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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

Analysis by RT-PCR of the endogenous splicing pattern of control and patients SFCP, SFC3, SFC6 and SFC13 derived fibroblasts after transfection with different U1 isoforms. (A), (C) and (E) The constitutive splicing of exons 2, 6 and 15 of the HGSNAT gene was not altered in control fibroblasts after overexpression of U1-WT or any of the modified U1 constructs. (B) In the patients SFCP and SFC3, bearing the homozygous mutation c.234 + 1G > A, only the fully adapted U1 (2.5 μg of U1-sup4) resulted in partial correction of exon 2 skipping. The same result was obtained with 1 or 3.5 μg of the U1-sup4 construct (data not shown). (D) and (F) For patients SFC6 and SFC13, bearing genotypes c.633 + 1G >A/c.1334T > C and c.1542 + 4dupA/c.1150C > T, respectively, the transfection of 2.5 or 3.5 μg of U1-WT or any generated U1 suppressor vector did not produced any change in the endogenous aberrant splicing pattern. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control; RFP: red fluorescent protein.
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Fig3: Analysis by RT-PCR of the endogenous splicing pattern of control and patients SFCP, SFC3, SFC6 and SFC13 derived fibroblasts after transfection with different U1 isoforms. (A), (C) and (E) The constitutive splicing of exons 2, 6 and 15 of the HGSNAT gene was not altered in control fibroblasts after overexpression of U1-WT or any of the modified U1 constructs. (B) In the patients SFCP and SFC3, bearing the homozygous mutation c.234 + 1G > A, only the fully adapted U1 (2.5 μg of U1-sup4) resulted in partial correction of exon 2 skipping. The same result was obtained with 1 or 3.5 μg of the U1-sup4 construct (data not shown). (D) and (F) For patients SFC6 and SFC13, bearing genotypes c.633 + 1G >A/c.1334T > C and c.1542 + 4dupA/c.1150C > T, respectively, the transfection of 2.5 or 3.5 μg of U1-WT or any generated U1 suppressor vector did not produced any change in the endogenous aberrant splicing pattern. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control; RFP: red fluorescent protein.

Mentions: Despite the data of minigene assays showing that the three ss defects were not corrected by the expression of the different modified U1 vectors, we explored the feasibility of this approach to correct the endogenously misspliced HGSNAT transcripts through transfection of the same U1 variants in patients’ derived cell lines. The spliced transcripts obtained after transfection of each fibroblast cell line were analysed by RT-PCR (Figure 3A-F).Figure 3


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)

Analysis by RT-PCR of the endogenous splicing pattern of control and patients SFCP, SFC3, SFC6 and SFC13 derived fibroblasts after transfection with different U1 isoforms. (A), (C) and (E) The constitutive splicing of exons 2, 6 and 15 of the HGSNAT gene was not altered in control fibroblasts after overexpression of U1-WT or any of the modified U1 constructs. (B) In the patients SFCP and SFC3, bearing the homozygous mutation c.234 + 1G > A, only the fully adapted U1 (2.5 μg of U1-sup4) resulted in partial correction of exon 2 skipping. The same result was obtained with 1 or 3.5 μg of the U1-sup4 construct (data not shown). (D) and (F) For patients SFC6 and SFC13, bearing genotypes c.633 + 1G >A/c.1334T > C and c.1542 + 4dupA/c.1150C > T, respectively, the transfection of 2.5 or 3.5 μg of U1-WT or any generated U1 suppressor vector did not produced any change in the endogenous aberrant splicing pattern. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control; RFP: red fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279800&req=5

Fig3: Analysis by RT-PCR of the endogenous splicing pattern of control and patients SFCP, SFC3, SFC6 and SFC13 derived fibroblasts after transfection with different U1 isoforms. (A), (C) and (E) The constitutive splicing of exons 2, 6 and 15 of the HGSNAT gene was not altered in control fibroblasts after overexpression of U1-WT or any of the modified U1 constructs. (B) In the patients SFCP and SFC3, bearing the homozygous mutation c.234 + 1G > A, only the fully adapted U1 (2.5 μg of U1-sup4) resulted in partial correction of exon 2 skipping. The same result was obtained with 1 or 3.5 μg of the U1-sup4 construct (data not shown). (D) and (F) For patients SFC6 and SFC13, bearing genotypes c.633 + 1G >A/c.1334T > C and c.1542 + 4dupA/c.1150C > T, respectively, the transfection of 2.5 or 3.5 μg of U1-WT or any generated U1 suppressor vector did not produced any change in the endogenous aberrant splicing pattern. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control; RFP: red fluorescent protein.
Mentions: Despite the data of minigene assays showing that the three ss defects were not corrected by the expression of the different modified U1 vectors, we explored the feasibility of this approach to correct the endogenously misspliced HGSNAT transcripts through transfection of the same U1 variants in patients’ derived cell lines. The spliced transcripts obtained after transfection of each fibroblast cell line were analysed by RT-PCR (Figure 3A-F).Figure 3

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