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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

Minigene therapeutic approaches with U1 adaptations to correct the pathogenic effect of different donor ss mutations on theHGSNATgene. (A), (D) and (G) Schematic illustrations of base pairing between the wild type U1 (U1-WT) and the 5’ ss of wild type and mutant exons 2, 6 and 15 of the HGSNAT gene. The position of each mutation in the 5’ ss is marked in grey and it is in italics. The different U1 snRNAs used for each mutated 5’ ss of HGSNAT (designated as U1-sup, for suppressor) are also shown. The U1 sequence changes are illustrated in bold. (B), (C), (E), (F), (H) and (I) RT-PCR analysis of COS-7 cells in which splicing reporter minigenes for each mutation were co-transfected with the different modified U1 constructs as appropriate. Neither the U1-WT nor the various adapted U1s showed an effect on the splicing of exon 2, 6 or 15 wild type minigenes (B, E and H). In the case of the mutant minigenes, some of the U1 co-transfections [U1-sup2, 3 and 4 (C); U1-sup6 (F); U1-sup7, 8 and 9 (I)] changed the splicing pattern, not to the correct one, but mainly towards an alternative pattern using a “gt” 5’ ss immediately downstream of the constitutive one. Importantly, the wild type minigene for exon 2 (Mini WT ex2) shows an extra lower band due to the amount of empty vector added to adjust the total quantity of DNA. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control.
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Fig2: Minigene therapeutic approaches with U1 adaptations to correct the pathogenic effect of different donor ss mutations on theHGSNATgene. (A), (D) and (G) Schematic illustrations of base pairing between the wild type U1 (U1-WT) and the 5’ ss of wild type and mutant exons 2, 6 and 15 of the HGSNAT gene. The position of each mutation in the 5’ ss is marked in grey and it is in italics. The different U1 snRNAs used for each mutated 5’ ss of HGSNAT (designated as U1-sup, for suppressor) are also shown. The U1 sequence changes are illustrated in bold. (B), (C), (E), (F), (H) and (I) RT-PCR analysis of COS-7 cells in which splicing reporter minigenes for each mutation were co-transfected with the different modified U1 constructs as appropriate. Neither the U1-WT nor the various adapted U1s showed an effect on the splicing of exon 2, 6 or 15 wild type minigenes (B, E and H). In the case of the mutant minigenes, some of the U1 co-transfections [U1-sup2, 3 and 4 (C); U1-sup6 (F); U1-sup7, 8 and 9 (I)] changed the splicing pattern, not to the correct one, but mainly towards an alternative pattern using a “gt” 5’ ss immediately downstream of the constitutive one. Importantly, the wild type minigene for exon 2 (Mini WT ex2) shows an extra lower band due to the amount of empty vector added to adjust the total quantity of DNA. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control.

Mentions: To reproduce the splicing defects in a cellular model that could be used to test U1 snRNA overexpression as a therapeutic strategy we constructed several mutant minigenes in the pSPL3 vector and expressed them in COS-7 cells. Each of the minigenes bore either one of the three mutations that affect the donor ss or the WT allele. The post-transfection cDNA analysis and sequencing revealed the skipping of exon 2 and exon 6 in the cases of the c.234 + 1G > A and c.633 + 1G > A mutant minigenes, respectively (Figure 2C,F). For the c.1542 + 4dupA minigene, the cDNA showed a band that corresponded to the skipping of exon 15 and another band that corresponded to the inclusion of this exon plus the first five nucleotides of intron 15 (Figure 2I). Meanwhile, the constructs with the WT sequences (relative to each mutation) revealed a single transcript for each of the three cases with the inclusion of exon 2, exon 6 or exon 15, respectively. These results show that the minigene-derived splicing patterns closely resemble the patterns observed in control and patients’ cDNAs obtained from fibroblasts. Thus, the minigenes are reliable tools to test and optimize the overexpression of modified U1 snRNAs in our attempts to correct the splicing defect.Figure 2


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)

Minigene therapeutic approaches with U1 adaptations to correct the pathogenic effect of different donor ss mutations on theHGSNATgene. (A), (D) and (G) Schematic illustrations of base pairing between the wild type U1 (U1-WT) and the 5’ ss of wild type and mutant exons 2, 6 and 15 of the HGSNAT gene. The position of each mutation in the 5’ ss is marked in grey and it is in italics. The different U1 snRNAs used for each mutated 5’ ss of HGSNAT (designated as U1-sup, for suppressor) are also shown. The U1 sequence changes are illustrated in bold. (B), (C), (E), (F), (H) and (I) RT-PCR analysis of COS-7 cells in which splicing reporter minigenes for each mutation were co-transfected with the different modified U1 constructs as appropriate. Neither the U1-WT nor the various adapted U1s showed an effect on the splicing of exon 2, 6 or 15 wild type minigenes (B, E and H). In the case of the mutant minigenes, some of the U1 co-transfections [U1-sup2, 3 and 4 (C); U1-sup6 (F); U1-sup7, 8 and 9 (I)] changed the splicing pattern, not to the correct one, but mainly towards an alternative pattern using a “gt” 5’ ss immediately downstream of the constitutive one. Importantly, the wild type minigene for exon 2 (Mini WT ex2) shows an extra lower band due to the amount of empty vector added to adjust the total quantity of DNA. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Minigene therapeutic approaches with U1 adaptations to correct the pathogenic effect of different donor ss mutations on theHGSNATgene. (A), (D) and (G) Schematic illustrations of base pairing between the wild type U1 (U1-WT) and the 5’ ss of wild type and mutant exons 2, 6 and 15 of the HGSNAT gene. The position of each mutation in the 5’ ss is marked in grey and it is in italics. The different U1 snRNAs used for each mutated 5’ ss of HGSNAT (designated as U1-sup, for suppressor) are also shown. The U1 sequence changes are illustrated in bold. (B), (C), (E), (F), (H) and (I) RT-PCR analysis of COS-7 cells in which splicing reporter minigenes for each mutation were co-transfected with the different modified U1 constructs as appropriate. Neither the U1-WT nor the various adapted U1s showed an effect on the splicing of exon 2, 6 or 15 wild type minigenes (B, E and H). In the case of the mutant minigenes, some of the U1 co-transfections [U1-sup2, 3 and 4 (C); U1-sup6 (F); U1-sup7, 8 and 9 (I)] changed the splicing pattern, not to the correct one, but mainly towards an alternative pattern using a “gt” 5’ ss immediately downstream of the constitutive one. Importantly, the wild type minigene for exon 2 (Mini WT ex2) shows an extra lower band due to the amount of empty vector added to adjust the total quantity of DNA. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control.
Mentions: To reproduce the splicing defects in a cellular model that could be used to test U1 snRNA overexpression as a therapeutic strategy we constructed several mutant minigenes in the pSPL3 vector and expressed them in COS-7 cells. Each of the minigenes bore either one of the three mutations that affect the donor ss or the WT allele. The post-transfection cDNA analysis and sequencing revealed the skipping of exon 2 and exon 6 in the cases of the c.234 + 1G > A and c.633 + 1G > A mutant minigenes, respectively (Figure 2C,F). For the c.1542 + 4dupA minigene, the cDNA showed a band that corresponded to the skipping of exon 15 and another band that corresponded to the inclusion of this exon plus the first five nucleotides of intron 15 (Figure 2I). Meanwhile, the constructs with the WT sequences (relative to each mutation) revealed a single transcript for each of the three cases with the inclusion of exon 2, exon 6 or exon 15, respectively. These results show that the minigene-derived splicing patterns closely resemble the patterns observed in control and patients’ cDNAs obtained from fibroblasts. Thus, the minigenes are reliable tools to test and optimize the overexpression of modified U1 snRNAs in our attempts to correct the splicing defect.Figure 2

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