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Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.

Matos L, Canals I, Dridi L, Choi Y, Prata MJ, Jordan P, Desviat LR, Pérez B, Pshezhetsky AV, Grinberg D, Alves S, Vilageliu L - Orphanet J Rare Dis (2014)

Bottom Line: Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Research and Development Unit, INSA, Porto, Portugal. liliana.matos@insa.min-saude.pt.

ABSTRACT

Background: Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.

Methods: In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.

Results: Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.

Conclusions: We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

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Related in: MedlinePlus

Enzymatic activity of mutant HGSNAT-L125_R128del protein can be partially restored by the pharmacological chaperone,glucosamine. (A) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid is significantly increased compared to that of cells transfected with the empty pcDNA plasmid (mock). The data show means (±S.D.) of individual measurements. Three transfections (each in duplicate) were performed on separate occasions.** and ***: statistically different from mock-transfected cells (p < 0.01 and p < 0.001, respectively) according to unpaired t-test. (B) COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid produce 160 kDa dimmers and 78 kDa monomers of HGSNAT precursor protein but show drastically reduced amounts of 44 kDa and 25 kDa mature HGSNAT chains produced by intra-lysosomal enzymatic cleavage. Panel shows representative data of 3 independent transfections. (C) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid and of cultured primary fibroblasts of the patient homozygous for the c.372-2A > G mutation is significantly increased after treating the cells in culture with 10 mM glucosamine for 72 h (+GA). The data show means (±S.D.) of individual measurements. Three independent experiments measurements were performed each of them with 2 cell plates. * and **: statistically different from untreated cells (p < 0.05 and p < 0.01, respectively) by unpaired t-test.
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Fig4: Enzymatic activity of mutant HGSNAT-L125_R128del protein can be partially restored by the pharmacological chaperone,glucosamine. (A) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid is significantly increased compared to that of cells transfected with the empty pcDNA plasmid (mock). The data show means (±S.D.) of individual measurements. Three transfections (each in duplicate) were performed on separate occasions.** and ***: statistically different from mock-transfected cells (p < 0.01 and p < 0.001, respectively) according to unpaired t-test. (B) COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid produce 160 kDa dimmers and 78 kDa monomers of HGSNAT precursor protein but show drastically reduced amounts of 44 kDa and 25 kDa mature HGSNAT chains produced by intra-lysosomal enzymatic cleavage. Panel shows representative data of 3 independent transfections. (C) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid and of cultured primary fibroblasts of the patient homozygous for the c.372-2A > G mutation is significantly increased after treating the cells in culture with 10 mM glucosamine for 72 h (+GA). The data show means (±S.D.) of individual measurements. Three independent experiments measurements were performed each of them with 2 cell plates. * and **: statistically different from untreated cells (p < 0.05 and p < 0.01, respectively) by unpaired t-test.

Mentions: In order to test whether mutant cDNA with a 12-nucleotide in-frame deletion (encoding p.[L125_R128del]) produces a stable protein and, if so, whether it is enzymatically active, we transfected cultured COS-7 cells with a corresponding pcDNA-HGSNAT-L125_R128del plasmid and measured N-acetyltransferase activity in the cell homogenate. Cells transfected with an empty plasmid or those transfected with the pcTAP-HGSNAT plasmid described previously [6] encoding WT human HGSNAT were used as positive and negative controls, respectively. Our data (Figure 4A) showed a small but significant increase of N-acetyltransferase activity in the cells transfected with the mutant HGSNAT compared to that in the cells transfected with the empty plasmid. The activity in the cells transfected with WT pcTAP-HGSNAT increased about 10-fold (Figure 4A).Figure 4


Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.

Matos L, Canals I, Dridi L, Choi Y, Prata MJ, Jordan P, Desviat LR, Pérez B, Pshezhetsky AV, Grinberg D, Alves S, Vilageliu L - Orphanet J Rare Dis (2014)

Enzymatic activity of mutant HGSNAT-L125_R128del protein can be partially restored by the pharmacological chaperone,glucosamine. (A) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid is significantly increased compared to that of cells transfected with the empty pcDNA plasmid (mock). The data show means (±S.D.) of individual measurements. Three transfections (each in duplicate) were performed on separate occasions.** and ***: statistically different from mock-transfected cells (p < 0.01 and p < 0.001, respectively) according to unpaired t-test. (B) COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid produce 160 kDa dimmers and 78 kDa monomers of HGSNAT precursor protein but show drastically reduced amounts of 44 kDa and 25 kDa mature HGSNAT chains produced by intra-lysosomal enzymatic cleavage. Panel shows representative data of 3 independent transfections. (C) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid and of cultured primary fibroblasts of the patient homozygous for the c.372-2A > G mutation is significantly increased after treating the cells in culture with 10 mM glucosamine for 72 h (+GA). The data show means (±S.D.) of individual measurements. Three independent experiments measurements were performed each of them with 2 cell plates. * and **: statistically different from untreated cells (p < 0.05 and p < 0.01, respectively) by unpaired t-test.
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Fig4: Enzymatic activity of mutant HGSNAT-L125_R128del protein can be partially restored by the pharmacological chaperone,glucosamine. (A) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid is significantly increased compared to that of cells transfected with the empty pcDNA plasmid (mock). The data show means (±S.D.) of individual measurements. Three transfections (each in duplicate) were performed on separate occasions.** and ***: statistically different from mock-transfected cells (p < 0.01 and p < 0.001, respectively) according to unpaired t-test. (B) COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid produce 160 kDa dimmers and 78 kDa monomers of HGSNAT precursor protein but show drastically reduced amounts of 44 kDa and 25 kDa mature HGSNAT chains produced by intra-lysosomal enzymatic cleavage. Panel shows representative data of 3 independent transfections. (C) N-acetyltransferase activity of COS-7 cells transfected with the pcDNA-HGSNAT-L125_R128del plasmid and of cultured primary fibroblasts of the patient homozygous for the c.372-2A > G mutation is significantly increased after treating the cells in culture with 10 mM glucosamine for 72 h (+GA). The data show means (±S.D.) of individual measurements. Three independent experiments measurements were performed each of them with 2 cell plates. * and **: statistically different from untreated cells (p < 0.05 and p < 0.01, respectively) by unpaired t-test.
Mentions: In order to test whether mutant cDNA with a 12-nucleotide in-frame deletion (encoding p.[L125_R128del]) produces a stable protein and, if so, whether it is enzymatically active, we transfected cultured COS-7 cells with a corresponding pcDNA-HGSNAT-L125_R128del plasmid and measured N-acetyltransferase activity in the cell homogenate. Cells transfected with an empty plasmid or those transfected with the pcTAP-HGSNAT plasmid described previously [6] encoding WT human HGSNAT were used as positive and negative controls, respectively. Our data (Figure 4A) showed a small but significant increase of N-acetyltransferase activity in the cells transfected with the mutant HGSNAT compared to that in the cells transfected with the empty plasmid. The activity in the cells transfected with WT pcTAP-HGSNAT increased about 10-fold (Figure 4A).Figure 4

Bottom Line: Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Research and Development Unit, INSA, Porto, Portugal. liliana.matos@insa.min-saude.pt.

ABSTRACT

Background: Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.

Methods: In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.

Results: Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.

Conclusions: We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

Show MeSH
Related in: MedlinePlus