B4GALT1 variants and uses thereof

Patent metadata

Field	Value
Publication number	US-12480103-B2
Application number	US-202016919423-A
Country	US
Kind code	B2
Filing date	Jul 2, 2020
Priority date	Jun 5, 2017
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

Variant B4GALT1 genomic, mRNA, and cDNA nucleic acid molecules, and polypeptides, methods of detecting the presence of these molecules, methods of modulating endogenous B4GALT1 genomic, mRNA, and cDNA nucleic acid molecules, and polypeptides, methods of ascertaining the risk of developing cardiovascular conditions by detecting the presence or absence of the variant B4GALT1 genomic, mRNA, and cDNA nucleic acid molecules, and polypeptides, and methods of treating cardiovascular conditions are provided herein.

First claim

What is claimed is:

1 . A method of lowering levels of low density lipoprotein cholesterol (LDL) and/or fibrinogen in a human subject who is not a carrier of a Beta-1,4-galactosyltransferase 1 (B4GALT1) variant having a serine at the position corresponding to position 352 in the B4GALT1 polypeptide having SEQ ID NO:8, comprising introducing into muscle tissue of the subject:
a) a Cas protein or a nucleic acid encoding the Cas protein; b) a guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA forms a complex with the Cas protein and hybridizes to a guide RNA recognition sequence within an endogenous B4GALT1 gene, wherein the guide RNA recognition sequence includes or is within about 1000 nucleotides to a position corresponding to positions 53575 to 53577 of SEQ ID NO: 1, comprises the start codon for the endogenous B4GALT1 gene, is within about 1,000 nucleotides of the start codon, or is selected from SEQ ID NOS: 9-12; and c) an expression vector comprising an exogenous donor sequence comprising a 5′ homology arm that hybridizes to a target sequence 5′ of the positions corresponding to positions 53575 to 53577 of SEQ ID NO:1, a 3′ homology arm that hybridizes to a target sequence 3′ of the positions corresponding to positions 53575 to 53577 of SEQ ID NO:1, and a nucleic acid insert comprising a nucleotide sequence encoding a serine at positions corresponding to positions 53575 to 53577 of SEQ ID NO:2 flanked by the 5′ homology arm and the 3′ homology arm, wherein the Cas protein cleaves the endogenous B4GALT1 gene in a cell in the subject and the exogenous donor sequence recombines with the endogenous B4GALT1 gene in the cell, wherein upon recombination of the exogenous donor sequence with the endogenous B4GALT1 gene, the serine is inserted at nucleotides corresponding to positions 53575 to 53577 of SEQ ID NO: 1.

2 . The method according to claim 1 , wherein the guide RNA recognition sequence is selected from SEQ ID NOS: 9-12.

3 . The method according to claim 1 , wherein the guide RNA recognition sequence includes the position corresponding to positions 53575 to 53577 of SEQ ID NO: 1.

4 . The method according to claim 1 , wherein the exogenous donor sequence is from about 50 nucleotides to about 1 kb in length.

5 . The method according to claim 4 , wherein the exogenous donor sequence is from about 80 nucleotides to about 200 nucleotides in length.

6 . The method according to claim 1 , wherein the exogenous donor sequence is a single-stranded oligodeoxynucleotide.

7 . A method of lowering levels of low density lipoprotein cholesterol (LDL) and/or fibrinogen in a human subject who is not a carrier of a Beta-1,4-galactosyltransferase 1 (B4GALT1) variant having a serine at the position corresponding to position 352 in the B4GALT1 polypeptide having SEQ ID NO:8, comprising introducing into muscle tissue of the subject:
a) a Cas protein or a nucleic acid encoding the Cas protein; b) a guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA forms a complex with the Cas protein and hybridizes to a guide RNA recognition sequence within an endogenous B4GALT1 gene, wherein the guide RNA recognition sequence comprises the start codon for the endogenous B4GALT1 gene or is within about 1,000 nucleotides of the start codon or is selected from SEQ ID NOS: 9-12; and c) an expression vector comprising a recombinant B4GALT1 gene comprising a nucleotide sequence encoding a serine at positions corresponding to positions 53575 to 53577 of SEQ ID NO:2, wherein the Cas protein cleaves or alters expression of the endogenous B4GALT1 gene in a cell in the subject and the expression vector expresses the recombinant B4GALT1 gene in the cell in the subject.

8 . The method according to claim 7 , wherein the guide RNA recognition sequence is selected from SEQ ID NOS: 9-12.

9 . The method according to claim 7 , wherein the Cas protein is a nuclease-active Cas protein.

10 . The method according to claim 7 , wherein the Cas protein is a nuclease-inactive Cas protein fused to a transcriptional repressor domain.

11 . The method according to claim 1 , wherein the introduction of the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the nucleic acid encoding the guide RNA into muscle tissue is carried out by vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or hydrodynamic delivery (HDD).

12 . The method according to claim 11 , wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the nucleic acid encoding the guide RNA are introduced into muscle tissue in a carrier comprising a poly (lactic acid) (PLA) microsphere, a poly (D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule.

13 . The method according to claim 7 , wherein the introduction of the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the nucleic acid encoding the guide RNA into muscle tissue is carried out by vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell-penetrating-peptide-mediated delivery, or hydrodynamic delivery (HDD).

14 . The method according to claim 13 , wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the nucleic acid encoding the guide RNA are introduced into muscle tissue in a carrier comprising a poly (lactic acid) (PLA) microsphere, a poly (D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule.

Assignees

• Regeneron Pharma
• Univ Maryland

Inventors

• Montasser May
• Van Hout Cristopher
• Shuldiner Alan
• Gatta Giusy Della
• Healy Matthew
• Puurunen Marja

Classifications

• C12Y204/01133

  Xylosylprotein 4-beta-galactosyltransferase (2.4.1.133) · CPC title

• C12N15/86

  Viral vectors · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title