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Inhibition of MDR1 expression with altritol-modified siRNAs.

Fisher M, Abramov M, Van Aerschot A, Xu D, Juliano RL, Herdewijn P - Nucleic Acids Res. (2007)

Bottom Line: Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases.Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs.These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina Chapel Hill NC 27599, USA.

ABSTRACT
Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3' end of the sense or antisense strands, while modification at the 5' end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

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Screening of ANA-modified siRNAs for activity versus MDR1. NIH 3T3-MDR cells were treated with 50 nM ORF1 sequence siRNA complexed to lipofectamine 2000 for 4 h at 37°C in complete medium. Thereafter the cells were placed in fresh medium (2% FBS/DMEM-H) and cultured for an additional 72 h. Cell surface P-glycoprotein levels were quantitated by immunostaining and flow cytometery as described in methods. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells. The positions of the ANA modifications are indicated in the figure. Results are means of three determinations. Figure 2 inset: a typical flow cytometry analysis is shown comparing Pgp levels in untreated control cells with cells treated with ANA-modified or control (unmodified) siRNAs. In this and subsequent figures unmodified anti-MDR1 siRNA is designated simply as ‘siRNA’ or ‘siRNA control’.
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Figure 2: Screening of ANA-modified siRNAs for activity versus MDR1. NIH 3T3-MDR cells were treated with 50 nM ORF1 sequence siRNA complexed to lipofectamine 2000 for 4 h at 37°C in complete medium. Thereafter the cells were placed in fresh medium (2% FBS/DMEM-H) and cultured for an additional 72 h. Cell surface P-glycoprotein levels were quantitated by immunostaining and flow cytometery as described in methods. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells. The positions of the ANA modifications are indicated in the figure. Results are means of three determinations. Figure 2 inset: a typical flow cytometry analysis is shown comparing Pgp levels in untreated control cells with cells treated with ANA-modified or control (unmodified) siRNAs. In this and subsequent figures unmodified anti-MDR1 siRNA is designated simply as ‘siRNA’ or ‘siRNA control’.

Mentions: ANA-modified strands of the ORF1 sequence were complexed with conventional RNA to form siRNA duplexes and screened for activity in NIH-3T3-MDR cells by measuring the reduction in cell surface expression of P-glycoprotein, the MDR1 gene product. As seen in Figure 2, a wide range of efficacy was observed. Modifications at the 5′ end of the antisense strand abolished activity, while extensive modification within the central region of the antisense strand also lowered activity. By contrast, modifications at the 3′ end of either the sense or antisense strand resulted in efficacies that were substantially greater than that of unmodified siRNA (for example, oligonucleotides 2392, 2193). Multiple modifications within the sense strand were also tolerated with only modest reductions in siRNA effectiveness (e.g. oligonucleotides 2312, 2313). The activity of the various modified oligonucleotides was not correlated in any way with their thermal stability; for example, multiply modified oligonucleotide 2365 had the highest Tm when complexed with unmodified RNA (73°C versus 67°C for conventional ORF1 siRNA) but had little activity (Tm data not shown).Figure 2.


Inhibition of MDR1 expression with altritol-modified siRNAs.

Fisher M, Abramov M, Van Aerschot A, Xu D, Juliano RL, Herdewijn P - Nucleic Acids Res. (2007)

Screening of ANA-modified siRNAs for activity versus MDR1. NIH 3T3-MDR cells were treated with 50 nM ORF1 sequence siRNA complexed to lipofectamine 2000 for 4 h at 37°C in complete medium. Thereafter the cells were placed in fresh medium (2% FBS/DMEM-H) and cultured for an additional 72 h. Cell surface P-glycoprotein levels were quantitated by immunostaining and flow cytometery as described in methods. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells. The positions of the ANA modifications are indicated in the figure. Results are means of three determinations. Figure 2 inset: a typical flow cytometry analysis is shown comparing Pgp levels in untreated control cells with cells treated with ANA-modified or control (unmodified) siRNAs. In this and subsequent figures unmodified anti-MDR1 siRNA is designated simply as ‘siRNA’ or ‘siRNA control’.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Figure 2: Screening of ANA-modified siRNAs for activity versus MDR1. NIH 3T3-MDR cells were treated with 50 nM ORF1 sequence siRNA complexed to lipofectamine 2000 for 4 h at 37°C in complete medium. Thereafter the cells were placed in fresh medium (2% FBS/DMEM-H) and cultured for an additional 72 h. Cell surface P-glycoprotein levels were quantitated by immunostaining and flow cytometery as described in methods. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells. The positions of the ANA modifications are indicated in the figure. Results are means of three determinations. Figure 2 inset: a typical flow cytometry analysis is shown comparing Pgp levels in untreated control cells with cells treated with ANA-modified or control (unmodified) siRNAs. In this and subsequent figures unmodified anti-MDR1 siRNA is designated simply as ‘siRNA’ or ‘siRNA control’.
Mentions: ANA-modified strands of the ORF1 sequence were complexed with conventional RNA to form siRNA duplexes and screened for activity in NIH-3T3-MDR cells by measuring the reduction in cell surface expression of P-glycoprotein, the MDR1 gene product. As seen in Figure 2, a wide range of efficacy was observed. Modifications at the 5′ end of the antisense strand abolished activity, while extensive modification within the central region of the antisense strand also lowered activity. By contrast, modifications at the 3′ end of either the sense or antisense strand resulted in efficacies that were substantially greater than that of unmodified siRNA (for example, oligonucleotides 2392, 2193). Multiple modifications within the sense strand were also tolerated with only modest reductions in siRNA effectiveness (e.g. oligonucleotides 2312, 2313). The activity of the various modified oligonucleotides was not correlated in any way with their thermal stability; for example, multiply modified oligonucleotide 2365 had the highest Tm when complexed with unmodified RNA (73°C versus 67°C for conventional ORF1 siRNA) but had little activity (Tm data not shown).Figure 2.

Bottom Line: Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases.Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs.These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina Chapel Hill NC 27599, USA.

ABSTRACT
Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3' end of the sense or antisense strands, while modification at the 5' end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5'-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

Show MeSH