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-2022049247-A1
Application number	US-202117391862-A
Country	US
Kind code	A1
Filing date	Aug 2, 2021
Priority date	Dec 21, 2017
Publication date	Feb 17, 2022
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 . A method for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299, the method comprising:
introducing into a 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 one or more of: intron 12-13, exon 13, and intron 13-14 of the USH2A gene that results in a correction thereby creating an edited human cell.

2 . The method of claim 1 , wherein the guanine deletion at nucleotide position c.2299 is located in exon 13 of the USH2A gene.

3 - 5 . (canceled)

6 . The method of claim 1 , wherein the one or more DNA endonuclease is a Cas1, Cas1 B, Cas2, Cas3, Cas4, Cas5, Casio, 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.

7 . The method of claim 6 , wherein the method comprises introducing into the cell one or more polynucleotides encoding the one or more DNA endonuclease.

8 . The method of claim 6 , wherein the method comprises introducing into the cell one or more ribonucleic acids (RNAs) encoding the one or more DNA endonuclease.

9 - 10 . (canceled)

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

12 . The method of claim 11 , wherein the one or more gRNAs are single-molecule guide RNA (sgRNAs).

13 . The method of claim 11 , wherein the one or more gRNAs or one or more sgRNAs is one or more modified gRNAs or one or more modified sgRNAs.

14 . The method of claim 10 , wherein the one or more DNA endonucleases is pre-complexed with one or more gRNAs or one or more sgRNAs.

15 . The method of claim 1 , wherein the restoration of usherin protein function is a result of exon 13 skipping during mRNA processing.

16 . The method of claim 1 , further comprising: introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type USH2A gene, or cDNA.

17 . The method of claim 16 , wherein the at least a portion of the wild-type USH2A gene or cDNA comprises exon 13, intronic regions, or combinations thereof.

18 . The method of claim 16 , wherein the polynucleotide donor template is either a single or double-stranded polynucleotide.

19 . The method of claim 16 , wherein the polynucleotide donor template has homologous arms to exon 13.

20 . The method of claim 1 , further comprising:
introducing into the cell one gRNA; wherein the one or more DNA endonuclease is one or more Cas9 or Cpf1 endonuclease that effect one SSB or DSB at a locus located within or near one or more of: the intron 12-13, the exon 13, and the intron 13-14 of the USH2A gene; and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus located within or near one or more of: the intron 12-13, the exon 13, and the intron 13-14 of the USH2A gene.

21 . The method of claim 1 , further comprising:
introducing into the cell one or more gRNAs; wherein the one or more DNA endonuclease is one or more Cas9 or Cpf1 endonuclease that effect a pair of SSBs or DSBs, the first at a 5′ locus within or near the intron 12-13 or the exon 13 of the USH2A gene and the second at a 3′ locus, within or near the exon 13 or the intron 13-14 of the USH2A gene; and wherein the first gRNA comprises a spacer sequence that is complementary to a segment of the 5′ locus and the second gRNA comprises a spacer sequence that is complementary to a segment of the 3′ locus.

22 . The method of claim 20 , wherein the guanine deletion at nucleotide position c.2299 is located in exon 13 of the USH2A gene.

23 - 25 . (canceled)

26 . The method of claim 20 , further comprising: a polynucleotide donor template comprising at least a portion of the wild-type USH2A gene;
wherein a new sequence from the polynucleotide donor template is inserted into the chromosomal DNA at the locus that results in a correction of the guanine deletion at nucleotide position c.2299 of the USH2A gene.

27 - 46 . (canceled)

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

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

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

50 - 53 . (canceled)

54 . The method of claim 1 , wherein the human cell is a photoreceptor cell or retinal progenitor cell.

55 - 59 . (canceled)

60 . One or more gRNAs for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299 in a cell from a patient with one or more of Usher Syndrome Type 2A and ARRP, the one or more gRNAs comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5272-5314.

61 . The one or more gRNAs of claim 60 , wherein the guanine deletion at nucleotide position c.2299 is located in exon 13 of the USH2A gene.

62 . The one or more gRNAs of claim 60 , wherein the one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs).

63 - 68 . (canceled)

69 . A therapeutic for treating a patient with one or more of Usher Syndrome Type 2A and ARRP, the therapeutic comprising:
one or more DNA endonuclease; one or more gRNA or one or more sgRNA for editing an USH2A gene containing a guanine deletion at nucleotide position c.2299; optionally one or more donor template; wherein the one or more gRNAs or sgRNAs comprise a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5272-5314.

70 . The therapeutic of claim 69 , wherein the guanine deletion at nucleotide position c.2299 is located in exon 13 of the USH2A gene.

71 - 77 . (canceled)

78 . A method for treating a patient with one or more of Usher Syndrome Type 2A and non-syndromic autosomal recessive Retinitis Pigmentosa (ARRP), wherein the patient has an USH2A gene containing a guanine deletion at nucleotide position c.2299, the method comprising editing the USH2A gene containing a guanine deletion at nucleotide position c.2299 by administering a first gRNA and a second qRNA to the patient.

79 - 195 . (canceled)

196 . The method of claim 7 , wherein the one or more gRNA are 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 compr

Assignees

• Crispr Therapeutics Ag
• Bayer Healthcare Llc

Inventors

• Allocca Mariacarmela
• Noma Akiko
• Scaria Abraham

Classifications

• C12N9/22

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

• A61K35/12

  Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00) · CPC title

• C12N2310/20

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

• C12N2750/14132

  Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent · CPC title

• C12N2800/80

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