Effective Anti-Hepatitis B Virus Hammerhead Ribozymes Derived from Multimeric Precursors

Weinberg, Marc S.; Ely, Abdullah; Passman, Marc; Mufamadi, Steven; Arbuthnot, Patrick

doi:10.1089/oli.2006.0049

Cited by 11 publications

(6 citation statements)

References 35 publications

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“…As an initial assessment of pri-miR-31 trimer-mediated inhibition of HBV replication, Huh7 liver-derived cells were transfected with the pCH-9-3091 HBV replication competent plasmid (14), together with the panel of RNAi expression plasmids. Secreted HBV surface antigen (HBsAg), which is a reliable indicator of HBV replication in our hands (3,19,21), was determined thereafter in the culture medium (Figure 4C). Knockdown of ∼90% was achieved.…”

Section: Resultsmentioning

confidence: 91%

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles

Ely

Naidoo

Arbuthnot

2009

Nucleic Acids Research

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Expressed polycistronic microRNA (miR) cassettes have useful properties that can be utilized for RNA interference (RNAi)-based gene silencing. To advance their application we generated modular trimeric anti-hepatitis B virus (HBV) Pol II cassettes encoding primary (pri)-miR-31-derived shuttles that target three different viral genome sites. A panel of six expression cassettes, comprising each of the possible ordering combinations of the pri-miR-31 shuttles, was initially tested. Effective silencing of individual target sequences was achieved in transfected cells and transcribed pri-miR trimers generated intended guide strands. There was, however, variation in processing and silencing by each of the shuttles. In some cases the monomers’ position within the trimers influenced processing and this correlated with target silencing. Compromised efficacy could be compensated by substituting the pri-miR-31 backbone with a pri-miR-30a scaffold. Inhibition of HBV replication was achieved in vivo, and in cell culture without disruption of endogenous miR function or induction of the interferon response. A mutant HBV target sequence, with changes in one of the guide cognates, was also silenced by the trimeric cassettes. The modular nature of the cassettes together with compatibility with expression from Pol II promoters should be advantageous for gene silencing applications requiring simultaneous targeting of different sites.

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Section: Resultsmentioning

confidence: 91%

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles

Ely

Naidoo

Arbuthnot

2009

Nucleic Acids Research

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“…The HHR has gained attention as a potential anti-HIV-1 therapeutic agent, 46–49 an inhibitor of BCR-ABL1 gene expression, 50 an inhibitor of hepatitis-B virus gene expressions, 51,52 and as a tool in drug design and target discovery for other diseases. 50,53,54 The hammerhead-like motif is distributed throughout various genomes, 55 and recently a discontinuous HHR motif has been found embedded in the 3′ untranslated regions of a mammalian messenger RNA, suggesting HHR’s possible common role in posttranscriptional gene regulation. 56 …”

Section: Introductionmentioning

confidence: 99%

Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

T¹,

Wong²,

Giambaşu³

et al. 2013

Progress in Molecular Biology and Translational Science

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Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg2+ ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg2+ ion or multiple Na+ ions, one of which is also in a bridging coordination pattern. In the case of a single Mg2+ ion bound in the active site, the Mg2+ ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2′). As the reaction proceeds, the Mg2+ ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2′. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis.

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“…Studies on the regulatory mechanisms of HBV gene expression and replication are, therefore, necessary for the development of promising antiviral medications. Interventions against HBV include novel nucleic acid‐based approaches employing antisense RNA and DNA,6–8 ribozymes,9, 10 and RNA interference 11–13…”

mentioning

confidence: 99%

Requirement of the cyclic adenosine monophosphate response element‐binding protein for hepatitis B virus replication†

2008

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The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequencecomparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Conclusion: Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection. (HEPATOLOGY 2008;48:361-373.) T he World Health Organization has reported that about 4 million people worldwide are acutely infected with hepatitis B virus (HBV) each year, and there are estimated to be 350 million chronic carriers worldwide. 1 The HBV is a major cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. [1][2][3] The agents that are currently available for the treatment of chronic HBV infection are interferon-␣ and nucleos(t)ide analogs, such as lamivudine (L-2Ј,3Ј-dideoxy-thiacytidene), adefovir dipivoxil, and entecavir. 2-5 However, there are some limitations to these therapeutics: interferon-␣ has adverse effects, and nucleos(t)ide derivatives, although safe and efficacious, can lead to the emergence of resistant mutants. [2][3][4][5] Furthermore, none of these therapeutic agents can effectively eradicate the virus. 4,5 Studies on the regulatory mechanisms of HBV gene expression and replication are, therefore, necessary for the development of promising antiviral medications. Interventions against HBV include novel nucleic acid-based approaches employing antisense RNA and DNA, 6-8 ribozymes, 9,10 and RNA interference. [11][12][13]

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Effective Anti-Hepatitis B Virus Hammerhead Ribozymes Derived from Multimeric Precursors

Cited by 11 publications

References 35 publications

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles

Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

Requirement of the cyclic adenosine monophosphate response element‐binding protein for hepatitis B virus replication†

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