Materials and methods for treatment of usher syndrome type 2a and/or non-syndromic autosomal recessive retinitis pigmentosa (arrp)

Patent metadata

Field	Value
Publication number	US-2020318108-A1
Application number	US-202016905258-A
Country	US
Kind code	A1
Filing date	Jun 18, 2020
Priority date	Dec 21, 2017
Publication date	Oct 8, 2020
Grant date	—

Abstract

The present application provides materials and methods for treating a patient with one or more of Usher Syndrome Type 2A and ARRP, both ex vivo and in vivo; materials and methods for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299. In addition, the present application provides one or more gRNAs or sgRNAs for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299; a therapeutic comprising at least one or more gRNAs or sgRNAs for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299; and a therapeutic for treating a patient with one or more of Usher Syndrome Type 2A and ARRP. The present application also provides a kit for treating a patient with one or more of Usher Syndrome Type 2A and ARRP.

First claim

1 .- 195 . (canceled)

196 . A method for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299 located in exon 13, the method comprising:
introducing into a human cell (i) one or more S. pyogenes Cas9 endonucleases, and (ii) one or more guide RNAs (gRNAs) comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5291-5314, thereby effecting one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near one or more of: intron 12-13, exon 13, and intron 13-14 of the USH2A gene that results in an edited human cell.

197 . The method of claim 196 , wherein the method comprises introducing into the cell one or more polynucleotides encoding the one or more Cas9 endonucleases.

198 . The method of claim 196 , wherein the one or more gRNAs are single-molecule guide RNAs (sgRNAs).

199 . The method of claim 196 , wherein the one or more DNA endonucleases is pre-complexed with one or more gRNAs.

200 . The method of claim 198 , wherein the one or more DNA endonucleases is pre-complexed with one or more sgRNAs.

201 . The method of claim 196 , further comprising introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type USH2A gene, or cDNA.

202 . A method for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299 located in exon 13, the method comprising:
introducing into a human cell (i) one or more S. pyogenes Cas9 endonucleases, and (ii) a first gRNA and a second gRNA selected from the group consisting of: (a) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5313; (b) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5314; (c) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5313; (d) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5314; (e) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5276; (f) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5275; (g) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5277; (h) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5278; (i) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5287; (j) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5286; (k) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5290; (l) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5291; (m) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5292; (n) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5294; (o) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5296; (p) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5302; (q) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5310; (r) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5276; (s) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5275; (t) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5277; (u) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5278; (v) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5287; (w) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5286; (x) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5290; (y) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5291; (z) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5292; (aa) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5294; (bb) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5296; (cc) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5302; and (dd) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5310, thereby effecting one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near one or more of: intron 12-13, exon 13, and intron 13-14 of the USH2A gene that results in an edited human cell.

203 . The method of claim 202 , wherein the method comprises introducing into the cell one or more polynucleotides encoding the one or more Cas9 endonucleases.

204 . The method of claim 202 , wherein the one or more gRNAs are single-molecule guide RNAs (sgRNAs).

205 . The method of claim 202 , wherein the one or more DNA endonucleases is pre-complexed with one or more gRNAs.

206 . The method of claim 204 , wherein the one or more DNA endonucleases is pre-complexed with one or more sgRNAs.

207 . The method of claim 202 , further comprising introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type USH2A gene, or cDNA.

208 . A gRNA comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5291-5314.

209 . The gRNA of claim 208 , wherein the gRNA is a sgRNA.

210 . A method of treating a patient with Usher Syndrome Type 2A or non-syndromic autosomal recessive Retinitis Pigmentosa (ARRP), comprising administering to the patient (i) one or more S. pyogenes Cas9 endonucleases and (ii) one or more gRNAs of claim 208 , wherein the patient comprises a guanine deletion at nucleotide position c.2299 located in exon 13 in an USH2A gene.

211 . A method for treating a patient with Usher Syndrome Type 2A or ARRP, comprising administering to the patient a cell edited ex vivo by a method comprising introducing into the cell (i) one or more S. pyogenes Cas9 endonucleases and (ii) one or more gRNAs of claim 208 , thereby effecting one or more single stranded breaks (SSBs) or double stranded breaks (DSBs) within one or more of: intron 12-13, exon 13, and intron 13-14 of an USH2A gene that results in an edited cell, wherein the patient comprises a guanine deletion at nucleotide position c.2299 located in exon 13 in the USH2A gene.

212 . A composition comprising at least two gRNAs selected from the group consisting of:
(a) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5313; (b) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5314; (c) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5313; (d) a first gRNA comprising SEQ ID NO: 5299 and a second gRNA comprising SEQ ID NO: 5314; (e) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5276; (f) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5275; (g) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5277; (h) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5278; (i) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5287; (j) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5286; (k) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5290; (l) a first gRNA comprising SEQ ID NO: 5295 and a second gRNA comprising SEQ ID NO: 5291;

Assignees

• Crispr Therapeutics Ag
• Bayer Healthcare Llc

Inventors

• Allocca Mariacarmela
• Noma Akiko
• Scaria Abraham

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/86

  Viral vectors · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/1138 Primary

  against receptors or cell surface proteins · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title