US 12,018,275 B2
Delivery and use of the CRISPR-CAS systems, vectors and compositions for hepatic targeting and therapy
Feng Zhang, Cambridge, MA (US); Le Cong, Cambridge, MA (US); and Fei Ran, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Feb. 25, 2020, as Appl. No. 16/800,988.
Application 16/800,988 is a division of application No. 14/971,356, filed on Dec. 16, 2015, granted, now 10,577,630.
Application 14/971,356 is a continuation in part of application No. PCT/US2014/041804, filed on Jun. 10, 2014.
Claims priority of provisional application 61/836,123, filed on Jun. 17, 2013.
Claims priority of provisional application 61/979,733, filed on Apr. 15, 2014.
Claims priority of provisional application 61/915,325, filed on Dec. 12, 2013.
Claims priority of provisional application 61/871,301, filed on Aug. 28, 2013.
Claims priority of provisional application 61/862,355, filed on Aug. 5, 2013.
Claims priority of provisional application 61/847,537, filed on Jul. 17, 2013.
Prior Publication US 2020/0392541 A1, Dec. 17, 2020
Int. Cl. A61K 48/00 (2006.01); A01K 67/0275 (2024.01); C12N 7/00 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01); C12N 15/86 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/907 (2013.01) [A01K 67/0275 (2013.01); A61K 48/0058 (2013.01); A61K 48/0091 (2013.01); C12N 7/00 (2013.01); C12N 9/22 (2013.01); C12N 15/1082 (2013.01); C12N 15/63 (2013.01); C12N 15/86 (2013.01); A01K 2217/052 (2013.01); A01K 2227/105 (2013.01); A01K 2267/0312 (2013.01); A01K 2267/0331 (2013.01); A01K 2267/0362 (2013.01); A61K 48/00 (2013.01); C12N 2710/24144 (2013.01); C12N 2740/15043 (2013.01); C12N 2750/14132 (2013.01); C12N 2750/14143 (2013.01); C12N 2750/14152 (2013.01); C12Y 301/00 (2013.01)] 16 Claims
 
1. A method of modifying a mammalian subject by editing in vivo a DNA target sequence in a genomic locus of interest of a hepatic cell in the mammalian subject, said method comprising in vivo delivering to the hepatic cell a single dose of a composition comprising a stable nucleic acid-lipid particle (SNALP) comprising a CRISPR-Cas system, wherein the CRISPR-Cas system comprises:
I. a CRISPR-Cas system RNA comprising a guide sequence that hybridizes to the DNA target sequence, and
II. a Cas9 comprising at least one nuclear localization sequence (NLS) or an mRNA encoding the Cas9,
wherein the guide sequence directs sequence-specific binding of a CRISPR complex to the DNA target sequence, the CRISPR complex comprises the Cas9 complexed with the CRISPR-Cas system RNA, and wherein the CRISPR complex introduces a double-stranded break in vivo in the DNA target sequence in the genomic locus of interest of the hepatic cell which forms a targeted indel in the genomic locus of interest and produces a phenotypic change in said mammalian subject, and wherein the delivering results in greater than 20% indel formation in the genomic locus of interest of the hepatic cell.