Limits...
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.

Show MeSH

Related in: MedlinePlus

Suppressive activities measured with fluorescent reporters. Suppressive activities were screened using gene-specific fluorescent fusion reporters in a transient expression assay. The 96 hairpins were individually transfected with a target-specific GFP fusion reporter, and an AsRed-1 non-specific reporter. Specific activity is shown as percentage fluorescence of the unsuppressed control (black bars above; control shown in blue), and non-specific activity is shown as the fold difference (normalization factor) relative to the baseline non-specific activity of the unsuppressed control (red bars below, control shown in blue). E.g. a normalization factor of 1 is no non-specific activity, a value of 2 is twice as much non-specific activity of the baseline control (set at 1). Categorical markers divide the hairpins into inactive, active and highly active groups. The reporter used for each hairpin (green bars), and the 22 targeted regions (brown bars) are also shown. (a) Suppressive activities measured with the first round 'long' (core genes) and 'short' (accessory genes) reporters. (b) The previous long reporters were replaced with a series of shorter fragment reporters and suppressive activities were remeasured. The suppressive activities form the previous short accessory gene reporters are included for comparison (hollow bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2698899&req=5

Figure 4: Suppressive activities measured with fluorescent reporters. Suppressive activities were screened using gene-specific fluorescent fusion reporters in a transient expression assay. The 96 hairpins were individually transfected with a target-specific GFP fusion reporter, and an AsRed-1 non-specific reporter. Specific activity is shown as percentage fluorescence of the unsuppressed control (black bars above; control shown in blue), and non-specific activity is shown as the fold difference (normalization factor) relative to the baseline non-specific activity of the unsuppressed control (red bars below, control shown in blue). E.g. a normalization factor of 1 is no non-specific activity, a value of 2 is twice as much non-specific activity of the baseline control (set at 1). Categorical markers divide the hairpins into inactive, active and highly active groups. The reporter used for each hairpin (green bars), and the 22 targeted regions (brown bars) are also shown. (a) Suppressive activities measured with the first round 'long' (core genes) and 'short' (accessory genes) reporters. (b) The previous long reporters were replaced with a series of shorter fragment reporters and suppressive activities were remeasured. The suppressive activities form the previous short accessory gene reporters are included for comparison (hollow bars).

Mentions: The average suppressive activity across the 96 hairpins was 63%; i.e. the presence of the shRNA reduced the average level of fluorescence to 37% of the unsuppressed control (Figure 4a). Twenty two hairpins were highly active (> 75% suppressive activity), 56 were active (between 50 and 75% activity) and 18 were inactive (< 50% activity). Non-specific activities varied widely and mostly enhanced the fluorescence levels, but did not appear to correlate to suppressive activity. While the mechanism and significance of non-specific signal enhancement is not known, it is a phenomenon that we have commonly observed and have previously determined to be sequence-specific, dose-dependent and highly reproducible [44].


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

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

Suppressive activities measured with fluorescent reporters. Suppressive activities were screened using gene-specific fluorescent fusion reporters in a transient expression assay. The 96 hairpins were individually transfected with a target-specific GFP fusion reporter, and an AsRed-1 non-specific reporter. Specific activity is shown as percentage fluorescence of the unsuppressed control (black bars above; control shown in blue), and non-specific activity is shown as the fold difference (normalization factor) relative to the baseline non-specific activity of the unsuppressed control (red bars below, control shown in blue). E.g. a normalization factor of 1 is no non-specific activity, a value of 2 is twice as much non-specific activity of the baseline control (set at 1). Categorical markers divide the hairpins into inactive, active and highly active groups. The reporter used for each hairpin (green bars), and the 22 targeted regions (brown bars) are also shown. (a) Suppressive activities measured with the first round 'long' (core genes) and 'short' (accessory genes) reporters. (b) The previous long reporters were replaced with a series of shorter fragment reporters and suppressive activities were remeasured. The suppressive activities form the previous short accessory gene reporters are included for comparison (hollow bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Suppressive activities measured with fluorescent reporters. Suppressive activities were screened using gene-specific fluorescent fusion reporters in a transient expression assay. The 96 hairpins were individually transfected with a target-specific GFP fusion reporter, and an AsRed-1 non-specific reporter. Specific activity is shown as percentage fluorescence of the unsuppressed control (black bars above; control shown in blue), and non-specific activity is shown as the fold difference (normalization factor) relative to the baseline non-specific activity of the unsuppressed control (red bars below, control shown in blue). E.g. a normalization factor of 1 is no non-specific activity, a value of 2 is twice as much non-specific activity of the baseline control (set at 1). Categorical markers divide the hairpins into inactive, active and highly active groups. The reporter used for each hairpin (green bars), and the 22 targeted regions (brown bars) are also shown. (a) Suppressive activities measured with the first round 'long' (core genes) and 'short' (accessory genes) reporters. (b) The previous long reporters were replaced with a series of shorter fragment reporters and suppressive activities were remeasured. The suppressive activities form the previous short accessory gene reporters are included for comparison (hollow bars).
Mentions: The average suppressive activity across the 96 hairpins was 63%; i.e. the presence of the shRNA reduced the average level of fluorescence to 37% of the unsuppressed control (Figure 4a). Twenty two hairpins were highly active (> 75% suppressive activity), 56 were active (between 50 and 75% activity) and 18 were inactive (< 50% activity). Non-specific activities varied widely and mostly enhanced the fluorescence levels, but did not appear to correlate to suppressive activity. While the mechanism and significance of non-specific signal enhancement is not known, it is a phenomenon that we have commonly observed and have previously determined to be sequence-specific, dose-dependent and highly reproducible [44].

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