Chemically modified guide RNAS for CRISPR/Cas-mediated gene regulation

Patent metadata

Field	Value
Publication number	US-11535846-B2
Application number	US-202217694310-A
Country	US
Kind code	B2
Filing date	Mar 14, 2022
Priority date	Apr 6, 2015
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

Provided herein are methods for inducing CRISPR/Cas-based gene regulation (e.g., genome editing or gene expression) of a target nucleic acid (e.g., target DNA or target RNA) in a cell. The methods include using modified single guide RNAs (sgRNAs) that enhance gene regulation of the target nucleic acid in a primary cell for use in ex vivo therapy or in a cell in a subject for use in in vivo therapy. Additionally, provided herein are methods for preventing or treating a genetic disease in a subject by administering a sufficient amount of a modified sgRNA to correct a mutation in a target gene associated with the genetic disease.

First claim

What is claimed is:

1. A primary cell comprising:
(a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid within the primary cell and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein:
(i) about 10% to about 30% of nucleotides in the first nucleotide sequence and/or about 1% to about 10% of nucleotides in the second nucleotide sequence are modified nucleotides;
(ii) the modified sgRNA comprises a modified nucleotide within five nucleotides from the 5′ end of the first nucleotide sequence and/or within five nucleotides from the 3′ end of the second nucleotide sequence; and
(iii) the modified nucleotides are selected from the group consisting of a 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, a 2′-O-methyl 3′-thioPACE (MSP) nucleotide, and a combination thereof; and
(b) a Cas polypeptide, an mRNA encoding the Cas polypeptide, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas polypeptide,
wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid, and wherein the modified sgRNA induces a gene regulation of the target nucleic acid with an enhanced activity relative to a corresponding unmodified sgRNA.

2. The primary cell of claim 1 , wherein the enhanced activity comprises increased stability of the modified sgRNA and/or increased specificity of the modified sgRNA for the target nucleic acid.

3. The primary cell of claim 1 , wherein the target nucleic acid comprises a target DNA or a target RNA.

4. The primary cell of claim 3 , wherein the gene regulation comprises genome editing of the target DNA.

5. The primary cell of claim 4 , wherein the genome editing comprises homologous-directed repair (HDR) or nonhomologous end joining (NHEJ) of the target DNA.

6. The primary cell of claim 4 , further comprising a recombinant donor repair template.

7. The primary cell of claim 6 , wherein the recombinant donor repair template comprises two nucleotide sequences comprising two non-overlapping, homologous portions, wherein one of the two homologous portions is located at the 5′ e n d and the other homologous portion is located at the 3′ end of the recombinant donor template, wherein each homologous portion is homologous to a corresponding region of the target DNA to undergo genome editing.

8. The primary cell of claim 6 , wherein the recombinant donor repair template comprises a synthetic single-stranded oligodeoxynucleotide (ssODN) template comprising a nucleotide sequence encoding a mutation to correct a single nucleotide polymorphism (SNP) and two nucleotide sequences comprising two non-overlapping, homologous portions that are homologous to corresponding regions of the target DNA, wherein one of the two homologous portions is located at the 5′ e n d and the other homologous portion is located at the 3′ end of the recombinant donor template.

9. The primary cell of claim 3 , wherein the Cas polypeptide is endonuclease-deficient.

10. The primary cell of claim 1 , wherein the primary cell is isolated from a multicellular organism.

11. The primary cell of claim 10 , wherein the multicellular organism is a plant, a multicellular protist, a multicellular fungus, or an animal.

12. The primary cell of claim 1 , wherein the primary cell is a stem cell or an immune cell.

13. The primary cell of claim 12 , wherein the stem cell is a hematopoietic stem and progenitor cell (HSPC), a mesenchymal stem cell, a neural stem cell, or an organ stem cell.

14. The primary cell of claim 12 , wherein the immune cell is a T cell, a natural killer cell, a monocyte, a peripheral blood mononuclear cell (PBMC), or a peripheral blood lymphocyte (PBL).

15. A population of primary cells comprising the primary cell of claim 1 .

16. The population of primary cells of claim 15 , wherein the modified sgRNA induces the gene regulation of the target nucleic acid in at least about 30% of the population of primary cells.

17. The primary cell of claim 1 , wherein the first nucleotide sequence is about 20 nucleotides in length.

18. The primary cell of claim 1 , wherein at least two nucleotides in the first nucleotide sequence are modified nucleotides.

19. The primary cell of claim 1 , wherein one or more modified nucleotides are located within five nucleotides from the 5′-end of the first nucleotide sequence.

20. The primary cell of claim 1 , wherein from about 10% to about 30% of the nucleotides in the first nucleotide sequence are modified nucleotides.

21. The primary cell of claim 1 , wherein the second nucleotide sequence is about 80 nucleotides in length.

22. The primary cell of claim 1 , wherein at least two nucleotides in the second nucleotide sequence are modified nucleotides.

23. The primary cell of claim 1 , wherein one or more modified nucleotides are located within five nucleotides from the 3′-end of the second nucleotide sequence.

24. The primary cell of claim 1 , wherein from about 1% to about 10% of the nucleotides in the second nucleotide sequence are modified nucleotides.

25. The primary cell of claim 1 , wherein the modified sgRNA comprises one, two, or three consecutive or non-consecutive modified nucleotides at or near the 5′-end of the first nucleotide sequence and one, two, or three consecutive or non-consecutive modified nucleotides at or near the 3′-end of the second nucleotide sequence.

26. The primary cell of claim 25 , wherein the modified sgRNA comprises three consecutive modified nucleotides at the 5′-end of the first nucleotide sequence and three consecutive modified nucleotides at the 3′-end of the second nucleotide sequence.

27. The primary cell of claim 1 , wherein the primary cell comprises mRNA encoding the Cas polypeptide.

28. The primary cell of claim 1 , wherein the Cas polypeptide is a Cas9 polypeptide, a variant thereof, or a fragment thereof.

29. The primary cell of claim 1 , wherein the Cas polypeptide is a nickase.

30. A primary cell comprising:
(a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid within the primary cell and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein:
(i) about 10% to about 30% of nucleotides in the first nucleotide sequence and/or about 1% to about 10% of nucleotides in the second nucleotide sequence are modified nucleotides;
(ii) the modified sgRNA comprises a modified nucleotide within five nucleotides from the 5′ end of the first nucleotide sequence and/or within five nucleotides from the 3′ end of the second nucleotide sequence; and
(iii) the modified nucleotides are selected from the group consisting of a 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, a 2′-O-methyl 3′-thioPACE (MSP) nucleotide, and a combination thereof; and
(b) a Cas polypeptide,
wherein the modified sgRNA and the Cas polypeptide are formed in a ribonucleoprotein (RNP) complex,
wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid, and wherein the modified sgRNA induces a gene regulation of the target nucleic acid with an enhanced activity relative to a corresponding unmodified sgRNA.

Assignees

• Univ Leland Stanford Junior
• Agilent Technologies Inc

Inventors

• Porteus Matthew H
• Hendel Ayal
• Clark Joe
• Bak Rasmus O
• Ryan Daniel E
• Dellinger Douglas J
• Kaiser Robert
• Myerson Joel

Classifications

• A61P19/00

  Drugs for skeletal disorders · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P31/18

  for HIV · CPC title

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• A61P27/02

  Ophthalmic agents · CPC title