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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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Conservations and the 22 targeted regions. The position and length of each gene in NL4-3 is shown above the corresponding position in the conservation graph for NL4-3. Conservations were calculated for 8, 846 19 mers from the NL4-3 gene sets against 24.8 million 19 mers in the corresponding HIV-1 variant gene sets, and plotted as conservations for all subtypes, and the clade B only subtypes. The NL4-3 genomic co-ordinates for the starting position of the 22 regions covering the 96 selected targets are given below the conservation graphs. The 22 regions and their component targets (divided into the targets made as hairpins with 20 and 21 bp stems) are shown at the bottom. Region numbers in red are the regions that have overlapping gene targets (e.g. region 1 hairpins include #1, #95 and #96, and target both the LTR and Nef). Regions 1, 2 and 3 within the LTR are repeated in light grey to indicate their overlapping target positions.
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Figure 2: Conservations and the 22 targeted regions. The position and length of each gene in NL4-3 is shown above the corresponding position in the conservation graph for NL4-3. Conservations were calculated for 8, 846 19 mers from the NL4-3 gene sets against 24.8 million 19 mers in the corresponding HIV-1 variant gene sets, and plotted as conservations for all subtypes, and the clade B only subtypes. The NL4-3 genomic co-ordinates for the starting position of the 22 regions covering the 96 selected targets are given below the conservation graphs. The 22 regions and their component targets (divided into the targets made as hairpins with 20 and 21 bp stems) are shown at the bottom. Region numbers in red are the regions that have overlapping gene targets (e.g. region 1 hairpins include #1, #95 and #96, and target both the LTR and Nef). Regions 1, 2 and 3 within the LTR are repeated in light grey to indicate their overlapping target positions.

Mentions: 96 targets were selected and came from 22 distinct regions containing highly conserved sequence (Figure 2) (Additional file 2). Fourteen of these regions were unique when compared with previously published anti-HIV siRNAs and shRNAs. There was at least one core targeted to all gene fragment sets, excluding Vpr. The 96 targets consisted of 8 to the LTR, 9 to Gag, 32 to Pol, 7 to Pol/Vif (overlapping gene targets), 7 to Tat/Rev, 12 to Vpu, 16 to Env, 2 to Env/Rev, 3 to Nef/LTR (Figure 3). The average conservation profile of the 96 cores was 65 – 67% for all clades, and 78 – 80% for the LANL clade B sequences. While the average conservations of all 5 positions in our top ranking target profiles were intentionally close, there were several profiles with as much as a 70% difference in conservation between p0 and one of the flanking positions. The conservation estimates using the Virco data set were within 2% of the corresponding LANL data set across all clades. For comparison, we calculated the conservations for 100+ previously published targets and found that less than a 1/4 were greater than 70% conserved, being on average only ~34 – 45% conserved across all clades (Additional file 1).


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

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

Conservations and the 22 targeted regions. The position and length of each gene in NL4-3 is shown above the corresponding position in the conservation graph for NL4-3. Conservations were calculated for 8, 846 19 mers from the NL4-3 gene sets against 24.8 million 19 mers in the corresponding HIV-1 variant gene sets, and plotted as conservations for all subtypes, and the clade B only subtypes. The NL4-3 genomic co-ordinates for the starting position of the 22 regions covering the 96 selected targets are given below the conservation graphs. The 22 regions and their component targets (divided into the targets made as hairpins with 20 and 21 bp stems) are shown at the bottom. Region numbers in red are the regions that have overlapping gene targets (e.g. region 1 hairpins include #1, #95 and #96, and target both the LTR and Nef). Regions 1, 2 and 3 within the LTR are repeated in light grey to indicate their overlapping target positions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Conservations and the 22 targeted regions. The position and length of each gene in NL4-3 is shown above the corresponding position in the conservation graph for NL4-3. Conservations were calculated for 8, 846 19 mers from the NL4-3 gene sets against 24.8 million 19 mers in the corresponding HIV-1 variant gene sets, and plotted as conservations for all subtypes, and the clade B only subtypes. The NL4-3 genomic co-ordinates for the starting position of the 22 regions covering the 96 selected targets are given below the conservation graphs. The 22 regions and their component targets (divided into the targets made as hairpins with 20 and 21 bp stems) are shown at the bottom. Region numbers in red are the regions that have overlapping gene targets (e.g. region 1 hairpins include #1, #95 and #96, and target both the LTR and Nef). Regions 1, 2 and 3 within the LTR are repeated in light grey to indicate their overlapping target positions.
Mentions: 96 targets were selected and came from 22 distinct regions containing highly conserved sequence (Figure 2) (Additional file 2). Fourteen of these regions were unique when compared with previously published anti-HIV siRNAs and shRNAs. There was at least one core targeted to all gene fragment sets, excluding Vpr. The 96 targets consisted of 8 to the LTR, 9 to Gag, 32 to Pol, 7 to Pol/Vif (overlapping gene targets), 7 to Tat/Rev, 12 to Vpu, 16 to Env, 2 to Env/Rev, 3 to Nef/LTR (Figure 3). The average conservation profile of the 96 cores was 65 – 67% for all clades, and 78 – 80% for the LANL clade B sequences. While the average conservations of all 5 positions in our top ranking target profiles were intentionally close, there were several profiles with as much as a 70% difference in conservation between p0 and one of the flanking positions. The conservation estimates using the Virco data set were within 2% of the corresponding LANL data set across all clades. For comparison, we calculated the conservations for 100+ previously published targets and found that less than a 1/4 were greater than 70% conserved, being on average only ~34 – 45% conserved across all clades (Additional file 1).

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