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  1. Article ; Online: A convenient method to generate and maintain poly(A)-encoding DNA sequences required for in vitro transcription of mRNA

    Patrick Arbuthnot / Abdullah Ely / Kristie Bloom

    BioTechniques, Vol 66, Iss 1, Pp 37-

    2019  Volume 39

    Abstract: Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3′ poly(A) sequence. However, ... ...

    Abstract Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3′ poly(A) sequence. However, reproducibly generating and maintaining templates from circular plasmids to have consistent lengths of the homo poly(A) sequences is challenging. The procedure reported here entails repeated restriction digestion with type IIS enzymes, ligation and circular plasmid propagation. The homopolymeric sequence of approximately 100 bp that is generated using the method is approximately equal to the number of 3′ A residues found in the mRNA of mammalian cells. Evaluating expression in vivo of a reporter transcript produced using this method showed efficient expression in vivo.
    Keywords in vitro transcription ; mRNA ; plasmid ; poly(A) ; type IIS restriction enzymes ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2019-01-01T00:00:00Z
    Publisher Future Science Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article: Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus

    Moyo, Buhle / Abdullah Ely / Kristie Bloom / Patrick Arbuthnot / Tristan Scott

    Virus research. 2018 Jan. 15, v. 244

    2018  

    Abstract: Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent ... ...

    Abstract Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas.
    Keywords antiviral agents ; circular DNA ; cultured cells ; drugs ; Francisella tularensis subsp. novicida ; gene transfer ; genes ; hepatitis B ; Hepatitis B virus ; hepatitis C ; Hepatitis C virus ; hepatoma ; mice ; RNA ; viruses
    Language English
    Dates of publication 2018-0115
    Size p. 311-320.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 605780-9
    ISSN 1872-7492 ; 0168-1702
    ISSN (online) 1872-7492
    ISSN 0168-1702
    DOI 10.1016/j.virusres.2017.01.003
    Database NAL-Catalogue (AGRICOLA)

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  3. Article: Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus

    Dreyer, Timothy / Samantha Nicholson / Abdullah Ely / Patrick Arbuthnot / Kristie Bloom

    Biochemical and biophysical research communications. 2016 Sept. 30, v. 478

    2016  

    Abstract: Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication ... ...

    Abstract Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific endonucleases and use of artificial activators of the RNA interference pathway have shown anti-HBV therapeutic promise. Although results from these gene therapies are encouraging, maximizing durable antiviral effects is important. To address this goal, a strategy that entails combining gene editing with homology-directed DNA recombination (HDR), to introduce HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported here. Previously described transcription activator-like effector nucleases (TALENs) that target the core and surface sequences of HBV were used to introduce double stranded breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted integration and the length of the flanking homologous arms of donor DNA had a minimal effect on antiviral efficiency. These results support the notion that gene editing and silencing may be combined to effect improved inhibition of HBV gene expression.
    Keywords Hepatitis B virus ; RNA interference ; antiviral properties ; circular DNA ; gene expression ; gene therapy ; genes ; hepatitis B ; microRNA ; nucleases ; sequence homology ; virus replication
    Language English
    Dates of publication 2016-0930
    Size p. 1563-1568.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 205723-2
    ISSN 0006-291X ; 0006-291X
    ISSN (online) 0006-291X
    ISSN 0006-291X
    DOI 10.1016/j.bbrc.2016.08.152
    Database NAL-Catalogue (AGRICOLA)

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