Intracellular application of hairpin ribozyme genes against hepatitis B virus

Abstract

HBV, a partially double-stranded DNA virus, replicates modifications were performed on these two Rzs, with the through a pregenomic RNA (pgRNA) intermediate, which goal of increasing catalytic efficiency both in vitro and in provides a therapeutic opportunity for a novel antiviral gene cells. To determine the Rz activities in liver cells, the therapy based on ribozyme RNA cleavage. Three hairpin cDNAs for each of the anti-HBV Rzs (and their catalytically ribozymes (Rzs) were designed which have the potential disabled negative controls) were cloned into retroviral vecto disrupt HBV replication by targeting the pgRNA as well tors. Unmodified ribozymes co-expressed with HBV in as specific mRNAs encoding the HBV surface antigen human liver Huh7 cells reduced the level of viral particle (HBsAg), the polymerase and the X protein. The ability of production by up to 66% based on the endogenous polyeach ribozyme to cleave approximately 0.3 kb HBV merase assay, while the structurally modified ribozymes subgenomic RNA fragments was tested in vitro. Two of the inhibited HBV production up to 83%. These encouraging three Rzs tested (BR1 and BR3) were capable of cleaving results indicate the feasibility of ribozyme-mediated gene their respective RNA substrates, while their catalytically therapy for the treatment of HBV infections. disabled mutated counterpart Rzs were not. Structural Keywords: plasmid-independent transcription; endogenous polymerase assay; ribozyme kinetics; RNA cleavage; antiviral problems associated with the direct injection of antisense Introduction DNA. Stable expression of the anti-HBV ribozymes in Hepatitis B is a major worldwide health problem.liver cells could theoretically lead to lifelong intracellular Although a vaccine is currently available for hepatitis B immunity against HBV. virus (HBV), it is estimated that no less than two billion HBV undergoes replication through reverse transcrippeople are infected globally and 300 million are chrontion of a pregenomic RNA intermediate 5,6 and is thus ically infected carriers. 1,2 Approximately 10% of infected somewhat analogous to HIV and other retroviruses. This adults will develop a chronic, life-long infection often offers an excellent rationale for the use of ribozymes as with devastating consequences. 1,2 About 2 million deaths antiviral agents for HBV based on the successful preclinioccur each year as a direct result of chronic HBV infection cal studies of ribozymes against HIV infections (for due to either cirrhosis or primary liver cancer. Presently review see Refs 7 and 8). An anti-HBV ribozyme can the only partially effective treatment for hepatitis B is potentially be multifunctional, targeting both the pregeninterferon-␣, which is effective in only less than half of omic RNA as well as the viral mRNAs (see Figure 1a). all patients.In addition, by targeting the ribozymes to small highly One recently investigated approach to the treatment of conserved regions of the viral genome, the possibility of HBV infection has been the utilization of antise...

Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

“…In addition, in vitro cleavage of several different short RNA transcripts by a comparable ribozyme library confirmed the randomness (data not shown). Plasmid pLHPM-BR1 (expressing a ribozyme directed against human hepatitis B virus), pLHPM-CR2A (directed against position 323 within the HCV 5Ј-UTR), as well as target validation ribozymes were constructed by annealing of overlapping ribozyme-specific oligonucleotides as described (11). Plasmids gag-pol (retroviral helper functions) and VSV-G (vesicular stomatitis virus G-protein) for retroviral production were a kind gift of Ted Friedman (University of California, San Diego).…”

“…The neomycin-phosphotransferase gene confers resistance against G418. The retroviral library plasmid pLHPM was constructed by inserting a ClaI-XhoI fragment (containing a cassette with tRNA val promoter and the sequence of an unrelated Rz) into pLNL-PUR-HCV vector (10) in place of BstBI-XhoI. The library insert was generated by annealing three chemically synthesized and 5Ј-end phosphorylated oligonucleotides Lib1, 5Ј-cgcgtaccaggtaatataccacaacgtgtgtttctctggtnnnnttctnnnnnnnnggatcctgtttccgcccggttt-3Ј, Lib2 5Ј-cgttgtggtatattacctggta-3Ј, and Lib3 5Ј-cgaaaccgggcggaaacagg-3Ј (IDT, Coralville, IA).…”

Gene therapy as an alternative to liver transplantation

“…55 Hammerhead ribozymes also have been used to inhibit hepatitis B replication. The messenger RNA (mRNA) for hepatitis X protein, which is implicated in viral genomic integration and development of hepatocellular carcinoma, was successfully targeted and reduced in cultured cells.…”

The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA

“…11,17 The native sCYMV1 ribozyme was prepared with loop 3 replaced by a thermostable tetraloop (tetraloop form in Figure 3). The specific tetraloop used was the 12 nt sequence GGAC(UUCG)GUCC determined to form an exceptionally thermostable stem/loop structure in solution.…”

“…6 The sTRSV hairpin ribozyme prefers to cleave substrates containing a BN*GUC sequence where B is any nucleotide except A and * is the site of cleavage. 7,8 By using these targeting rules, the sTRSV system has been successfully applied to down-regulate gene expression in HIV-1, 6,9,10 hepatitis B virus, 11 hepatitis C virus 12 and human papillomavirus. 13 In this report we determined the sCYMV1-based hairpin ribozyme can be engineered to cleave heterologous substrates as well.…”