SUPPLEMENTATION OF LIVER ENZYME EXPRESSION
Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful supplementing liver enzyme deficiencies.
1 . An engineered nuclease system, comprising:
a) an endonuclease comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96;
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and
c) a donor template comprising a nucleic acid sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof.
2 . The engineered nuclease system of claim 1 , wherein the target nucleic acid sequence within the albumin gene is within intron 1 of the albumin gene.
3 . The engineered nuclease system of any one of claims 1-2 , wherein the sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof is linked to a splice acceptor sequence targeting exon 1 of the albumin gene.
4 . The engineered nuclease system of any one of claims 1-3 , wherein the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96.
5 . The engineered nuclease system of any one of claims 1-3 , wherein the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96.
6 . The engineered nuclease system of any one of claims 1-5 , wherein the endonuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95.
7 . The engineered nuclease system of any one of claims 1-5 , wherein the endonuclease is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95.
8 . The engineered nuclease system of any one of claims 1-5 , wherein the endonuclease is encoded by a nucleic acid sequence having 100% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95.
9 . The engineered nuclease system of any one of claims 1-8 , wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.
10 . The engineered nuclease system of any one of claims 1-8 , wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.
11 . The engineered nuclease system of any one of claims 1-10 , wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.
12 . The engineered nuclease system of any one of claims 1-10 , wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.
13 . The engineered nuclease system of any one of claims 1-10 , wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.
14 . The engineered nuclease system of any one of claims 1-10 , wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-6.
15 . The engineered nuclease system of any one of claims 1-10 , wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-6.
16 . The engineered nuclease system of any one of claims 1-10 , wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 3-6.
17 . The engineered nuclease system of any one of claims 1-16 , wherein the donor template further comprises a polyadenylation signal.
18 . The engineered nuclease system of any one of claims 1-17 , wherein the donor template further comprises a nucleus-targeting sequence.
19 . The engineered nuclease system of claim 18 , wherein the nucleus-targeting sequence comprises a plurality of transcription factor binding sites.
20 . The engineered nuclease system of claim 19 , wherein the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β/E47, or MyoD.
21 . The engineered nuclease system of any one of claims 1-20 , wherein the nucleus-targeting sequence is on a 5′ end and a 3′ end of the donor template.
22 . The engineered nuclease system of any one of claims 1-21 , wherein the donor template further comprises a recognition site sequence for the endonuclease on a 5′ end or a 3′ end.
23 . The engineered nuclease system of claim 22 , wherein the nucleus-targeting sequence is 5′ to the recognition site sequence when the donor template is flanked on a 5′ end.
24 . The engineered nuclease system of claim 22 , wherein the nucleus-targeting sequence is 3′ to the recognition site sequence when the donor template is flanked on a 3′ end.
25 . The engineered nuclease system of any one of claims 1-24 , wherein the donor template comprises, from 5′ to 3′:
NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2),
wherein
NTS(1) denotes a first nucleus-targeting sequence;
NTS(2) denotes a second nucleus-targeting sequence;
NRS(1) denotes a first nuclease recognition site sequence;
NRS(2) denotes a second nuclease recognition site sequence;
SA denotes the splice acceptor sequence targeting exon 1 of said albumin gene; and
FVIII denotes the Factor VIII gene or fragment thereof.
26 . The engineered nuclease system of claim 25 , wherein a 5′ to 3′ orientation of NRS(1) and NRS(2) is according to:
(a) forward, forward;
(b) reverse, reverse;
(c) forward, reverse;
(d) reverse, forward;
wherein forward denotes a same 5′ to 3′ orientation as the target nucleic acid sequence, and reverse denotes an opposite 5′ to 3′ orientation as the target nucleic acid sequence.
27 . The engineered nuclease system of any one of claims 1-26 , wherein the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.
28 . The engineered nuclease system of any one of claims 1-26 , wherein the donor template comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.
29 . The engineered nuclease system of any one of claims 1-26 , wherein the donor template comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.
30 . The engineered nuclease system of any one of claims 1-29 , wherein the FVIII gene or functional fragment thereof is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif.
31 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 10, 71-79, and 89.
32 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 10, 71-79, and 89.
33 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79, and 89.
34 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 80% identity to SEQ ID NO: 10.
35 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 10.
36 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof comprises a sequence having 100% identity to SEQ ID NO: 10.
37 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof is modified to comprise a B-domain comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 71-79 and 89.
38 . The engineered nuclease system of any one of claims 1-30 , wherein the FVIII gene or functional fragment thereof is modified to comprise a B-domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89.
39 . The engineered nuclease system of any one of claims 37-38 , wherein the FVIII gene or functional fragment thereof comprising a modified B-domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 86-87 and 90.
40 . The engineered nuclease system of any one of claims 37-38 , wherein the FVIII gene or functional fragment thereof comprising a modified B-domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.
41 . A method for supplementing liver enzyme expression in a subject in need thereof, comprising administering to the subject:
a) an endonuclease comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96;
b) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence within an albumin gene or within an intron of the albumin gene; and
c) a donor template comprising a nucleic acid sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof, thereby supplementing liver enzyme expression in said subject.
42 . The method of claim 41 , wherein the target nucleic acid sequence within the albumin gene is within intron 1 of the albumin gene.
43 . The method of any one of claims 41-42 , wherein the sequence encoding a Factor VIII (FVIII) gene or a functional fragment thereof is operably linked to a splice acceptor sequence targeting exon 1 of the albumin gene.
44 . The method of any one of claims 41-43 , wherein the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96.
45 . The method of any one of claims 41-43 , wherein the endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO: 54 or SEQ ID NO: 96.
46 . The method of any one of claims 41-45 , wherein the endonuclease is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95.
47 . The method of any one of claims 41-46 , wherein the endonuclease is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95.
48 . The method of any one of claims 41-46 , wherein the endonuclease is encoded by a nucleic acid sequence having 100% sequence identity to any one of SEQ ID NOs: 30, 31, 53, and 95.
49 . The method of any one of claims 41-48 , wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.
50 . The method of any one of claims 41-48 , wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 14-15, 24-27, 43-45, 50, 55, 60-68, 97-98.
51 . The method of any one of claims 41-50 , wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.
52 . The method of any one of claims 41-50 , wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.
53 . The method of any one of claims 41-50 , wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1, 2, and 8.
54 . The method of any one of claims 41-50 , wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-6.
55 . The method of any one of claims 41-50 , wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-6.
56 . The method of any one of claims 41-50 , wherein the target nucleic acid sequence comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 3-6.
57 . The method of any one of claims 41-56 , wherein the donor template further comprises a polyadenylation signal.
58 . The method of any one of claims 41-57 , wherein the donor template further comprises a nucleus-targeting sequence.
59 . The method of claim 58 , wherein the nucleus-targeting sequence comprises a plurality of transcription factor binding sites.
60 . The engineered nuclease system of claim 59 , wherein the transcription factor is TCF1, HNF1, NFY, CEBP, OCT1, AP1, HNF1-α, HNF1-β, CEBPA, LEF-1, FOX D1, IRF1, HNF3, HNF4, HNF5, Tal1β/E47, or MyoD.
61 . The method of any one of claims 41-60 , wherein the nucleus-targeting sequence is on a 5′ end and a 3′ end of the donor template.
62 . The method of any one of claims 41-61 , wherein the donor template further comprises a recognition site sequence for the endonuclease on a 5′ end or a 3′ end.
63 . The method of claim 62 , wherein the nucleus-targeting sequence is 5′ to the recognition site sequence when the donor template is flanked on a 5′ end.
64 . The method of claim 62 , wherein the nucleus-targeting sequence is 3′ to the recognition site sequence when the donor template is flanked on a 3′ end.
65 . The method of any one of claims 41-64 , wherein the donor template comprises, from 5′ to 3′:
NTS(1)-NRS(1)-SA-FVIII-NRS(2)-NTS(2),
wherein
NTS(1) denotes a first nucleus-targeting sequence;
NTS(2) denotes a second nucleus-targeting sequence;
NRS(1) denotes a first nuclease recognition site sequence;
NRS(2) denotes a second nuclease recognition site sequence;
SA denotes the splice acceptor sequence targeting exon 1 of said albumin gene; and
FVIII denotes the Factor VIII gene or fragment thereof.
66 . The method of claim 65 , wherein a 5′ to 3′ orientation of NRS(1) and NRS(2) is according to:
(a) forward, forward;
(b) reverse, reverse;
(c) forward, reverse;
(d) reverse, forward;
wherein forward denotes a same 5′ to 3′ orientation as the target nucleic acid sequence, and reverse denotes an opposite 5′ to 3′ orientation as the target nucleic acid sequence.
67 . The method of any one of claims 41-66 , wherein the donor template comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.
68 . The method of any one of claims 41-66 , wherein the donor template comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.
69 . The method of any one of claims 41-66 , wherein the donor template comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 12-13, 16-23, 32-33, 56-59, 81-88, and 90-94.
70 . The method of any one of claims 41-69 , wherein the FVIII gene or functional fragment thereof is codon-optimized to remove at least one cytosine-guanine (CG or CpG) motif.
71 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 10, 71-79, and 89.
72 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 10, 71-79, and 89.
73 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof comprises a sequence having 100% identity to any one of SEQ ID NOs: 10, 71-79, and 89.
74 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 80% identity to SEQ ID NO: 10.
75 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof comprises a sequence having at least 90% identity to SEQ ID NO: 10.
76 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof comprises a sequence having 100% identity to SEQ ID NO: 10.
77 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof is modified to comprise a B-domain comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 71-79 and 89.
78 . The method of any one of claims 41-70 , wherein the FVIII gene or functional fragment thereof is modified to comprise a B-domain comprising a sequence having 100% identity to any one of SEQ ID NOs: 71-79 and 89.
79 . The method of any one of claims 77-78 , wherein the FVIII gene or functional fragment thereof comprising a modified B-domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 86-87 and 90.
80 . The method of any one of claims 77-78 , wherein the FVIII gene or functional fragment thereof comprising a modified B-domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 86-87 and 90.
81 . A cell comprising the engineered nuclease system of any one of claims 1-40 .
82 . The cell of claim 81 , wherein the cell is a liver cell.
83 . The cell of claim 81 , wherein the cell is a eukaryotic cell.
84 . The cell of claim 81 , wherein the cell is a mammalian cell.
85 . The cell of claim 81 , wherein the cell is an immortalized cell.
86 . The cell of claim 81 , wherein the cell is an insect cell.
87 . The cell of claim 81 , wherein the cell is a yeast cell.
88 . The cell of claim 81 , wherein the cell is a plant cell.
89 . The cell of claim 81 , wherein the cell is a fungal cell.
90 . The cell of claim 81 , wherein the cell is a prokaryotic cell.
91 . The cell of claim 81 , wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.
92 . The cell of claim 81 , wherein the cell is an engineered cell.
93 . The cell of claim 81 , wherein the cell is a stable cell.
94 . A lipid nanoparticle (LNP) comprising components (a) and (b) or components (a), (b), and (c) of the engineered nuclease system of any one of claims 1-40 .
95 . The lipid nanoparticle of claim 94 , wherein the LNP comprises a cationic lipid, a neutral lipid, cholesterol or a cholesterol analog, and a PEG-linked lipid.
96 . The lipid nanoparticle of claim 95 , wherein the cationic lipid comprises C12-200 (1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)), said neutral lipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or said PEG-linked lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).
97 . A viral vector comprising the engineered nuclease system of any one of claims 1-40 .
98 . The viral vector of claim 97 , wherein the viral vector is an adeno-associated viral (AAV) vector.
99 . The viral vector of claim 98 , wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof.
100 . The viral vector of claim 98 , wherein the AAV is AAV6.
101 . The viral vector of claim 98 , wherein the AAV is AAV8.