Granted Patent B2

US 12,317,874 · App. 18/175,010 · Granted Jun 3, 2025

Inventors: Qing Fang (Chappaqua, NY); Chia-Jen Siao (New York, NY); Dan Chalothorn (New York, NY); KehDih Lai (Yardley, PA); Leah Sabin (Goldens Bridge, NY); Rachel Sattler (New York, NY); Brian Zambrowicz (Sleepy Hollow, NY); Lori Morton (Chappaqua, NY)

Assignee: REGENERON PHARMACEUTICALS, INC.

A01K67/0278A61K49/0008C07K14/76C12N15/8509A01K2207/15A01K2227/105A01K2267/03C12N2015/8527

Abstract

Claims (41)

1. A method of determining whether a genome-editing reagent modifies a humanized albumin gene in vivo, comprising:

(a) administering the genome-editing reagent to a genetically modified mouse whose genome comprises a humanized albumin gene in which the region from the start codon to the stop codon of an endogenous albumin gene is replaced with a human nucleic acid sequence encoding human albumin and a human albumin signal peptide,

wherein the human nucleic acid sequence is operably linked to an endogenous albumin promoter and comprises the region from the start codon to the stop codon of a human albumin gene, and

wherein the mouse functionally expresses human albumin and has a serum level of human albumin that is at least as high as the serum level of mouse albumin in a wild type mouse; and

(b) determining whether the genome-editing reagent modified the humanized albumin gene in the mouse.

2. The method of claim 1 , wherein the humanized albumin gene comprises a human albumin 3′ untranslated region.

3. The method of claim 1 , wherein the humanized albumin gene comprises the nucleic acid sequence of SEQ ID NO: 17, 18, or 35 or encodes the protein of SEQ ID NO: 5.

4. The method of claim 1 , wherein the humanized albumin gene further comprises a selection cassette.

5. The method of claim 1 , wherein the mouse is homozygous for the humanized albumin gene.

6. The method of claim 1 , wherein the mouse is heterozygous for the humanized albumin gene.

7. The method of claim 1 , wherein the mouse comprises serum albumin levels of at least about 10 mg/mL.

8. The method of claim 1 , wherein the mouse further comprises an inactivated factor 9 (F9) gene.

9. The method of claim 1 , wherein the genome-editing reagent is administered by adeno-associated virus (AAV)-mediated delivery, lipid nanoparticle (LNP)-mediated delivery, or hydrodynamic delivery (HDD).

10. The method of claim 9 , wherein the genome-editing reagent is administered by LNP-mediated delivery.

11. The method of claim 9 , wherein the genome-editing reagent is administered by AAV8-mediated delivery.

12. The method of claim 1 , wherein step (b) comprises isolating a liver from the genetically modified mouse and determining whether the genome-editing reagent modified the humanized albumin gene in liver tissue of the mouse.

13. The method of claim 1 , wherein step (b) comprises determining whether the genome-editing reagent caused insertions or deletions within the humanized albumin gene in the mouse.

14. The method of claim 1 , wherein the genome-editing reagent comprises a nuclease that targets a human albumin gene or a nucleic acid encoding the nuclease.

15. The method of claim 14 , wherein the genome-editing reagent comprises a Cas protein and a guide RNA (gRNA) that targets a human albumin gene.

16. The method of claim 15 , wherein the gRNA targets intron 1 of the human albumin gene.

17. The method of claim 15 , wherein the Cas protein is a Cas9 protein.

18. The method of claim 1 , wherein the genome-editing reagent is an exogenous nucleic acid sequence that targets a human albumin gene.

19. The method of claim 18 , wherein the exogenous nucleic acid sequence:

(I) is a single-stranded oligodeoxynucleotide;

(II) does not comprise homology arms; or

(III) comprises a donor sequence flanked by 5′ and 3′ homology arms.

20. The method of claim 19 , wherein the 5′ and 3′ homology arms target intron 1 of a human albumin gene.

21. The method of claim 18 , wherein the exogenous nucleic acid sequence encodes a protein.

22. The method of claim 21 , wherein the protein encoded by a humanized albumin gene that has been targeted with the exogenous nucleic acid sequence is a heterologous protein comprising a human albumin signal peptide fused to the protein encoded by the exogenous nucleic acid sequence.

23. The method of claim 21 , wherein the protein encoded by the exogenous nucleic acid sequence is a factor IX protein or a human factor IX protein.

24. The method of claim 23 , wherein the assessing further comprises measuring serum levels of the factor IX protein or the human factor IX protein in the genetically modified mouse and/or comprises assessing activated partial thromboplastin time or performing a thrombin generation assay.

25. The method of claim 21 , wherein the assessing further comprises measuring expression of a messenger RNA encoded by the exogenous nucleic acid sequence.

26. The method of claim 25 , wherein the measuring expression of the messenger RNA encoded by the exogenous nucleic acid sequence comprises an in situ hybridization assay to quantify expression of the messenger RNA at single-cell resolution.

27. The method of claim 25 , wherein the measuring expression of the messenger RNA encoded by the exogenous nucleic acid sequence comprises measuring expression of the messenger RNA in multiple lobes from the liver of the genetically modified mouse.

28. The method of claim 21 , wherein the assessing further comprises measuring expression of the protein.

29. The method of claim 28 , wherein the measuring expression of the protein comprises measuring:

(a) serum levels of the protein in the genetically modified mouse; or

(b) expression in the liver of the genetically modified mouse.

30. The method of claim 1 , wherein the genome-editing reagent comprises (1) a nuclease that targets a human albumin gene or a nucleic acid encoding the nuclease and (2) an exogenous nucleic acid sequence that targets the human albumin gene,

wherein the exogenous nucleic acid sequence encodes a protein, and

wherein the protein encoded by a humanized albumin gene that has been targeted with the exogenous nucleic acid sequence is a heterologous protein comprising a human albumin signal peptide fused to the protein encoded by the exogenous nucleic acid sequence.

Assignments (1)

Continuity (4)

References Cited (231)