Granted Patent B2

US 12,214,023 · App. 16/657,961 · Granted Feb 4, 2025

Inventors: Jonathan Douglas Finn (Cambridge, MA); Hon-Ren Huang (Cambridge, MA); Moitri Roy (Cambridge, MA); KehDih Lai (Tarrytown, NY); Rachel Sattler (Tarrytown, NY); Christos Kyratsous (Tarrytown, NY); Cheng Wang (Tarrytown, NY)

Assignees: Intellia Therapeutics, Inc.; Regeneron Pharmaceuticals, Inc.

A61K38/4846A61K38/465A61K48/005A61P7/00C12N15/113C12N15/86C12N15/90C12Y304/21022

Abstract

Claims (33)

1. A method of expressing Factor IX in a cell or population of cells, comprising administering:

i) a nucleic acid construct comprising a Factor IX protein coding sequence;

ii) an RNA-guided DNA binding agent; and

iii) a guide RNA (gRNA) comprising the sequence of SEQ ID NO: 8,

thereby expressing Factor IX in a cell or population of cells.

2. The method of claim 1 , wherein the RNA-guided DNA binding agent is a Cas9 or a nucleic acid encoding a Cas9.

3. The method of claim 1 , wherein the RNA-guided DNA binding agent is a nucleic acid encoding the RNA-guided DNA binding agent, and wherein the nucleic acid encoding the RNA-guided DNA binding agent is an mRNA.

4. The method of claim 1 , wherein the nucleic acid construct and/or the gRNA is administered as a nucleic acid vector, and wherein the nucleic acid vector is a viral vector.

5. The method of claim 4 , wherein the viral vector is selected from the group consisting of an adeno associate viral (AAV) vector, adenoviius vector, retrovirus vector, and lentivirus vector.

6. The method of claim 1 , wherein the nucleic acid construct encodes a Factor IX protein having a mutation R338L.

7. A composition for use in expressing Factor IX in a cell, wherein the composition comprises:

i) a nucleic acid construct comprising a Factor IX protein coding sequence;

ii) an RNA-guided DNA binding agent; and

iii) a guide RNA (gRNA) comprising the sequence of SEQ ID NO: 8.

8. The composition of claim 7 , wherein the RNA-guided DNA binding agent is a Cas nuclease or a nucleic acid encoding a Cas nuclease.

9. The composition of claim 8 , wherein the Cas nuclease is an S. pyogenes Cas9 nuclease or variant thereof.

10. A method of treating a Factor IX deficiency comprising administering to an individual with the Factor IX deficiency:

i) a nucleic acid construct comprising a Factor IX protein coding sequence;

ii) an RNA-guided DNA binding agent; and

iii) a guide RNA (gRNA) comprising the sequence of SEQ ID NO: 8,

thereby expressing the Factor IX protein in the individual.

11. The method of claim 1 , wherein the gRNA comprises the sequence of SEQ ID NO: 40.

12. The method of claim 1 , wherein the gRNA comprises the sequence of:

mU*mA*mA*AGCAUAGUGCAAUGGAUGUUUUAGAmGmCmUmAmGmAmAmAm UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 72),

wherein “mA,” “mC,” “mU,” and “mG” represent nucleotides that have been substituted with 2′-O-Me and * indicates a phosphorothioate (PS) bond.

13. The composition of claim 7 , wherein the gRNA comprises the sequence of SEQ ID NO: 40.

14. The composition of claim 7 , wherein the gRNA comprises the sequence of:

mU*mA*mA*AGCAUAGUGCAAUGGAUGUUUUAGAmGmCmUmAmGmAmAmAm UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 72),

wherein “mA,” “mC,” “mU,” and “mG” represent nucleotides that have been substituted with 2′-O-Me and * indicates a phosphorothioate (PS) bond.

15. The method of claim 10 , wherein the gRNA comprises the sequence of SEQ ID NO: 40.

16. The method of claim 10 , wherein the gRNA comprises the sequence of:

mU*mA*mA*AGCAUAGUGCAAUGGAUGUUUUAGAmGmCmUmAmGmAmAmAm UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmA mAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 72),

wherein “mA,” “mC,” “mU,” and “mG” represent nucleotides that have been substituted with 2′-O-Me and * indicates a phosphorothioate (PS) bond.

Assignments (4)

Continuity (5)

References Cited (355)