US 12,480,103 B2
B4GALT1 variants and uses thereof
May Montasser, Baltimore, MD (US); Cristopher Van Hout, Tarrytown, NY (US); Alan Shuldiner, Tarrytown, NY (US); Giusy Della Gatta, Tarrytown, NY (US); Matthew Healy, Tarrytown, NY (US); and Marja Puurunen, Tarrytown, NY (US)
Assigned to Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US); and University of Maryland, Baltimore, Baltimore, MD (US)
Filed by Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US); and University of Maryland, Baltimore, Baltimore, MD (US)
Filed on Jul. 2, 2020, as Appl. No. 16/919,423.
Application 16/919,423 is a continuation of application No. 15/996,892, filed on Jun. 4, 2018, granted, now 10,738,284.
Claims priority of provisional application 62/659,344, filed on Apr. 18, 2018.
Claims priority of provisional application 62/550,161, filed on Aug. 25, 2017.
Claims priority of provisional application 62/515,140, filed on Jun. 5, 2017.
Prior Publication US 2020/0399617 A1, Dec. 24, 2020
Int. Cl. C12N 9/10 (2006.01); C12N 15/86 (2006.01)
CPC C12N 9/1051 (2013.01) [C12N 15/86 (2013.01); C12Y 204/01133 (2013.01)] 14 Claims
 
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.