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Targeting nuclear RNA for in vivo correction of myotonic dystrophy.

Wheeler TM, Leger AJ, Pandey SK, MacLeod AR, Nakamori M, Cheng SH, Wentworth BM, Bennett CF, Thornton CA - Nature (2012)

Bottom Line: The effect was sustained for up to 1 year after treatment was discontinued.Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus.These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, USA.

ABSTRACT
Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUG(exp)) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG(exp) RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

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Differential sensitivity of transcripts to ASO knockdown in skeletal musclea, In HSALR or FVB/n wild-type mice, ASOs targeting Srb1 (353382) or Pten (116847) were effective for knockdown in liver but not in quadriceps muscle (qRT-PCR, ± SD; n = 4 per group). * P = 0.02; *** P < 0.0001 (t-test). b,HSALR and FVB/n wild-type mice were treated with ASO 399462 targeting Malat-1, a nuclear-retained lncRNA. Levels of Malat-1 transcript in the indicated tissues were determined by qRT-PCR (± SD). (n = 4 ASO, 3 saline) * P = 0.035; ** P < 0.007; and *** P = 0.001 for ASO vs. saline (t-test). c, Dose response of Malat-1 knockdown in BALB/c wild-type mice. BALB/c wild-type mice were treated with saline or ASO 399462 targeting Malat-1 at 12.5, 25, and 50 mg/kg twice per week for 3.5 weeks (7 total doses; n = 4 per group). Tissues were collected for RNA isolation two days after the final dose. Malat-1 transcript levels were determined by qRT-PCR (± SEM). * P < 0.01; ** P < 0.001; *** P < 0.0001 (2-way ANOVA).
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Figure 3: Differential sensitivity of transcripts to ASO knockdown in skeletal musclea, In HSALR or FVB/n wild-type mice, ASOs targeting Srb1 (353382) or Pten (116847) were effective for knockdown in liver but not in quadriceps muscle (qRT-PCR, ± SD; n = 4 per group). * P = 0.02; *** P < 0.0001 (t-test). b,HSALR and FVB/n wild-type mice were treated with ASO 399462 targeting Malat-1, a nuclear-retained lncRNA. Levels of Malat-1 transcript in the indicated tissues were determined by qRT-PCR (± SD). (n = 4 ASO, 3 saline) * P = 0.035; ** P < 0.007; and *** P = 0.001 for ASO vs. saline (t-test). c, Dose response of Malat-1 knockdown in BALB/c wild-type mice. BALB/c wild-type mice were treated with saline or ASO 399462 targeting Malat-1 at 12.5, 25, and 50 mg/kg twice per week for 3.5 weeks (7 total doses; n = 4 per group). Tissues were collected for RNA isolation two days after the final dose. Malat-1 transcript levels were determined by qRT-PCR (± SEM). * P < 0.01; ** P < 0.001; *** P < 0.0001 (2-way ANOVA).

Mentions: A uniform finding in previous studies of MOE gapmer ASOs was that systemic administration failed to cause significant target reduction in muscle, despite efficient knockdown in liver (n = 12 different mRNA targets; Supplementary Table 3), raising the possibility that muscle tissue in our model is unusually susceptible to antisense silencing. We examined the functional integrity of the muscle membrane, a physiological barrier to ASO uptake24, and found that muscle penetration of the extracellular dye, Evans Blue, was similar in HSALR and wild-type mice (Supplementary Fig. 11a). Direct analysis of muscle tissue indicated that ASO accumulation was no greater in HSALR mice than in wild-type controls (Supplementary Fig. 11b, c). The mRNA level for RNase H1 likewise was similar in HSALR and WT muscle (Supplementary Fig. 12). We tested ASOs targeting other muscle-expressed transcripts. ASOs for Pten phosphatase or Srb1 scavenger receptor showed efficient target knockdown in liver, but no appreciable knockdown in HSALR or WT muscle (Fig. 3a). Taken together with previous studies, our results argue for a specific sensitivity of hACTA1-CUGexp transcripts rather than a general enhancement of ASO activity in HSALR muscle.


Targeting nuclear RNA for in vivo correction of myotonic dystrophy.

Wheeler TM, Leger AJ, Pandey SK, MacLeod AR, Nakamori M, Cheng SH, Wentworth BM, Bennett CF, Thornton CA - Nature (2012)

Differential sensitivity of transcripts to ASO knockdown in skeletal musclea, In HSALR or FVB/n wild-type mice, ASOs targeting Srb1 (353382) or Pten (116847) were effective for knockdown in liver but not in quadriceps muscle (qRT-PCR, ± SD; n = 4 per group). * P = 0.02; *** P < 0.0001 (t-test). b,HSALR and FVB/n wild-type mice were treated with ASO 399462 targeting Malat-1, a nuclear-retained lncRNA. Levels of Malat-1 transcript in the indicated tissues were determined by qRT-PCR (± SD). (n = 4 ASO, 3 saline) * P = 0.035; ** P < 0.007; and *** P = 0.001 for ASO vs. saline (t-test). c, Dose response of Malat-1 knockdown in BALB/c wild-type mice. BALB/c wild-type mice were treated with saline or ASO 399462 targeting Malat-1 at 12.5, 25, and 50 mg/kg twice per week for 3.5 weeks (7 total doses; n = 4 per group). Tissues were collected for RNA isolation two days after the final dose. Malat-1 transcript levels were determined by qRT-PCR (± SEM). * P < 0.01; ** P < 0.001; *** P < 0.0001 (2-way ANOVA).
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Figure 3: Differential sensitivity of transcripts to ASO knockdown in skeletal musclea, In HSALR or FVB/n wild-type mice, ASOs targeting Srb1 (353382) or Pten (116847) were effective for knockdown in liver but not in quadriceps muscle (qRT-PCR, ± SD; n = 4 per group). * P = 0.02; *** P < 0.0001 (t-test). b,HSALR and FVB/n wild-type mice were treated with ASO 399462 targeting Malat-1, a nuclear-retained lncRNA. Levels of Malat-1 transcript in the indicated tissues were determined by qRT-PCR (± SD). (n = 4 ASO, 3 saline) * P = 0.035; ** P < 0.007; and *** P = 0.001 for ASO vs. saline (t-test). c, Dose response of Malat-1 knockdown in BALB/c wild-type mice. BALB/c wild-type mice were treated with saline or ASO 399462 targeting Malat-1 at 12.5, 25, and 50 mg/kg twice per week for 3.5 weeks (7 total doses; n = 4 per group). Tissues were collected for RNA isolation two days after the final dose. Malat-1 transcript levels were determined by qRT-PCR (± SEM). * P < 0.01; ** P < 0.001; *** P < 0.0001 (2-way ANOVA).
Mentions: A uniform finding in previous studies of MOE gapmer ASOs was that systemic administration failed to cause significant target reduction in muscle, despite efficient knockdown in liver (n = 12 different mRNA targets; Supplementary Table 3), raising the possibility that muscle tissue in our model is unusually susceptible to antisense silencing. We examined the functional integrity of the muscle membrane, a physiological barrier to ASO uptake24, and found that muscle penetration of the extracellular dye, Evans Blue, was similar in HSALR and wild-type mice (Supplementary Fig. 11a). Direct analysis of muscle tissue indicated that ASO accumulation was no greater in HSALR mice than in wild-type controls (Supplementary Fig. 11b, c). The mRNA level for RNase H1 likewise was similar in HSALR and WT muscle (Supplementary Fig. 12). We tested ASOs targeting other muscle-expressed transcripts. ASOs for Pten phosphatase or Srb1 scavenger receptor showed efficient target knockdown in liver, but no appreciable knockdown in HSALR or WT muscle (Fig. 3a). Taken together with previous studies, our results argue for a specific sensitivity of hACTA1-CUGexp transcripts rather than a general enhancement of ASO activity in HSALR muscle.

Bottom Line: The effect was sustained for up to 1 year after treatment was discontinued.Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus.These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, USA.

ABSTRACT
Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUG(exp)) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG(exp) RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

Show MeSH
Related in: MedlinePlus