Methods for treating spinal muscular atrophy

摘要

Described herein are methods for the identification or validation of compounds capable of causing ribosomal frameshifting and the use of the compounds identified by the methods described herein to produce a stabilized SMNΔEx7 protein and treat Spinal Muscular Atrophy.

主权项

1. A method for the identification of a compound that produces a stabilized SMNΔEx7 protein comprising:
(A) contacting a compound with either a host cell containing an mRNA transcript transcribed from a nucleic acid construct, or a composition comprising a cell-free extract and an mRNA transcript transcribed from a nucleic acid construct, wherein the nucleic acid construct comprises, in 5′ to 3′ order:
(a) a start codon;(b) a fragment of the nucleic acid residues of exon 8 of SMN; and(c) a reporter gene coding sequence lacking a start codon, wherein (i) the reporter gene coding sequence is fused to the fragment of the nucleic acid residues of exon 8 of SMN such that the first codon of the reporter gene coding sequence and the first codon of the fragment are out of frame with each other in the mRNA transcript transcribed from the nucleic acid construct and a stop codon is upstream of the reporter gene coding sequence in the mRNA transcript; and (ii) the start codon and the stop codon upstream of the reporter gene coding sequence in the mRNA transcript are in the same contiguous open reading frame; (B) detecting the activity or amount of a fusion protein translated from the mRNA transcript, wherein an increase in the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a compound when compared to:
(i) a previously determined reference range for a negative control,(ii) the activity or amount of the fusion protein translated from the mRNA transcript in the absence of the compound, or(iii) the activity or amount of the fusion protein translated from the mRNA transcript in the presence of a negative control indicates that the compound produces a stabilized SMNΔEx7 protein; and (C) contacting a compound that increases the activity or amount of the fusion protein translated from the mRNA transcript with a cell that produces SMNΔEx7 protein and assaying the ability of the compound to increase the level of stabilized SMNΔEx7 protein in the cell in the presence of the compound as compared to the level of SMNΔEx7 protein in the cell in the presence of the compound, wherein an increase in the level of stabilized SMNΔEx7 protein relative to the level of SMNΔEx7 protein indicates that a compound that produces a stabilized SMNΔEx7 protein is identified, wherein said stabilized SMNΔEx7 protein comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.