Dataset Information

In vivo correction of a Menkes disease model using antisense oligonucleotides.

ABSTRACT: Although the molecular basis of many inherited metabolic diseases has been defined, the availability of effective therapies in such disorders remains problematic. Menkes disease is a fatal neurodegenerative disorder due to loss-of-function mutations in the ATP7A gene encoding a copper-transporting P-type Atpase. To develop therapeutic approaches in affected patients, we have identified a zebrafish model of Menkes disease termed calamity that results from splicing defects in the zebrafish orthologue of the ATP7A gene. Embryonic-recessive lethal mutants have impaired copper homeostasis that results in absent melanin pigmentation, impaired notochord formation, and hindbrain neurodegeneration. In this current study, we have attempted to rescue these striking phenotypic alterations by using a series of antisense morpholino oligonucleotides directed against the splice-site junctions of two mutant calamity alleles. Our findings reveal a robust and complete correction of the copper-deficient defects of calamity in association with the generation of the WT Menkes protein in all rescued mutants. Interestingly, a quantitative analysis of atp7a-specific transcripts suggests that competitive translational regulation may account for the synthesis of WT protein in these embryos. This in vivo correction of Menkes disease through the rescue of aberrant splicing may provide therapeutic options in this fatal disease and illustrates the potential for zebrafish models of human genetic disease in the development of treatments based on the principles of interactions of synthetic oligonucleotide analogues with mRNA.

SUBMITTER: Madsen EC

PROVIDER: S-EPMC2268804 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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In vivo correction of a Menkes disease model using antisense oligonucleotides.

Madsen Erik C EC Morcos Paul A PA Mendelsohn Bryce A BA Gitlin Jonathan D JD

Proceedings of the National Academy of Sciences of the United States of America 20080303 10

Although the molecular basis of many inherited metabolic diseases has been defined, the availability of effective therapies in such disorders remains problematic. Menkes disease is a fatal neurodegenerative disorder due to loss-of-function mutations in the ATP7A gene encoding a copper-transporting P-type Atpase. To develop therapeutic approaches in affected patients, we have identified a zebrafish model of Menkes disease termed calamity that results from splicing defects in the zebrafish ortholo ...[more]

PMID: 18316734

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Project description:(1) Background: Autosomal recessive polycystic kidney disease (ARPKD) is a rare ciliopathy characterized by progressively enlarged kidneys with fusiform dilatation of the collecting ducts. Loss-of-function mutations in the PKHD1 gene, which encodes fibrocystin/polyductin, cause ARPKD; however, an efficient treatment method and drug for ARPKD have yet to be found. Antisense oligonucleotides (ASOs) are short special oligonucleotides which function to regulate gene expression and alter mRNA splicing. Several ASOs have been approved by the FDA for the treatment of genetic disorders, and many are progressing at present. We designed ASOs to verify whether ASOs mediate the correction of splicing further to treat ARPKD arising from splicing defects and explored them as a potential treatment option. (2) Methods: We screened 38 children with polycystic kidney disease for gene detection using whole-exome sequencing (WES) and targeted next-generation sequencing. Their clinical information was investigated and followed up. The PKHD1 variants were summarized and analyzed, and association analysis was carried out to analyze the relationship between genotype and phenotype. Various bioinformatics tools were used to predict pathogenicity. Hybrid minigene analysis was performed as part of the functional splicing analysis. Moreover, the de novo protein synthesis inhibitor cycloheximide was selected to verify the degraded pathway of abnormal pre-mRNAs. ASOs were designed to rescue aberrant splicing, and this was verified. (3) Results: Of the 11 patients with PKHD1 variants, all of them exhibited variable levels of complications of the liver and kidneys. We found that patients with truncating variants and variants in certain regions had a more severe phenotype. Two splicing variants of the PKHD1 genotypes were studied via the hybrid minigene assay: variants c.2141-3T>C and c.11174+5G>A. These cause aberrant splicing, and their strong pathogenicity was confirmed. We demonstrated that the abnormal pre-mRNAs produced from the variants escaped from the NMD pathway with the use of the de novo protein synthesis inhibitor cycloheximide. Moreover, we found that the splicing defects were rescued by using ASOs, which efficiently induced the exclusion of pseudoexons. (4) Conclusion: Patients with truncating variants and variants in certain regions had a more severe phenotype. ASOs are a potential drug for treating ARPKD patients harboring splicing mutations of the PKHD1 gene by correcting the splicing defects and increasing the expression of the normal PKHD1 gene.

Dataset Information

In vivo correction of a Menkes disease model using antisense oligonucleotides.

Publications

In vivo correction of a Menkes disease model using antisense oligonucleotides.

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