96 shRNAs designed for maximal coverage of HIV-1 variants
Identifieur interne : 002824 ( Main/Exploration ); précédent : 002823; suivant : 00282596 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.
Descripteurs français
- KwdFr :
- ARN viral (antagonistes et inhibiteurs), ARN viral (génétique), Analyse de séquence d'ADN (), Données de séquences moléculaires, Gènes rapporteurs, Humains, Inactivation virale (), Infections à VIH (), Infections à VIH (génétique), Interférence par ARN, Lignée cellulaire, Petit ARN interférent (génétique), Petit ARN interférent (pharmacologie), Séquence conservée, Séquences répétées inversées, Thérapie génétique (), VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (), VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (génétique), Variation génétique.
- MESH :
- antagonistes et inhibiteurs : ARN viral.
- génétique : ARN viral, Infections à VIH, Petit ARN interférent, VIH-1 (Virus de l'Immunodéficience Humaine de type 1).
- pharmacologie : Petit ARN interférent.
- Analyse de séquence d'ADN, Données de séquences moléculaires, Gènes rapporteurs, Humains, Inactivation virale, Infections à VIH, Interférence par ARN, Lignée cellulaire, Séquence conservée, Séquences répétées inversées, Thérapie génétique, VIH-1 (Virus de l'Immunodéficience Humaine de type 1), Variation génétique.
English descriptors
- KwdEn :
- Cell Line, Conserved Sequence, Genes, Reporter, Genetic Therapy (methods), Genetic Variation, HIV Infections (genetics), HIV Infections (therapy), HIV-1 (drug effects), HIV-1 (genetics), Humans, Inverted Repeat Sequences, Molecular Sequence Data, RNA Interference, RNA, Small Interfering (genetics), RNA, Small Interfering (pharmacology), RNA, Viral (antagonists & inhibitors), RNA, Viral (genetics), Sequence Analysis, DNA (methods), Virus Inactivation (drug effects).
- MESH :
- chemical , antagonists & inhibitors : RNA, Viral.
- chemical , genetics : RNA, Small Interfering, RNA, Viral.
- drug effects : HIV-1, Virus Inactivation.
- genetics : HIV Infections, HIV-1.
- methods : Genetic Therapy, Sequence Analysis, DNA.
- chemical , pharmacology : RNA, Small Interfering.
- therapy : HIV Infections.
- Cell Line, Conserved Sequence, Genes, Reporter, Genetic Variation, Humans, Inverted Repeat Sequences, Molecular Sequence Data, RNA Interference.
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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<term>Conserved Sequence</term>
<term>Genes, Reporter</term>
<term>Genetic Therapy (methods)</term>
<term>Genetic Variation</term>
<term>HIV Infections (genetics)</term>
<term>HIV Infections (therapy)</term>
<term>HIV-1 (drug effects)</term>
<term>HIV-1 (genetics)</term>
<term>Humans</term>
<term>Inverted Repeat Sequences</term>
<term>Molecular Sequence Data</term>
<term>RNA Interference</term>
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<term>RNA, Small Interfering (pharmacology)</term>
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<term>RNA, Viral (genetics)</term>
<term>Sequence Analysis, DNA (methods)</term>
<term>Virus Inactivation (drug effects)</term>
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<term>ARN viral (génétique)</term>
<term>Analyse de séquence d'ADN ()</term>
<term>Données de séquences moléculaires</term>
<term>Gènes rapporteurs</term>
<term>Humains</term>
<term>Inactivation virale ()</term>
<term>Infections à VIH ()</term>
<term>Infections à VIH (génétique)</term>
<term>Interférence par ARN</term>
<term>Lignée cellulaire</term>
<term>Petit ARN interférent (génétique)</term>
<term>Petit ARN interférent (pharmacologie)</term>
<term>Séquence conservée</term>
<term>Séquences répétées inversées</term>
<term>Thérapie génétique ()</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) ()</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1) (génétique)</term>
<term>Variation génétique</term>
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<term>HIV-1</term>
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<term>Infections à VIH</term>
<term>Petit ARN interférent</term>
<term>VIH-1 (Virus de l'Immunodéficience Humaine de type 1)</term>
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<term>Sequence Analysis, DNA</term>
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<keywords scheme="MESH" qualifier="pharmacologie" xml:lang="fr"><term>Petit ARN interférent</term>
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<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>RNA, Small Interfering</term>
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<keywords scheme="MESH" qualifier="therapy" xml:lang="en"><term>HIV Infections</term>
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<term>Conserved Sequence</term>
<term>Genes, Reporter</term>
<term>Genetic Variation</term>
<term>Humans</term>
<term>Inverted Repeat Sequences</term>
<term>Molecular Sequence Data</term>
<term>RNA Interference</term>
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<term>Données de séquences moléculaires</term>
<term>Gènes rapporteurs</term>
<term>Humains</term>
<term>Inactivation virale</term>
<term>Infections à VIH</term>
<term>Interférence par ARN</term>
<term>Lignée cellulaire</term>
<term>Séquence conservée</term>
<term>Séquences répétées inversées</term>
<term>Thérapie génétique</term>
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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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