Inhibition of MDR1 expression with altritol-modified siRNAs

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.

Figure 1.

The chemistry and structure of altritol-modified oligonucleotides. (A) The chemical structure of ANA oligonucleotides. (B) Model of an ANA–RNA duplex; the green strand represents the ANA–modified oligonucleotide.

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’.

Figure 3.

(A) Effects of ANA modifications in both strands of the duplex. NIH 3T3-MDR cells were treated with 50 nM siRNA and monitored for Pgp expression by immunostaining and flow cytometry as in Figure 2. A comparison is shown between siRNAs with ANA modifications in one strand (gray bars = modified in sense strand, white = modified in antisense strand) duplexed with a conventional RNA complementary strand, or siRNAs with ANA modifications in both strands (hatched bars). Results are the means of triplicate determinations. (B) Effects of mismatches. ANA-modified ORF2 siRNAs (50 nM) with (2531/2532) or without (2485/2487) 4 mismatches to the target sequence were tested for inhibition of Pgp expression versus control siRNA. The 2531/2532 (sense/antisense) mismatch duplex has ANAs in exactly the same positions as 2485/2487 (2532-uuc gUa uag GuC ucU aua* c*dtdt; mismatches indicated by capital letters, positions of ANAs indicated by *). Results are the means and standard errors of triplicate determinations.

Figure 4.

Dose–response curves for ANA-modified siRNAs. NIH 3T3-MDR cells were treated with various concentrations of siRNA complexed with 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. Cells were monitored for Pgp expression by immunostaining and flow cytometry as in Figure 2. Ordinate: percentage reduction in Pgp expression versus untreated control cells. Abscissa: concentration of siRNA (nM). Results are the means of triplicate determinations.

Figure 5.

Rescue of siRNA effectiveness by 5′ phosphorylation. Antisense oligonucleotide 2361 which has ANA modifications at its 5′end (and is therefore inactive) was synthesized including a 5′-terminal phosphate group and now termed 2516-P. NIH 3T3 MDR cells were treated with 50 nM siRNAs for 4 h, washed, and then analyzed for Pgp expression after 72 h. Results are expressed as the percentage reduction in Pgp expression compared to untreated control cells (means and standard errors of 3 determinations). (Inset) Assays after long exposures. The analysis was exactly the same as above except that the siRNAs and transfection agent were left in contact with the cells for the entire 76-h period.

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.

Figure 7.

(A) Effects of siRNAs on Rh123 accumulation. NIH 3T3-MDR cells were treated with 50 nM ANA-modified or conventional siRNAs as in Figure 2. After 72 h the cells were exposed to 1 μg/ml rhodamine 123 for 1 h. The cells were washed, harvested and analyzed for Rh123 levels by flow cytometry, as described in methods. Results are normalized based on a value of 1 for untreated cells. The data represents means and standard errors for three determinations. The inset in 7A illustrates cell surface Pgp levels in the cells used in one of the Rh123 experiments. (B) Effects of siRNAs on doxorubicin toxicity. NIH 3T3-MDR cells were treated with 50 nM ANA-modified or conventional siRNAs as in Figure 2. After 72 h the cells were exposed for 24 h to various concentrations of doxorubicin. After a further 48 h, cells were harvested and cell numbers were determined using a particle counter as described in methods. Results are expressed as the percentage of the cell number in controls not treated with siRNA. Ordinate: cell number as percentage of control. Abscissa: doxorubicin concentration (nM). Results are means of triplicate determinations.

Figure 8.

Persistent effects of ANA-modified siRNAs. (A) Control and ANA-modified ORF1 oligonucleotides. NIH 3T3-MDR cells were treated with 50 nM ANA-modified or conventional ORF1 siRNAs as in Figure 2. Cells were recovered on days 4–8 after siRNA treatment and monitored for P-glycoprotein expression by immunostaining and flow cytometry. The percentage reduction in Pgp expression on day 4 was taken as 1 and the results for subsequent days were expressed as a fraction of that value. Thus a decline in the value of the ordinate represents an increase in the level of cell surface Pgp. Results are means of triplicate determinations. (B) Control and ANA-modified ORF2 oligonucleotides. The experiment was the same as in (A) except that we compared conventional ORF2 siRNA to an ANA-modified ORF2 oligonucleotide duplex (2485/2487).

Figure 9.

Nuclease stability of ANA-modified siRNAs. Standard MDR1 siRNA or the 2385/2470 ANA-modified siRNA were exposed to nuclease activity and the degradation of the siRNAs monitored by agarose gel electophoresis with ethidium bromide staining. Upper panel: exposure to 10% fetal calf serum for various times. Lower panel: exposure to various doses of micrococcal nuclease.