COMPOSITIONS AND METHODS FOR MODULATING APOLIPOPROTEIN B (APOB) GENE EXPRESSION
The present invention provides agents and compositions for modulating expression (e.g., enhanced or reduced expression) of an apolipoprotein B (APOB) gene by targeting an APOB expression control region and methods of use thereof for treating an APOB associated disorder, e.g., hypercholesterolemia.
1 . A site-specific apolipoprotein B (APOB) disrupting agent, comprising a site-specific APOB targeting moiety which targets an APOB expression control region.
2 - 4 . (canceled)
5 . The site-specific APOB disrupting agent of claim 1 ,
(a) wherein the site specific APOB targeting moiety comprises a polymeric molecule, optionally a polyamide or a polynucleotide; optionally wherein the polymeric molecule comprises a peptide nucleic acid (PNA);
(b) wherein the expression control region comprises an APOB-associated anchor sequence; optionally, wherein the APOB-associated anchor sequence comprises a CCCTC-binding factor (CTCF) binding motif, and/or the nucleotide sequence of APOB CTCF site 3;
(c) wherein the expression control region comprises an APOB-associated anchor sequence-mediated conjunction, optionally, wherein the APOB-associated anchor sequence-mediated conjunction comprises one or more transcriptional control elements internal to the conjunction, or one or more transcriptional control elements external to the conjunction;
(d) wherein the APOB-associated anchor sequence is located within about 300 kb or within 10 kb of the transcriptional control element; and/or
(e) wherein the disrupting agent comprises a modification.
6 - 12 . (canceled)
13 . The site-specific APOB disrupting agent of claim 1 , wherein the expression control region comprises an APOB-specific transcriptional control element; optionally, wherein the transcriptional control element comprises an APOB promoter, a transcriptional enhancer, or a transcriptional repressor.
14 - 16 . (canceled)
17 . The site-specific APOB disrupting agent of claim 1 , wherein the APOB targeting moiety comprises
(a) a nucleotide sequence having at least 85% nucleotide identity to the entire nucleotide sequence of any of the nucleotide sequences in Table 2; and/or
(b) a polynucleotide encoding a DNA-binding domain, or fragment thereof, of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the APOB expression control region.
18 - 23 . (canceled)
24 . The site-specific APOB disrupting agent of claim 1 , wherein the site-specific APOB disrupting agent is present in a composition,
optionally, wherein the composition comprises a pharmaceutical composition, optionally,
wherein the pharmaceutical composition comprises (a) a lipid formulation comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or combinations of any of the foregoing, and/or
(b) a lipid nanoparticle.
25 - 28 . (canceled)
29 . A site-specific APOB disrupting agent, comprising a nucleic acid molecule encoding a fusion protein, the fusion protein comprising a site-specific APOB targeting moiety which targets an APOB expression control region and an effector molecule.
30 . The site-specific APOB disrupting agent of claim 29 , wherein the site-specific APOB targeting moiety comprises a polynucleotide encoding a DNA-binding domain, or fragment thereof, of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the APOB expression control region.
31 . The site-specific APOB disrupting agent of claim 29 ,
(a) wherein the effector molecule comprises a nucleic acid molecule encoding a polypeptide; and/or further comprising a catalytically active domain of human exonuclease 1 (hEXO1);
(b) wherein the effector molecule is selected from the group consisting of a nuclease, a physical blocker, an epigenetic recruiter, and an epigenetic CpG modifier, and combinations of any of the foregoing; optionally, wherein the epigenetic recruiter comprises a transcriptional enhancer or a transcriptional repressor; and/or wherein the epigenetic CpG modifier comprises a DNA methylase, a DNA demethylase, a histone modifying agent, or a histone deacetylase;
(c) wherein the effector comprises a CRISPR associated protein (Cas) polypeptide or nucleic acid molecule encoding the Cas polypeptide; optionally, wherein the Cas polypeptide is an enzymatically inactive Cas polypeptide;
(d) wherein the effector molecule comprises a zinc finger polypeptide;
(e) wherein the effector molecule comprises a Transcription activator-like effector nuclease (TALEN) polypeptide; and/or
(f) wherein the fusion protein comprises a peptide nucleic acid fusion.
32 - 40 . (canceled)
41 . A vector comprising a nucleic acid molecule encoding the site-specific APOB disrupting agent of claim 1 ; optionally, wherein the vector is a viral expression vector.
42 . (canceled)
43 . A cell comprising the site-specific APOB disrupting agent of claim 1 .
44 . The site-specific APOB disrupting agent of claim 29 , wherein the site-specific APOB disrupting agent is present in a composition
optionally, wherein the composition comprises a pharmaceutical composition,
optionally, wherein the pharmaceutical composition comprises (a) a lipid formulation comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or combinations of any of the foregoing, and/or
(b) a lipid nanoparticle.
45 - 48 . (canceled)
49 . A method of modulating expression of Apolipoprotein B (APOB) in a cell, the method comprising contacting the cell with the site-specific APOB disrupting agent of claim 1 , and an effector molecule, thereby modulating expression of APOB in the cell.
50 . The method of claim 49 , wherein the modulation of expression is enhanced or reduced expression of APOB in the cell; optionally, wherein the cell is a mammalian cell;
optionally wherein the cell is a somatic cell or a primary cell; and/or
optionally, wherein the contacting is performed in vitro; in vivo; or ex vivo.
51 - 100 . (canceled)
101 . The method of claim 49 , further comprising administering the cell to a subject; optionally, wherein the cell is within a subject, and/or wherein the subject has an APOB-associated disease selected from the group consisting of a hyperlipidemia, a hypercholesterolemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia, insulin resistance not related to an immune response to insulin, type 2 diabetes, hypertension, endothelial cell dysfunction, heart disease, and atherosclerosis.
102 - 104 . (canceled)
105 . A method for treating a subject having an APOB-associated disease, comprising administering to the subject a therapeutically effective amount of the site-specific APOB disrupting agent of claim 1 , and an effector molecule, thereby treating the subject.
106 . The method of claim 105 , wherein the APOB-associated disease is hypercholesterolemia and the site-specific APOB disrupting agent reduces expression of APOB in the subject; optionally
(a) wherein the site-specific APOB disrupting agent and the effector molecule are administered to the subject concurrently,
(b) wherein the site-specific APOB disrupting agent and the effector molecule are administered to the subject sequentially;
(c) wherein the effector molecule is administered to the subject prior to administration of the site-specific APOB disrupting agent; and/or
(d) wherein the site-specific APOB disrupting agent is administered to the subject prior to administration of the effector molecule.
107 - 110 . (canceled)