CRISPR/Cas9-mediated exon-skipping approach for USH2A-associated usher syndrome
Inventors
Pierce, Eric A. • MARGULIES, CARRIE MARIE • PENDSE, NACHIKET D. • GLOSKOWSKI, SEBASTIAN W. • Liu, Qin • Maeder, Morgan L.
Assignees
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Publication Number
US-12331328-B2
Publication Date
2025-06-17
Expiration Date
Abstract
Compositions for use in treating subjects with USH2A-associated retinal and/or cochlear degeneration that result from mutations in exon 13 of the USH2A gene by deletion of exon 13 from the USH2A gene or transcripts, and methods of use thereof, as well as genetically modified animals and cells.
Core Innovation
The invention relates to CRISPR/Cas9 exon-skipping for USH2A exon 13 mutations associated with Usher syndrome type 2 and/or autosomal recessive retinitis pigmentosa, with degeneration of the retina and/or cochlea as a disease context. It is based on removing exon 13 of the USH2A gene or enabling exon 13 removal/splice skipping so that an in-frame USH2A transcript is produced while retaining partial protein function. The approach is grounded in splice acceptor disruption and exon 13 removal to restore an altered USH2A transcript.
The core strategy uses CRISPR/Cas9 with guide RNAs that either disrupt exon 13 splice acceptor or enable exon 13 removal/splice skipping. A dual gRNA dual-cut concept is described using gRNAs targeting intron 12 and intron 13 to create a genome alteration that removes exon 13, and a single-gRNA disruption concept is also described for enabling exon 13 removal via splice acceptor disruption. The resulting edited USH2A transcript is stated to lack portions corresponding to laminin EGF-like domains while maintaining partial protein function.
The disclosed system specifies nucleic acids and genome-altering methods using a Cas9 protein and guide RNAs with defined sequence identifiers. Delivery is described using AAV-based delivery, including AAV vector elements such as ITRs and a U6 promoter, and expression is described with promoters selected from CMV, EFS, or hGRK1. Documented support includes generation and rescue of USH2A knockout OC-K1 cell models, mouse exon-skip models with retina and cochlea context and transcript changes, and off-target evaluation approaches.
Claims Coverage
The partial claim set includes two independent claims. Across these claims, the inventive coverage centers on a nucleic acid encoding Cas9 plus two specified gRNAs for exon 13 targeting, and a CRISPR-Cas9 genome-altering method that creates two double strand breaks in USH2A intron 12 and intron 13 to remove USH2A exon 13.
Dual gRNA nucleic acid encoding defined exon 13-targeting guides
A nucleic acid comprising a sequence encoding a Cas9 protein, and sequences encoding a first gRNA and a second gRNA, wherein the first and second gRNAs comprise SEQ ID NO: 54 and SEQ ID NO: 125; SEQ ID NO: 54 and SEQ ID NO: 129; SEQ ID NO: 54 and SEQ ID NO: 130; SEQ ID NO: 54 and SEQ ID NO: 134; SEQ ID NO: 54 and SEQ ID NO: 135; SEQ ID NO: 68 and SEQ ID NO: 125; SEQ ID NO: 68 and SEQ ID NO: 129; SEQ ID NO: 68 and SEQ ID NO: 130; SEQ ID NO: 68 and SEQ ID NO: 134; or SEQ ID NO: 68 and SEQ ID NO: 135, respectively.
Dual intron double-strand-break genome editing removes USH2A exon 13
A method of altering the genome of a cell comprising contacting the cell with a CRISPR-Cas9 nuclease and gRNAs to form a first double strand break within intron 12 of the human USH2A gene and a second double strand break within intron 13 of the human USH2A gene, wherein the first and second double strand breaks are then repaired by the cell, thereby removing exon 13 of the USH2A gene on human chromosome 1, and wherein the first gRNA and the second gRNA comprise SEQ ID NO: 54 and SEQ ID NO: 125; SEQ ID NO: 54 and SEQ ID NO: 129; SEQ ID NO: 54 and SEQ ID NO: 130; SEQ ID NO: 54 and SEQ ID NO: 134; SEQ ID NO: 54 and SEQ ID NO: 135; SEQ ID NO: 68 and SEQ ID NO: 125; SEQ ID NO: 68 and SEQ ID NO: 129; SEQ ID NO: 68 and SEQ ID NO: 130; SEQ ID NO: 68 and SEQ ID NO: 134; or SEQ ID NO: 68 and SEQ ID NO: 135, respectively.
Across the independent claims, the coverage is defined by specified Cas9/gRNA combinations that target intron 12 and intron 13 of the human USH2A gene to produce repair outcomes that remove exon 13, and by corresponding nucleic acids that encode the Cas9 protein and the two defined gRNAs.
Stated Advantages
Enables exon 13 removal/splice skipping to produce an in-frame USH2A transcript lacking portions of laminin EGF-like domains while retaining partial protein function.
Restores an altered USH2A transcript as part of a CRISPR/Cas9 exon-skipping approach for USH2A exon 13 mutations associated with Usher syndrome type 2 and/or autosomal recessive retinitis pigmentosa.
Documented Applications
Treatment or therapeutic use for subjects with Usher Syndrome type 2 or autosomal recessive retinitis pigmentosa caused by a mutation in USH2A exon 13, by administering a nucleic acid as defined in the claims.
A genome-altering method applied to a mammalian eye or inner ear cell, including retinal cell or photoreceptor cell, with specified narrowing to cone, rod, or macular cone photoreceptor cells.
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