96 shRNAs designed for maximal coverage of HIV-1 variants
Identifieur interne : 001387 ( Pmc/Checkpoint ); précédent : 001386; suivant : 00138896 shRNAs designed for maximal coverage of HIV-1 variants
Auteurs : Glen John Mcintyre [Australie] ; Jennifer Lynne Groneman [Australie] ; Yi-Hsin Yu [Australie] ; Angel Jaramillo [Australie] ; Sylvie Shen [Australie] ; Tanya Lynn Applegate [Australie]Source :
- Retrovirology [ 1742-4690 ] ; 2009.
Abstract
The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains.
We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 – 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains.
In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.
Url:
DOI: 10.1186/1742-4690-6-55
PubMed: 19497094
PubMed Central: 2698899
Affiliations:
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains.</p>
</sec>
<sec><title>Results</title>
<p>We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 – 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains.</p>
</sec>
<sec><title>Conclusion</title>
<p>In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.</p>
</sec>
</div>
</front>
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Retrovirology</journal-id>
<journal-title>Retrovirology</journal-title>
<issn pub-type="epub">1742-4690</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
</publisher>
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<article-meta><article-id pub-id-type="pmid">19497094</article-id>
<article-id pub-id-type="pmc">2698899</article-id>
<article-id pub-id-type="publisher-id">1742-4690-6-55</article-id>
<article-id pub-id-type="doi">10.1186/1742-4690-6-55</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research</subject>
</subj-group>
</article-categories>
<title-group><article-title>96 shRNAs designed for maximal coverage of HIV-1 variants</article-title>
</title-group>
<contrib-group><contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Mcintyre</surname>
<given-names>Glen John</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>glen@madebyglen.com</email>
</contrib>
<contrib id="A2" contrib-type="author"><name><surname>Groneman</surname>
<given-names>Jennifer Lynne</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>j_groneman@hotmail.com</email>
</contrib>
<contrib id="A3" contrib-type="author"><name><surname>Yu</surname>
<given-names>Yi-Hsin</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>yyu11@its.jnj.com</email>
</contrib>
<contrib id="A4" contrib-type="author"><name><surname>Jaramillo</surname>
<given-names>Angel</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>a.jaramillo@unsw.edu.au</email>
</contrib>
<contrib id="A5" contrib-type="author"><name><surname>Shen</surname>
<given-names>Sylvie</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>swshen@optusnet.com.au</email>
</contrib>
<contrib id="A6" contrib-type="author"><name><surname>Applegate</surname>
<given-names>Tanya Lynn</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>tanya.applegate@gmail.com</email>
</contrib>
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<aff id="I1"><label>1</label>
Johnson and Johnson Research Pty Ltd, Level 4 Biomedical Building, 1 Central Avenue, Australian Technology Park, Eveleigh, NSW, 1430, Australia</aff>
<pub-date pub-type="collection"><year>2009</year>
</pub-date>
<pub-date pub-type="epub"><day>4</day>
<month>6</month>
<year>2009</year>
</pub-date>
<volume>6</volume>
<fpage>55</fpage>
<lpage>55</lpage>
<ext-link ext-link-type="uri" xlink:href="http://www.retrovirology.com/content/6/1/55"></ext-link>
<history><date date-type="received"><day>2</day>
<month>3</month>
<year>2009</year>
</date>
<date date-type="accepted"><day>4</day>
<month>6</month>
<year>2009</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2009 Mcintyre et al; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2009</copyright-year>
<copyright-holder>Mcintyre et al; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0"></ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
<pmc-comment>
Mcintyre
John
Glen
glen@madebyglen.com
96 shRNAs designed for maximal coverage of HIV-1 variants
2009 Retrovirology 6(1): 55-. (2009) 1742-4690(2009)6:1<55> urn:ISSN:1742-4690 </pmc-comment>
</license>
</permissions>
<abstract><sec><title>Background</title>
<p>The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains.</p>
</sec>
<sec><title>Results</title>
<p>We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 – 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains.</p>
</sec>
<sec><title>Conclusion</title>
<p>In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.</p>
</sec>
</abstract>
</article-meta>
</front>
</pmc>
<affiliations><list><country><li>Australie</li>
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<tree><country name="Australie"><noRegion><name sortKey="Mcintyre, Glen John" sort="Mcintyre, Glen John" uniqKey="Mcintyre G" first="Glen John" last="Mcintyre">Glen John Mcintyre</name>
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<name sortKey="Applegate, Tanya Lynn" sort="Applegate, Tanya Lynn" uniqKey="Applegate T" first="Tanya Lynn" last="Applegate">Tanya Lynn Applegate</name>
<name sortKey="Groneman, Jennifer Lynne" sort="Groneman, Jennifer Lynne" uniqKey="Groneman J" first="Jennifer Lynne" last="Groneman">Jennifer Lynne Groneman</name>
<name sortKey="Jaramillo, Angel" sort="Jaramillo, Angel" uniqKey="Jaramillo A" first="Angel" last="Jaramillo">Angel Jaramillo</name>
<name sortKey="Shen, Sylvie" sort="Shen, Sylvie" uniqKey="Shen S" first="Sylvie" last="Shen">Sylvie Shen</name>
<name sortKey="Yu, Yi Hsin" sort="Yu, Yi Hsin" uniqKey="Yu Y" first="Yi-Hsin" last="Yu">Yi-Hsin Yu</name>
</country>
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