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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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Related in: MedlinePlus

shRNA target sites mapped to the 13 fluorescent reporters. The target sites of the 96 shRNAs were mapped to the 13 fluorescent reporters. The 4 short accessory genes and LTR reporters are shown down the left, and the 3 long core gene reporters are shown down the right. Mapped onto the long core gene reporters were the corresponding 6 short gene fragment reporters made to retest the core gene shRNAs. Lengths are to scale to show the relative distances between the target sites, fusion junctions and total reporter lengths. n.b. some shRNAs were assayed with a different reporter to that of the gene targeted due to overlapping target sequences, e.g. the Vif matched shRNAs #51 – 57 were assayed with Pol reporters.
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Figure 5: shRNA target sites mapped to the 13 fluorescent reporters. The target sites of the 96 shRNAs were mapped to the 13 fluorescent reporters. The 4 short accessory genes and LTR reporters are shown down the left, and the 3 long core gene reporters are shown down the right. Mapped onto the long core gene reporters were the corresponding 6 short gene fragment reporters made to retest the core gene shRNAs. Lengths are to scale to show the relative distances between the target sites, fusion junctions and total reporter lengths. n.b. some shRNAs were assayed with a different reporter to that of the gene targeted due to overlapping target sequences, e.g. the Vif matched shRNAs #51 – 57 were assayed with Pol reporters.

Mentions: While we observed an expected spread of suppressive activities, the average level for the shRNAs measured with the core-gene reporters was generally lower than that observed for the shRNAs measured with the accessory-gene reporters. We observed an average percentage fluorescence of 38% for Gag, 42% Pol, 52% Env reporters vs. 46% Nef-LTR, 19% Tat exons 1 and 2, 10% Vpu and 13% Rev exon 2 reporters. We also noted that the target domains for the core-gene reporters were longer than the accessory gene reporters. The lengths of the target domains ranged from 1, 503 – 3, 012 bp for the core gene reporters but only 243 – 921 bp for the accessory genes. The shRNA target sites in the long-core reporters were also generally further away from the fusion junction between the GFP and gene-target domains. A second set of shorter core-gene reporters were constructed to determine if the length of the target domain and or the distance of the target site from the fusion junction was affecting apparent suppressive activities (Figure 5). The length of these shortened reporters ranged from 425 – 650 bp and all matching hairpins were re-assayed.


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

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

shRNA target sites mapped to the 13 fluorescent reporters. The target sites of the 96 shRNAs were mapped to the 13 fluorescent reporters. The 4 short accessory genes and LTR reporters are shown down the left, and the 3 long core gene reporters are shown down the right. Mapped onto the long core gene reporters were the corresponding 6 short gene fragment reporters made to retest the core gene shRNAs. Lengths are to scale to show the relative distances between the target sites, fusion junctions and total reporter lengths. n.b. some shRNAs were assayed with a different reporter to that of the gene targeted due to overlapping target sequences, e.g. the Vif matched shRNAs #51 – 57 were assayed with Pol reporters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: shRNA target sites mapped to the 13 fluorescent reporters. The target sites of the 96 shRNAs were mapped to the 13 fluorescent reporters. The 4 short accessory genes and LTR reporters are shown down the left, and the 3 long core gene reporters are shown down the right. Mapped onto the long core gene reporters were the corresponding 6 short gene fragment reporters made to retest the core gene shRNAs. Lengths are to scale to show the relative distances between the target sites, fusion junctions and total reporter lengths. n.b. some shRNAs were assayed with a different reporter to that of the gene targeted due to overlapping target sequences, e.g. the Vif matched shRNAs #51 – 57 were assayed with Pol reporters.
Mentions: While we observed an expected spread of suppressive activities, the average level for the shRNAs measured with the core-gene reporters was generally lower than that observed for the shRNAs measured with the accessory-gene reporters. We observed an average percentage fluorescence of 38% for Gag, 42% Pol, 52% Env reporters vs. 46% Nef-LTR, 19% Tat exons 1 and 2, 10% Vpu and 13% Rev exon 2 reporters. We also noted that the target domains for the core-gene reporters were longer than the accessory gene reporters. The lengths of the target domains ranged from 1, 503 – 3, 012 bp for the core gene reporters but only 243 – 921 bp for the accessory genes. The shRNA target sites in the long-core reporters were also generally further away from the fusion junction between the GFP and gene-target domains. A second set of shorter core-gene reporters were constructed to determine if the length of the target domain and or the distance of the target site from the fusion junction was affecting apparent suppressive activities (Figure 5). The length of these shortened reporters ranged from 425 – 650 bp and all matching hairpins were re-assayed.

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