US2019127713A1PendingUtilityA1
Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use
Est. expiryApr 13, 2036(~9.7 yrs left)· nominal 20-yr term from priority
C07K 14/4703C07K 2319/80C07K 2319/09C12N 15/113C12N 2310/20C12N 2320/32C07K 2319/71C12N 9/22A61P 9/00C12N 2800/80A61K 9/0019C12N 7/00C12N 2750/14143C12N 15/11C12N 15/63A61K 48/00C12N 15/86A61K 48/0058C12N 15/111C12N 15/52
55
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
The present disclosure provides Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A method of modulating expression of a gene, in vivo, in a subject comprising administering to, or providing in, the subject:
(a) (i) a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; or (ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; and (b) (i) a gRNA which targets the fusion molecule to the gene; or (ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene, in an amount sufficient to modulate expression of the gene.
2 . The method of claim 1 , comprising administering to, or provided in, the subject any of: (a)(ii) and (b)(ii), (a)(i) and (b)(i), (a)(i) and (b)(ii), or (a)(ii) and (b)(i).
3 . The method of claim 1 , comprising administering to, or provided in, the subject:
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; and (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene.
4 . The method of claim 1 , wherein the nucleic acid of (a)(ii) comprises DNA.
5 . The method of claim 1 , wherein the nucleic acid of (b)(ii) comprises DNA.
6 . The method of claim 1 , wherein the nucleic acid of (a)(ii) comprises RNA.
7 . The method of claim 1 , wherein the nucleic acid of (b)(ii) comprises RNA.
8 . The method of claim 1 , wherein one or both of (a) and (b) are packaged in a viral vector.
9 . The method of claim 1 , wherein (a) is packaged in a viral vector.
10 . The method of claim 1 , wherein (b) is packaged in a viral vector.
11 . The method of claim 1 , wherein (a) and (b) are packaged in the same viral vector.
12 . The method of claim 8 , wherein the viral vector comprises an AAV vector.
13 . The method of claim 8 , wherein the viral vector comprises a lentiviral vector.
14 . The method of claim 1 , wherein (a) is packaged in a first viral vector and (b) is packaged in a second viral vector.
15 . The method of claim 14 , wherein the first viral vector comprises an AAV vector and the second viral vector comprises an AAV vector.
16 . The method of claim 1 , wherein the dCas9 molecule comprises a gRNA binding domain of a Cas9 molecule.
17 . The method of claim 1 , wherein the dCas9 molecule comprises one, two or all of: a Rec1 domain, a bridge helix domain, or a PAM interacting domain, of a Cas9 molecule.
18 . The method of claim 1 , wherein the dCas9 molecule is a mutant of a wild-type Cas9 molecule, e.g., in which the Cas9 nuclease activity is inactivated.
19 . The method of claim 1 , wherein the dCas9 molecule comprises a mutation that inactivates a Cas9 nuclease activity, e.g., a mutation in a DNA-cleavage domain of a Cas9 molecule.
20 . The method of claim 1 , wherein the dCas9 molecule comprises a mutation that inactivates a Cas9 nuclease activity, e.g., a mutation in a RuvC domain and/or a mutation in a HNH domain.
21 . The method of claim 1 , wherein the dCas9 molecule comprises a Staphylococcus aureus dCas9 molecule, a Streptococcus pyogenes dCas9 molecule, a Campylobacter jejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, a Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillus farciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (e.g., strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria cinerea dCas9 molecule, a Roseburia intestinalis dCas9 molecule, a Parvibaculum lavamentivorans dCas9 molecule, a Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9 molecule, a Campylobacter lari (e.g., strain CF89-12) dCas9 molecule, or a Streptococcus thermophilus (e.g., strain LMD-9) dCas9 molecule.
22 . The method of claim 1 , wherein the dCas9 molecule comprises an S. aureus dCas9 molecule, e.g., comprising an S. aureus dCas9 sequence described herein.
23 . The method of claim 1 , wherein the S. aureus dCas9 molecule comprises a mutation at an amino acid position, corresponding to position 10, 580, or both (e.g., D10A, N580A, or both), relative to a wild-type S. aureus dCas9 molecule, numbered according to SEQ ID NO: 25.
24 . The method of claim 1 , wherein the S. aureus dCas9 molecule comprises the amino acid sequence of SEQ ID NO: 35 or 36, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 35 or 36, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 35 or 36, or any fragment thereof.
25 . The method of claim 1 , wherein the dCas9 molecule comprises an S. pyogenes dCas9 molecule, e.g., comprising an S. pyogenes dCas9 sequence described herein.
26 . The method of claim 1 , the S. pyogenes dCas9 molecule comprises a mutation at an amino acid position, corresponding to position 10, 840, or both (e.g., D10A, H840A, or both), relative to a wild-type S. pyogenes dCas9 molecule, numbered according to SEQ ID NO: 24.
27 . The method of claim 1 , wherein the dCas9 molecule is less than 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, or 500 amino acids in length.
28 . The method of claim 1 , wherein the dCas9 molecule is 500-1300, 600-1200, 700-1100, 800-1000, 500-1200, 500-1000, 500-800, 500-600, 1000-1200, 800-1200, or 600-1200 amino acids in length.
29 . The method of claim 1 , wherein the dCas9 molecule has a size that is less than 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the size of a wild-type Cas9 molecule, e.g., a wild-type S. pyogenes Cas9 molecule or a wild-type S. aureus dCas9 molecule.
30 . The method of claim 1 , wherein the modulator of gene expression comprises a modulator of gene expression described herein.
31 . The method of claim 1 , wherein the modulator of gene expression comprises a repressor of gene expression, e.g., a Krüppel associated box (KRAB) molecule, an mSin3 interaction domain (SID) molecule, four concatenated mSin3 interaction domains (SID4X), MAX-interacting protein 1 (MXI1), or any fragment thereof.
32 . The method of claim 1 , wherein the modulator of gene expression comprises a Krüppel associated box (KRAB) molecule comprising the sequence of SEQ ID NO: 34, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, or any fragment thereof.
33 . The method of claim 1 , wherein the modulator of gene expression comprises an activator of gene expression, e.g., a VP16 transcription activation domain, a VP64 transcriptional activation domain, a p65 activation domain, an Epstein-Barr virus R transactivator Rta molecule, a VP64-p65-Rta fusion (VPR), Ldb1 self-association domain, or any fragment thereof.
34 . The method of claim 1 , wherein the modulator of gene expression comprises a modulator of epigenetic modification, e.g., a histone acetyltransferase (e.g., p300 catalytic domain), a histone deacetylase, a histone methyltransferase (e.g., SUV39H1 or G9a (EHMT2)), a histone demethylase (e.g., Lys-specific histone demethylase 1 (LSD1)), a DNA methyltransferase (e.g., DNMT3a or DNMT3a-DNMT3L), a DNA demethylase (e.g., TET1 catalytic domain or TDG), or fragment thereof.
35 . The method of claim 1 , wherein the modulator of gene expression is fused to the C-terminus, N-terminus, or both, of the dCas9 molecule.
36 . The method of claim 1 , wherein the modulator of gene expression is fused to the dCas9 molecule directly.
37 . The method of claim 1 , wherein the modulator of gene expression is fused to the dCas9 molecule indirectly, e.g., via a non-modulator or a linker, or a second modulator.
38 . The method of claim 1 , wherein a plurality of modulators of gene expression, e.g., two or more identical, substantially identical, or different modulators, are fused to the dCas9 molecule.
39 . The method of claim 1 , wherein the fusion molecule further comprises a nuclear localization sequence.
40 . The method of claim 39 , wherein one or more nuclear localization sequences are fused to the C-terminus, N-terminus, or both, of the dCas9 molecule, e.g., directly or indirectly, e.g., via a linker.
41 . The method of claim 40 , wherein the one or more nuclear localization sequences comprise the amino acid sequence of SEQ ID NO: 37 or 38, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 37 or 38, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 37 or 38, or any fragment thereof.
42 . The method of claim 1 , wherein the fusion molecule comprises the amino acid sequence of SEQ ID NO: 39, 40, or 41, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 39, 40, or 41, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 39, 40, or 41, or any fragment thereof.
43 . The method of claim 1 , wherein the nucleic acid that encodes the fusion molecule comprises the sequence of SEQ ID NO: 23, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 23, or a sequence having one, two, three, four, five or more changes, e.g., substitutions, insertions, or deletions, relative to SEQ ID NO: 23, or any fragment thereof.
44 . The method of claim 1 , wherein the gRNA comprises a unimolecular gRNA.
45 . The method of claim 1 , wherein the gRNA comprises a bimolecular gRNA.
46 . The method of claim 1 , wherein the gRNA comprises a gRNA sequence described herein.
47 . The method of claim 1 , wherein gene expression is modulated in a cell, tissue, or organ described herein, e.g., Table 2 or 3.
48 . The method of claim 1 , wherein gene expression is modulated in the liver.
49 . The method of claim 1 , wherein the modulation is sufficient to alter a function of the gene, or a symptom of a disorder associated with the gene, as described herein, e.g., in Table 2 or 3.
50 . The method of claim 1 , wherein the modulation comprises modulation of transcription.
51 . The method of claim 1 , wherein the modulation comprises down-regulation of transcription.
52 . The method of claim 1 , wherein the modulation comprises up-regulation of transcription.
53 . The method of claim 1 , wherein the modulation comprises modulating the temporal pattern of expression of the gene.
54 . The method of claim 1 , wherein the modulation comprises modulating the spatial pattern of expression of the gene.
55 . The method of claim 1 , wherein the modulation comprises modulating a post-transcriptional or co-transcriptional modification, e.g., splicing, 5′ capping, 3′ cleavage, 3′ polyadenylation, or RNA export.
56 . The method of claim 1 , wherein the modulation comprises modulating the expression of an isoform, e.g., an increase or decrease in the expression of an isoform, the increase or decrease in the expression of a first isoform over a second isoform.
57 . The method of claim 1 , wherein the modulation comprises modulating chromatin structure, e.g., increasing or decreasing methylation, acetylation, phosphorylation, or ubiquitination, e.g., at a preselected site, or altering the spatial pattern, cell specificity, or temporal occurrence of methylation, acetylation, phosphorylation, or ubiquitination.
58 . The method of claim 1 , wherein the modulation comprises modulating a post-translational modification (e.g., indirectly), e.g., glycosylation, lipidation, acetylation, phosphorylation, amidation, hydroxylation, methylation, ubiquitination, sulfation, nitrosylation, or proteolysis.
59 . The method of claim 1 , wherein the modulation does not comprise cleaving the subject's DNA.
60 . The method of claim 1 , wherein the modulation comprises an inducible modulation.
61 . The method of claim 1 , wherein the gene is selected from Table 2, optionally wherein the method down-regulates the expression of the gene.
62 . The method of any of claims 1 - 60 , wherein the gene is selected from Table 3, optionally wherein the method up-regulates the expression of the gene.
63 . The method of claim 1 , wherein the gene comprises PCSK9.
64 . The method of claim 1 , wherein the dCas9 molecule does not cleave the genome of the subject.
65 . A method of modulating expression of a gene, in vivo, in a subject comprising administering to, or providing in, the subject:
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising an S. aureus dCas9 molecule fused to a KRAB molecule; and (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene, and wherein one or both of (a)(i) and (b)(ii) are packaged in an AAV vector.
66 . The method of claim 65 , wherein the fusion molecule (e.g., a fusion molecule described herein) comprises a sequence described herein, e.g., the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or any fragment thereof.
67 . The method of claim 65 , wherein the gRNA comprises a gRNA sequence described herein.
68 . The method of claim 65 , wherein the gene is selected from Table 2 or 3.
69 . The method of claim 65 , wherein the gene comprises PCSK9.
70 . The method of claim 65 , wherein (a)(ii) and (b)(ii) are packaged in different AAV vectors.
71 . The method of claim 65 , wherein (a)(ii) and (b)(ii) are packaged in the same AAV vector.
72 . A pharmaceutical composition, or unit dosage form, comprising, in an amount sufficient for modulating a gene in a human subject, or in an amount sufficient for a therapeutic effect in a human subject,
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; and/or (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene, wherein one or both of (a)(ii) and (b)(ii) are packaged in a viral vector.
73 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the fusion molecule (e.g., a fusion molecule described herein) comprises a sequence described herein, e.g., the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or any fragment thereof.
74 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gRNA comprises a gRNA sequence described herein.
75 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gene is selected from Table 2 or 3.
76 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gene comprises PCSK9.
77 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein (a)(ii) and (b)(ii) are packaged in the same viral vector, e.g., an AAV vector.
78 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein (a)(ii) and (b)(ii) are packaged in different viral vectors, e.g., AAV vectors.
79 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the viral vector (e.g., AAV vector) comprising (a)(ii), and the viral vector (e.g., AAV vector) comprising (b)(ii), are provided in separate containers.
80 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the viral vector (e.g., AAV vector) comprising (a)(ii) and the viral vector (e.g., AAV vector) comprising (b)(ii), are provided in the same container.
81 . The pharmaceutical composition, or unit dosage form, of claim 72 , which is formulated for administration, e.g., oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, or intraarticular administration, or administration via inhalation or via buccal administration, or any combination thereof, to the subject.
82 . The pharmaceutical composition, or unit dosage form, of claim 72 , which is formulated for intravenous administration to the subject.
83 . The pharmaceutical composition, or unit dosage form, of claim 72 , which is disposed in a device suitable for administration, e.g., oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, or intraarticular administration, or administration via inhalation or via buccal administration, or any combination thereof, to the subject.
84 . The pharmaceutical composition, or unit dosage form, of claim 72 , which is disposed in a device suitable for intravenous administration to the subject.
85 . The pharmaceutical composition, or unit dosage form, of claim 72 , which is disposed in a volume of at least 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 ml.
86 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the nucleic acid of (a)(ii) comprises DNA.
87 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the nucleic acid of (b)(ii) comprises DNA.
88 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the nucleic acid of (a)(ii) comprises RNA.
89 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the nucleic acid of (b)(ii) comprises RNA.
90 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises a gRNA binding domain of a Cas9 molecule.
91 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises one, two or all of: a Rec1 domain, a bridge helix domain, or a PAM interacting domain, of a Cas9 molecule.
92 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule is a mutant of a wild-type Cas9 molecule, e.g., in which the Cas9 nuclease activity is inactivated.
93 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises a mutation that inactivates a Cas9 nuclease activity, e.g., a mutation in a DNA-cleavage domain of a Cas9 molecule.
94 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises a mutation that inactivates a Cas9 nuclease activity, e.g., a mutation in a RuvC domain and/or a mutation in a HNH domain.
95 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises a Staphylococcus aureus dCas9 molecule, a Streptococcus pyogenes dCas9 molecule, a Campylobacter jejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, a Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillus farciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (e.g., strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria cinerea dCas9 molecule, a Roseburia intestinalis dCas9 molecule, a Parvibaculum lavamentivorans dCas9 molecule, a Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9 molecule, a Campylobacter lari (e.g., strain CF89-12) dCas9 molecule, or a Streptococcus thermophilus (e.g., strain LMD-9) dCas9 molecule.
96 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises an S. aureus dCas9 molecule, e.g., comprising an S. aureus dCas9 sequence described herein.
97 . The pharmaceutical composition, or unit dosage form, of claim 96 , wherein the S. aureus dCas9 molecule comprises a mutation at an amino acid position, corresponding to position 10, 580, or both (e.g., D10A, N580A, or both), relative to a wild-type S. aureus dCas9 molecule, numbered according to SEQ ID NO: 25.
98 . The pharmaceutical composition, or unit dosage form, of claim 96 , wherein the S. aureus dCas9 molecule comprises the amino acid sequence of SEQ ID NO: 35 or 36, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 35 or 36, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 35 or 36, or any fragment thereof.
99 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule comprises an S. pyogenes dCas9 molecule, e.g., comprising an S. pyogenes dCas9 sequence described herein.
100 . The pharmaceutical composition, or unit dosage form, of claim 99 , wherein the S. pyogenes dCas9 molecule comprises a mutation at an amino acid position, corresponding to position 10, 840, or both (e.g., D10A, H840A, or both), relative to a wild-type S. pyogenes dCas9 molecule, numbered according to SEQ ID NO: 24.
101 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule is less than 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, or 500 amino acids in length.
102 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule is 500-1300, 600-1200, 700-1100, 800-1000, 500-1200, 500-1000, 500-800, 500-600, 1000-1200, 800-1200, or 600-1200 amino acids in length.
103 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 molecule has a size that is less than 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the size of a wild-type Cas9 molecule, e.g., a wild-type S. pyogenes Cas9 molecule or a wild-type S. aureus dCas9 molecule.
104 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein modulator of gene expression comprises a modulator of gene expression described herein.
105 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein modulator of gene expression comprises a KRAB molecule, e.g., comprising the sequence of SEQ ID NO: 34, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, or any fragment thereof.
106 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gRNA comprises a unimolecular gRNA.
107 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gRNA comprises a bimolecular gRNA.
108 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gRNA comprises a gRNA sequence described herein.
109 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein gene expression is modulated in a cell, tissue, or organ described herein, e.g., Table 2 or 3.
110 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein gene expression is modulated in the liver.
111 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation is sufficient to alter a function of the gene, or a symptom of a disorder associated with the gene, as described herein, e.g., in Table 2 or 3.
112 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulation of transcription.
113 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises down-regulation of transcription.
114 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises up-regulation of transcription.
115 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulating the temporal pattern of expression of the gene.
116 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulating the spatial pattern of expression of the gene.
117 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulating a post-transcriptional or co-transcriptional modification, e.g., splicing, 5′ capping, 3′ cleavage, 3′ polyadenylation, or RNA export.
118 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulating the expression of an isoform, e.g., an increase or decrease in the expression of an isoform, the increase or decrease in the expression of a first isoform over a second isoform.
119 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulating chromatin structure, e.g., increasing or decreasing methylation, acetylation, phosphorylation, or ubiquitination, e.g., at a preselected site, or altering the spatial pattern, cell specificity, or temporal occurrence of methylation, acetylation, phosphorylation, or ubiquitination.
120 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the modulation comprises modulating a post-translational modification (e.g., indirectly), e.g., glycosylation, lipidation, acetylation, phosphorylation, amidation, hydroxylation, methylation, ubiquitination, sulfation, nitrosylation, or proteolysis.
121 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gene is selected from Table 2, optionally wherein the method down-regulates the expression of the gene.
122 . The pharmaceutical composition, or unit dosage form, of any of claim 72 , wherein the gene is selected from Table 3, optionally wherein the method up-regulates the expression of the gene.
123 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the gene comprises PCSK9.
124 . The pharmaceutical composition, or unit dosage form, of claim 72 , wherein the dCas9 does not cleave the genome of the subject.
125 . A pharmaceutical composition, or unit dosage form, comprising, in an amount sufficient for modulating a gene in a human subject, or in an amount sufficient for a therapeutic effect in a human subject,
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising an S. aureus dCas9 molecule fused to a KRAB molecule; and/or (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to the gene, wherein one or both of (a)(ii) and (b)(ii) are packaged in a viral vector.
126 . The pharmaceutical composition, or unit dosage form, of claim 125 , wherein the fusion molecule comprises a sequence described herein, e.g., the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or any fragment thereof.
127 . The pharmaceutical composition, or unit dosage form, of claim 125 , wherein the gRNA comprises a gRNA sequence described herein.
128 . The pharmaceutical composition, or unit dosage form, of claim 125 , wherein the gene is selected from Table 2 or 3.
129 . The pharmaceutical composition, or unit dosage form, of claim 125 , wherein the gene comprises PCSK9.
130 . The pharmaceutical composition, or unit dosage form, of claim 125 , wherein (a)(ii) and (b)(ii) are packaged in different AAV vectors.
131 . The pharmaceutical composition, or unit dosage form, of claim 125 , wherein (a)(ii) and (b)(ii) are packaged in the same AAV vector.
132 . A viral vector comprising:
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; and/or (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to a gene.
133 . The viral vector of claim 132 , wherein the viral vector is an AAV vector, the fusion molecule comprises a fusion molecule described herein, the dCas9 molecule comprises a dCas9 molecule described herein (e.g., an S. aureus dCas9 molecule), and/or the modulator of gene expression comprises a modulator described herein.
134 . The viral vector of claim 132 , comprising:
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising an S. aureus dCas9 molecule fused to a KRAB molecule; and (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to PCSK9, wherein one or both of (a)(ii) and (b)(ii) are packaged in an AAV vector.
135 . The viral vector of claim 132 , wherein the fusion molecule comprises a sequence described herein, e.g., the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or any fragment thereof.
136 . The viral vector of claim 132 , wherein the gRNA comprises a gRNA sequence described herein.
137 . The viral vector of claim 132 , wherein the gene is selected from Table 2 or 3.
138 . The viral vector of claim 132 , wherein the gene comprises PCSK9.
139 . A method of treating a disorder, comprising administering to a subject:
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; and (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to a gene associated with the disorder, thereby treating the disorder.
140 . The method of claim 139 , wherein the disorder is selected from Table 2 or 3, the fusion molecule comprises a fusion molecule described herein, the dCas9 molecule comprises a dCas9 molecule described herein, the modulator of gene expression comprises a modulator described herein, and/or the gRNA comprises a gRNA sequence described herein.
141 . The method of claim 139 , wherein the gene is selected from Table 2 or 3.
142 . The method of claim 139 , wherein one or both of (a)(ii) and (b)(ii) are provided in an AAV vector.
143 . A method of treating a cardiovascular disease, comprising administering to a subject:
(a)(ii) a nucleic acid that encodes a fusion molecule comprising a sequence comprising a dCas9 molecule fused to a modulator of gene expression; and (b)(ii) a nucleic acid that encodes a gRNA which targets the fusion molecule to a PCSK9 gene, thereby treating the cardiovascular disease.
144 . The method of claim 143 , wherein the fusion molecule comprises a fusion molecule described herein, the dCas9 molecule comprises a dCas9 molecule described herein, e.g., an S. aureus dCas9 molecule, and/or the modulator of gene expression comprises a modulator described herein.
145 . The method of claim 143 , wherein the fusion molecule comprises a sequence described herein, e.g., the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or a sequence having one, two, three, four, five or more changes, e.g., amino acid substitutions, insertions, or deletions, relative to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, or 41, or any fragment thereof.
146 . The method of claim 143 , wherein the gRNA comprises a gRNA sequence described herein.
147 . The method of claim 143 , wherein one or both of (a)(ii) and (b)(ii) are provided in an AAV vector.Join the waitlist — get patent alerts
Track US2019127713A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.