Materials and methods for treatment of usher syndrome type 2a

Patent metadata

Field	Value
Publication number	US-2021403905-A1
Application number	US-202117360578-A
Country	US
Kind code	A1
Filing date	Jun 28, 2021
Priority date	Dec 21, 2017
Publication date	Dec 30, 2021
Grant 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.

First claim

1 . A method for editing an USH2A gene in a human cell, the method comprising:
introducing into the human cell one or more deoxyribonucleic acid (DNA) endonuclease, thereby effecting one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the USH2A gene or a DNA sequence encoding a regulatory sequence of the USH2A gene that results in a correction thereby creating an edited human cell.

2 . A method for editing an USH2A gene containing an IVS40 mutation, the method comprising:
introducing into the human cell one or more DNA endonuclease, thereby effecting one or more SSBs or DSBs within or near intron 40 of the USH2A gene that results in a correction thereby creating an edited human cell.

3 . The method of claim 2 , wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

4 . An in vivo method for treating a patient with Usher Syndrome Type 2A, the method comprising: editing an USH2A gene containing an IVS40 mutation in a cell of the patient, wherein the editing comprises introducing into the cell one or more DNA endonucleases to effect one or more SSBs or DSBs within or near intron 40 of the USH2A gene that results in a correction and results in restoration of usherin protein function.

5 . (canceled)

6 . The method of claim 4 , wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

7 . The method of claim 1 , wherein the one or more DNA endonucleases is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof.

8 . The method of claim 7 , wherein the method comprises introducing into the cell one or more polynucleotides encoding the one or more DNA endonucleases; or (ii) one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases.

9 - 11 . (canceled)

12 . The method of claim 1 , wherein the method further comprises: introducing into the cell one or more guide ribonucleic acids (gRNAs).

13 - 16 . (canceled)

17 . The method of claim 16 , wherein the at least a portion of the wild-type USH2A gene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, intronic regions, fragments or combinations thereof, or the entire USH2A gene or cDNA.

18 - 19 . (canceled)

20 . The method of claim 2 , further comprising:
introducing into the cell one gRNA; wherein the one or more DNA endonucleases is one or more Cas9 or Cpf1 endonucleases that effect one SSB or DSB at a locus located within or near intron 40 of the USH2A gene; and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus located within intron 40.

21 - 30 . (canceled)

31 . The method of claim 20 , wherein the SSB or DSB is located within intron 40, 0 to 1800 nucleotides upstream of the IVS40 mutation.

32 - 35 . (canceled)

36 . The method of claim 2 , further comprising:
introducing into the cell two gRNAs; wherein the one or more DNA endonucleases is one or more Cas9 or Cpf1 endonucleases that effect a pair of DSBs, the first at a 5′ DSB locus and the second at a 3′ DSB locus, within or near intron 40 of the USH2A gene that causes a deletion of the chromosomal DNA between the 5′ DSB locus and the 3′ DSB locus that results in a deletion of the chromosomal DNA between the 5′ DSB locus and the 3′ DSB locus within or near intron 40 of the USH2A gene; and wherein the first gRNA comprises a spacer sequence that is complementary to a segment of the 5′ DSB locus and the second gRNA comprises a spacer sequence that is complementary to a segment of the 3′ DSB locus.

37 - 42 . (canceled)

43 . The method of claim 36 , wherein the deletion is a deletion of 50 bp to 2900 bp.

44 - 48 . (canceled)

49 . The method of claim 20 , wherein the Cas9 or Cpf1 mRNA, gRNA, and donor template are either each formulated into separate lipid nanoparticles or all co-formulated into a lipid nanoparticle.

50 . The method of claim 20 , wherein the Cas9 or Cpf1 mRNA, gRNA, and donor template are either each formulated into separate adeno-associated virus (AAV) vectors or all co-formulated into an AAV vector.

51 . The method of claim 20 , wherein the Cas9 or Cpf1 mRNA is formulated into a lipid nanoparticle, and both the gRNA and donor template are delivered to the cell by an AAV vector.

52 . The method of claim 20 , wherein the Cas9 or Cpf1 mRNA is formulated into a lipid nanoparticle, and the gRNA is delivered to the cell by electroporation and donor template is delivered to the cell by an AAV vector.

53 . The method of claim 50 , wherein the AAV vector is a self-inactivating AAV vector.

54 . The method of claim 1 , wherein the USH2A gene is located on Chromosome 1: 215,622,893-216,423,395 (Genome Reference Consortium—GRCh38/hg38).

55 - 61 . (canceled)

62 . The method of claim 4 , wherein the correction results in a modulation of expression or function of the USH2A gene and results in restoration of usherin protein function.

63 - 66 . (canceled)

67 . One or more gRNAs for editing an USH2A gene containing an IVS40 mutation in a cell from a patient with Usher Syndrome Type 2A, the one or more gRNAs comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461 of the Sequence Listing.

68 . The one or more gRNAs of claim 67 , wherein the IVS40 mutation is located within intron 40 of the USH2A gene.

69 . The one or more gRNAs of claim 67 , wherein the one or more gRNAs are one or more sgRNAs.

70 . The one or more gRNAs or sgRNAs of claim 67 , wherein the one or more gRNAs or one or more sgRNAs is one or more modified gRNAs or one or more modified sgRNAs.

71 . The one or more gRNAs or sgRNAs of claim 67 , wherein the cell is a photoreceptor cell, retinal progenitor cell, mesenchymal stem cell (MSC), or induced pluripotent stem cell (iPSC).

72 . A therapeutic comprising at least one or more gRNAs for editing an USH2A gene containing an IVS40 mutation, the one or more gRNAs comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5272-5319, 5321, 5323, 5325, 5327-5328, 5443, and 5446-5461.

73 - 83 . (canceled)

84 . A gRNA or sgR

Assignees

• Crispr Therapeutics Ag
• Bayer Healthcare Llc

Inventors

• Kantardzhieva Albena
• Noma Akiko
• Scaria Abraham

Classifications

• 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

• A61P27/00

  Drugs for disorders of the senses · CPC title

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N2310/20

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