Trophin genome editing for treating Duchenne Muscular Dystrophy (DMD)
View Patent ↗The invention relates to compositions and methods for improving a dystrophic phenotype in a human subject having myopathies, such as Duchenne Muscular Dystrophy (DMD). In one embodiment, the invention relates to compositions comprising an adenoviral vector targeting the let-7c miRNA binding sequence in 3′-UTR genome editing of the utrophin gene and methods of treatment comprising administration thereof.
1 . A method for improving a dystrophic phenotype in a human subject having Duchenne Muscular Dystrophy (DMD), the method comprising:
CRISPR-cas9 based genome editing a 3′ untranslated region (UTR) of a utrophin gene to delete a let-7c microRNA (miRNA) binding sequence in it,
wherein deletion of the let-7c miRNA binding sequence alleviates miRNA-mediated repression and upregulates utrophin expression, thereby improving the dystrophic phenotype in the human subject.
2 . The method of claim 1 , wherein the CRISPR-cas9 based genome editing comprises:
(a) constructing an adenoviral vector comprising a pair of short guide RNAs (sgRNAs), the pair of sgRNAs targeting the let-7c miRNA binding sequence in 3′-UTR for deletion; and
(b) administering the constructed adenoviral vector to the human subject.
3 . The method of claim 2 , wherein the pair of sgRNAs is sgRNA1 and sgRNA4, wherein the sgRNA1 and the sgRNA4 are each within 1-15 base pairs of the let-7c miRNA binding sequence.
4 . The method of claim 2 , wherein the pair of sgRNAs is sgRNA2 and sgRNA4 wherein the sgRNA2 and the sgRNA4 are each within 1-15 base pairs of the let-7c miRNA binding sequence.
5 . The method of claim 2 , further comprising inserting an enhanced green fluorescent protein (EGFP) gene in the constructed adenoviral vector in step (a) to EGFP-tag the constructed adenoviral vector.
6 . The method of claim 2 or 5 , wherein the constructed adenoviral vector is transfected into human stem cells prior to administration to the human subject.
7 . The method of claim 6 , wherein the stem cells are human induced pluripotent stem cells (hiPSCs).
8 . The method of claim 6 , wherein the stem cells are human muscle stem cells.
9 . The method of claim 1 , wherein the let-7c microRNA binding sequence is either SEQ ID NO: 62 or SEQ ID NO: 18.
10 . The method claim 1 , wherein the CRISPR-cas9 based genome editing further comprises deleting one or more microRNAs selected from the group consisting of miR-133b, miR-150, miR-196b, miR-206, and miR-296-5p.
11 . The method of claim 2 or 5 , wherein the constructed adenoviral vector is administered to the human subject in vivo.
12 . The method of claim 2 or 5 , wherein the constructed adenoviral vector is administered to the human subject in utero.
13 . The method of claim 1 , wherein the dystrophic phenotype is selected from the group consisting of skeletal or cardiac muscle degeneration, skeletal or cardiac muscle weakness, skeletal muscle cramps or pain, respiratory impairment, cardiomyopathy and dystrophin abnormalities in the brain.
14 . The method of claim 13 , wherein the dystrophin abnormalities in the brain are selected from the group consisting of attention focusing, verbal learning and memory and emotional interaction.
15 . A method for treating Duchenne Muscular Dystrophy (DMD) in a human subject, the method comprising:
CRISPR-cas9 based genome editing a 3′ untranslated region (UTR) of a utrophin gene to delete a let-7c microRNA (miRNA) binding sequence in it,
wherein deletion of the let-7c miRNA binding sequence alleviates miRNA-mediated repression and upregulates utrophin expression, thereby treating DMD in the human subject.
16 . The method of claim 15 , wherein the CRISPR-cas9 based genome editing comprises:
(a) constructing an adenoviral vector comprising a pair of short guide RNAs (sgRNAs), the pair of sgRNAs targeting the let-7c miRNA binding sequence in 3′-UTR for deletion; and
(b) administering the constructed adenoviral vector to the human subject.
17 . The method of claim 16 , wherein the pair of sgRNAs is sgRNA1 and sgRNA4, wherein the sgRNA1 and the sgRNA4 are each within 1-15 base pairs of the let-7c miRNA binding sequence.
18 . The method of claim 16 , wherein the pair of sgRNAs is sgRNA2 and sgRNA4 wherein the sgRNA2 and the sgRNA4 are each within 1-15 base pairs of the let-7c miRNA binding sequence.
19 . The method of claim 16 , further comprising inserting an enhanced green fluorescent protein (EGFP) gene in the constructed adenoviral vector in step (a) to EGFP-tag the constructed adenoviral vector.
20 . The method of claim 16 or 19 , wherein the constructed adenoviral vector is transfected into human stem cells prior to administration to the human subject.
21 . The method of claim 20 , wherein the stem cells are human induced pluripotent stem cells (hiPSCs).
22 . The method of claim 20 , wherein the stem cells are human muscle stem cells.
23 . The method of claim 15 , wherein the let-7c microRNA binding sequence is either SEQ ID NO: 62 or SEQ ID NO: 18.
24 . The method of claim 15 , wherein the CRISPR-cas9 based genome editing further comprises deleting one or more microRNAs selected from the group consisting of miR-133b, miR-150, miR-196b, miR-206, and miR-296-5p.
25 . The method of claim 16 or 19 , wherein the constructed adenoviral vector is administered to the human subject in vivo.
26 . The method of claim 16 or 19 , wherein the constructed adenoviral vector is administered to the human subject in utero.
27 . The method of claim 16 , wherein the treatment improves or alleviates skeletal or cardiac muscle degeneration, skeletal or cardiac muscle weakness, skeletal muscle cramps or pain, respiratory impairment, cardiomyopathy and dystrophin abnormalities in the brain.
28 . The method of claim 27 , wherein the dystrophin abnormalities in the brain are selected from the group consisting of attention focusing, verbal learning and memory and emotional interaction.