Methods for treating spinal muscular atrophy
The present invention provides nucleic acid constructs, methods for identifying and validating compounds that increase the inclusion of exon 7 of SMN2 into mRNA transcribed from the SMN2 gene, compounds and pharmaceutical compositions that increase levels of SMN protein produced from the SMN2 gene, and methods for use thereof in treating of SMA.
1. A nucleic acid construct comprising a minigene which comprises, in 5′ to 3′ order: a start codon, the nucleic acid residues of exon 6 of human survival motor neuron (SMN), the nucleic acid residues of intron 6 of human SMN, the nucleic acid residues of exon 7 of human SMN2, the nucleic acid residues of intron 7 of human SMN, a fragment of exon 8 of human SMN, and the nucleic acid residues of the coding sequence of a reporter gene lacking a start codon, wherein a single adenine residue is inserted after nucleic acid residue 48 and before nucleic acid residue 49 of the nucleic acid residues of exon 7 of human SMN2, and wherein the first codon of the coding sequence of the reporter gene and the start codon of the minigene are in the same open reading frame.
2. The nucleic acid construct of claim 1 , wherein the fragment of exon 8 of human SMN consists of 23 nucleic acid residues from the 5′ end of exon 8 of human SMN.
3. The nucleic acid construct of claim 1 , wherein the coding sequence of the reporter gene encodes firefly luciferase, renilla luciferase, click beetle luciferase, a genetically modified luciferase, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, blue fluorescent protein, beta-galactosidase, beta-glucoronidase, beta-lactamase, chloramphenicol acetyltransferase or alkaline phosphatase.
4. The nucleic acid construct of claim 2 , wherein the coding sequence of the reporter gene encodes firefly luciferase, renilla luciferase, click beetle luciferase, a genetically modified luciferase, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, blue fluorescent protein, beta-galactosidase, beta-glucoronidase, beta-lactamase, chloramphenicol acetyltransferase or alkaline phosphatase.
5. An isolated host cell containing the nucleic acid construct of any one of claims 1 , 2 , 3 or 4 .
6. A method for the identification of a compound that increases the inclusion of exon 7 of human SMN2 into mRNA transcribed from the human SMN2 gene comprising:
(a) contacting a compound with the host cell of claim 5 ; and
(b) detecting the activity or amount of a fusion protein encoded by the minigene,
wherein an increase in the activity or amount of the fusion protein expressed by the host cell in the presence of the compound relative to the activity or amount of the fusion protein expressed by the host cell in the absence of the compound or the presence of a negative control compound, or relative to a previously determined reference range indicates that the compound increases the inclusion of exon 7 of human SMN2 into mRNA transcribed from the human SMN2 gene.
7. A method for the identification of a compound that increases the inclusion of exon 7 of human SMN2 into mRNA transcribed from the human SMN2 gene comprising:
(a) contacting a compound with a composition comprising a cell-free extract and a pre-mRNA transcript encoded by the minigene of the nucleic acid construct of claim 1 ; and
(b) detecting the activity or amount of a fusion protein encoded by the minigene, wherein an increase in the activity or amount of the fusion protein expressed by the host cell in the presence of the compound relative to the activity or amount of the fusion protein expressed by the host cell in the absence of the compound or the presence of a negative control compound, or relative to a previously determined reference range indicates that the compound that increases the inclusion of exon 7 of human SMN2 into mRNA transcribed from the human SMN2 gene.