Atherosclerotic plaque reduction treatments and compositions

Abstract

Disclosed herein are therapeutic methods and compositions for the treatment of atherosclerosis and regression of a distal atherosclerotic plaque, wherein the composition is administered to a subject and targeted to the liver.

Description

ATHEROSCLEROTIC PLAQUE REDUCTION TREATMENTS AND COMPOSITIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority pursuant to 35 U.S.C. § 119(e) of U.S. provisional patent application No. 63 / 569,437 entitled “ATHEROSCLEROTIC PLAQUE REDUCTION TREATMENTS AND COMPOSITIONS,” filed on 25 March 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml file, created on 25 March 2025, is named P318597WO01.xml and is 20,589 bytes in size.BACKGROUND

[0003] Atherosclerosis is a chronic maladaptive inflammatory response triggered by the retention of cholesterol-rich low-density lipoproteins (LDL-C) within the walls of arteries. Buildup of these lipoproteins results in the formation of atherosclerotic plaques in the artery walls. Rupture and mobilization of the plaques result in ischemic stroke and myocardial infarction, and is therefore a leading cause of mortality in the United States.

[0004] Circulating monocytes / macrophages enter the arterial wall, ingest excess LDL-C and pass the cholesterol to high-density lipoproteins (HDL-C) in an attempt to prevent further accumulation by returning cholesterol to the circulation and ultimately the liver for excretion. This process is known as reverse cholesterol transport.

[0005] Currently, medical treatments for atherosclerosis are focused on altering aspects of normal lipoprotein metabolism to slow the accumulation of lipoproteins (principally LDLs) in the vasculature. For example, statins target and impair de novo synthesis of cholesterol in while PCSK9 inhibitors increase cell surface LDL receptor availability resulting in increased uptake of circulating LDL-C.

[0006] Adverse cardiovascular events and mortality are determined by atherosclerotic plaque burden. Human studies show that the level of plaque present in an individual corresponds to 67-100% of cardiovascular disease (CVD) risk. Thus, plaque regression is associated with lowering of CVD risk. For example, 1% regression in percent atheroma volume has been associated with a -20% reduction in major cardiovascular events.

[0007] While statins and PCSK9 inhibitors may slow the growth of atherosclerotic plaques, by reducing circulating LDL-C levels, they cannot effectively regress the size of existing plaques. Thus, these treatments produce very modest (-15%) reductions in cardiovascular-based mortality in humans.

[0008] Thus, mortality associated with atherosclerotic CVD remains high and there is a great need for therapeutic compositions and methods that result in meaningful regression of atherosclerotic measures such as plaque size.SUMMARY

[0009] Disclosed herein are methods and systems for reducing at least one measurable aspect of an atherosclerotic plaque in a subject, comprising: identifying at least one atherosclerotic plaque in the subject with a measured aspect having a first value; administering a composition comprising; at least one cholesterol degrading protein, and at least one molecule or compound configured to bind a surface protein of a hepatocyte; allowing the at least one cholesterol degrading protein to be expressed in the hepatocyte; measuring the measured aspect to determine a second value, wherein the first and second value differ by greater than 5%. In many embodiments, the subject may suffer from hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, or homozygous familial hypercholesterolemia, and the measurable aspect may be selected from area, volume, and collagen content. In some embodiments, the method further comprises the step of administering a statin or PCSK9 inhibitor to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a schematic representations of A) a liver lobule showing the central vein, the portal triad (hepatic artery, hepatic vein and bile duct) and the targeted hepatocytes, B) CDP1 mRNA encapsulated in LNPs that have an average diameter of 80 nm, pegylated lipids (PEG-lipids) and N-acetylgalactosamine (GalNAc) lipids on their surface and carry a low surface charge. C) The compact size of the LNPs allows their passage through liver sinusoidal fenestrae that normally range between 100-200 nm in diameter while the surface GalNAc facilitates their binding and uptake via ASGPR, allowing the ionizable lipids of the LNP to release CDP1 mRNA into the hepatocyte cytoplasm where it can be translated by ribosomes.

[0011] FIG. 2 Kinetics of CDP1 expression following a single LNP-mRNA treatment. Total proteins extracted from the right liver lobe of mice were separated by SDS-PAGE and the level of CDP1 expression relative to the housekeeping protein, a-tubulin, wasquantified by Western blot. The approximate half-life of CDP1 expression was assessed to be 37.5 hours.

[0012] FIG. 3 shows results from 6 weeks of LNP-CDP1 mRNA treatment, wherein the treatment significantly reduces plaque lipids in the aortic root without reducing serum LDL-cholesterol. (A) Compared to controls (G1 - LNP-Empty), mice treated with CDP1 (G3 - LNP-CDP1 mRNA) exhibited a significant (P = 0.05) 19% reduction in plaque lipids in the aortic root. Atorvastatin treatment produced no significant reduction. Reduction in plaque lipids acts to stabilize unstable plaques, reducing risk of rupture and cardiovascular event. (B) Atorvastatin treatment rapidly and significantly lowers serum LDL-cholesterol, while LNP-CDP1 mRNA treatment did not affect serum LDL- cholesterol. Nonetheless, LNP-CDP1 mRNA treatment produced profound and significant changes in the plaque environment, while atorvastatin did not.

[0013] FIG. 4 shows that CDP1 treatment significantly increases plaque collagen content in the aortic roots of ApoE-null mice. Cholesterol-rich plaques are unstable and acute coronary events such as myocardial infarction are frequently caused by the rupture of unstable atherosclerotic plaque. Collagen plays a key role in determining plaque stability. In addition to significantly lowering plaque lipid content, treatment with LNP-CDP1 mRNA significantly increased plaque collagen content (P<0.05 vs vehicle), while treatment with atorvastatin alone did not affect collagen content. LNP-CDP1 mRNA monotherapy and the combination (Atorvastatin + LNP-CDP1 mRNA) therapy increased collagen content to a similar extent, suggesting that this response was specifically mediated by CDP.

[0014] FIG. 5 shows CDP1 treatment significantly decreases serum free cholesterol and triglycerides. It is expected that CDP1 reduces circulating free cholesterol, given that intracellular free cholesterol is the target of CDP1. Serum triglyceride reduction is broadly considered to be beneficial, as evidence shows raised triglyceride levels associate with atherosclerotic heart disease.

[0015] FIG. 6 shows CDP1 treatment significantly increases serum IL-10 levels. IL- 10 has potent anti-inflammatory properties in macrophages and T cells. It modulates many cellular processes that can interfere with the development and stability of the atherosclerotic plaque. Increased expression may contribute to the observed effects on plaque resulting from CDP1 treatment.

[0016] FIG. 7 shows CDP1 treatment significantly increases SRA1 expression in spleen tissue. The spleen harbors monocytes that enter circulation to migrate to areas of plaque and endothelial damage, where they become macrophages. Increased SRA1expression is known to beneficially affect macrophage function and reduce inflammation.

[0017] FIG. 8 shows that CDP1 treatment reduces the appearance of serum lipids. LDLR-null mice exhibit lipidemic, cloudy serum. In this set of samples, serum becomes visibly less lipidemic in the time course following a single LNP-CDP1 mRNA injection, up to the 96 hour time point.

[0018] FIG. 9 shows that CDP1 treatment significantly decreases serum triglycerides. CDP1 treatment produces as large a relative effect on serum triglyceride levels in LDLR-null mice as it does in ApoE-null mice, despite the much higher levels. Serum triglyceride reduction is broadly considered to be beneficial, as evidence shows raised triglyceride levels to associate with atherosclerotic heart disease.

[0019] FIG. 10 shows that CDP1 treatment significantly decreases serum total cholesterol. CDP treatment results in a lowering of serum total cholesterol in LDLR-null mice.

[0020] FIG. 11 shows that CDP1 treatment rapidly and significantly decreases serum alanine transaminase (ALT). Raised serum ALT is characteristic of liver pathology, and a reduction in serum ALT is an indication of reduced cell stress and reduced cell death in the liver.

[0021] FIG. 12 shows that CDP1 treatment significantly reduces the total cholesterol present in liver tissue. Excess cholesterol in liver tissue is pathological, contributing to dysfunction by forcing cells into bearing a toxic excess of intracellular free cholesterol.

[0022] FIG. 13 shows that CDP1 treatment of LDLR mice leads to plaque regression

[0023] FIG. 14 shows that low does of CDP1 treatment of LDLR mice leads to plaque regression.

[0024] FIG. 15 shows that all groups treated with REP-0003 / CDP1 significantly outperformed the vehicle treated group, indicating improved cardiovascular function.DETAILED DESCRIPTION

[0025] Disclosed herein are methods and systems for systemic treatment of atherosclerosis by targeting intracellular cholesterol metabolism of liver hepatocytes. In many embodiments, the disclosed treatments result in regression of atherosclerotic plaques at sites distal to the liver, for example plaque at or near the aorta and / or increase the plaques’ collagen content. In many embodiments, the disclosed therapeutic treatments and compositions may also affect serum levels of various biomarkers, including cholesterol, triglycerides, cytokines, and transcription co-factors.

[0026] Surprisingly, Applicants’ disclosed therapeutic treatments and composition may result in rapid and measurable systemic beneficial results despite targeting liver tissue. In some embodiments, these surprising results involve regression of atherosclerotic plaques without changing levels of serum lipoprotein concentrations, for example low and very low density lipoproteins.

[0027] Disclosed herein are therapeutic methods and compositions targeted to liver cells, for example hepatocytes. In many embodiments, the composition comprises a lipid nanoparticle (LNP). In many embodiments, the LNP may be sized to easily pass through liver sinusoidal fenestrae that normally range between 100-200 nm in diameter. In many embodiments, the LNP are about 80 nm in diameter. The disclosed LNPs may comprise one or more targeting molecules that may aid in targeting liver cells. In various embodiments, the targeting molecule may comprise N-acetylgalactosamine (GalNAc). GalNAc may aid in targeting the LNP to liver cells by binding to asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes. ASGPR is expressed only by hepatocytes and some circulating monocytes. However, because monocytes are circulating cells, their uptake of GalNAc-targetted LNPs is minimal. Efficient endocytosis and recycling (circa 15 minutes) make ASGPR an ideal protein to target liver specific therapies. LNPs targeted to ASGPR by inclusion of GalNAc rapidly bind to and are endocytosed by hepatocytes. LNP cargos, for example sequences coding for cholesterol degrading proteins (CDPs), then enter the cytoplasm where they can be translated by ribosomes. FIG. 1 is a schematic diagram showing one embodiment of the disclosed therapeutic compounds targeting liver cells.

[0028] The disclosed therapeutic compositions and methods may aid in converting cholesterol into another compound. In various embodiments, the disclosed proteins and enzymes may help to convert cholesterol to pregnenolone. Pregnenolone is shown to have anti-inflammatory effects in relevant cell populations, for one example macrophages. In macrophages, pregnenolone has been shown to promote the ubiquitination and degradation of the Toll-like receptor (TLR)2 / 4 adaptor protein, TLRAP, and TLR2 in macrophages and microglial cells. This leads to decreased secretion of pro-inflammatory cytokines, TNF-a and IL-6, that would otherwise be enhanced via TLR2 and TLR4 signaling.

[0029] Disclosed herein are in-vivo experimental results demonstrating that the disclosed therapeutic compounds and treatments, which involve targeting lipid- nanoparticles comprising cholesterol degrading protein coding sequences to hepatocytes, produces significant, rapid reductions in atherosclerotic plaque lipids inmice with established atherosclerosis and affected other biomarkers associated with atherosclerosis.

[0030] Surprisingly the present studies indicated that the disclosed compositions and methods were effective in mice with hypercholesterolemia, and whose liver cells lack functional apolipoprotein E (involved in forming lipoproteins) and low density lipoprotein receptors. Moreover, the extent and speed of plaque regression was far greater than is currently possible with the present standard of care. Surprisingly, considering the therapies are targeted to the liver, the plaque area at or near the aortic route is greatly affected by the disclosed treatement.

[0031] At present, the standard of care consists of interventions with compounds such as statins and PCSK9 inhibitors that target serum LDL cholesterol, with little or no impact on the size of established plaques.Therapeutic compositions

[0032] Disclosed herein are various therapeutic compositions for the treatment of a disease or condition associated with increased levels of cholesterol and / or triglyceride in serum and tissues. In many embodiments, the therapeutic compositions may deliver one or more active ingredients to a specific cell type, for example a hepatocyte. In many embodiments, the disclosed compositions comprise lipid-nanoparticles (LNP) surrounding the active ingredient. In many embodiments, the disclosed LNP may comprise at least one molecule with affinity for a liver-specific surface protein, for example ASPGR. In some embodiments, the disclosed composition may be referred to as CDP, LNP-mRNA, LNP-CDP, or REP-0003.

[0033] Affinity may refer to the avidity of one molecule for another - for example a ligand and a ligand receptor, for example a surface receptor. In some embodiments, affinity can be presented as a relative binding - e.g. competition of two different ligands for the same receptor. In the present case, the disclosed therapeutic compositions display affinity for hepatocytes, in particular the ASPG receptor on hepatocytes.Active ingredient

[0034] The terms “active ingredient” or “active pharmaceutical ingredient” as used herein refer to a pharmaceutical agent, compound, substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, or ameliorative effect. In various embodiments, the active ingredient is a nucleic acid, for example a deoxyribonucleotide or ribonucleotide. In many embodiments the active ingredient is a polynucleotide, for example an mRNA. In many embodiments, the active ingredient may code for a fusion protein, which, in most embodiments, comprises a cytochrome P450.Ameliorate

[0035] Amelioration as used herein refers to any improvement of a disease state (for example hypercholesterolemia or atherosclerosis) of a patient suffering therefrom, by the administration of one or more therapeutic compositions or active ingredients, according to the present disclosure, to such patient or subject in need thereof. Such an improvement may be seen as a slowing down of the progression, a cessation of the progression, or a regression of an aspect or symptom of the disease of the patient, a decrease in the frequency, duration, and / or severity of any symptom, and / or an increase in frequency or duration of disease symptom-free periods or a prevention of impairment or disability due to the disease.Co-Administer

[0036] Co-administering refers to administering two (or more) therapeutically active ingredients or drugs. In some embodiments, the co-administration may be simultaneous, such as combining the active ingredients in an intraveneous delivery. In other embodiments, the co-administration may be sequential (i.e. one after the other), for example infusions of a first active ingredient followed by a second active ingredient. In some embodiments, co-administration may involve combining the two (or more) drugs or active ingredients into the same syringe or IV bag, or adding the second drug to an I.V. bag comprising the first drug, prior to co-administration thereof. In other embodiments, one a drug may be delivered intraveneously and the other orally or otherwise. In some embodiments, the co-administered drugs (for example statins) may be delivered on dosing schedules that may be different than, for example, schedules for the presently disclosed thereapeutic compositions.Statin

[0037] Statins are cholesterol-lowering drugs that specifically inhibit HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; HMGCR) and help treat dyslipidemias. HMGCR is the rate-limiting enzyme of the mevalonate pathway that produces cholesterol and other isoprenoids. It catalyzes the conversion of HMG-CoA to mevalonic acid, which is an essential step in cholesterol synthesis. The first HMGCR inhibitor or statin to be approved was a natural product derived from a fungus, lovastatin in 1987, initially named mevinolin, it was followed by another naturally occuing statin simvastatin (Zocor; 1998) - synthtetic statins followed, including pravastatin (Pravachol; 1991), fluvastatin (Lescolin; 1994), atorvastatin (Lipitor; 1997), cerivastatin (Baycol; 1998), and rosuvastatin (Crestor; 2003).Gene Therapy

[0038] Disclosed herein are therapeutic methods and compositions for delivering one or more cholesterol related proteins or enzymes to a cell. In many embodiments, the proteins and enzymes may degrade or catabolize intracellular cholesterol. In many embodiments, the disclosed methods and compositions may help cause rapid and significant regression and / or stabilization of atherosclerotic plaques.

[0039] The disclosed cholesterol related proteins and enzymes may catalyze at least one chemical reaction of cholesterol. In many embodiments, the disclosed proteins and enzymes may be be useful in converting cholesterol into another compound, for example by catalyzing removal of the cholesterol side chain. In many embodiments, the disclosed proteins and enzymes may include one or more of cytochromes P450 (for example without limitation, cholesterol P450 side chain cleavage enzyme, CYP11A, CYP27A, CYP8, CYP7, CYP3), P450 oxiodoreductase / POR, Cytochrome b5, ferredoxin reductase (FdxR; AdxR / Adrenodoxin reductase), and ferredoxin (Fdx; Adx / Adrenodoxin). In many embodiments, the protein or enzyme is a fusion, for example (P450-FdxR-Fdx), and may catalyze conversion of cholesterol to pregnenolone. As used herein, the terms CYP and Cyp may designate the gene, coding sequence, amino acid sequence, and / or protein.

[0040] Humans possess two types of cytochrome P450 enzymes. Type 1 is found in the mitochondria (i.e. Cyp11A and Cyp27A) and type 2 is associated with microsomal fractions, for example the endoplasmic reticulum. Type 2 cytochromes (e.g. Cyp3A, Cyp7A, and Cyp8A) possess somewhat different electron-transport chains than type 1 cytochromes. In some embodiments, the disclosed fusion proteins may comprise a type 2 P450. These type 2 fusion proteins may include one or two additional protein modules. In some embodiments, the type 2 fusion protein may comprise one example Cyp7A and a membrane bound P450 oxidoreductase (POR), and, optionally, a cytochrome b protein, for example Cytochrome b5.P450 and P450 fusion proteins.

[0041] The disclosed side chain cleavage enzyme may include various cytochrome proteins. In many embodiments the cytochrome P450 protein may be selected from two-component, 3-component, mitochondrial, and microsomal systems.

[0042] Various P450 proteins may be uses with the disclosed compositions, methods and systems. In various embodiments, the P450 isotype may be selected from CYP3, CYP3A, CYP7, CYP7A (NM_000780), CYP8, CYP8A, CYP11 , CYP11A, CYP27, CYP27A (NM_000784.4 ). In many embodiments, the P450 isotype may be expressedas a fusion with FDXR / ADXR, FDX / ADX, cytochrome b5 (Cyb5A;NM_148923) and / or P450 oxidoreductase (POR;NM_001367562.3).

[0043] Various Type 1 cytochromes P450 may be used in the disclosed fusion proteins. In some embodiments, the Type 1 cytochromes P450 may be selected from Cyp11 and Cyp27. Cyp27A has a much higher affinity for cholesterol than Cyp11A. The km, i.e. concentration of cholesterol needed to reach half Vmax, for Cyp27A is in the order of 5-25uM, for example ~20pM. The Km of Cyp11 A for cholesterol may be greater than that of Cyp27A, for example ~50uM or greater. Additionally, Cyp27A may be less tightly regulated than Cyp11A, whose mRNA and protein may be quickly destabilized / turned-over. Applicants hypothesized that in some embodiments, a Cyp27A fusion protein, i.e. CDP2, expressed in the liver may be equivalent to or more efficient than CDP1 at degradation of excess cholesterol. Data shows that Cyp27-null mice lose expression of Cyp27a in all tissues - not just the liver. These Cyp-null mice show a -5-10 fold increase in hepatic CYP7A1, CYP3A, and CYP8B1 expression - these CYPs are contributors to cholesterol breakdown and removal through the conversion of cholesterol into bile acids.

[0044] The disclosed fusion protein may be referred to as CDP, for example CDP1 or CDP2. In many embodiments CDP / CDP1 / CDP11 may refer to a fusion of Cyp11 A- FDXR-FDX1 , while CDP2 / CDP27 may refer to a fusion of Cyp27A-FDXR-FDX1. CDP27, like CDP11 may be expressed on the matrix-facing side of the inner mitochondrial membrane, and can significantly reduce free cholesterol via its metabolizing precursors of bile salts. CDP may also refer to fusion proteins comprising a type 2 cytochrome.

[0045] In many embodiments, CDP1 / CDP11 fusion proteins coded for by the disclosed active ingredients may have an amino acid sequence at least 80% identical to MLAKGLPPRSVLVKGCQTFLSAPREGLGRLRVPTGEGAGISTRSPRPFNEIPSPGDNG WLNLYHFWRETGTHKVHLHHVQNFQKYGPIYREKLGNVESVYVIDPEDVALLFKSEGP NPERFLIPPWVAYHQYYQRPIGVLLKKSAAWKKDRVALNQEVMAPEATKNFLPLLDAV SRDFVSVLHRRIKKAGSGNYSGDISDDLFRFAFESITNVIFGERQGMLEEVVNPEAQRFI DAIYQMFHTSVPMLNLPPDLFRLFRTKTWKDHVAAWDVIFSKADIYTQNFYWELRQKG SVHHDYRGILYRLLGDSKMSFEDIKANVTEMLAGGVDTTSMTLQWHLYEMARNLKVQ DMLRAEVLAARHQAQGDMATMLQLVPLLKASIKETLRLHPISVTLQRYLVNDLVLRDYM IPAKTLVQVAIYALGREPTFFFDPENFDPTRWLSKDKNITYFRNLGFGWGVRQCLGRRI AELEMTIFLINMLENFRVEIQHLSDVGTTFNLILMPEKPISFTFWPFNQEATQQTDGTSS TQEKTPQICVVGSGPAGFYTAQHLLKHPQAHVDIYEKQPVPFGLVRFGVAPDHPEVKN VINTFTQTAHSGRCAFWGNVEVGRDVTVPELREAYHAVVLSYGAEDHRALEIPGEELPGVCSARAFVGWYNGLPENQELEPDLSCDTAVILGQGNVALDVARILLTPPEHLERTDIT KAALGVLRQSRVKTVWLVGRRGPLQVAFTIKELREMIQLPGARPILDPVDFLGLQDKIK EVPRPRKRLTELLLRTATEKPGPAEAARQASASRAWGLRFFRSPQQVLPSPDGRRAA GVRLAVTRLEGVDEATRAVPTGDMEDLPCGLVLSSIGYKSRPVDPSVPFDSKLGVIPN VEGRVMDVPGLYCSGWVKRGPTGVIATTMTDSFLTGQMLLQDLKAGLLPSGPRPGYA AIQALLSSRGVRPVSFSDWEKLDAEEVARGQGTGKPREKLVDPQEMLRLLGHTDGAS SSSEDKITVHFINRDGETLTTKGKVGDSLLDVVVENNLDIDGFGACEGTLACSTCHLIFE DHIYEKLDAITDEENDMLDLAYGLTDRSRLGCQICLTKSMDNMTVRVPETVADARQSID VGKTSDYKDDDDK (SEQ ID NO: 1). In many embodiments, the identity may be greater than about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and less than 100%, 99%, 98%, 97%, 96%, 95%, 93%, 94%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81%.

[0046] CDP1 may be coded for a nucleic acid with sequence SEQ ID NO: 2. One of skill in the art understands that such coding sequence can vary significantly without changing the sequence of the protein coded for, and / or producing a protein that is at least 80% identical to the protein coded for by such coding sequence.

[0047] In many embodiments, the disclosed CDP2 / CDP27 fusion proteins may have an amino acid sequence at least 80% identical to MAALGCARLRWALRGAGRGLCPHGARAKAAIPAALPSDKATGAPGAGPGVRRRQRS LEEIPRLGQLRFFFQLFVQGYALQLHQLQVLYKAKYGPMWMSYLGPQMHVNLASAPLL EQVMRQEGKYPVRNDMELWKEHRDQHDLTYGPFTTEGHHWYQLRQALNQRLLKPA EAALYTDAFNEVIDDFMTRLDQLRAESASGNQVSDMVQLFYYFALEAICYILFEKRIGCL QRSIPEDTVTFVRSIGLMFQNSLYATFLPKWTRPVLPFWKRYLDGWNAIFSFGKKLIDE KLEDMEAQLQAAGPDGIQVSGYLHFLLASGQLSPREAMGSLPELLMAGVDTTSNTLT WALYHLSKDPEIQEALHEEVVGWPAGQVPQHKDFAHMPLLKAVLKETLRLYPWPTN SRIIEKEIEVDGFLFPKNTQFVFCHYVVSRDPTAFSEPESFQPHRWLRNSQPATPRIQH PFGSVPFGYGVRACLGRRIAELEMQLLLARLIQKYKWLAPETGELKSVARIVLVPNKKV GLQFLQRQCTDGTSSTQEKTPQICWGSGPAGFYTAQHLLKHPQAHVDIYEKQPVPFG LVRFGVAPDHPEVKNVINTFTQTAHSGRCAFWGNVEVGRDVTVPELQEAYHAVVLSY GAEDHRALEIPGEELPGVCSARAFVGWYNGLPENQELEPDLSCDTAVILGQGNVALDV ARILLTPPEHLERTDITKAALGVLRQSRVKTVWLVGRRGPLQVAFTIKELREMIQLPGAR PILDPVDFLGLQDKIKEVPRPRKRLTELLLRTATEKPGPAEAARQASASRAWGLRFFRS PQQVLPSPDGRRAAGVRLAVTRLEGVDEATRAVPTGDMEDLPCGLVLSSIGYKSRPV DPSVPFDSKLGVIPNVEGRVMDVPGLYCSGWVKRGPTGVIATTMTDSFLTGQMLLQD LKAGLLPSGPRPGYAAIQALLSSRGVRPVSFSDWEKLDAEEVARGQGTGKPREKLVD PQEMLRLLGHTDGASSSSEDKITVHFINRDGETLTTKGKVGDSLLDVVVENNLDIDGFGACEGTLACSTCHLIFEDHIYEKLDAITDEENDMLDLAYGLTDRSRLGCQICLTKSMDNM TVRVPETVADARQSIDVGKTS (SEQ ID NO: 3). In many embodiments, the identity may be greater than about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and less than 100%, 99%, 98%, 97%, 96%, 95%, 93%, 94%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81%.

[0048] The disclosed CDP2 / CDP27 fusion protein may be coded for by a nucleic acid of sequence SEQ ID NO: 4. One of skill in the art understands that such coding sequence can vary significantly without changing the sequence of the protein coded for, and / or producing a protein that is at least 80% identical to the protein coded for by such coding sequence.

[0049] In many embodiments, the disclosed fusion protein may comprise CYP7 and have an amino acid sequence at least 80% identical to MMTTSLIWGIAIAACCCLWLILGIRRRQTGEPPLENGLIPYLGCALQFGANPLEFLRANQ RKHGHVFTCKLMGKYVHFITNPLSYHKVLCHGKYFDWKKFHFATSAKAFGHRSIDPMD GNTTENINDTFIKTLQGHALNSLTESMMENLQRIMRPPVSSNSKTAAWVTEGMYSFCY RVMFEAGYLTIFGRDLTRRDTQKAHILNNLDNFKQFDKVFPALVAGLPIHMFRTAHNAR EKLAESLRHENLQKRESISELISLRMFLNDTLSTFDDLEKAKTHLWLWASQANTIPATF WSLFQMIRNPEAMKAATEEVKRTLENAGQKVSLEGNPICLSQAELNDLPVLDSIIKESL RLSSASLNIRTAKEDFTLHLEDGSYNIRKDDIIALYPQLMHLDPEIYPDPLTFKYDRYLDE NGKTKTTFYCNGLKLKYYYMPFGSGATICPGRLFAIHEIKQFLILMLSYFELELIEGQAKC PPLDQSRAGLGILPPLNDIEFKYKFKHLTDGTSMGDSHVDTSSTVSEAVAEEVSLFSMT DM1LFSLIVGLLTYWFLFRKKKEEVPEFTKIQTLTSSVRESSFVEKMKKTGRNIIVFYGSQ TGTAEEFANRLSKDAHRYGMRGMSADPEEYDLADLSSLPEIDNALWFCMATYGEGD PTDNAQDFYDWLQETDVDLSGVKFAVFGLGNKTYEHFNAMGKYVDKRLEQLGAQRIF ELGLGDDDGNLEEDFITWREQFWPAVCEHFGVEATGEESSIRQYELVVHTDIDAAKVY MGEMGRLKSYENQKPPFDAKNPFLAAVTTNRKLNQGTERHLMHLELDISDSKIRYESG DHVAVYPANDSALVNQLGKILGADLDVVMSLNNLDEESNKKHPFPCPTSYRTALTYYL DITNPPRTNVLYELAQYASEPSEQELLRKMASSSGEGKELYLSWVVEARRHILAILQDC PSLRPPIDHLCELLPRLQARYYSIASSSKVHPNSVHICAWVEYETKAGRINKGVATNWL RAKEPAGENGGRALVPMFVRKSQFRLPFKATTPVIMVGPGTGVAPFIGFIQERAWLRQ QGKEVGETLLYYGCRRSDEDYLYREELAQFHRDGALTQLNVAFSREQSHKVYVQHLL KQDREHLWKLIEGGAHIYVCGDARNMARDVQNTFYDIVAELGAMEHAQAVDYIKKLMT KGRYSLDVWSTDGASMAEQSDEAVKYYTLEEIQKHNHSKSTWLILHHKVYDLTKFLEE HPGGEEVLREQAGGDATENFEDVGHSTDAREMSKTFIIGELHPDDRPKLNKPPETLITT I DSSSSWWTN WVI PAISAVAVALMYRLYMAED (SEQ ID NO: 5) (note that connecting residues are depicted in bold, and transmembrane residues underlined). In manyembodiments, the identity may be greater than about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and less than 100%, 99%, 98%, 97%, 96%, 95%, 93%, 94%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81%. In some embodiments, removal or mutation of transmembrane residues may aid in allowing the fused proteins to work together - for example, the first 27 residues of the POR protein may be deleted and / or connector sequences elongated in order to improve freedom / flexibility of indifidual proteins and / or the overall structure of the fusion protein.

[0050] The disclosed fusion protein comprising CYP7 may be coded for a nucleic acid with sequence SEQ ID NO: 6. One of skill in the art understands that such coding sequence can vary significantly without changing the sequence of the protein coded for, and / or producing a protein that is at least 80% identical to the protein coded for by such coding sequence.Ferredoxin Reductase / FDXR

[0051] Also referred to as adrenodoxin / ADXR reductase it is part of the mitochondrial P450 enzyme systems. Specifically, it functions as the first electron transfer protein, where its coenzyme FAD receives two electrons from NADPH and transfers them to ferredoxin / adrenodoxin.Ferredoxin / FDX

[0052] Also known as adrenodoxin / ADX, it functions to transfers electrons between ADXR and P450. FDX is an iron-sulfur containing protein that can accept / carry / transfer single electrons. Ferredoxin receives electrons from ferroodoxin reductase, reducing cytochrome P450.Cytochrome P450 Reductase (POR)

[0053] Cytochrome P450 reductatse is a membrane-bound enzyme that transfers electrons to cytochrome P450 in the endoplasmic reticulum, for example Type 2 cytochromes P450.Cytochrome b

[0054] Directed delivery of gene therapy

[0055] The disclosed therapeutic compositions may be targeted to and / or their contents expressed only in specific tissues. In most embodiments, the therapeutic compositions are targeted to and expressed in liver tissue. In many embodiments, the therapeutic compositions are targeted to and expressed in liver cells, for example hepatocytes.

[0056] In some embodiments, the therapeutic composition may comprise a nanoparticle or liposome and one or more nucleic acid coding sequences, for example mRNA. In many embodiments, a lipid nanoparticle (LNP) may be used to deliver a polynucleotide, for example mRNA or other therapeutic nucleic acids, coding for one or more proteins or enzymes related to cholesterol metabolism. In many cases, use of LNP may allow for delivery of multiple nucleic acids that are larger than may be delivered by other methods. In some embodiments, the disclosed LNP and / or LNP systems may include one or more of four components whose variations can optimize their integrity and organ-specificity, particularly to the liver. In some embodiments, the LNP variations may include one or more of ionizable cationic lipids, phospholipids (typically phosphatidylcholine), cholesterol, and PEG-lipids. Additionally, some LNP formulations may include one or more molecules that help target the LNP to a specific cell or tissue.Dosina

[0057] The terms “dosage” or “dose” as used herein denote any form of the active ingredient formulation that contains an amount sufficient to produce a therapeutic effect with a single administration. In some embodiments, dosing may also refer to the frequency the therapeutic amount is delivered to the patient.

[0058] The presently disclosed therapeutic compositions and methods may be dosed in various forms, amounts, and frequency. In some embodiments, the disclosed therapeutic dose may be between about 10 mg to about 0.01 mg per kg of patient weight. In many embodiments the dose is between about 5 mg and 0.1 mg / kg, for example 0.25, 0.5, 0.75, 1.0 or 1.5 mg / kg. The frequency may vary depending upon the needs of the patient and mode of delivery, but in most embodiments is from about once per month to twice per day. In one example the frequency may be twice weekly.Effective amount

[0059] The phrase “effective amount,” in terms of the disclosed therapeutic compositions and methods, may refer to an amount of a composition of the present disclosure or active ingredient sufficient to provide a therapeutic, ameliorative, or prophylactic benefit in the treatment or prevention of a disease or to delay, minimize, or relieve one or more symptoms or conditions associated with a disease. Further, a therapeutically effective amount with respect to a composition of the present disclosure means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a composition of the presently disclosed therapeutic compositions and methods, the term can encompass an amount or an administrative frequency thatimproves overall therapy, reduces, or avoids symptoms, conditions, or causes of disease, or enhances the therapeutic efficacy or synergies with another therapeutic agent.

[0060] The phrase “therapeutically effective amount” means an amount of a compound of the present disclosure that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In the case of atherosclerosis or hypercholesterolemia, the therapeutically effective amount of the drug may reduce the size, volume, area of a plaque, or affect the concentration of at least one component of the plaque, for example collagen or lipid; inhibit (i.e., slow to some extent and preferably stop) growth of atherosclerotic plaques; or reduce the concentration of one or more lipid in the serum or tissue of a patient. To the extent the drug may reduce serum levels of one or more toxic or detrimental compounds, efficacy can also be measured, for example, by assessing time to fatigue of a patient.Biomarkers

[0061] A “biomarker” is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers may be of several types: predictive, prognostic, or pharmacodynamics (PD). Predictive biomarkers predict which patients are likely to respond or benefit from a particular therapy. Prognostic biomarkers predict the likely course of the patient's disease and may guide treatment. Pharmacodynamic biomarkers confirm drug activity, and enables optimization of dose and administration schedule.

[0062] SRA1 , Steroid Receptor RNA Activator I, is involved in regulating activities such as metabolism and adipogenesis through regulation (typically repression) of nuclear receptors and other factors. Increases in SRA1 expression beneficially affect macrophage function and reduce inflammation. Thus, SRA1 expression, especially in the spleen, is useful in tracking therapeutic effect.

[0063] IL-10, Interleukin 10, is an anti-inflammatory cytokine that can block NF-KB activity, IFNy secretion from myeloid lineage cells, and regulation of the JAK-STAT signaling pathway. IL-10 also affects immunoregulation, downregulating expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. IL-10 also affects survival, proliferation, and antibody production of B-cells.

[0064] Low-density lipoprotein (LDL) is one type of lipoprotein. Other types include very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL). When a cellneeds to synthesize cholesterol, the cell takes up LDL via LDL receptors - LDLRs. The amount of LDLRs on a cell is partially regulated by the protein PCSK9, which marks the LDL receptor for degradation. Excess levels of LDL are involved in atherosclerosis.

[0065] Triglycerides are derived from glycerol and three fatty acids, and the main constituent of human body fat. High blood triglycerides are associated with lipid disorders like high blood cholesterol and cardiovascular disease.

[0066] Cholesterol is lipid sterol synthesized by most, if not all animal cells. Cholesterol synthesis is a complex and an energy-expensive process, requiring the coordinated activity of more than fifteen enzymes. The backbone of cholesterol consists of 27 carbon atoms that are assembled by multiple enzymes, with all carbons coming from acetyl-CoA. The cholestane ring cannot be opened enzymatically in human cells, meaning that the body has difficulty reducing excess levels of cholesterol.

[0067] Pregnenolone, formed from side chain cleavage of cholesterol, has been shown to have anti-inflammatory effects in cell populations relevant to atherosclerosis pathology. Pregnenolone constitutes the first product of steroid synthesis in steroidogenic cells of adrenal glands, ovaries and testes. In humans, it has no progestogenic, corticosteroid, estrogenic, androgenic, or anti-androgenic activities. Human non-steroidogenic cells do not possess enzymes that are capable of converting pregnenolone to downstream steroids and, if produced in these cells, pregnenolone is readily secreted into the extracellular milieu. Indeed, pregnenolone produced in-vitro in non-steroidogenic cells (e.g. hepatocytes, monocytes / macrophages, fibroblasts, kidney epithelial cells) using the disclosed compositions and methods is secreted into the extracellular media where it is readily detected.Therapeutic Outcomes

[0068] Plaque regression means a reduction of at least one measurable physical attribute of an atherosclerotic plaque. The physical attribute may include, without limitation, surface area, volume, or height. In some embodiments, the disclosed therapy may result in a reduction from about 5% to about 100%, for example from about 70% to about 90%, and by greater than about 30%, 40%, 50%, 60%, or more. For example, the disclosed therapeutic composition and treatments may reduce the plaque volume in a patient compared to measurements prior to treatment and / or to a population of untreated individuals, wherein the volume is based on imaging by one or more of invasive or non-invasive modalities such as intravascular ultrasound (IVUS), B-mode ultrasound, cardiac computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI). In many embodiments, the measured physical attribute may be reduced by greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and less than about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%. In many embodiments, the interval over which reduction is measured may be greater than about 1 month after treatment to about 24 months after treatment, for example more than about 1 week, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 7 wks, 8 wks, 2 mos., 3 mos., 4 mos., 5 mos., 6 mos., 7 mos., 8 mos., 9 mos., 10 mos., 11 mos., 12 mos., 13 mos., 14 mos., 15 mos., 16 mos., 17 mos., 18 mos., 19 mos., 20 mos., 21 mos., 22 mos., 23 mos., 24 mos., and less than about 36 mos., 30 mos., 25 mos., 24 mos., 23 mos., 22 mos., 21 mos., 20 mos., 19 mos., 18 mos., 17 mos., 16 mos., 15 mos., 14 mos., 13 mos., 12 mos., 11 mos., 10 mos., 9 mos., 8 mos., 7 mos., 6 mos., 5 mos., 4 mos., 3 mos., 2 mos., 4 wks, 3 wks, or 2 wks. In some embodiments, for example wherein the subjects are assessed for normalized plaque fraction, the normalized plaque fraction may have an average reduction greater than about 30%. In many cases, normalized plaque fraction may be assayed as percent atheroma volume, such as the percentage of the lumen volume for a single plaque that is occupied by the plaque. In many embodiments, normalized plaque fraction may be determined using Coronary Computed Tomography Angiography (CCTA). In many embodiment, normalized plaque fraction in a population of treated subjects (for example 2 or more subjects) may have an average reduction of more than about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and less than about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%, for example about 42%.

[0069] The disclosed therapeutic compositions and treatments may be useful in altering the amount of at least constituent of an atherosclerotic plaque. In most embodiments, the disclosed compositions and methods the constituent may be lipids, and the treatment may reduce the amount of lipid in an atherosclerotic plaque of a subject. In most embodiments, the disclosed compositions and methods may reduce the amount of lipid in an atherosclerotic plaque of a subject by more than 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 24%, 23%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, or 50%, and less than about 60%, 55%, 50%, 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5%. In many embodiments, the interval over which the decrease is measured may be greater than about 1 month after treatment to about 24 months after treatment, for example more than about 1 week, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 7 wks, 8 wks, 2 mos., 3 mos., 4mos., 5 mos., 6 mos., 7 mos., 8 mos., 9 mos., 10 mos., 11 mos., 12 mos., 13 mos., 14 mos., 15 mos., 16 mos., 17 mos., 18 mos., 19 mos., 20 mos., 21 mos., 22 mos., 23 mos., 24 mos., and less than about 36 mos., 30 mos., 25 mos., 24 mos., 23 mos., 22 mos., 21 mos., 20 mos., 19 mos., 18 mos., 17 mos., 16 mos., 15 mos., 14 mos., 13 mos., 12 mos., 11 mos., 10 mos., 9 mos., 8 mos., 7 mos., 6 mos., 5 mos., 4 mos., 3 mos., 2 mos., 4 wks, 3 wks, or 2 wks.

[0070] In some embodiments, the constituent is collagen. In embodiments wherein the constituent is collagen, the amount of collagen in the plaque may increase. In many embodiments, the amount of collagen in a plaque may increase by more than about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and less than about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%. In many embodiments, the interval over which the increase is measured may be greater than about 1 month after treatment to about 24 months after treatment, for example more than about 1 week, 2 wks, 3 wks, 4 wks, 5 wks, 6 wks, 7 wks, 8 wks, 2 mos., 3 mos., 4 mos., 5 mos., 6 mos., 7 mos., 8 mos., 9 mos., 10 mos., 11 mos., 12 mos., 13 mos., 14 mos., 15 mos., 16 mos., 17 mos., 18 mos., 19 mos., 20 mos., 21 mos., 22 mos., 23 mos., 24 mos., and less than about 36 mos., 30 mos., 25 mos., 24 mos., 23 mos., 22 mos., 21 mos., 20 mos., 19 mos., 18 mos., 17 mos., 16 mos., 15 mos., 14 mos., 13 mos., 12 mos., 11 mos., 10 mos., 9 mos., 8 mos., 7 mos., 6 mos., 5 mos., 4 mos., 3 mos., 2 mos., 4 wks, 3 wks, or 2 wks.

[0071] The disclosed therapeutic compositions and treatments may be useful in altering the amount or concentration of at least serum constituent of a treated subject. In most embodiments, the disclosed serum constituent is selected from total cholesterol, free cholesterol, lipids, lipoproteins, triglycerides, cytokines, SRA1 , IL-10, etc. In one embodiment, the disclosed therapeutic compositions and methods may reduce the free cholesterol and / or triglycerides in the serum of a treated subject. In these embodiments, the disclosed reduction may be more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and less than about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, or 5%.

[0072] The disclosed therapeutic compositions and methods may have little or no effect on the concentration of low density or very low density lipoproteins concentrations in the serum of a treated subject. In these embodiments, the concentration of serumLDL and / or VLDL may change by less than about 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% and more than about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, or 4% compared to the same subject prior to treatment. In many embodiments the time for this change may be more than about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 wks, 3 wks, or 4 wks, and less than about 2 mos., 4 wks, 3 wks, 2 wks, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days.Indications / Conditions / Diseases / Disorders

[0073] Various diseases and conditions may be treated or prevented with the presently disclosed therapeutic compositions and methods. In most embodiments, the disease or conditions treated with the disclosed methods and compositions are related to diseases and conditions associated with hypercholesterolemia and / or atherosclerosis. While, in one embodiment the disease or condition may be any form of atherosclerosis, in many embodiments, the disclosed therapeutic compositions and methods are useful in treating genetic forms of accelerated atherosclerosis, for example familial hypercholesterolemia.

[0074] Autosomal homozygous familial hypercholesterolemia (FH) is a rare genetic disease, which leads to the rapid onset of coronary heart disease due to a persistent elevation in low density lipoprotein (LDL) cholesterol concentration. Heterozygous FH (HeFH) is more common (1 in 200 to 1 in 500 people), effecting between 14 and 34 million individuals worldwide. Patients with heterozygous FH demonstrate a clinical phenotype characterized by severely elevated plasma levels of total cholesterol, low density lipoprotein cholesterol, tendinous xanthomata, and have a high predisposition for cardiovascular disease. Most forms of familial hypercholesterolemia are genetic disorders disrupting normal lipid metabolism, often due to mutations in genes encoding the LDL receptor (LDLR), apolipoprotein B-100 (apoB), or the proprotein convertase subtilisin / kexin type 9 (PCSK9). Defects in any of these genes encoding proteins integral to lipoprotein metabolism result in a significant increase in levels of low density lipoprotein cholesterol (LDL-C).

[0075] Homozygous FH (HoFH) is frequently associated with the loss of LDL receptor expression or function. Although FH patients lack functional LDL receptors, uptake in macrophages still occurs via scavenger receptors (SR), and lack of LDL-R in other tissues yields more LDLs for macrophages. Therefore, FH patients are at a greater risk for a myocardial infarction or stroke, which often occur within the first two decades of life. Furthermore, these patients do not respond to life style modification or statin therapy. Common treatment for homozygous FH currently depends on routinesessions of lipid apheresis. Homozygous FH is rare (-1 :1,000,000) and usually leads to advanced CVD or death before the age of 20.

[0076] At the fundamental biochemical level, the increased risk of cardiovascular disease is due to the inability of the human body to clear excess cholesterol from the bloodstream. Interestingly, several enzymes endogenous to human cells are present that can likely degrade the intermediate metabolites produced after ring opening. This suggests that ring opening may be the missing step in preventing humans from clearing excess cholesterol and preventing its accumulation.Prevention

[0077] “Prevention” as used herein means the avoidance of the occurrence or of the re-occurrence of a disease, disorder, or condition as specified herein, by the administration of a composition, compound, treatment, or therapy according to the present disclosure to a subject in need thereof.Amino acid

[0078] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: leucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into lie or into Leu.

[0079] “Amino acid identity,” “residue identity,” “identity,” and the like, as used herein refers to the structure of the functional group (R group) on the poly peptide backbone at a given position. Naturally occurring amino acid identities are (name / 3- letter code / one-letter code): alanine / ala / A; arginine / arg / R; asparagine / asn / N; aspartic acid / asp / D; cysteine / cys / C; glutamine / gln / Q; glutamic acid / glu / E; glycine / gly / G; histidine / his / H; isoleucine / ile / l; leucine / leu / L; lysine / lys / K; methionine / met / M;phenylalanine / phe / F; proline / pro / P; serine / ser / S; threonine / thr / T; tryptophan / trp / W; tyrosine / tyr / Y; and valine / val / V.

[0080] An amino acid within a molecule may be substituted to create an engineered molecule or variant. The amino acid (aa or a. a.) residue can be replaced by a residue having similar physiochemical characteristics, that is a ‘conservative substitution’ - e.g., substituting one aliphatic residue for another (such as lie, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, for example based on size, charge, polarity, hydrophobicity, chain rigidity / orientation, etc., are well known in the art of protein engineering. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. binding, specificity, and / or function of a native or reference polypeptide is achieved.

[0081] While conservative substitutions within a protein, i.e. buried or non-solvent accessible residues / positions, may in some cases alter the structure of the protein or affect folding of the protein, conservative substitutions at or near the protein’s surface, i.e. exposed or solvent accessible residues / positions may cause little or no discernable change to the protein’s structure and / or function, unless the altered surface protein is necessary for an interaction with another molecule, peptide, or protein. It is well within the abilities of the skilled artisan to alter the disclosed protein sequences by introducing conservative substitutions at up to 20% of the residues / positions without disrupting or changing the protein’s structure and / or function.

[0082] Similarity between amino acid or peptide sequences is expressed in terms of the homology of two sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (percentage of identical residues for peptides or bases for nucleic acids; or similarity or homology); the higher the percentage, the more similar the two sequences are. Complete identity is 100% identical over a given sequence, for example 50, 100, 150, or 200 bases or residues.Protein

[0083] As used herein, the terms “protein” and “polypeptide” are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used inreference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.Variant / Homologs

[0084] “Variant,” as used herein refers to a polypeptide, nucleic acid, gene, sequence, or molecule that is substantially homologous to a naturally occurring or reference member, but which is different from that of the native or reference member because of one or a plurality of deletions, insertions, substitutions, molecules, expression levels, etc. Variant polypeptide-encoding nucleic acid sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference nucleic acid sequence, but that encode a variant protein or fragment thereof. A wide variety of cloning, PCR-based sitespecific mutagenesis, and gene editing approaches are known in the art, and can be applied by the ordinarily skilled artisan.

[0085] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and understood by those of skill in the art.

[0086] Variant amino acid or nucleic acid sequences can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings). In many cases, evolutionarily conserved amino acids, in a protein, may not be modified in the disclosed variants - for example, in the case of cytochromes P450, residues that interact and holdthe heme or the sulfated amino acid positioned near the Fe in the heme group. Conserved amino acids may be readily identified by those of skill in the art.Prevent

[0087] “Prevention” as used herein means the avoidance of the occurrence or of the re-occurrence of a disease, disorder, or condition as specified herein, by the administration of a composition, compound, treatment, or therapy according to the present disclosure to a subject in need thereof.Modulate

[0088] The terms “modulate”, “modulation” and the like refer to the ability of a compound to increase or decrease the function, or activity of an organism, cell, protein, peptide, gene, biomarker, etc (“target”). “Modulation”, in its various forms, is intended to encompass inhibition, antagonism, partial antagonism, activation, agonism and / or partial agonism of the activity associated with the target. Inhibitors compounds may bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate signal transduction. The ability of a compound to modulate a target’s activity can be demonstrated in various ways, such as nucleic acid quantitation (northern analysis), an enzymatic assay or a cell-based assay.Subject

[0089] Subject, subject in need, patient and those “in need of treatment” include those with an existing disease (i.e. hypercholesterolemia, for example, without limitation, atherosclerosis), as well as those at risk of or susceptible to the disease. The terms also include human and other mammalian subjects that receive either prophylactic or therapeutic treatments as disclosed herein.Treat

[0090] As used herein, “treat”, “treating” and “treatment” refer to eliminating, reducing, suppressing, or ameliorating, either temporarily or permanently, either partially or completely, a clinical symptom, manifestation or progression of an event, disease or condition associated with the disorders and diseases described herein. As is recognized in the pertinent field, methods and drugs employed as therapies may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful. Simply reducing the impact of a disease (for example, as disclosed herein, serum lipoprotein levels, plaque burden, etc.) and / or reducing the number or severity of associated symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect, or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient. One embodiment of thedisclosed therapeutic compositions and methods is directed to a method for determining the efficacy of treatment comprising administering to a patient therapeutic treatment in an amount, duration, and repetition sufficient to induce a sustained improvement over pre-existing conditions, or a baseline indicator that reflects the severity of the particular disorder.Diagnose

[0091] “Diagnose” or “diagnostic” refers identifying the presence or absence of or nature of a disease or disorder. Such detection methods can be used, for example, for early diagnosis of the condition, to determine whether a subject is predisposed to a disease or disorder, to monitor the progress of the disease or disorder or the progress of treatment protocols, to assess the severity of the disease or disorder, to forecast the an outcome of a disease or disorder and / or prospects of recovery, or to aid in the determination of a suitable treatment for a subject.Disease / Condition

[0092] Disclosed herein are compositions and methods useful in treating various diseases, disorders, and conditions, which may be characterized by one or more symptoms, for example hypercholesterolemia, atherosclerotic heart disease, atherosclerosis, hyperlipidemia, coronary artery disease, .Expression

[0093] “Expression” as used herein, refers to cellular processes involved in producing, displaying (e.g., on or at a cell’s surface / outer membrane), or secreting RNA and proteins including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., mRNA) or antisense RNA derived from a nucleic acid fragment or fragments and / or to the translation of mRNA into a polypeptide.About

[0094] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1 , 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term “about” or “approximately” precedes the first numerical value in aseries of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series.EXAMPLES

[0095] Applicants studied treatment of several mouse lines with the disclosed LNP- mRNA CDP gene therapies.

[0096] C57BL / 6 mice are a typical inbred mouse strain with a high degree of uniformity in their inherited characteristics, or phenotypes. Thus, this strain is often used to investigate “typical” responses to experimental treatments.Example 1 - Targeted delivery of LNP-mRNA therapy to liver tissue

[0097] REP-0003 is a liver-targeted LNP-mRNA therapy conjugated with a N- acetylgalactosamine (GalNAc) ligand. GalNAc binds to the asialoglycoprotein receptor, ASGPR, which is highly expressed by hepatocytes in the liver. Thus, the disclosed LNP-mRNA therapeutic compounds are targeted to liver cells. The active ingredient of REP-0003 is a fusion protein comprising CDP1.Example 2 - Pharmacokinetic Study of LNP-CDP mRNA in C57BL / 6 Mice [RP277]

[0098] REP-0003 was administered, intravenously, to 4-week old C57BL / 6J mice(TABLE 1). Mice were fed a GAN diet (40% Fat, 20% Fructose, 2% Cholesterol, Research Diets: D09100310) for 6 weeks. Mice were assigned into 7 study groups (A-G; n=3 mice / group) based on body weight, and were housed in metabolic cages. At study outset, mice were removed from the GAN diet and placed on normal chow.

[0099] At study start, mice received a single injection of PBS (group A) or REP-0003 (groups B-G) via tail vein. At the time of sacrifice (0-72 hours post injection), mice were euthanized and blood and organs including the heart and aortic tree were removed and either frozen for future biochemical analyses or immediately processed for histology.TABLE 1

[0100] Total proteins extracted from tissue of the right liver lobe of mice. Proteins were separated by SDS-PAGE and assayed for CDP and tubulin expression by Western blot. CDP relative to the housekeeping protein, a-tubulin, was quantified.

[0101] As shown in FIG. 2, this study demonstrated that CDP protein has an approximate half-life of about 37.5 hours.Example 3 - ApoE-null mice treated with liver-targeted LNP-CDP mRNA gene therapy

[0102] ApoE-null mice are atherosclerosis-prone mice deficient in apolipoprotein E (ApoE- / -). ApoE-null mice display poor lipoprotein clearance and accumulate cholesterol ester-enriched particles in their blood. These particles promote the development of atherosclerotic plaques.

[0103] For these studies, sixty four ApoE-null mice (4 week-old, JAX 002052) were fed a Western diet (0.2% Cholesterol, Research Diets: D12079B) for 12 weeks and then randomly assigned into 4 groups (G1-G4; TABLE 2).

[0104] This study was designed as a reversal study, such that all mice were switched to a low-fat control diet with cornstarch and no added cholesterol (Research Diets, D14042701).

[0105] Beginning on Day 0, mice were intravenously administered with either LNP- Empty (G1 , volume equivalent dose) or 1 mg / kg of LNP-CDP mRNA qw over the next 6 weeks (G3 and G4). On a daily basis, ApoE-null mice in G2 and G4 were given 5 mg / kg atorvastatin via oral gavage (po).TABLE 2

[0106] At 6 weeks post-treatment, mice were euthanized and blood and organs including the heart and aortic tree were removed and either frozen for future biochemical analyses or immediately processed for histology.

[0107] Serum and organs were assayed to determine lipid plaque area, free cholesterol, triglycerides, LDL, VLDL, collagen, IL-10, and SRA1.REP-0003 treatment significantly decreased area of atherosclerotic plaques without reducing serum LDL levels

[0108] As shown in FIG. 3, LNP-CDP mRNA treatment significantly reduces the area of plaque lipids in the aortic root (left panel). However, and in contrast to atorvastatin treatment, serum levels of LDLA / LDAL-cholesterol were unchanged. Specifically, in comparison to control mice (G1 - LNP-Empty), mice treated with REP- 0003 (G3 - LNP-CDP mRNA) exhibited a significant (P = 0.05) 19% reduction in plaque lipid area at the aortic root. In contrast, atorvastatin treatment alone showed no significant reduction.

[0109] Reduction in plaque lipids may help stabilize plaques, especially unstable plaques. This stabilization may result in reduced risk of plaque rupture and subsequent cardiovascular event.

[0110] While REP-0003 treatment left serum LDL / VLDL levels unchanged, mice treated with atorvastatin alone showed significantly lower serum LDLA / LDL-cholesterol levels.Liver-targeted LNP-mRNA stabilizes distal plagues by increasing fibrosis, reducing risk of rupture and / or release of plaques

[0111] FIG. 4 shows that REP-0003 treatment significantly increased collagen content of atherosclerotic plaques of the aortic roots of treated mice. Cholesterol-rich atherosclerotic plaques are unstable and may rupture leading to mobilization of plaques. This rupture and mobilization frequently result in acute coronary events such as myocardial infarction etc. Because collagen plays a key role in determining plaque stability, aortic root plaques were analyzed for collagen content.

[0112] These studies showed that REP-0003 treatment, in addition to significantly lowering plaque lipid content as discussed above, significantly increased plaque collagen content (P<0.05 vs vehicle). In contrast, treatment with atorvastatin alone did not affect collagen content. REP-0003 monotherapy and the combination (Atorvastatin + LNP-CDP mRNA) therapy increased collagen content to a similar extent, suggesting that this response was specifically mediated by REP-0003 treatment.Liver-targeted gene therapy reduces serum levels of free cholesterol and triglycerides

[0113] As shown in FIG. 5, REP-0003 treatment reduced serum levels of free cholesterol as well as triglycerides. While mice treated with atorvastatin alone showed no change in serum levels of free cholesterol and triglycerides, REP-0003 treatment reduced these markers significantly compared to the control mice. Combining atorvastatin and REP-0003 further reduced triglyceride levels without affecting free cholesterol levels.

[0114] Given that intracellular free cholesterol is targeted by REP-0003 treatment, Applicant hypothesized that treatment with REP-0003 may reduce circulating free cholesterol. Serum triglyceride reduction is broadly considered to be beneficial, as evidence shows raised triglyceride levels are associated with atherosclerotic heart disease.CDP Treatment Increases Serum IL- 10 Levels

[0115] IL-10 serum levels were also tested because of IL-10’s potent antiinflammatory properties in macrophages and T cells. IL-10 is also known to modulate many cellular processes that can interfere with the development and stability of the atherosclerotic plaque.

[0116] While atorvastatin treatment alone had a modest effect on IL- 10 levels (see FIG. 6), REP-0003 treatment (with and without atorvastatin) significantly increased levels of IL-10 in the serum of treated mice.

[0117] Without wishing to be limited by theory, Applicants hypothesized that increased IL-10 expression may contribute to the observed effects of REP-0003 treatment on atherosclerotic plaques.CDP Treatment Increases SRA1 Expression in Spleen Tissue

[0118] The spleen harbors monocytes that enter circulation and migrate to areas of endothelial damage, such as atherosclerotic plaques. At atherosclerotic plaques, the splenic monocytes become macrophages.

[0119] Steroid Receptor RNA Activator I, has been found to be involved in regulating activities such as metabolism and adipogenesis by regulating (typicallyrepressing) nuclear receptors and other factors. Increases in SRA1 expression beneficially affects macrophage function and reduces inflammation. Thus, Applicants hypothesized that REP-0003 treatment may affect SRA1 expression in the spleen.

[0120] FIG. 7 shows that SRA1 expression in the spleen is elevated in response to REP-0003 treatment. This elevated expression is similar to that seen with atorvastatin treatment, while the combination of REP-0003 and atorvastatin appears to be somewhat additive.Low dose gene therapy in ApoE-null Mice is effective in treating atherosclerosis

[0121] Applicants investigated the dosing of REP-0003. In these studies, vascular plague regression and liver pathology in ApoE-null mice, in response to various dosages of REP with or without atorvastatin was investigated. 84 4-week old ApoE-null mice were fed a Western diet (0.2% Cholesterol, Research Diets: D12079B) for 11 weeks and then randomly assigned into 6 groups (A to F).TABLE 3

[0122] As this was a reversal study, all mice were switched to normal chow i.e. a lower-fat control diet on Day 0, and were intravenously administered with either PBS (groups A and B, volume eguivalent dose) or various dosages of REP-0003 every 4 days (q4d) over the next 6 weeks (groups C, D, and E; 0.25, 0.50 or 1.00 mg / kg of REP- 0003, respectively).

[0123] Mice in groups B and F were also administered 5 mg / kg daily (qd) of atorvastatin via oral gavage, while mice in the remaining groups (A, C, D, and E) were administered, by gavage, an equivalent volume of atorvastatin vehicle.

[0124] After 6 weeks, and following their final dose on Day 41 , mice were fasted for 6 hours, euthanized on Days 42-44, and assessed for blood chemistry / biomarkers, atherosclerotic plaque, and liver pathology.

[0125] These results show reduced serum triglyceride and increased serum IL-10 concentrations at LNP-CDP mRNA doses as low as 0.25 mg / kg. These results are similar to those recorded in the study described in Table 2 and LNP-CDP mRNA also show significant efficacy in reducing aortic root plaque lipid and increasing plaque collagen composition at doses as low as 0.25mg / kg.Example 3 - Treatment of LDLR-null Mice

[0126] LDLR (Low Density Lipoprotein, LDL, Receptor)-null mice are models for studying familial hypercholesterolemia (FH). LDLR-null mice have elevated serum cholesterol level of 200-400 mg / dL on a typical diet compared to normal mouse levels of 80-100 mg / dL. Serum levels of LDLR-null mice can exceed 2,000 mg / dL on high fat diets.

[0127] This study determined the pharmacokinetics of intravenously injected LNP- CDP mRNA and its derived CDP protein and markers of activity in male 4-week old LDLR-null mice over 168 hours. Mice were fed a Western diet (0.2% Cholesterol, Research Diets: D12079B) for 6 weeks. Mice were assigned into 10 study groups (A-J; n=3 mice / group) based on body weight, and were housed in metabolic cages. Mice remained on the Western diet throughout the study.

[0128] At study start, mice received a single injection of PBS (group A) or 1.00 mg / kg LNP-CDP mRNA (groups B-J) via tail vein. At the time of sacrifice, mice were euthanized and blood and organs including the heart and aortic tree were removed and either frozen for future biochemical analyses or immediately processed for histology.TABLE 4REP-0003 Treatment Rapidly Reduces Serum Lipids

[0129] REP-0003 treatment of LDLR-null mice results in visually detectable reduction in serum lipid levels. LDLR-null mice exhibit lipidemic, cloudy serum. As shown in in FIG. 8 the serum of LDLR-null mice becomes visibly less lipidemic over time following a single administration of REP-0003. After one administration of REP-0003 to LDLR-null mice, serum was prepared at various time points up to 96 hours.

[0130] Serum triglyceride reduction is broadly considered to be beneficial, as evidence shows raised triglyceride levels are associated with atherosclerotic heart disease.

[0131] Serum triglyceride levels were measured in REP-0003 treated mice. Despite having much higher initial serum triglyceride levels, as shown in FIG. 9, these studies demonstrate that REP-0003 treatment produces as large a relative effect on serum triglyceride levels in LDLR-null mice as it does in ApoE-null mice.

[0132] FIG. 10 also shows that REP-0003 treatment results in a lowering of serum total cholesterol in LDLR-null mice (**P<0.01).Serum ALT levels are rapidly decreased after REP-0003 treatment

[0133] Raised serum ALT is characteristic of liver pathology. As depicted in FIG.11 , LDLR-null mice have high SLT levels that rapidly decline post-treatment with REP- 0003. A reduction in serum ALT is associated with reduced cell stress and reduced cell death in the liver.REP-0003 treatment lowers total cholesterol levels in liver tissue

[0134] Excess cholesterol in liver tissue is pathological, contributing to dysfunction by forcing cells into bearing a toxic excess of intracellular free cholesterol.

[0135] As shown in FIG. 12, treatment with REP-0003 resulted in a significant decrease in cholesterol in liver tissue by day 3 (***P<0.001).REP-0003 mRNA Therapy Rapidly Reverses Atherosclerosis in LDLR-null Mice

[0136] This study aimed to determine the effect of liver-specific delivery of LNP- CDP mRNA on vascular plaque regression and liver pathology in LDLR-null mice. 784 week-old LDLR-null mice were fed a Western diet (0.2% Cholesterol, Research Diets: D12079B) for 16 weeks and then randomly assigned into 6 groups (A to F).

[0137] As this was a reversal study, all mice were switched to normal chow i.e. a lower-fat control diet on Day 0, and were intravenously administered with either PBS (Group A, volume equivalent dose) or 0.25, 0.50, 0.75, 1.00 and 1.50 mg / kg of LNP- CDP mRNA once weekly (qw) over the next 6 weeks (groups B to F). After 6 weeks, and following their final dose on Day 40, mice were fasted for 6 hours, euthanized on Days 41-44, and assessed for blood chemistry / biomarkers, atherosclerotic plaque, and liver pathology.TABLE 5Aortic root plaque size is rapidly reduced in REP-0003 treated LDLR-null mice

[0138] LDLR-null mice were placed on a high-fat Western diet for 16 weeks. Mice with existing plaque were then switched to a low-fat control diet with cornstarch and no added cholesterol (Research Diets, D14042701) and dosed once weekly for 6 weeks with LNP-CDP mRNA. Mice were divided into six groups receiving either vehicle or LNP-CDP mRNA at doses ranging from 0.25 mg / kg to 1.50 mg / kg.

[0139] As shown in FIG. 13, the area of plaque in the aortic root was significantly reduced in REP-0003 treated mice compared to mice treated with vehicle. When averaging across all treated groups, the present treatment produced a significant 17% reduction in plaque cross-sectional area relative to aortic root cross-sectional area, versus the vehicle control group.

[0140] Additional treatments produce greater plaque regression.Treatment of LDLR-null mice at 0.25 mg / kg was sufficient for plaque regression.

[0141] As shown in FIG. 14, while the present study indicated that significant regression occurred at once weekly dosing of 0.25 mg / kg REP-0003, additional dosing intervals or concentrations may result in additional gains. Inter-group variation was determined by the random distribution of responsiveness to treatment.

[0142] Focal variabilities in the degree of liver pathology, a feature of LDLR-null mice, may be a factor in determining the efficiency of 1) LNP uptake by hepatocytes and 2) translation of CDP mRNA and 3) optimal cellular location of CDP protein.REP-0003 treatment also significantly improved treadmill performance

[0143] The endurance capacity of sedentary LDLR-null study mice was assessed at study end. Mice were prompted to run on a treadmill set to a 10-degree incline and maximum speed of 14 m / min. Time and distance were marked at the point at which the mouse ceased to run for at least 10 seconds, indicating exhaustion.

[0144] FIG. 15 shows that all groups treated with REP-0003 significantly outperformed the vehicle-treated group, indicating improved cardiovascular function.

[0145] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive.

[0146] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control.

[0147] Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.

Claims

CLAIMSWe claim:

1. A method of reducing at least one measurable aspect of an atherosclerotic plaque in a subject, comprising: identifying at least one atherosclerotic plaque in the subject with a measured aspect having a first value; administering a composition comprising; at least one cholesterol degrading protein; at least one molecule or compound configured to bind a surface protein of a hepatocyte; and a lipid; allowing the at least one cholesterol degrading protein to be expressed in the hepatocyte; measuring the measured aspect to determine a second value, wherein the first and second value differ by greater than 5%.

2. The method of claim 1, wherein the subject suffers from hypercholesterolemia.

3. The method of claim 1 or 2, wherein the subject suffers from familial hypercholesterolemia.

4. The method of claim 3, wherein the subject suffers from heterozygous familial hypercholesterolemia.

5. The method of claim 3, wherein the subject suffers from homozygous familial hypercholesterolemia.

6. The method of any one of claims 1-5, wherein the measurable aspect is selected from area, volume, and lipid or collagen content.

7. The method of any one of claims 1-6, further comprising the step of administering a statin or PCSK9 inhibitor to the subject.

8. The method of any one of claims 1-7, wherein the cholesterol degrading protein is a fusion protein.

9. The method of claim 8, wherein the fusion protein comprises a cytochrome P450.

10. The method of claim 9, wherein the cytochrome P450 is a Type 1 or Type 2 cytochrome p450.

11. The method of claim 10, wherein the Type 1 cytochrome P450 is selected from CYP11 and CYP27.

12. The method of claim 11 , wherein the the Type 1 cytochrome P450 is CYP11.

13. The method of claim 11 , wherein the the Type 1 cytochrome P450 is CYP27.

14. The method of any one of claims 11-13, wherein the fusion protein comprisesFerredoxin Reductase and Ferredoxin.

15. The method of claim 14, wherein the fusion protein is CDP1 or CDP27.

16. The method of claim 10, wherein the cytochrome P450 is Type 2.

17. The method of claim 16, wherein the Type 2 cytochrome P450 is selected fromCYP3, CYP7, and CYP8.

18. The method of claim 17, wherein the fusion protein comprises P450 reductase (POR).

19. The method of claim 17, wherein the fusion protein comprises P450 reductase (POR) and cytochrome b.

20. A composition for use in treating or preventing a disease associated with hypercholesterolemia, the composition comprising; at least one cholesterol degrading protein (CDP); a lipid encapsulating the at least one CDP; and CDP; and a molecule or compound configured to bind a surface protein of a hepatocyte.

21. The method of treating hyperlipidemia in a subject in need thereof, comprising: administering to the subject, a composition comprising; at least one cholesterol degrading protein (CDP); a lipid encapsulating the at least one CDP; and CDP; and a molecule or compound configured to bind a surface protein of a hepatocyte; and affecting the concentration of at least one serum biomarker of the subject.

22. The method of claim 21 , wherein the biomarker is selected from total cholesterol, free cholesterol, lipids, lipoproteins, triglycerides, cytokines, SRA1, IL-10.

23. The method of claim 21 or 22, wherein the biomarker is free cholesterol.

24. The method of claim 21 or 22, wherein the biomarker is triglycerides.

25. The method of claim 23 or 24, wherein the biomarker is reduced after administration by greater than 5%.

26. The method of claim 21 or 22, wherein the biomarker is IL-10.

27. The method of claim 6, wherein the biomarker is increase by more than 50% after administration.

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