further applied as therapeutic agents for the treatment of chronic HBV infection.