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ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy.

Liu J, Yu D, Aiba Y, Pendergraff H, Swayze EE, Lima WF, Hu J, Prakash TP, Corey DR - Nucleic Acids Res. (2013)

Bottom Line: We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity.We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing.These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA and Department of Medicinal Chemistry and Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.

ABSTRACT
Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

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A benchmark ss-siRNA is an allele-selective inhibitor of ATX-3 expression in GM06151 patient-derived fibroblasts. (A) Structures of chemically modified bases and PS linkages in ss-siRNA. Underlined bases are mismatched relative to the CAG repeat. Subscript ‘s’ indicates PS linkage; Green, 2′-Fluoro; Blue, 2′-O-methyl; Orange, 2′-O-methoxyethyl. All other sugars are ribose and all other linkages are phosphate. (B) Sequence and inhibitory effect of ss-siRNA ISIS 537775 on protein or (C) RNA expression. Error bars on ATX-3 mRNA levels are standard deviations (SD) from independent replicate data. Western analysis data are representative of triplicate experiments. CM: non-complementary duplex RNA. siATX: positive control duplex RNA that is complementary to a sequence with ATX3 mRNA outside of the trinucleotide repeat. Statistic significance was calculated by t-test. **P < 0.01 relative to negative control CM.
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gkt693-F1: A benchmark ss-siRNA is an allele-selective inhibitor of ATX-3 expression in GM06151 patient-derived fibroblasts. (A) Structures of chemically modified bases and PS linkages in ss-siRNA. Underlined bases are mismatched relative to the CAG repeat. Subscript ‘s’ indicates PS linkage; Green, 2′-Fluoro; Blue, 2′-O-methyl; Orange, 2′-O-methoxyethyl. All other sugars are ribose and all other linkages are phosphate. (B) Sequence and inhibitory effect of ss-siRNA ISIS 537775 on protein or (C) RNA expression. Error bars on ATX-3 mRNA levels are standard deviations (SD) from independent replicate data. Western analysis data are representative of triplicate experiments. CM: non-complementary duplex RNA. siATX: positive control duplex RNA that is complementary to a sequence with ATX3 mRNA outside of the trinucleotide repeat. Statistic significance was calculated by t-test. **P < 0.01 relative to negative control CM.

Mentions: In 2012, Lima and colleagues (11) discovered a pattern of phosphorothioate (PS) (Figure 1A), 2′-fluoro (2′-F), and 2′-O-methyl (2′-O-Me) modifications that yielded RNA single-strands capable of entering the protein machinery of the RNA-induced silencing complex and inhibiting gene expression with potencies approaching those of RNA duplexes. They termed these compounds single-stranded siRNAs (ss-siRNAs). Introduction of a metabolically stable 5′-(E)-vinylphosphonate moiety to mimic a natural 5′ phosphate allowed efficient gene silencing inside animals. This study showed that iterative design optimization could achieve dramatic improvements in the properties of single-stranded RNA.Figure 1.


ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy.

Liu J, Yu D, Aiba Y, Pendergraff H, Swayze EE, Lima WF, Hu J, Prakash TP, Corey DR - Nucleic Acids Res. (2013)

A benchmark ss-siRNA is an allele-selective inhibitor of ATX-3 expression in GM06151 patient-derived fibroblasts. (A) Structures of chemically modified bases and PS linkages in ss-siRNA. Underlined bases are mismatched relative to the CAG repeat. Subscript ‘s’ indicates PS linkage; Green, 2′-Fluoro; Blue, 2′-O-methyl; Orange, 2′-O-methoxyethyl. All other sugars are ribose and all other linkages are phosphate. (B) Sequence and inhibitory effect of ss-siRNA ISIS 537775 on protein or (C) RNA expression. Error bars on ATX-3 mRNA levels are standard deviations (SD) from independent replicate data. Western analysis data are representative of triplicate experiments. CM: non-complementary duplex RNA. siATX: positive control duplex RNA that is complementary to a sequence with ATX3 mRNA outside of the trinucleotide repeat. Statistic significance was calculated by t-test. **P < 0.01 relative to negative control CM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814390&req=5

gkt693-F1: A benchmark ss-siRNA is an allele-selective inhibitor of ATX-3 expression in GM06151 patient-derived fibroblasts. (A) Structures of chemically modified bases and PS linkages in ss-siRNA. Underlined bases are mismatched relative to the CAG repeat. Subscript ‘s’ indicates PS linkage; Green, 2′-Fluoro; Blue, 2′-O-methyl; Orange, 2′-O-methoxyethyl. All other sugars are ribose and all other linkages are phosphate. (B) Sequence and inhibitory effect of ss-siRNA ISIS 537775 on protein or (C) RNA expression. Error bars on ATX-3 mRNA levels are standard deviations (SD) from independent replicate data. Western analysis data are representative of triplicate experiments. CM: non-complementary duplex RNA. siATX: positive control duplex RNA that is complementary to a sequence with ATX3 mRNA outside of the trinucleotide repeat. Statistic significance was calculated by t-test. **P < 0.01 relative to negative control CM.
Mentions: In 2012, Lima and colleagues (11) discovered a pattern of phosphorothioate (PS) (Figure 1A), 2′-fluoro (2′-F), and 2′-O-methyl (2′-O-Me) modifications that yielded RNA single-strands capable of entering the protein machinery of the RNA-induced silencing complex and inhibiting gene expression with potencies approaching those of RNA duplexes. They termed these compounds single-stranded siRNAs (ss-siRNAs). Introduction of a metabolically stable 5′-(E)-vinylphosphonate moiety to mimic a natural 5′ phosphate allowed efficient gene silencing inside animals. This study showed that iterative design optimization could achieve dramatic improvements in the properties of single-stranded RNA.Figure 1.

Bottom Line: We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity.We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing.These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA and Department of Medicinal Chemistry and Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.

ABSTRACT
Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

Show MeSH
Related in: MedlinePlus