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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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Inhibition of ATX-3 expression in GM06151 patient-derived fibroblasts by ss-siRNAs with one or more mismatched bases. (A) List of ss-siRNAs. Subscript ‘s’ indicates PS linkage; Green, 2′-F; Blue, 2′-O-Me; Orange, 2′-MOE. The terminal T has a 5′ phosphate. All other sugars are ribose and all other linkages are phosphate. (B) Effects of ss-siRNAs tested at 25 nM. Effect on ATX-3 expression of increasing concentrations of (C) ISIS 553822, (D) ISIS 557409, (E) a duplex RNA with no chemically modified bases or PS linkages analogous to ISIS 557409 and (F) ISIS 557426. Western analysis data (C–F) is representative of triplicate experiments that were averaged to yield IC50 and selectivity values. CM: non-complementary duplex RNA.
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gkt693-F2: Inhibition of ATX-3 expression in GM06151 patient-derived fibroblasts by ss-siRNAs with one or more mismatched bases. (A) List of ss-siRNAs. Subscript ‘s’ indicates PS linkage; Green, 2′-F; Blue, 2′-O-Me; Orange, 2′-MOE. The terminal T has a 5′ phosphate. All other sugars are ribose and all other linkages are phosphate. (B) Effects of ss-siRNAs tested at 25 nM. Effect on ATX-3 expression of increasing concentrations of (C) ISIS 553822, (D) ISIS 557409, (E) a duplex RNA with no chemically modified bases or PS linkages analogous to ISIS 557409 and (F) ISIS 557426. Western analysis data (C–F) is representative of triplicate experiments that were averaged to yield IC50 and selectivity values. CM: non-complementary duplex RNA.

Mentions: We screened additional ss-siRNAs to help understand how placement of mismatches affected potency and allele selectivity. Single mismatches were systematically placed throughout the ss-siRNAs or introduced in groups of two or three (Figure 2A). All compounds contained a 5′-phosphate rather than a 5′-(E)-vinyl phosphonate to simplify the synthesis of multiple compounds. Previously, we had tested these ss-siRNAs for inhibiting expression of HTT and identified many compounds with allele selectivities from 5 to greater than 30-fold selectivity (12).Figure 2.


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)

Inhibition of ATX-3 expression in GM06151 patient-derived fibroblasts by ss-siRNAs with one or more mismatched bases. (A) List of ss-siRNAs. Subscript ‘s’ indicates PS linkage; Green, 2′-F; Blue, 2′-O-Me; Orange, 2′-MOE. The terminal T has a 5′ phosphate. All other sugars are ribose and all other linkages are phosphate. (B) Effects of ss-siRNAs tested at 25 nM. Effect on ATX-3 expression of increasing concentrations of (C) ISIS 553822, (D) ISIS 557409, (E) a duplex RNA with no chemically modified bases or PS linkages analogous to ISIS 557409 and (F) ISIS 557426. Western analysis data (C–F) is representative of triplicate experiments that were averaged to yield IC50 and selectivity values. CM: non-complementary duplex RNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt693-F2: Inhibition of ATX-3 expression in GM06151 patient-derived fibroblasts by ss-siRNAs with one or more mismatched bases. (A) List of ss-siRNAs. Subscript ‘s’ indicates PS linkage; Green, 2′-F; Blue, 2′-O-Me; Orange, 2′-MOE. The terminal T has a 5′ phosphate. All other sugars are ribose and all other linkages are phosphate. (B) Effects of ss-siRNAs tested at 25 nM. Effect on ATX-3 expression of increasing concentrations of (C) ISIS 553822, (D) ISIS 557409, (E) a duplex RNA with no chemically modified bases or PS linkages analogous to ISIS 557409 and (F) ISIS 557426. Western analysis data (C–F) is representative of triplicate experiments that were averaged to yield IC50 and selectivity values. CM: non-complementary duplex RNA.
Mentions: We screened additional ss-siRNAs to help understand how placement of mismatches affected potency and allele selectivity. Single mismatches were systematically placed throughout the ss-siRNAs or introduced in groups of two or three (Figure 2A). All compounds contained a 5′-phosphate rather than a 5′-(E)-vinyl phosphonate to simplify the synthesis of multiple compounds. Previously, we had tested these ss-siRNAs for inhibiting expression of HTT and identified many compounds with allele selectivities from 5 to greater than 30-fold selectivity (12).Figure 2.

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