2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity - PubMed (original) (raw)

Xianbin Yang 2, Kshipra M Gharpure 3, Hiroto Hatakeyama 3, Martin Egli 4, Michael H McGuire 3, Archana S Nagaraja 3, Takahito M Miyake 3, Rajesha Rupaimoole 3, Chad V Pecot 5, Morgan Taylor 3, Sunila Pradeep 3, Malgorzata Sierant 6, Cristian Rodriguez-Aguayo 7, Hyun J Choi 3, Rebecca A Previs 3, Guillermo N Armaiz-Pena 3, Li Huang 8, Carlos Martinez 9, Tom Hassell 9, Cristina Ivan 10, Vasudha Sehgal 11, Richa Singhania 12, Hee-Dong Han 13, Chang Su 3, Ji Hoon Kim 14, Heather J Dalton 3, Chandra Kovvali 3, Khandan Keyomarsi 15, Nigel A J McMillan 16, Willem W Overwijk 17, Jinsong Liu 18, Ju-Seog Lee 11, Keith A Baggerly 19, Gabriel Lopez-Berestein 7, Prahlad T Ram 11, Barbara Nawrot 6, Anil K Sood 20

Affiliations

2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity

Sherry Y Wu et al. Nat Commun. 2014.

Abstract

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2'-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.

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Conflict of interest statement

Conflict of interest

X.Y. is an employee of AM Biotechnologies LLC. The remaining authors declare no competing financial interests

Figures

Figure 1

Figure 1. Chemical structures of modified siRNAs

Chemical structures of modified siRNAs used for the initial screen.

Figure 2

Figure 2. Stability and gene silencing activity of chemically modified siRNA

(a) Knockdown of EphA2 in SKOV3ip1 cells using chemically modified siRNAs (100 nM, serum-free condition). (b) Knockdown of EphA2 using selected siRNA sequences in HeyA8 cells (100 nM, serum-free condition). (c) Knockdown of EphA2 using chemically modified siRNAs in SKOV3ip1 cells (10, 20, and 50 nM, 10% FBS-containing media). (a, b, and c) Cells were treated with siRNAs for 4 h and EphA2 protein levels were examined 48 h post-transfection. (d) Stability of chemically modified siRNAs in 10% FBS at 37 °C. (e) Intracellular stability of UM and MePS2-1 modified siEphA2 in SKOV3ip1 cells (50 nM, serum-free condition). (f) Duration of EphA2 knockdown following UM and MePS2-1 modified siEphA2 treatment in SKOV3ip1 cells (50 nM, 10% FBS-containing media). [_P_-values obtained with Student’s _t_-test; *, P<0.05; **, P<0.01; ***, P<0.001; or ****, P<0.0001; compared to UM siEphA2 sequence; bars and error bars represent mean values and the corresponding S.E.M.s (n=3)].

Figure 3

Figure 3. Biophysical properties of MePS2-1 siRNA

(a) Circular dichroism spectra of Me-1, PS-1, MePS-1, PS2-1, and MePS2-1 modified siEphA2s. (b) Intracellular binding of UM, PS2-1, MePS2-1, and Me2 modified siEphA2 to Ago2 protein in SKOV3ip1 cells (50 nM, serum-free condition). (c) Ago2 association with UM, PS2-1, and MePS2-1 biotin-labeled siRNAs. Apart from mock-treated (no treatment control) sample, each sample contains the same amount of siRNA (quantified using stem loop PCR). Transfection was performed using 50 nM biotin-labeled siRNA in serum-free condition. The bottom two panels show the effect of transfection on total Ago2 level in cells. (d) Computational modeling of the siRNA:PAZ interaction. UM, MePS2-1, Me-1, and PS2-1 modified siRNAs were modeled, all shown in the same orientation. (e) Induction of IFN-α by UM and MePS2-1 modified siEphA2 in C57BL/6-derived dendritic cells (75 nM, serum-free condition). A high IFN-α-inducing siRNA sequence and CpG2216 were used as positive controls. (f) Knockdown of EphA2 protein in tumors following a single dose of siRNA-DOPC treatment in SKOV3ip1 ovarian cancer mouse model (1.25 and 2.5 μg per dose). Effect of UM-siEphA2-DOPC and MePS2-1-siEphA2-DOPC on tumor burden (g) and body weight (h) in SKOV3ip1 orthotopic ovarian cancer mouse model following 4 weeks of siRNA treatment (n=10). (i) Biodistribution of MePS2-1-siEphA2-DOPC. SiRNA levels were measured in various organs at 24 h post intraperitoneal administration. Stemloop PCR technique was employed to assess intact siRNA levels. The total amount of siRNA in each organ was measured (n=4) and was expressed as percentage of injected dose (ID). [_P_-values obtained with Student’s _t_-test; (b) **, P<0.01; ****, P<0.0001; compared to UM; (c) ****, P<0.0001; compared to respective controls; (e) **, P<0.01; ***, P<0.001; compared to UM siCon; (f, g) *, P<0.05 or **, P<0.01; compared to the corresponding control groups; bars and error bars represent mean values and the corresponding S.E.M.s (n= 2-3)].

Figure 4

Figure 4. Targeting GRAMD1B for ovarian cancer treatment

(a) A Venn diagram for identification of novel targets for the treatment of ovarian cancer. (b) Expression of GRAMD1B, RBBP6, and SLC23A1, in a panel of chemosensitive and chemoresistant ovarian cancer cell lines. (c) Protein expression of GRAMD1B in taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (HeyA8-MDR and SKOV3-TR) ovarian cancer cells (n=1). Effect of GRAMD1B downregulation on (d) cell viablity and (e) apoptosis in HeyA8-MDR cells. (f) Effect of knockdown of GRAMD1B on the sensitivity of HeyA8-MDR cells to paclitaxel or docetaxel treatment. Cell viability is expressed as a percentage of that measured in siRNA monotherapy treated wells. (g) Effect of GRAMD1B protein expression on overall survival in ovarian cancer patients (n=156). Scale bar represents 100 μm. [_P_-values obtained with (a-f) Student’s _t_-test or (g) log-rank test; (b) ***, P<0.001 or ****, P<0.0001; compared to the corresponding chemosensitive cell line; (d, e, f) *, P<0.05 or **, P<0.01; compared to cells treated with siCon; bars and error bars represent mean values and the corresponding S.E.M.s (n=3)].

Figure 5

Figure 5. Therapeutic effect of MePS2-1 siGRAMD1B in vitro and in vivo

(a) Knockdown of GRAMD1B using UM and MePS2-1 modified siRNAs (40 nM, 10% FBS-containing media) (n=1). (b) Intracellular binding of UM and MePS2-1 siGRAMD1B to Ago2 in HeyA8-MDR cells (50 nM, serum-free condition). (c) Effect of MePS2-1 siGRAMD1B on tumoral response to paclitaxel treatment in HeyA8-MDR orthotopic ovarian cancer mouse model. (d) Knockdown of GRMAD1B by MePS2-1-siGRAMD1B-DOPC in HeyA8-MDR tumors. (e) Caspase 3 activity in tumors treated with MePS2-1-siCon-DOPC or MePS2-1-siGRAMD1B-DOPC +/- paclitaxel. Scale bar represents 100 μm. (f) Effect of MePS2-1 siGRAMD1B on tumoral response to paclitaxel treatment in SKOV3-TR orthotopic ovarian cancer mouse model. [_P_-values obtained with Student’s _t_-test; (b) ***, P<0.001, compared to UM si GRAMD1B (n=3); (c, f) *, P<0.05 or **, P<0.01, compared to the corresponding control groups (n=10); (d) **, P<0.01, compared to MePS2-1-siCon-DOPC group (n=2-3); (e), ****, P<0.0001, compared to paclitaxel or MePS2-1-siGRAMD1B-DOPC monotherapy groups (n=10); bars and error bars represent mean values and the corresponding S.E.M.s].

Figure 6

Figure 6. A schematic representation of mechanisms by which MePS2 modification enhances siRNA activity

MePS2-1 modification enhances serum stabilty of siRNAs and promotes the loading of the antisense strand into RISC.

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