Materials and methods for treatment of autosomal dominant retinitis pigmentosa

Patent metadata

Field	Value
Publication number	US-10662425-B2
Application number	US-201816198361-A
Country	US
Kind code	B2
Filing date	Nov 21, 2018
Priority date	Nov 21, 2017
Publication date	May 26, 2020
Grant date	May 26, 2020

Abstract

The present application provides materials and methods for treating a patient with autosomal dominant RP, both ex vivo and in vivo; materials and methods for editing a RHO gene in a human cell; and materials and methods for editing a P23H mutation in a RHO gene in a human cell. In addition, the present application provides one or more gRNAs or sgRNAs for editing a RHO gene; one or more gRNAs or sgRNAs for editing a P23H mutation in a RHO gene; and a therapeutic comprising at least one or more gRNAs or sgRNAs for editing a P23H mutation in a RHO gene. The present application provides a therapeutic for treating a patient with autosomal dominant RP. The present application provides a kit for treating a patient with autosomal dominant RP. In addition, the present application provides a self-inactivating CRISPR-Cas system.

First claim

What is claimed is:

1. A self-inactivating CRISPR-Cas system comprising:
a first segment comprising a nucleotide sequence that encodes Cas9;
a second segment comprising a nucleotide sequence that encodes a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA) wherein the gRNA or the sgRNA comprises SEQ ID NO: 5290 or 5291; and
one or more third segments comprising a self-inactivating (SIN) site, wherein the SIN site is:
a 5′ SIN site located upstream of the nucleotide sequence that encodes Cas9 and downstream of a SV40 nuclear localization signal (NLS); or
a 3′ SIN site located within a naturally occurring or chimeric inserted intron located within the nucleotide sequence that encodes Cas9;

wherein the gRNA or sgRNA is complementary to the SIN site;
wherein the gRNA or sgRNA is complementary to a genomic target sequence.

2. The self-inactivating CRISPR-Cas system of claim 1 , wherein the Cas9 is Staphylococcus aureus Cas9 (SaCas9).

3. The self-inactivating CRISPR-Cas system of claim 1 , wherein the 5′ SIN site comprises SEQ ID NO: 5300 or 5301.

4. The self-inactivating CRISPR-Cas system of claim 1 , wherein the 3′ SIN site comprises SEQ ID NO: 5280 or 5281.

5. The self-inactivating CRISPR-Cas system of claim 1 , where the SIN site comprises a protospacer adjacent motif (PAM).

6. The self-inactivating CRISPR-Cas system of claim 5 , wherein the PAM is NNGRRT.

7. The self-inactivating CRISPR-Cas system of claim 1 , wherein the genomic target sequence is a P23H mutation in a rhodopsin (RHO) gene.

8. The self-inactivating CRISPR-Cas system of claim 1 , wherein the first segment comprising a nucleotide sequence that encodes Cas9, further comprises a start codon, a stop codon, and a poly(A) termination site.

9. The self-inactivating CRISPR-Cas system of claim 1 , wherein the first segment and the third segment are provided together in a first vector and the second segment is provided in a second vector.

10. The self-inactivating CRISPR-Cas system of claim 1 , wherein the first segment, second segment, and third segment are provided together in a vector.

11. The self-inactivating CRISPR-Cas system of claim 9 , wherein the first vector comprises SEQ ID NO: 5341 or 5342.

12. The self-inactivating CRISPR-Cas system of claim 9 , wherein the second vector comprises SEQ ID NO: 5339 or 5340.

13. The self-inactivating CRISPR-Cas system of claim 1 , wherein the third segment is less than 100 nucleotides in length.

14. The self-inactivating CRISPR-Cas system of claim 1 , wherein the gRNA or the sgRNA is complementary to the nucleotide sequence of the SIN site except for in at least one location.

15. The self-inactivating CRISPR-Cas system of claim 1 , wherein a nucleic acid sequence encoding a promoter is operably linked to the first segment.

16. The self-inactivating CRISPR-Cas system of claim 15 , wherein the promoter is a spatially-restricted promoter, bidirectional promoter, or an inducible promoter.

17. The self-inactivating CRISPR-Cas system of claim 16 , wherein the spatially-restricted promoter is selected from the group consisting of: any tissue or cell type specific promoter, a hepatocyte-specific promoter, a neuron-specific promoter, an adipocyte-specific promoter, a cardiomyocyte-specific promoter, a skeletal muscle-specific promoter, lung progenitor cell specific promoter, a photoreceptor-specific promoter, and a retinal pigment epithelial (RPE) selective promoter.

18. The self-inactivating CRISPR-Cas system of claim 9 , wherein the first vector and the second vector are adeno-associated virus (AAV) vectors.

19. The self-inactivating CRISPR-Cas system of claim 18 , wherein the AAV vectors are AAVS serotype capsid vectors.

20. The self-inactivating CRISPR-Cas system of claim 10 , wherein the vector is an AAV vector.

21. The self-inactivating CRISPR-Cas system of claim 20 , wherein the AAV vector is an AAV5 serotype capsid vector.

22. A kit for treating a patient with autosomal dominant Retinitis Pigmentosa in vivo, the kit comprising:
the self-inactivating CRISPR-Cas system of claim 1 ; and
optionally, one or more donor template.

23. A therapeutic for treating a patient with autosomal dominant Retinitis Pigmentosa, the therapeutic comprising the self-inactivating CRISPR-Cas system of claim 1 .

24. The self-inactivating CRISPR-Cas system of claim 1 , wherein the gRNA or the sgRNA is complementary to the genomic target sequence except for in at least one location.

25. A self-inactivating CRISPR-Cas system comprising:
a first segment comprising a nucleotide sequence that encodes Cas9;
a second segment comprising a nucleotide sequence that encodes a gRNA or a sgRNA wherein the gRNA or the sgRNA comprises SEQ ID NO: 5290 or 5291; and
one or more third segments comprising a SIN site, wherein the SIN site is:
a 5′ SIN site located upstream of the nucleotide sequence that encodes Cas9 and upstream of a SV40 NLS within a 5′ UTR; or
a 3′ SIN site located within a naturally occurring or chimeric inserted intron located within the nucleotide sequence that encodes Cas9;

wherein the gRNA or sgRNA is complementary to the SIN site;
wherein the gRNA or sgRNA is complementary to a genomic target sequence.

26. The self-inactivating CRISPR-Cas system of claim 25 , wherein the gRNA or the sgRNA is complementary to the nucleotide sequence of the SIN site except for in at least one location.

27. The self-inactivating CRISPR-Cas system of claim 25 , wherein the gRNA or the sgRNA is complementary to the genomic target sequence except for in at least one location.

28. A method for editing a P23H mutation within a RHO gene, the method comprising: administering the self-inactivating CRISPR-Cas system of claim 1 .

29. A method for treating a patient with a P23H mutation within a RHO gene, the method comprising: administering the self-inactivating CRISPR-Cas system of claim 1 .

30. A method of controlling Cas9 expression in a cell comprising: contacting the cell with the self-inactivating CRISPR-Cas system of claim 1 .

Assignees

• Crispr Therapeutics Ag
• Bayer Healthcare Llc

Inventors

• Kantardzhieva Albena
• Noma Akiko
• Scaria Abraham
• Takeuchi Ryo

Classifications

• A61K38/465

  acting on ester bonds (3.1), e.g. lipases, ribonucleases · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N2320/34

  Allele or polymorphism specific uses · 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