Compositions and methods comprising a TTR guide RNA and a polynucleotide encoding an RNA-guided DNA binding agent
Compositions and methods for editing, e.g., introducing double-stranded breaks, within the TTR gene are provided. Compositions and methods for treating subjects having amyloidosis associated with transthyretin (ATTR), are provided.
1 . A method of treating amyloidosis associated with deposition of transthyretin (ATTR) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising:
a single guide RNA (sgRNA) comprising the sequence mA*mA*mA*GGCUGCUGAUGACACCUGUUUUAGAmGmCmUmAmGmAmAmAmUmA mGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 87), wherein a * indicates a phosphorothioate (PS) linkage and a lower case “m” indicates that the nucleotide is 2′-O-Me modified; and
a messenger RNA (mRNA) comprising an open reading frame (ORF) that is at least 98% identical to SEQ ID NO: 311, wherein the ORF encodes a Cas9 and each thymidine in SEQ ID NO: 311 is replaced with a modified or unmodified uridine.
2 . The method of claim 1 , wherein the ORF comprises SEQ ID NO: 311, wherein each thymidine in SEQ ID NO: 311 is replaced with a modified or unmodified uridine.
3 . The method of claim 2 , wherein each thymidine in SEQ ID NO: 311 is replaced with N1-methyl-pseudouridine.
4 . The method of claim 1 , wherein the mRNA comprises:
a 5′ UTR with at least 90% identity to any one of SEQ ID NOs: 232, 234, 236, 238, 241, or 275-277, wherein each thymidine in any one of SEQ ID NOs: 232, 234, 236, 238, 241, and 275-277 is replaced with a modified or unmodified uridine; and/or
a 3′ UTR with at least 90% identity to any one of SEQ ID NOs: 233, 235, 237, 239, or 240, wherein each thymidine in any one of SEQ ID NOs: 233, 235, 237, 239, or 240 is replaced with a modified or unmodified uridine.
5 . The method of claim 1 , wherein the mRNA comprises a 5′ UTR and a 3′ UTR from the same source, wherein the source is a constitutively expressed mRNA.
6 . The method of claim 1 , wherein the mRNA comprises a 5′ cap selected from Cap0, Cap1, and Cap2.
7 . The method of claim 1 , wherein at least 10% of the uridine in the mRNA is modified uridine.
8 . The method of claim 7 , wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.
9 . The method of claim 1 , wherein the mRNA is at least 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 377, wherein each uridine in SEQ ID NO: 377 is modified or unmodified.
10 . The method of claim 9 , wherein each uridine in SEQ ID NO: 377 is N1-methyl-pseudouridine.
11 . The method of claim 1 , wherein the Cas9 comprises a nuclear localization signal (NLS).
12 . The method of claim 1 , wherein the sgRNA and the mRNA are associated with a lipid nanoparticle (LNP).
13 . The method of claim 12 , wherein the LNP comprises a CCD lipid, a neutral lipid, a helper lipid, and a stealth lipid.
14 . The method of claim 13 , wherein the CCD lipid is Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate).
15 . The method of claim 14 , wherein
the neutral lipid is distearoylphosphatidylcholine (DSPC);
the helper lipid is cholesterol; and
the stealth lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DMG).
16 . The method of claim 14 , wherein the LNP comprises:
about 50 mol-% Lipid A;
about 9 mol-% distearoylphosphatidylcholine (DSPC);
about 38 mol-% cholesterol; and
about 3 mol-% 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DMG).
17 . The method of claim 1 , wherein each thymidine in SEQ ID NO: 311 is replaced with N1-methyl-pseudouridine.
18 . The method of claim 1 , wherein the subject is human and
serum transthyretin (TTR) levels in the subject, measured eight weeks after administration of the pharmaceutical formulation, are reduced by at least 50% compared to serum TTR levels before administration of the pharmaceutical formulation to the subject; and/or
amyloid deposition in the subject is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% compared to the amyloid deposition in a negative control or to the level of amyloid deposition in the subject before administration of the pharmaceutical formulation to the subject.
19 . The method of claim 1 , wherein the ATTR is ATTR familial amyloidosis.
20 . The method of claim 1 , wherein the ATTR is wild-type ATTR.
21 . The method of claim 1 , wherein the subject has been diagnosed with familial amyloid cardiomyopathy or exhibits symptoms of restrictive cardiomyopathy or congestive heart failure.
22 . The method of claim 1 , wherein the subject has been diagnosed with familial amyloid polyneuropathy or exhibits symptoms of sensorimotor neuropathy.
23 . A method of treating amyloidosis associated with deposition of transthyretin (ATTR) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising:
a single guide RNA (sgRNA) comprising the sequence mA*mA*mA*GGCUGCUGAUGACACCUGUUUUAGAmGmCmUmAmGmAmAmAmUmA mGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 87) wherein a * indicates a phosphorothioate (PS) linkage and a lower case “m” indicates that the nucleotide is 2′-O-Me modified; and
a messenger RNA (mRNA) comprising SEQ ID NO: 377, wherein each uridine in SEQ ID NO: 377 is modified or unmodified.
24 . The method of claim 23 , wherein the mRNA further comprises a 5′ cap selected from Cap0, Cap1, and Cap2 and each uridine in SEQ ID NO: 377 is modified.
25 . The method of claim 23 , wherein each uridine in SEQ ID NO:377 is N1-methyl-pseudouridine.
26 . The method of claim 23 , wherein the subject is human and
serum transthyretin (TTR) levels in the subject, measured eight weeks after administration of the pharmaceutical formulation, are reduced by at least 50% compared to serum TTR levels before administration of the pharmaceutical formulation to the subject; and/or
amyloid deposition in the subject is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% compared to the amyloid deposition in a negative control or to the level of amyloid deposition in the subject before administration of the pharmaceutical formulation to the subject.
27 . The method of claim 23 , wherein the ATTR is ATTR familial amyloidosis.
28 . The method of claim 23 , wherein the ATTR is wild-type ATTR.
29 . The method of claim 23 , wherein the subject has been diagnosed with familial amyloid cardiomyopathy or exhibits symptoms of restrictive cardiomyopathy or congestive heart failure.
30 . The method of claim 23 , wherein the subject has been diagnosed with familial amyloid polyneuropathy or exhibits symptoms of sensorimotor neuropathy.