Materials and methods for treatment of usher syndrome type 2A
Inventors
Kantardzhieva, Albena • Noma, Akiko • Scaria, Abraham
Assignees
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Publication Number
US-12215316-B2
Publication Date
2025-02-04
Expiration Date
Abstract
The present application provides materials and methods for treating a patient with Usher Syndrome Type 2A, both ex vivo and in vivo; materials and methods for editing a USH2A gene in a human cell; materials and methods for editing an USH2A gene containing an IVS40 mutation; materials and methods for treating a patient with an USH2A gene containing an IVS40 mutation; and a method for deleting a sequence comprising an IVS40 mutation within a USH2A gene of a cell. The present application also provides one or more gRNAs or sgRNAs for editing an USH2A gene containing an IVS40 mutation. The present application provides a therapeutic for treating a patient with Usher Syndrome Type 2A. The present application also provides a kit for treating a patient with Usher Syndrome Type 2A.
Core Innovation
The invention relates to treating a patient with Usher Syndrome Type 2A by editing an USH2A gene containing an IVS40 mutation in eye cells of the patient. The editing introduces one or more DNA endonucleases selected from Cas9 and Cpf1 endonucleases and nickases together with one or more guide ribonucleic acids (gRNAs) comprising a spacer sequence complementary to a sequence with or near intron 40 of the USH2A gene. The spacer targets sequences within or near intron 40 to effect one or more SSBs or DSBs within or near intron 40 or the USH2A gene, resulting in correction and restoration of usherin protein function.
The described gRNA/sgRNA families define compositions by spacer sequence sets identified by specific SEQ ID NOs targeting intron 40 or sequences with or near intron 40. Multiple guide RNA compositions and guide pair combinations are defined using enumerated SEQ ID NOs, including single guide selections and first/second guide pair selections.
The document further describes chemically modified RNA, including Cas9 mRNA, to enhance translation and stability and to reduce innate immune activation. It also outlines nucleic-acid system components and delivery/administration frameworks for CRISPR-based editing in human cells, including edited human cells, iPSCs, and eye-related cell types including retinal progenitor/photoreceptor cells.
Claims Coverage
The independent claims in the provided set cover in vivo editing of an USH2A IVS40 mutation in eye cells using Cas9/Cpf1, including nickases, with intron 40-targeting gRNAs to create SSBs or DSBs and restore usherin protein function. The claims also cover sequence-defined gRNA/sgRNA compositions using listed SEQ ID NOs, including single-sequence options and paired-sequence combinations. In total, inventive features are present in 7 independent claims.
In vivo editing of USH2A IVS40 in eye cells to restore usherin function
Editing an USH2A gene containing an IVS40 mutation in eye cells of the patient by introducing one or more DNA endonucleases selected from Cas9 and Cpf1 endonucleases and nickases and one or more gRNAs comprising a spacer sequence complementary to a sequence with or near intron 40 of the USH2A gene to effect one or more SSBs or DSBs within or near intron 40 or the USH2A gene, resulting in correction and restoration of usherin protein function.
Guide RNA defined by specific SEQ ID NOs for spacer sequences
A gRNA or sgRNA comprising a nucleotide sequence set forth in SEQ ID NO: 5321, SEQ ID NO: 5323, SEQ ID NO: 5325, SEQ ID NO: 5327, or SEQ ID NO: 5328.
Guide RNA with paired SEQ ID NO combinations
A gRNA or sgRNA comprising SEQ ID NO: 5321, 5323, 5325, 5327, or 5328 together with any one of SEQ ID NOs: 5267-5269.
Treatment by administering Cas9/Cpf1 endonuclease and SEQ ID-defined single gRNA/sgRNA
Administering an endonuclease selected from Cas9 endonuclease and nickases and a Cfp1 endonuclease and a gRNA or sgRNA to eye cells of the patient, wherein the gRNA or sgRNA comprises SEQ ID NO: 5321, SEQ ID NO: 5323, SEQ ID NO: 5325, SEQ ID NO: 5327, or SEQ ID NO: 5328.
Treatment using paired first and second gRNA/sgRNA SEQ ID combinations
Administering an endonuclease selected from Cas9 and Cfp1 endonucleases and nickases and a first gRNA or sgRNA and second gRNA or sgRNA to eye cells of the patient, wherein the first and second gRNA or sgRNA comprise one of the listed SEQ ID NO pairings including 5295/5279, 5294/5300, 5295/5300, 5290/5300, 5277/5300, 5452/5449, 5453/5449, 5455/5457, 5452/5451, or 5448/5449.
The claim set centers on in vivo editing of an USH2A gene containing an IVS40 mutation in eye cells to achieve correction and restoration of usherin protein function. Coverage is provided for sequence-defined gRNA/sgRNA compositions by enumerated SEQ ID NOs and for treatment methods that administer Cas9/Cpf1 endonucleases, including nickases, with specified single gRNAs/sgRNAs or paired first/second gRNA/sgRNA combinations.
Stated Advantages
Restoration of usherin protein function.
Correction of the IVS40 mutation and restoration of usherin protein function.
Reduction of innate immune activation is described for chemically modified RNA.
Enhancement of translation and stability is described for chemically modified RNA, including Cas9 mRNA.
Documented Applications
Treating a patient with Usher Syndrome Type 2A by editing an USH2A gene containing an IVS40 mutation in eye cells.
Use of gRNA or sgRNA nucleotide sequences defined by listed SEQ ID NOs, including 5321, 5323, 5325, 5327, and 5328, as guide RNAs for the described editing approach.
Treatment methods administering Cas9/Cpf1 endonucleases, including nickases, together with a gRNA or sgRNA to eye cells of the patient for an USH2A gene containing an IVS40 mutation.
Guide RNA pairing configurations for treatment methods that administer a first and second gRNA or sgRNA to eye cells using specified SEQ ID NO pairings.
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