Oligonucleotide therapy for wilson disease
A technology of antisense oligonucleotides and nucleotides, applied in the field of antisense oligonucleotides for the treatment of Wilson's disease, can solve problems such as splicing effects that are not considered
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[0236] The following materials, methods, and examples are illustrative only and not intended to be limiting.
[0237] Materials and methods
[0238] In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA techniques and standard techniques of electrophoresis. See, eg, Sambrook, Fritsch, and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, Eds.: Ausubel et al., John Wiley & Sons (1992).
[0239] Oligonucleotides. All antisense oligonucleotides used were obtained from Integrated DNA Technologies Inc. (USA). All bases in the antisense oligonucleotides are 2'-O-methoxyethyl modified (MOE) with an intact phosphorothioate backbone.
[0240] Construction of the ATP7B minigene. The minigene plasmid for ATP7B exon 6 was designed to contain a fragment corresponding to exons 5 to 7 of the locus of the ATP7B gene, incl...
Embodiment 2
[0256] Example 2 Characterization of target regions (hot spots) for increased ATP7B exon 6 inclusion
[0257] To explore the possible use of splice-switching oligonucleotides as a treatment for Wilson's disease patients carrying the M645R variant, the hepatic cell line HepG2 carrying the mutation 2F3 was derived. The 2F3 line carries the M645R variant at one allele , carrying a large plasmid insert at the other allele. This insertion was postulated to render the allele incapable of producing functional protein, creating a situation similar to compound heterozygosity, consistent with that found in Wilson's disease patients.
[0258] The oligonucleotides listed in Table 2 were transfected into 2F3 cells and the RT-PCR products were analyzed using capillary electrophoresis. These oligonucleotides were designed to hybridize to the hotspots identified above and to expand the search for additional hotspots. Then, the percent splicing (PSI) of exon 6 and the change in percent splic...
Embodiment 4
[0264] Example 4 Treatment of 2F3 cells with splicing-regulating antisense oligonucleotides increases protein levels and copper tolerance
[0265] To model the effectiveness of antisense oligonucleotides that induce exon 6 inclusion as a potential treatment for Wilson's disease, an in vitro copper sensitivity assay was used. like Figure 4A As shown, the M645R mutation reduces copper tolerance in 2F3 cells, reflecting Wilson's disease in which cells are unable to process copper due to mutations in ATP7B. Transfection with an antisense oligonucleotide having the sequence described in SEQ ID 29 increased the copper tolerance of 2F3 cells, indicating that by transfecting with the oligonucleotides shown herein to increase exon 6 inclusion, at least Partial rescue of the copper-sensitive phenotype. Western blotting against ATP7B protein was consistent with these results, as 2F3 cells produced significantly less ATP7B protein than wild-type HepG2 cells ( Figure 4B ). Transfecti...
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