Use of Tissue-Specific MicroRNA to Control Pathology of Wild-Type Adenovirus without Attenuation of Its Ability to Kill Cancer Cells
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{"title"=>"Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells", "type"=>"journal", "authors"=>[{"first_name"=>"Ryan", "last_name"=>"Cawood", "scopus_author_id"=>"24537130100"}, {"first_name"=>"Hannah H.", "last_name"=>"Chen", "scopus_author_id"=>"26022747300"}, {"first_name"=>"Fionnadh", "last_name"=>"Carroll", "scopus_author_id"=>"26648167200"}, {"first_name"=>"Miriam", "last_name"=>"Bazan-Peregrino", "scopus_author_id"=>"24069939600"}, {"first_name"=>"Nico", "last_name"=>"Van Rooijen", "scopus_author_id"=>"35428581800"}, {"first_name"=>"Leonard W.", "last_name"=>"Seymour", "scopus_author_id"=>"7102949097"}], "year"=>2009, "source"=>"PLoS Pathogens", "identifiers"=>{"issn"=>"15537366", "sgr"=>"67249084276", "scopus"=>"2-s2.0-67249084276", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "pui"=>"354743721", "doi"=>"10.1371/journal.ppat.1000440", "pmid"=>"19461878"}, "id"=>"0bcf4228-0761-314e-ad6e-f601cfd6c69d", "abstract"=>"Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.", "link"=>"http://www.mendeley.com/research/tissuespecific-microrna-control-pathology-wildtype-adenovirus-without-attenuation-ability-kill-cance", "reader_count"=>70, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Researcher"=>22, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>17, "Student > Postgraduate"=>2, "Student > Master"=>10, "Other"=>7, "Student > Bachelor"=>2, "Professor"=>3, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Researcher"=>22, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>17, "Student > Postgraduate"=>2, "Student > Master"=>10, "Other"=>7, "Student > Bachelor"=>2, "Professor"=>3, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>37, "Medicine and Dentistry"=>12, "Chemistry"=>3, "Computer Science"=>2, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>12}, "Chemistry"=>{"Chemistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>37}, "Computer Science"=>{"Computer Science"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>4}}, "reader_count_by_country"=>{"United States"=>3, "China"=>1, "Denmark"=>1, "South Africa"=>1, "Germany"=>2}, "group_count"=>2}

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  • {"files"=>["https://ndownloader.figshare.com/files/896588"], "description"=>"<p>pCIK-Lux (referred to as pCMV-Luc) was cleaved with Not1 and concatamers of mir-122 binding sites (4 or 8 sense, or 4 antisense; the sequence of the 4 sense insert is shown at the top of the figure) inserted into the luciferase 3′UTR. Both pCMV-Luc and the version containing 4 microRNA sites (pCMV-Luc-mir) were modified with the C terminal half of E1A expression cassette, isolated from pAd5WT (Ad5 wild-type) by PCR. Both resulting constructs were then cloned into pAd5Kpn1, which contains the E1A promoter and coding sequence, to produce E1A promoter regulated E1A-luciferase fusion constructs termed pE1A-Luc and pE1A-Luc-mir.</p>", "links"=>[], "tags"=>["microbiology/applied microbiology"], "article_id"=>567040, "categories"=>["Microbiology"], "users"=>["Ryan Cawood", "Hannah H. Chen", "Fionnadh Carroll", "Miriam Bazan-Peregrino", "Nico Van Rooijen", "Leonard W. Seymour"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000440.g001", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Plasmid_construction_/567040", "title"=>"Plasmid construction.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:49:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/896653"], "description"=>"<p>Cells were seeded in triplicate in 12 well plates. After 24 h 0.5 µg of plasmid DNA (containing 0 (black), 4 (light grey) or 8 (white) sense mir-122 binding sites, or 4 antisense binding sites (dark grey)) was mixed with 2.5 ul DOTAP (Roche) reagent. 24 h following transfection cells were lysed and relative luminescence was measured using 25 µl cell lysate. N = 3, Error bars +/−standard deviation and data is shown as RLU/µg cell protein, determined by BCA assay. (** P<0.005).</p>", "links"=>[], "tags"=>["microrna", "binding", "sites", "cmv", "promoter", "driven", "luciferase", "plasmids"], "article_id"=>567104, "categories"=>["Microbiology"], "users"=>["Ryan Cawood", "Hannah H. Chen", "Fionnadh Carroll", "Miriam Bazan-Peregrino", "Nico Van Rooijen", "Leonard W. Seymour"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000440.g002", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_microRNA_binding_sites_on_expression_of_CMV_promoter_driven_luciferase_plasmids_in_vitro_/567104", "title"=>"Effects of microRNA binding sites on expression of CMV promoter driven luciferase plasmids <i>in vitro</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:49:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/897189"], "description"=>"<p>(A) Measurement of serum ALT (black bars) and AST (grey bars) 72 h following intravenous administration of 5×10<sup>10</sup> viral particles of wild-type Ad5 and Ad5-mir122. Analysis was performed as described in the <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#s4\" target=\"_blank\">Methods</a> section. (B) Adenovirus genomes in murine liver were measured by real time PCR 72 h following intravenous administration of 5×10<sup>10</sup> viral particles of wild-type Ad5 WT and Ad5-mir122, as described in the <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#s4\" target=\"_blank\">Methods</a> section. (C) Assessment of liver histology. The left liver lobe from each mouse was immersed in 10% buffered formalin overnight at room temperature, embedded in wax and sectioned using a vibratome. Sections were stained with haematoxylin and eosin and analysed by light microscopy at ×40 magnification. Mice were treated with PBS, non-replicating E1, E3-deleted Ad5 expressing GFP (Ad5-GFP), wild-type Ad5, or wild-type Ad5 modified to contain 4 mir-122 binding sites, as indicated. (*** P<0.0005).</p>", "links"=>[], "tags"=>["hepatotoxicity", "wild-type", "ad5", "modified", "microrna", "binding"], "article_id"=>567642, "categories"=>["Microbiology"], "users"=>["Ryan Cawood", "Hannah H. Chen", "Fionnadh Carroll", "Miriam Bazan-Peregrino", "Nico Van Rooijen", "Leonard W. Seymour"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000440.g006", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assessment_of_hepatotoxicity_of_wild_type_Ad5_modified_with_microRNA_binding_sites_/567642", "title"=>"Assessment of hepatotoxicity of wild-type Ad5 modified with microRNA binding sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:52:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/896737"], "description"=>"<p>(A) Imaging luminescence (8 h from mice administered pCMV-Luc not containing (left panel) and containing (right panel) four binding sites for mir-122 (plasmids pCMV-Luc and pCMV-Luc-mir in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#ppat-1000440-g001\" target=\"_blank\">Figure 1</a>).The animal on the right side of all images is a control treated with PBS, but mock injected with luciferin, used to provide a background reading. (B) Imaging luminescence (8 h from mice administered pE1A-Luc fusion constructs not containing (left panel) and containing (right panel) four binding sites for microRNA122a (plasmids pE1A-Luc and pE1A-luc-mir in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#ppat-1000440-g001\" target=\"_blank\">Figure 1</a>). The animal on the right is an untreated control. The two images in A are directly comparable with each other, as also the two images in B; however scaling is different between (A) and (B) in order to accommodate substantially different signal intensities from these plasmids. (C) Time course of luciferase expression from CMV promoter-driven and E1A promoter driven constructs shown in A and B. Black = 8 h, Grey = 24 h, White = 48 h. n = 4 throughout (except pCMV-Luc-mir122 where n = 3), error bars show standard deviation.</p>", "links"=>[], "tags"=>["binding", "sites", "microrna122a", "plasmids", "vivo", "hydrodynamic"], "article_id"=>567188, "categories"=>["Microbiology"], "users"=>["Ryan Cawood", "Hannah H. Chen", "Fionnadh Carroll", "Miriam Bazan-Peregrino", "Nico Van Rooijen", "Leonard W. Seymour"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000440.g003", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_including_binding_sites_for_microRNA122a_on_expression_of_plasmids_in_vivo_following_hydrodynamic_delivery_to_mice_/567188", "title"=>"Effects of including binding sites for microRNA122a on expression of plasmids in vivo following hydrodynamic delivery to mice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:49:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/896849"], "description"=>"<p>(A) Structures of viruses engineered and used in this study. (i) Ad5-WT (ii) Four tandem repeats of binding sites for mir-122 were inserted into the 3′ UTR of E1A. (iii) Luciferase coding sequence was inserted into Ad5 to generate a fusion with the E1A coding sequence. (iv) Four tandem repeats of binding sites for mir-122 were inserted into 3′ UTR of luciferase in the virus shown in iii. (B) A549 cells were seeded at 5×10<sup>4</sup> cells per well and transfected with pre-mir122 (Ambion) or pre-mir negative control (Ambion). Immediately following transfection Ad-E1A-Luc-mir122 was added at 10 vp/cell in 450 µl DMEM media (10% FCS). 18 h later, 30 pmol/well of pre-cursor mir122 and negative control precursor microRNAs were added to each well in addition to the 500 µl described above. Luciferase readings were performed at 24 h (p = <0.0005). (C–E) Time course of luciferase expression of Ad5-E1A-Luc (solid squares) and Ad5-E1A-Luc-mir (open squares) in mir-122-negative OVCAR3 (C) and A549 (D) cells, and in mir-122-positive Huh7 cells (E) in vitro. (*** P<0.0005).</p>", "links"=>[], "tags"=>["e1a"], "article_id"=>567311, "categories"=>["Microbiology"], "users"=>["Ryan Cawood", "Hannah H. Chen", "Fionnadh Carroll", "Miriam Bazan-Peregrino", "Nico Van Rooijen", "Leonard W. Seymour"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000440.g004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Regulation_of_E1A_expression_/567311", "title"=>"Regulation of E1A expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:50:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/897039"], "description"=>"<p>Groups of four mice were administered intravenously 5×10<sup>10</sup> virus particles of Ad5-E1A-Luc (left hand group of each pair of images) or Ad5-E1A-Luc-mir122 (right hand group of each pair) and luminescence was quantified using an Ivis100 imaging system after 6 h–96 h. The mouse on the right of all images is an untreated control, mock injected with luciferin for background levels. Images within pairs can be directly compared, although the scaling is different between time points (see scale bars for details). The graph summarises the expression profile as a function of time.</p>", "links"=>[], "tags"=>["transgene", "intravenous", "viruses", "mice"], "article_id"=>567498, "categories"=>["Microbiology"], "users"=>["Ryan Cawood", "Hannah H. Chen", "Fionnadh Carroll", "Miriam Bazan-Peregrino", "Nico Van Rooijen", "Leonard W. Seymour"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1000440.g005", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Luciferase_transgene_expression_following_intravenous_administration_of_reporter_viruses_to_mice_in_vivo_/567498", "title"=>"Luciferase transgene expression following intravenous administration of reporter viruses to mice <i>in vivo</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 05:51:28"}

PMC Usage Stats | Further Information

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