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Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

Clayton NP, Nelson CA, Weeden T, Taylor KM, Moreland RJ, Scheule RK, Phillips L, Leger AJ, Cheng SH, Wentworth BM - Mol Ther Nucleic Acids (2014)

Bottom Line: GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver.Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity.Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice.

View Article: PubMed Central - PubMed

Affiliation: Genzyme, A Sanofi Company, Framingham, Massachusetts, USA.

ABSTRACT
Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

No MeSH data available.


Related in: MedlinePlus

Gys1 mRNA levels are reduced in skeletal and cardiac muscles of Pompe mice treated with repeated intravenous injections of GS-PPMO. (a) Gel electrophoresis of semi-quantitative polymerase chain reaction (PCR) products derived from samples of RNA isolated from tissues of wild-type and Pompe mice that received the indicated treatments to determine Gys1 transcript levels. Gys1 mRNA levels (normalized to β-actin mRNA levels) were quantified in the (b) quadriceps, (c) diaphragm, and (d) heart tissues. (e) Liver was examined for the impact of GS-PPMO on Gys2 mRNA levels. Data represent mean ± SEM, n = 9–10 mice per group as indicated in the Materials and Methods. P < 0.05, (*) compared to WT, (^) compared to vehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.
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fig1: Gys1 mRNA levels are reduced in skeletal and cardiac muscles of Pompe mice treated with repeated intravenous injections of GS-PPMO. (a) Gel electrophoresis of semi-quantitative polymerase chain reaction (PCR) products derived from samples of RNA isolated from tissues of wild-type and Pompe mice that received the indicated treatments to determine Gys1 transcript levels. Gys1 mRNA levels (normalized to β-actin mRNA levels) were quantified in the (b) quadriceps, (c) diaphragm, and (d) heart tissues. (e) Liver was examined for the impact of GS-PPMO on Gys2 mRNA levels. Data represent mean ± SEM, n = 9–10 mice per group as indicated in the Materials and Methods. P < 0.05, (*) compared to WT, (^) compared to vehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.

Mentions: The selected PMO was synthesized with a 5′ primary amine to facilitate conjugation of a well-characterized arginine-rich cell penetrating sequence that had been shown previously to facilitate muscle delivery.29 This conjugated PMO (GS-PPMO) was injected through a tail vein into Pompe mice once every two weeks for a total of 12 weeks. Age-matched Pompe mice administered saline vehicle or 20 mg/kg recombinant α-glucosidase (rhGAA) on the same schedule served as treatment controls. Age-matched wild-type (C57Bl/6) mice served as untreated controls. Gel electrophoresis analysis of RT-PCR products derived from tissue extracts from Pompe mice at the end of the study showed that treatment with either 15 or 30 mg/kg GS-PPMO significantly reduced Gys1 mRNA levels in the quadriceps and diaphragm muscle (Figure 1a–c). Gys1 mRNA levels were also dramatically reduced in the heart, but only after treatment with the higher dose (Figure 1d). No significant changes were seen in the steady-state levels of Gys2 mRNA in the liver, indicating that GS-PPMO-mediated knockdown was specific for the muscle isoform of glycogen synthase (Figure 1e). As expected, treating animals with rhGAA had no impact on Gys1 mRNA levels in skeletal muscle or heart nor the Gys2 mRNA levels in liver.


Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

Clayton NP, Nelson CA, Weeden T, Taylor KM, Moreland RJ, Scheule RK, Phillips L, Leger AJ, Cheng SH, Wentworth BM - Mol Ther Nucleic Acids (2014)

Gys1 mRNA levels are reduced in skeletal and cardiac muscles of Pompe mice treated with repeated intravenous injections of GS-PPMO. (a) Gel electrophoresis of semi-quantitative polymerase chain reaction (PCR) products derived from samples of RNA isolated from tissues of wild-type and Pompe mice that received the indicated treatments to determine Gys1 transcript levels. Gys1 mRNA levels (normalized to β-actin mRNA levels) were quantified in the (b) quadriceps, (c) diaphragm, and (d) heart tissues. (e) Liver was examined for the impact of GS-PPMO on Gys2 mRNA levels. Data represent mean ± SEM, n = 9–10 mice per group as indicated in the Materials and Methods. P < 0.05, (*) compared to WT, (^) compared to vehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217081&req=5

fig1: Gys1 mRNA levels are reduced in skeletal and cardiac muscles of Pompe mice treated with repeated intravenous injections of GS-PPMO. (a) Gel electrophoresis of semi-quantitative polymerase chain reaction (PCR) products derived from samples of RNA isolated from tissues of wild-type and Pompe mice that received the indicated treatments to determine Gys1 transcript levels. Gys1 mRNA levels (normalized to β-actin mRNA levels) were quantified in the (b) quadriceps, (c) diaphragm, and (d) heart tissues. (e) Liver was examined for the impact of GS-PPMO on Gys2 mRNA levels. Data represent mean ± SEM, n = 9–10 mice per group as indicated in the Materials and Methods. P < 0.05, (*) compared to WT, (^) compared to vehicle, (#) compared to rhGAA, ($) compared to GS-PPMO at 15 mg/kg.
Mentions: The selected PMO was synthesized with a 5′ primary amine to facilitate conjugation of a well-characterized arginine-rich cell penetrating sequence that had been shown previously to facilitate muscle delivery.29 This conjugated PMO (GS-PPMO) was injected through a tail vein into Pompe mice once every two weeks for a total of 12 weeks. Age-matched Pompe mice administered saline vehicle or 20 mg/kg recombinant α-glucosidase (rhGAA) on the same schedule served as treatment controls. Age-matched wild-type (C57Bl/6) mice served as untreated controls. Gel electrophoresis analysis of RT-PCR products derived from tissue extracts from Pompe mice at the end of the study showed that treatment with either 15 or 30 mg/kg GS-PPMO significantly reduced Gys1 mRNA levels in the quadriceps and diaphragm muscle (Figure 1a–c). Gys1 mRNA levels were also dramatically reduced in the heart, but only after treatment with the higher dose (Figure 1d). No significant changes were seen in the steady-state levels of Gys2 mRNA in the liver, indicating that GS-PPMO-mediated knockdown was specific for the muscle isoform of glycogen synthase (Figure 1e). As expected, treating animals with rhGAA had no impact on Gys1 mRNA levels in skeletal muscle or heart nor the Gys2 mRNA levels in liver.

Bottom Line: GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver.Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity.Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice.

View Article: PubMed Central - PubMed

Affiliation: Genzyme, A Sanofi Company, Framingham, Massachusetts, USA.

ABSTRACT
Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

No MeSH data available.


Related in: MedlinePlus