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96 shRNAs designed for maximal coverage of HIV-1 variants.

McIntyre GJ, Groneman JL, Yu YH, Jaramillo A, Shen S, Applegate TL - Retrovirology (2009)

Bottom Line: 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.Assay performance was improved by dividing large targets into several shorter domains.Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd, Australian Technology Park, Eveleigh, NSW, Australia. glen@madebyglen.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Impact on HIV-1 expression. Anti-HIV-1 suppressive activity was tested by transfecting each shRNA expression plasmid with the NL4-3 expression plasmid. Activities were measured as a reduction in, and expressed as percentage of, p24 production (measured as pg/ml) relative to p24 production from the unsuppressed control (shown in blue). The 96 shRNAs were measured in 3 sets (as indicated in purple) with 2 or more replicate experiments in each set (accessory gene shRNAs in one set, Gag plus Env shRNAs together, and Pol shRNAs separately).
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Figure 7: Impact on HIV-1 expression. Anti-HIV-1 suppressive activity was tested by transfecting each shRNA expression plasmid with the NL4-3 expression plasmid. Activities were measured as a reduction in, and expressed as percentage of, p24 production (measured as pg/ml) relative to p24 production from the unsuppressed control (shown in blue). The 96 shRNAs were measured in 3 sets (as indicated in purple) with 2 or more replicate experiments in each set (accessory gene shRNAs in one set, Gag plus Env shRNAs together, and Pol shRNAs separately).

Mentions: All shRNAs were also tested in the NL4-3 HIV-1 expression assay. In this assay, shRNA expression plasmids were co-transfected with the pNL4-3 HIV-1 expression plasmid and total p24 levels were measured 2 days later. Activity was calculated as a relative percentage of the baseline p24 levels present in the plasmid backbone sample. In this assay, the average percentage p24 for the 96 hairpins was 29% of the unsuppressed control. Sixty five were classed as highly active, 16 were active and 15 were inactive (Figure 7). These groupings closely matched those from the gene fusion assay using the shorter reporters, with several exceptions. These exceptions could be due to differences in target structure and accessibility to the target site, or to influences from non-specific activities which were not distinguishable in the current format of this assay. It should also be noted that the suppressive activities for the highly active hairpins in the NL4-3 assay generally appeared greater than those observed in the gene fusion assay. However, we consider that this was likely due to differences in assay dynamics such as different shRNA to target ratios, target production times, etc. Importantly, the overall categorical and relative ranking of hairpins based on suppressive activities were similar in both assays.


96 shRNAs designed for maximal coverage of HIV-1 variants.

McIntyre GJ, Groneman JL, Yu YH, Jaramillo A, Shen S, Applegate TL - Retrovirology (2009)

Impact on HIV-1 expression. Anti-HIV-1 suppressive activity was tested by transfecting each shRNA expression plasmid with the NL4-3 expression plasmid. Activities were measured as a reduction in, and expressed as percentage of, p24 production (measured as pg/ml) relative to p24 production from the unsuppressed control (shown in blue). The 96 shRNAs were measured in 3 sets (as indicated in purple) with 2 or more replicate experiments in each set (accessory gene shRNAs in one set, Gag plus Env shRNAs together, and Pol shRNAs separately).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698899&req=5

Figure 7: Impact on HIV-1 expression. Anti-HIV-1 suppressive activity was tested by transfecting each shRNA expression plasmid with the NL4-3 expression plasmid. Activities were measured as a reduction in, and expressed as percentage of, p24 production (measured as pg/ml) relative to p24 production from the unsuppressed control (shown in blue). The 96 shRNAs were measured in 3 sets (as indicated in purple) with 2 or more replicate experiments in each set (accessory gene shRNAs in one set, Gag plus Env shRNAs together, and Pol shRNAs separately).
Mentions: All shRNAs were also tested in the NL4-3 HIV-1 expression assay. In this assay, shRNA expression plasmids were co-transfected with the pNL4-3 HIV-1 expression plasmid and total p24 levels were measured 2 days later. Activity was calculated as a relative percentage of the baseline p24 levels present in the plasmid backbone sample. In this assay, the average percentage p24 for the 96 hairpins was 29% of the unsuppressed control. Sixty five were classed as highly active, 16 were active and 15 were inactive (Figure 7). These groupings closely matched those from the gene fusion assay using the shorter reporters, with several exceptions. These exceptions could be due to differences in target structure and accessibility to the target site, or to influences from non-specific activities which were not distinguishable in the current format of this assay. It should also be noted that the suppressive activities for the highly active hairpins in the NL4-3 assay generally appeared greater than those observed in the gene fusion assay. However, we consider that this was likely due to differences in assay dynamics such as different shRNA to target ratios, target production times, etc. Importantly, the overall categorical and relative ranking of hairpins based on suppressive activities were similar in both assays.

Bottom Line: 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.Assay performance was improved by dividing large targets into several shorter domains.Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Johnson and Johnson Research Pty Ltd, Australian Technology Park, Eveleigh, NSW, Australia. glen@madebyglen.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus