Limits...
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.

Show MeSH
Analysis of effects of ANA-modified siRNA by real-time PCR. NIH 3T3 MDR cells were treated with 50 nM siRNAs as in Figure 2 and cellular mRNA levels were analyzed by RT-PCR as described in methods. Ordinate: fraction of MDR1 mRNA as compared to untreated control cells. Results are means of three determinations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851637&req=5

Figure 6: Analysis of effects of ANA-modified siRNA by real-time PCR. NIH 3T3 MDR cells were treated with 50 nM siRNAs as in Figure 2 and cellular mRNA levels were analyzed by RT-PCR as described in methods. Ordinate: fraction of MDR1 mRNA as compared to untreated control cells. Results are means of three determinations.

Mentions: We examined whether the observed siRNA-mediated reductions in P-glycoprotein expression were accompanied by similar changes at the message level and by functional changes in drug transport. As seen in Figure 6, reductions MDR1 mRNA were observed that paralleled reduced expression of the protein product, suggesting that the action of the ANA-modified siRNAs is at the level of message degradation.Figure 6.


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)

Analysis of effects of ANA-modified siRNA by real-time PCR. NIH 3T3 MDR cells were treated with 50 nM siRNAs as in Figure 2 and cellular mRNA levels were analyzed by RT-PCR as described in methods. Ordinate: fraction of MDR1 mRNA as compared to untreated control cells. Results are means of three determinations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851637&req=5

Figure 6: Analysis of effects of ANA-modified siRNA by real-time PCR. NIH 3T3 MDR cells were treated with 50 nM siRNAs as in Figure 2 and cellular mRNA levels were analyzed by RT-PCR as described in methods. Ordinate: fraction of MDR1 mRNA as compared to untreated control cells. Results are means of three determinations.
Mentions: We examined whether the observed siRNA-mediated reductions in P-glycoprotein expression were accompanied by similar changes at the message level and by functional changes in drug transport. As seen in Figure 6, reductions MDR1 mRNA were observed that paralleled reduced expression of the protein product, suggesting that the action of the ANA-modified siRNAs is at the level of message degradation.Figure 6.

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