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.

Fig. 1.

Comparison of phenotype, genotype, and protein expression for two alleles of calamity. (A) calamity^vu69, characterized in ref. , displays the copper-deficient phenotype of loss of pigmentation, notochord abnormalities (arrowheads), and an enlarged ventricle and is due to a loss of function of atp7a. (B) calamity^gw246, a second allele of atp7a, has the same phenotype. (C) cal^gw246 fails to complement cal^vu69, confirming allelism. (D) Amino acid sequence changes caused by the mutations. cal^vu69 results in a frameshift and truncation of the C terminus of the protein. cal^gw246 contains an in-frame deletion in the highly conserved ATPase domain of Atp7a. (E) Immunoblot analysis using an antibody made to a C-terminal peptide from zebrafish Atp7a shows complete loss of WT protein in cal^vu69, cal^gw246, and in the compound heterozygote. (F) The mRNA and genomic sequences of cal^vu69 (Upper) and cal^gw246 (Lower), highlighting the mutation and subsequent changes in amino acid sequence due to missplicing of exons through the aberrant use of mutant splice sites (red boxes). Exon boundaries in the mRNA are illustrated by a change in color from magenta to blue. In the genomic sequence, introns are in lowercase and the mutations are the bases with the white background. The green boxes delineate the WT splice donor/acceptor pair and red boxes the mutant pair. For cal^gw246, a small percentage of transcripts splice at the WT splice junction retaining the nonsynonymous mutation, a Leu to Arg amino acid substitution (*).

Fig. 2.

A series of morpholinos causes alterations of splicing in cal^gw246. (A) Schematic and sequence information of the morpholino design for exploring alterations in splicing caused by variable morpholino placement with respect to WT and mutant splice donors. Exon sequence is uppercase. The mutation is red. Lines indicate the binding site of each listed morpholino. (B) Injection of the series of morpholinos in A into cal^gw246. Splice forms were amplified by PCR of cDNA using the primers illustrated in A. The WT and three alternate splice forms were seen on polyacrylamide gel stained with ethidium bromide. Each was sequenced to identify exact locations of splicing that are illustrated in A. All cryptic splice forms were located within exon 20.

Fig. 3.

Morpholinos rescue the phenotype of cal^vu69 embryos. (A) Schematic and sequence information for the design of morpholinos directed toward the rescue of the mutant splicing in cal^vu69. The WT exon is uppercase. The mutation is highlighted in red, and the WT splice acceptor is in green. Each morpholino-binding site is indicated by a line located 5′ to the polypyrimidine tract. Morpholinos that cause phenotypic rescue are highlighted in pink. Morpholino i1 served as a control to demonstrate that blocking of proper splicing at this exon–exon boundary would result in the calamity phenotype (data not shown). (B and C) Side-by-side comparisons of zebrafish 48 h after fertilization. (Top) WT sibling from cal^vu69 intercross clutch. (Bottom) cal^vu69/vu69-mutant embryo. (Middle) cal^vu69/vu69 embryo rescued by injection of morpholino i3. (D and E) Close-up view of cal^vu69/vu69-uninjected embryo showing lack of melanin pigmentation over head region and notochord abnormalities (arrowhead). (F and G) Close-up view of morpholino i3-rescued cal^vu69/vu69 embryo showing restoration of melanin pigmentation and a notochord absent of defects. (H) Quantification of the extent of rescue. A single group of embryos derived from several clutches, half injected with rescuing morpholino (red bars; uninjected, blue bars), was scored for the presence of any or a significant number of melanocytes (>20) and the presence of notochord defects. The scale of the graph reflects the expected 25% ratio of homozygous mutants derived from the heterozygous intercross. (I and J) Six days after fertilization, embryos were either uninjected (I) or injected (J) with rescuing morpholino i3.5. Rescued embryos maintain near-normal body morphology, whereas unrescued embryos swell, possibly because of decreased extracellular matrix integrity due to the loss of lysyl oxidase activity.

Fig. 4.

Rescue morpholinos cause restoration of full-length protein without detectable changes in mRNA. (A) Phenotypic rescue by morpholinos i3 and i3.5 is accompanied by the restoration of the WT protein. Forty-eight hours after fertilization, lysates from embryos of the indicated genotype were run on a SDS/6% PAGE gel and blotted for zebrafish Atp7a by using an antibody generated to the zebrafish protein. β-catenin was used as a loading control. Morpholino injection restored protein to 30–45% of WT levels in this experiment. (B) qRT-PCR using primers specific for WT and mutant atp7a transcript were used to measure the amounts of each in single embryos. Normalization of WT transcript was to cal^+/+ embryos and mutant transcript to cal^−/− embryos. Error bars indicate one standard deviation around the mean. (C) Polyacrylamide gel of RT-PCR products reveals a band containing 800 bp of intronic sequence caused by morpholino injection. This band was sequenced and represents use of a cryptic 3′ splice site located within the intron. Also seen are mutant and WT transcripts in uninjected and rescued embryos. A nonspecific band was present in both uninjected and injected mutant embryos (*).