Improved mitochondrial cholesterol fluidity and catabolism

Abstract

Disclosed herein are compositions, methods and systems for enhancing cholesterol degradation in cells, tissues or living organisms.In many embodiments, the disclosure describes the use of one or more proteins or sequences encoding the same to enhance cholesterol flow into mitochondria, where cholesterol is degraded by one or more proteins comprising bacterial-associated sequences.The compositions, methods and systems disclosed herein are useful for preventing or treating diseases, disorders and conditions associated with high cholesterol levels in patients' blood or cells.

Description

TECHNICAL FIELD

[0001] The processes, methods, and systems of the present disclosure aim to treat a subject with high levels of cholesterol or at risk thereof by enhancing the transport of cholesterol into mitochondria and its degradation therein.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 63 / 122,886, filed December 8, 2020, entitled "Enhancing mitochondrial-based flow and catabolism of cholesterol", the entire contents of which are incorporated herein by reference in their entirety.

[0003] SEQUENCE LISTING This application contains a Sequence Listing electronically submitted in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on December 7, 2021, named P289347_WO_01_SL.txt, and has a size of 52,905 bytes.

BACKGROUND ART

[0004] High cholesterol (or hypercholesterolemia) refers to the presence of elevated cholesterol levels in the blood, which may result from an unbalanced diet, lifestyle, disease (such as diabetes) or heredity. High intracellular cholesterol concentrations are characteristic of several diseases, such as fatty liver, arteriosclerosis and the like.

[0005] Atherosclerosis is associated with narrowing of arteries, resulting from the accumulation of cholesterol-rich lipoproteins within the artery walls. It is often a precursor to cardiovascular disease (CVD), including death, myocardial infarction, stroke, and peripheral vascular disease. Many current treatments for these diseases focus on preventing secondary events (or initial events in high-risk individuals) by reducing circulating levels of low-density lipoprotein (LDL) and / or increasing high-density lipoprotein (HDL), because mammalian cells are capable of synthesizing cholesterol but not of breaking it down (catabolizing) it.

[0006] What is needed are compositions and methods for reducing lipid and cholesterol levels in individuals suffering from lipid and cholesterol excess. [Overview of the project]

[0007] Compositions, methods, and systems for improving the transport of cholesterol (including various modified cholesterol compounds) into modified mammalian mitochondria in which cholesterol has been modified to be catabolic. In many embodiments, cells may be engineered to improve the flow of cholesterol across the mitochondrial membrane. The cells may contain one or more proteins that can assist in the transport of cholesterol across the mitochondrial membrane.

[0008] Methods for preparing the disclosed compositions, methods for using the disclosed compositions, and methods for treating subjects who have or are at risk of developing high cholesterol may help reduce cholesterol in the subjects. In various embodiments, the disclosed compositions may be administered to subjects who need treatment for high cholesterol. In many embodiments, the nucleic acids expressing the disclosed proteins may be integrated into the genome of mammalian cells or exist outside the genome. The disclosed cells may be selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells.

[0009] Compositions, methods, and systems for modified mammalian mitochondria that enhance cholesterol transport and degradation are disclosed herein. In many embodiments, mammalian mitochondria are engineered to contain one or more recombinant and / or non-endogenous proteins that can help improve the flow of cholesterol across the mitochondrial membrane. In many embodiments, mitochondria may also contain one or more proteins that can assist in cholesterol catabolism. The disclosed modified mitochondria may be useful for reducing intracellular cholesterol, increasing cholesterol transport across the mitochondrial membrane, and / or reducing blood cholesterol levels in patients or subjects at risk of or suffering from high cholesterol or hypercholesterolemia.

[0010] In another aspect, a method for modifying mammalian mitochondria involves inserting at least one non-native, engineered steroid-producing acute regulatory (StAR) protein or a portion thereof into the outer mitochondrial membrane of a mammal, wherein the mitochondria contain one or more cholesterol-degrading proteins (CDPs), including cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol-metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0011] In various aspects of the methods and compositions disclosed herein, the StAR protein may comprise residues 63 to 188 of SEQ ID NO: 7, its duplicates, or at least a portion of the TOMM20 protein. The CDP may comprise P450 or P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx). The StAR protein and CDP may comprise at least one mitochondrial signaling protein and / or be expressed from a cell-specific promoter sequence and / or from a gene integrated into the genome or an extragenomic gene, and / or at least about 10% of the total amount of StAR protein and CDP in the cell may be located within the mitochondrial membrane. The disclosed methods and compositions may be useful in treating subjects suffering from or at risk of developing high cholesterol, for example, by reducing the cholesterol of subjects who have high cholesterol. Cells may be selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the mean catabolic product concentrations in the culture medium of CDP-expressing cells in the presence and absence of StAR-StAR or Tom-StAR (n=3, independent experiments).

[0013] [Figure 2] Figure 2 shows Western blots of tubulin, CDP, StAR-StAR, and TomStAR protein expression levels in the cells from Figure 1 (n=3, independent experiments).

[0014] [Figure 3] Figure 3 is a bar graph showing that StAR expression in the CDP-expressing mouse RAW264.7 cell line significantly increases cholesterol degradation and subsequent catabolism production in vitro.

[0015] [Figure 4] Figure 4 is a Western blot showing the amount of protein in the cell line described in Figure 3.

[0016] [Figure 5] Figure 5 shows the sequences and nucleic acid coding sequences of various proteins disclosed herein. Specifically, the human protein sequence of P450-FdxR-Fdx (SEQ ID NO: 1) is shown in bold with a Flag tag: Cholesterol dehydrogenase, CholD (SEQ ID NO: 2) is shown; 3-ketosteroid Δ1-dehydrogenase, Δ1-KstD (SEQ ID NO: 3) is shown; anaerobic cholesterol metabolizing enzyme B, acmB (SEQ ID NO: 4) is shown; 3-ketosteroid 9α-hydroxylase, KshAB (SEQ ID NO: 5) is shown; 3β-hydroxysteroid dehydrogenase 2, HSD2 (SEQ ID NO: 6) is shown; human StAR protein sequence (SEQ ID NO: 7) is shown; human TOMM20 protein sequence (SEQ ID NO: 8) is shown; human Tom / Star fusion protein sequence (SEQ ID NO: 9; Tom sequence is shown in bold, linker sequence is shown in color and underlined) is shown; human StAR / StAR fusion protein sequence (with color and underlined linker sequence (SEQ ID NO: 10)); StAR mRNA complete sequence (SEQ ID NO: 11; The start codon is shown in uppercase, along with the underlined amino acid sequence and the coding sequence corresponding to sequence numbers 63-285 of sequence number 7, which is written in uppercase; and the complete TOMM20 mRNA sequence, CDS = 123-560 (sequence number 12; start codon in uppercase, stop codon in red). [Modes for carrying out the invention]

[0017] Disclosure herein relates to compositions, methods, and systems for reducing cholesterol in target cells and / or in the bloodstream by improving the transport of cholesterol into mitochondria, which is catalyzed by cholesterol catabolases.

[0018] The disclosure herein is the development of a unique approach to help manage hypercholesterolemia and other disorders associated with the presence of excess cholesterol. Humans lack the enzymes to break down cholesterol, and many mammalian cells take up excess cholesterol without adequate storage or metabolic mechanisms. In some cases, macrophages develop into foam cells when they take up cholesterol or cholesterol esters. This cholesterol accumulation can lead to the formation of arterial plaques, which can lead to atherosclerosis. Depending on their location, poor blood flow can cause plaques to cause ischemic heart disease, angina (chest pain), intermittent claudication (leg pain during exercise), or chronic kidney disease. Furthermore, many plaques can be unstable and prone to rupture even before they significantly affect blood flow. These structures typically show no clinical symptoms until the plaque ruptures (complex plaque), allowing the underlying tissue to form a blood clot that can suddenly block the affected blood vessel.

[0019] The applicant hereby indicates that human cells can be manipulated to assist in cholesterol reduction. In many embodiments, the cells may be any mammalian cells. The disclosed cells may be manipulated to express a variety of proteins that can assist in cholesterol transport and degradation / catabolism. As used herein, the manipulated cells may be cells accompanied by one or more non-natural and / or exogenous coding sequences or proteins; for example, a manipulated mammalian cell may contain one or more mammalian and / or bacterial-related sequences or proteins. As used herein, non-natural, exogenous, or non-intrinsic may mean sequences or proteins that are often introduced into cells by recombination. In most embodiments, bacterial-related may mean a protein or coding sequence with a similar bacterial sequence and a higher identity of about 50% or more. In many embodiments, the disclosed proteins may be fusion proteins and / or modified proteins, for example, proteins lacking and / or duplicate sequences found in natural proteins.

[0020] The disclosed proteins may be overexpressed with respect to endogenous genes and / or proteins. In one example, the endogenous gene or protein may be a mammalian p450scc, StAR, or TOMM20 protein. In most embodiments, the overexpression of the disclosed protein can result in 2X, 3X, 4X, 5X, 10X, 20X, 100X, 200X or more of the disclosed gene or protein in a given sample, based on weight or molar amount, as compared to the endogenous gene or protein. In most cases, overexpression relates to non-induced expression of the endogenous protein. In some cases, overexpression may relate to the induced level of the endogenous protein, and the expression may be similar or identical. In most embodiments, the degradation related to cholesterol degradation refers to the removal of various side chains of cholesterol and / or the ring opening of at least one ring.

[0021] The disclosed coding sequences and proteins for cholesterol degradation may be of bacterial and / or mammalian origin. In many embodiments, the disclosed proteins may be referred to as cholesterol degrading proteins (CDP), and may be selected from one or more of cholesterol dehydrogenase (CholD; SEQ ID NO: 2), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD; SEQ ID NO: 3), anaerobic cholesterol metabolism B enzyme (acmB; SEQ ID NO: 4), 3-ketosteroid 9α-hydroxylase (KshAB; SEQ ID NO: 5), 3β-hydroxysteroid dehydrogenase 2 (HSD2; SEQ ID NO: 6), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx; SEQ ID NO: 1; uniprot Nos. P05108, P22570, P10109), and steroidogenic acute regulatory protein (StAR; SEQ ID NO: 7). In many embodiments, the disclosed coding sequences and proteins may share greater than about 80% identity with the sequences disclosed herein.

[0022] The disclosed coding sequences and proteins can be delivered to cells in vivo or in vitro by various methods, including, for example, electroporation, transfection, viral vectors, nanoparticles, and the like. In some embodiments, the disclosed coding sequences and proteins may be encoded in one or more nucleic acids within cells, vectors, or particles. In some embodiments, the particles may be lipid nanoparticles (or lipo nanoparticles; LNP). In some embodiments, the vector may be a viral vector such as an adenovirus or a lentivirus. In many embodiments, the coding sequence may comprise one or more promoter and / or enhancer sequences that can support the expression of the encoded protein in a specific cell or tissue, or can support high expression of the encoded protein generally or in a specific cell or tissue.

[0023] The disclosed proteins, which may be collectively or individually referred to as cholesterol degrading proteins (CDPs), can be directed to mammalian mitochondria, for example, the outer membrane, inner membrane, intermembrane space, or matrix thereof.

[0024] The terms "about" or "approximately" mean an allowable error for a specific value as determined by one of ordinary skill in the art, and depend in part on how the value is measured or determined. In certain embodiments, the terms "about" or "approximately" mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms "about" or "approximately" mean within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of the stated value or range. Where the term "about" or "approximately" precedes the first of two or more consecutive numerical values, the term "about" or "approximately" applies to each of the consecutive numerical values.

[0025] The administration of the disclosed compositions, constructs, cells, methods, and systems may be via a variety of methods. In one embodiment, the administration may be “intravenous” administration, which means the introduction of the compositions, constructs, and / or cells into the patient’s veins, for example, by infusion (slow therapeutic introduction into a vein). In other embodiments, the administration may be “subcutaneous” administration, and the introduction is carried out under the patient’s skin. “Infusion” or “infusion” means introducing a solution into the body through a vein for therapeutic purposes. Generally this is done via an intravenous (IV) bag, for example, a bag capable of holding the solution. The disclosed compositions, constructs, cells, methods, and systems may be “concurrently administered” to a patient, for example by administering at least a second therapeutic agent by intravenous injection during the same administration. In some embodiments, concurrent administration is performed simultaneously, and in other embodiments, concurrent administration is performed sequentially.

[0026] As used herein, the term “prevention” means preventing the occurrence or recurrence of at least one disease or symptom associated therewith as defined herein by administering a composition, construct, method, or system according to the present invention to a subject in need thereof.

[0027] The term "patient" or "subject" includes a variety of animals, including humans, monkeys, cats, dogs, mice, rats, rabbits, or guinea pigs. The animals may be mammals, such as non-primates and primates (e.g., monkeys and humans). In one embodiment, the patient is a human, such as an infant, child, adolescent, or adult.

[0028] The terms “to treat,” “to treat,” and “treatment” mean to eliminate, reduce, suppress, or remit, either temporarily or permanently, partially or completely, at least one symptom, sign, or progression of an event, disease, disorder, or condition disclosed herein. As is recognized in the relevant art, methods, compositions, and systems used as treatment may reduce the severity of a given disease condition, but do not need to eliminate all associated symptoms to be considered useful. Similarly, a prophylactic treatment does not need to be completely effective in preventing the onset of a condition in order to constitute a viable prophylactic composition, drug, method, or system. It may suffice to simply reduce the effects of the disease (e.g., by reducing blood cholesterol levels and / or the number or severity of associated symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect, as disclosed herein), or by reducing the likelihood that the disease will develop or worsen in a subject.

[0029] The term "effective dose" means the amount of the compound or other active ingredient of the present invention that is sufficient to produce a therapeutic or preventive benefit in the treatment or prevention of a disease, or to delay or minimize symptoms associated with the disease. Furthermore, the therapeutic effective dose with respect to the compound of the present invention means the amount of the therapeutic agent alone or in combination with other treatments that produces a therapeutic benefit in the treatment or prevention of a disease. When used with respect to the compound of the present invention, the terms may include amounts that improve the overall treatment, reduce or avoid the cause of symptoms or disease, or enhance therapeutic efficacy and synergistic effects with other therapeutic agents.

[0030] The expression “therapeutic dose” means an amount of the composition of the present invention that (i) treats a particular disease, condition or disorder, (ii) relieves or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder as described herein. In the case of hypercholesterolemia, a therapeutic dose of the disclosed composition may reduce the cholesterol concentration in the patient’s blood; reduce intracellular cholesterol; inhibit the growth and / or development of cholesterol plaques (i.e., slow it down to some extent, preferably stop it); inhibit lipid accumulation in the liver (i.e., slow it down to some extent, preferably stop it); reduce the lipid content in the liver; and / or alleviate to some extent one or more symptoms associated with hypercholesterolemia.

[0031] As used herein, the term “remission” means any improvement in the patient’s disease state (e.g., high blood cholesterol) by administering one or more treatments and / or compositions in accordance with this disclosure to a patient or subject requiring treatment and / or compositions. Such improvement may be observed as a slowing or cessation of disease progression and / or a reduction in the severity of at least one disease symptom, an increase in the frequency or duration of disease-free periods, or prevention of disease-related functional impairment or physical disability.

[0032] target cell Various tissues and cell types capable of expressing CDP can be targeted by the disclosed compositions. In various embodiments, the cells may be smooth muscle, skeletal muscle, as well as endothelial and epithelial cells of all organs and tissues, including but not limited to cardiomyocytes, nerve cells, glandular cells, osteocytes, immune cells, hematopoietic cells, and their stem cells or progenitor cells. In some embodiments, the cells may be pluripotent stem cells, such as induced pluripotent stem cells. In many embodiments, the tissue may be cancerous or precancerous, and may be skin, as well as one or more cells of intestinal cancer (squamous epithelial cells), adenocarcinoma (glandular epithelial cell origin of the pancreas), gastrointestinal cancer (neuroendocrine cells), pancreatic cancer, small cell carcinoma (neuroendocrine cells), leiomyosarcoma (smooth muscle cells), and lymphoma (lymphocytes found in the digestive tract organs / walls).

[0033] Various cells and tissues can be targeted by the disclosed compositions and methods. In many embodiments, the disclosed compositions may target specific cells by various methods known to those skilled in the art. In some embodiments, the LNP may include one or more molecules, such as proteins that have affinity for receptors or surface proteins on the target cells, which help to target specific cells.

[0034] Cholesterol-degrading enzymes This specification discloses various cholesterol, cholesterol-related genes, and proteins. In some embodiments, the disclosed genes and proteins can assist in the transport and / or metabolism of cholesterol. In most embodiments, the disclosed genes and proteins can be expressed in and / or target mitochondria. In many embodiments, the disclosed genes and proteins enable cells to break down and / or catabolize cholesterol to open its ring, and then endogenous proteins and enzymes can further metabolize that molecule.

[0035] Mitochondria Mitochondria are found in most eukaryotic cells and eukaryotes and are composed of a double membrane. Mitochondria supply energy to cells and play a role in signaling, cell cycling, cell proliferation, as well as differentiation, apoptosis, and cell death. Energy-demanding cells such as heart, muscle, brain, and liver cells may have thousands of mitochondria.

[0036] The mitochondrial bimembranous structure has specialized compartments: the outer membrane, intermembrane space, inner membrane, cristae, and matrix. These compartments have specialized functions, such as ATP synthesis. The matrix contains the mitochondrial genome.

[0037] Steroid production acute regulatory protein (StAR) Steroids and steroid hormones are synthesized from cholesterol. The first step in steroid production is the cleavage of cholesterol side chains by P450scc located on the matrix side of the inner mitochondrial membrane (IMM). Typically, cells that produce steroid hormones store and maintain only small amounts of the hormone at all times. In other words, an increase in steroid secretion is made by an initial increase in its steroid synthesis. However, steroids have broad and potent effects in cells and in vivo - therefore steroid-producing cells tightly regulate the production of steroids and precursors to avoid overproduction and pathological conditions associated with excessive steroid levels. To avoid overproduction, synthesis can be started and terminated rapidly. One way cells perform this regulation (synthesis can be increased 10-100 times within minutes) is by regulating the flow of cholesterol into the mitochondrial matrix. Cholesterol is transported across the mitochondrial membrane by StAR proteins.

[0038] The StAR protein is synthesized as a 37 kDa protein containing a leader peptide sequence that targets mitochondria. Upon insertion into the mitochondrial membrane, the leader peptide is cleaved, resulting in a 30 kDa protein located inside the mitochondria. Mutations in the StAR gene can lead to congenital lipoid adrenal hyperplasia. Individuals with adrenal hyperplasia synthesize very small amounts of steroids.

[0039] StAR can interact with two additional proteins that support cholesterol importation into mitochondria. TSPO, a translocator protein, is an 18kDa protein that assists in the transport of steroid-producing cholesterol into mitochondria. Studies have shown that TSPO expression is upregulated in response to macrophage exposure to modified LDLs. In addition, the widely expressed PBR (peripheral benzodiazepine receptor) is a mitochondrial outer membrane protein involved in regulating cholesterol transport. Inhibition of PBR leads to a loss of cellular steroid production capacity.

[0040] The ability to produce large amounts of steroids from cholesterol can be conferred to non-steroid-producing cells (i.e., cells that typically do not produce steroids or produce very small amounts of steroids) by introducing StAR. Specifically, non-steroid-producing cells genetically modified with the P450scc enzyme system (i.e., H2N-P450scc-adrenodoxin reductase-adrenodoxin-COOH) can metabolize cholesterol at very low levels. The presence of StAR (i.e., co-expression of StAR and the P450scc enzyme system) increases cholesterol conversion in these cells several-fold. Steroid production in cells can help slow down the transport of StAR across the outer mitochondrial membrane (OMM) by OMM proteins (e.g., the TOM gene for mitochondrial translocases). t ranslocase, o uter mStudies have shown that this can be further enhanced by fixing StAR to the mitochondrial outer membrane through either fusion with the embrane or duplication of the StAR domain. Duplication of the StAR domain can be called the StAR-StAR protein. StAR-StAR is a fusion protein in which residues 1-188 of StAR are bound to residues 63-285 of StAR, thus dimerizing the protease resistance domain (residues 63-188) of StAR.

[0041] The StAR proteins used in the compositions and methods of the present invention may comprise various forms of StAR protein sequences. In some embodiments, the disclosed StAR protein may comprise one or more mutations, truncations, deletions, duplications, fusions, etc., or the disclosed protein may be a wild-type or native StAR protein. In some embodiments, the disclosed protein may be modified to reduce the transport time across the mitochondrial outer membrane, which may help improve cholesterol import into mitochondria.

[0042] As used herein, the term cholesterol means cholesterol or cholesteryl alcohol, and the formula for sterol is C 27 H 46 O, the IUPAC names are cholesterol-5-en-3-ol and (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-ol. As used herein, the term cholesterol refers to oxidized cholesterol, C 27 H 46 This can also refer to cholesterol derivatives including O2, oxycholesterol, or 5,6-epoxycholesterol, 7-ketocholesterol (7KC), cholestane-3β,5α,6β-triol, and 7-α / β-hydroxycholesterol.

[0043] The genes and proteins that can break down cholesterol may be selected from cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), and combinations thereof.

[0044] One or more of the disclosed genes, proteins, and enzymes may be packaged in one or more vectors, constructs, or cassettes. In various embodiments, a cassette containing one or more cholesterol-degrading enzymes may be called a cholesterol catabolic cassette (CCC). In some embodiments, the cassette may be a construct and may contain a nucleic acid sequence encoding at least one protein having about 80% or more identity with the protein encoded by either of the disclosed proteins or genes. In many embodiments, the percentage of identity with the disclosed protein or nucleotide sequence may be greater than about 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and less than about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, and 81%. Sequence identity may be calculated for protein sequences that are greater than approximately 40 aa (amino acid residues), 50 aa, 60 aa, 70 aa, 80 aa, 90 aa, 100 aa, 110 aa, 120 aa, 130 aa, 140 aa, 150 aa, 160 aa, 170 aa, 180 aa, 190 aa, 200 aa or more, and less than approximately 300 aa, 200 aa, 190 aa, 180 aa, 170 aa, 160 aa, 150 aa, 140 aa, 130 aa, 120 aa, 110 aa, 100 aa, 90 aa, 80 aa, 70 aa, or 60 aa. In many embodiments, sequence identity is calculated over approximately 80-150 amino acid residues. In most cases, any inserted or deleted amino acids compared to the reference sequence (i.e., sequence numbers 1-10) are counted as a single non-identical amino acid.In some embodiments, for example, in embodiments where the delivery of one or more compositions is via an LNP, such as when the composition is one or more nanoplasmids or mRNAs, the composition may include one or more cholesterol-degrading proteins, e.g., enzymes, and cholesterol transport proteins, e.g., StAR proteins or StAR variants. Those skilled in the art can construct mRNA from the proteins and nucleic acid sequences disclosed herein. In embodiments where one or more compositions are contained in therapeutic cells that can be administered to a target, the therapeutic cells may be administered by one or more vectors, constructs, and / or cassettes containing nucleic acids encoding cholesterol-degrading proteins, e.g., enzymes, and cholesterol transport proteins, e.g., StAR proteins or StAR variants. In the above case, the nucleic acids may be introduced into the cells by one or more viral vectors (one example being a lentivirus) that assist in incorporating or inserting the coding sequence into the cell's genomic or mitochondrial sequence. In some embodiments, the nucleic acids may not be incorporated, and the disclosed proteins are expressed from extragenomic sequences in the nucleus or mitochondria.

[0045] Cholesterol-related diseases or disorders Excessive natural LDL and an increased LDL:HDL ratio have been shown to play a significant role in cardiovascular diseases, atherosclerosis, stroke, and coronary heart disease and heart attacks. In some embodiments, subjects or patients at risk of or suffering from high cholesterol may have total cholesterol concentrations higher than approximately 200 mg of cholesterol per deciliter of blood (i.e., low-density lipoprotein + high-density lipoprotein) and / or total cholesterol higher than approximately 5.2 mmol / L. In some embodiments, subjects or patients at risk of or suffering from high cholesterol may have LDL concentrations higher than approximately 100 mg / dl and / or higher than approximately 2.6 mM LDL. In some embodiments, subjects or patients at risk of or suffering from high cholesterol may have HDL concentrations lower than approximately 50 mg / dl and / or lower than approximately 1.3 mM HDL. In some embodiments, subjects or patients at risk of or suffering from high cholesterol may have triglyceride concentrations higher than approximately 150 mg / dl and / or higher than approximately 1.7 mM triglycerides. In some embodiments, subjects or patients at risk of or suffering from high cholesterol may have an LDL:HDL ratio of approximately 4.0 (i.e., 4:1) or higher than 4.0.

[0046] Acetylated LDL is a form of LDL that is chemically modified in vitro and does not exist in vivo. Both acetylated and oxidized LDL are taken up by macrophages, which convert these cells into foam cells. In most cases, all components of LDL are easily oxidized, producing the oxidized form of LDL (oxLDL). Uptake of oxLDL by arterial macrophages is central to plaque formation. Unlike unmodified LDL, oxLDL is taken up by macrophages in the arterial wall in an unregulated manner via LDL scavenger receptors. Oxysterols are 10-100 times more reactive than natural cholesterol, with 7-ketocholesterol (7KC) being the most toxic, but also the most abundant form of oxLDL.

[0047] Studies have shown that higher circulating 7KC levels are significantly associated with an increased risk of future cardiovascular events and increased overall mortality. 7KC is a pro-inflammatory, pro-oxidative, pro-apoptotic, and fibrogenic molecule that disrupts critical ion transport pathways for cell membranes and vasodilation, altering endothelial cell function. In some embodiments, individuals or patients at risk of or suffering from high cholesterol may have 7KC concentrations higher than approximately 109.8 nmol / L.

[0048] Subjects or patients who have high cholesterol or are at risk of developing it may be considered to have hypercholesterolemia. In some embodiments, hypercholesterolemia can be assessed by measuring the percentage of plasma oxysterols, for example, the percentage of 7-KC in plasma oxysterols. In patients with hypercholesterolemia, 7-KC may account for approximately 57% of plasma oxysterols. 7-KC is metabolized to 7-α / β-hydroxycholesterol (21% of plasma oxysterols), which is the direct product of 7-KC metabolism. In arterial plaque, 55% of oxysterols have been reported to be 7-KC, followed by cholestane-3β,5α,6β-triol and 7-α / β-hydroxycholesterol, which are the second and third most abundant at 13% and 12%, respectively.

[0049] As mentioned above, NASH (non-alcoholic steatohepatitis) is another disease associated with excess cholesterol. When cholesterol homeostasis and transport are altered, free cholesterol accumulates in the liver, leading to NAFLD (non-alcoholic fatty liver disease) through liver cell damage and activation of non-parenchymal cells. In particular, an overload of free cholesterol in and around mitochondria induces mitochondrial dysfunction, promoting inflammation, fibrosis, and hepatocyte death.

[0050] Other cholesterol-related diseases include alveolar proteinosis (PAP), eye diseases, neurodegenerative diseases, Niemann-Pick disease type C (NPC), and lysosomal acid lipase (LAL) deficiency. This is because cholesterol content plays a role in regulating the fluidity and function of pulmonary surfactant in lunged animals, and this fluidity can change rapidly, especially under extreme temperatures, and reduced cholesterol clearance is a primary defect that leads to the development of PAP. In the case of eye diseases, oxysterols, and 7KC in particular, cause degeneration of retinal cells. In other words, increased oxysterol levels may be at play in a variety of eye diseases, including age-related macular degeneration (AMD), choroidal neovascularization (CNV), glaucoma, and cataracts.

[0051] Increased oxysterol levels can also lead to changes in cholesterol metabolism in the brain. Cholesterol metabolism may be a key component of several brain disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and dementia progression. Various oxysterols, including 7KC, produced by the auto-oxidation of cholesterol, have been identified in the postmortem brains of Alzheimer's patients. Chronic epilepsy may also share many of the above pathological conditions. Specifically, a link has been suggested between epilepsy and atherosclerosis. That is, treatment of atherosclerosis, such as the compositions, cells, and methods of this disclosure, may mitigate the effects of epilepsy. Furthermore, 7KC is highly cytotoxic to nerve cells and is suspected to be involved in the progression of various neurological diseases. Surprisingly, unlike cholesterol, oxysterols can cross the blood-brain barrier (BBB), accumulate in brain cells, and ultimately cause neurodegeneration.

[0052] Various other diseases can be associated with elevated cholesterol levels. For example, patients with Niemann-Pick disease type C (NPC) are unable to remove cholesterol, leading to the accumulation of cholesterol and oxysterols, primarily in the liver, pancreas, and brain. A positive correlation between the 7KC profile and disease severity has been reported. In addition, patients with lysosomal acid lipase (LAL) deficiency may accumulate cholesterol esters and triglycerides in lysosomes, exhibiting symptoms of hypercholesterolemia, hyperlipidemia, and / or atherosclerosis. These patients also have very high levels of oxysterols, including 7KC, in their plasma. Increased oxysterol formation further increases oxidative stress, which worsens the condition.

[0053] The disclosed compositions and methods are useful for diseases or conditions associated with excess cholesterol and / or fatty deposits in cells, tissues, and organs. In some embodiments, the disease or condition may be associated with the presence of excess cholesterol and / or one or more types of oxidized cholesterol, such as 7-ketocholesterol. In some embodiments, the disease or condition may be one or more, such as fatty liver disease, atherosclerosis, heart failure, stroke, ischemia, coronary heart disease, eye diseases, neurodegenerative and neurological diseases, eye diseases such as macular degeneration, and pulmonary dysfunction.

[0054] The disclosed compositions, cells, methods, and therapies may help treat, reduce, or reverse a variety of diseases, disorders, or conditions associated with excessive cholesterol. In one embodiment, the disease, disorder, or condition may be one or more of the following: early type 2 lesions (i.e., macrophage foam cell formation), type 3 lesions or preatheromas (i.e., having a small storage of extracellular lipids), type 4 lesions or atheromas (i.e., having a core of extracellular lipids), type 5 lesions or fibrous atheromas (i.e., atheromas with fibrous thickening).

[0055] The disclosed compositions, constructs, cells, methods, treatments, and systems may help reduce cholesterol levels in a patient's blood. In some embodiments, the reduction in blood cholesterol level (e.g., weight per volume such as mg / dl or molar concentration such as mmol / L) may be 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%, or 90%, and less than about 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. In many embodiments, the reduction in cholesterol levels following treatment reflects an increase in the concentration or amount of one or more cholesterol catabolic products, for example, an increase relative to the catabolic product concentration before treatment of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5X, 2.0X, 2.5X, 3.0X, 3.5X, 4.0X, 4.5X, 5.0X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X The increase may be greater than 200X, 300X, 400X, 500X, 600X, 700X, 800X, or more, and may be less than approximately 2000X, 1500X, 1000X, 500X, 400X, 300X, 200X, 100X, 90X, 80X, 70X, 60X, 50X, 40X, 30X, 20X, 10X, 5X, 4X, 3X, 2X, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%.

[0056] Examples Example 1 - Co-expression of cholesterol-degrading enzyme and StAR protein In vitro experiments were conducted to test the cholesterol-degrading activity of cholesterol-degrading enzymes combined with StAR proteins. In these experiments, human 293T kidney epithelial cells were placed in 12-well plates, DMEM, and 10% FBS, with 3 x 10 cells. 5Cells were seeded at a rate of one cell per well. At 75% confluence, the cells were transfected with Lipofectamine 3000. Next, under the following conditions, equimolar amounts of CDP or empty plasmid were mixed with StARStAR, TomSTaR, or empty plasmid along with P3000 in 50 μL of Opti-MEM. 1. Empty plasmid + Empty plasmid 2. CDP plasmid + empty plasmid 3. StARStAR plasmid + empty plasmid 4. TomStAR plasmid + empty plasmid 5. CDP plasmid + StARStAR plasmid 6. CDP plasmid + TomStAR plasmid

[0057] 3X μL of Lipofectamine reagent was mixed with 50 μL of Opti-MEM. Next, the DNA / P3000 tube was mixed with the Lipofectamine tube and incubated at room temperature for 15 minutes. The transfection mixture was added to the cells in droplet form and incubated for 48 hours.

[0058] At the end of the incubation period, the catabolic concentrations of the cell culture medium were assayed. Total cell protein was extracted with RIPA buffer, and the total protein concentration was quantified using a standard BCA kit.

[0059] result As shown in Figure 1 below, CDP expression in 293T kidney epithelial cells increased the catabolic product concentration in the culture medium by approximately 300-fold from baseline levels (culture medium of cells expressing only empty plasmids, i.e., cells not expressing CDP). Co-expression of this fusion protein with CDP increased catabolic products by approximately 4-fold compared to CDP alone. Tom20 has 50 N-terminal residues located in the mitochondrial outer membrane (OMM) and 95 residues in the cytoplasm. Fusion of StAR residues 63-285 to the C-terminus of Tom20 (i.e., Tom-StAR), which positions StAR on the cytoplasmic side of the OMM, increased catabolic products by approximately 30% compared to cells expressing CDP alone when co-expressed with CDP. Expression of either StAR-StAR or TomStAR alone did not affect catabolic product concentration. Cellular transfection with StAR-StAR cDNA led to strong expression of StAR-StAR protein, detected as a band of approximately 50 kD by SDS-PAGE and Western blotting (see Figure 2 below). Interestingly, co-expression of StAR-StAR and CDP (detected as a FLAG-tagged protein with anti-FLAG antibody) resulted in significant downregulation of steady-state StAR-StAR protein levels. While we do not wish to be bound by any theory, the above data suggest that the sequential StAR-StAR-mediated transport of CDP-substrates and cholesterol into the mitochondrial matrix led to the destabilization and degradation / consumption of StAR-StAR protein. Downregulation of Tom-StAR was not detected in CDP-co-expressing cells, and while we do not wish to be bound by any theory, this is likely due to the lower efficiency of Tom-StAR in cholesterol transport into the mitochondrial matrix. This decrease in efficiency was evidenced by the fact that cells co-expressing CDP and Tom-StAR had significantly lower levels of CDP-derived catabolites compared to cells co-expressing CDP and StAR-StAR (see Figure 1 above).Levels of StAR-StAR protein were higher in cells expressing StAR-StAR alone compared to cells expressing both StAR-StAR and CDP. This suggests that StAR-StAR activity levels and their resulting degradation may have been limited in the absence of CDP by increased cholesterol accumulation levels within the mitochondrial matrix. We hypothesize that the continuous conversion of cholesterol to its catabolites by CDP increases the cholesterol concentration gradient along the inner mitochondrial membrane, thereby further stimulating StAR-StAR-mediated cholesterol transport into the mitochondria.

[0060] Figure 1 shows the results of this experiment in bar graph format. Specifically, as described below the graph, Figure 1 shows the average catabolic concentrations in the culture medium of CDP-expressing cells in the presence and absence of StAR-StAR or Tom-StAR (n=3, independent experiments).

[0061] Figure 2 shows Western blots representing intracellular CDP protein levels in the presence and absence of StAR-StAR or Tom-StAR, with tubulin as a control (n=3, independent experiments). The lanes are as follows: 1, empty plasmid + empty plasmid; 2, CDP plasmid + empty plasmid; 3, StARStAR plasmid + empty plasmid; 4, TomStAR plasmid + empty plasmid; 5, CDP plasmid + StARStAR plasmid; 6, CDP plasmid + TomStAR plasmid.

[0062] Example 2 - Co-expression of cholesterol-degrading enzyme and StAR protein in mouse cells In vitro experiments were performed as described in Example 2. In these experiments, mouse RAW264.7 cells were placed in 12-well plates, DMEM, 10% FBS, with 3 x 10 cells per well. 5Cells were seeded at 1 cell / well. At 75% confluence, cells were transfected with Lipofectamine 3000. Next, equimolar amounts of CDP or empty plasmid and either StARStAR or empty plasmid were mixed with P3000 in 50 μL of Opti-MEM under the following conditions: empty plasmid + empty plasmid; CDP plasmid + empty plasmid; or CDP plasmid + StARStAR plasmid.

[0063] In cells co-expressing CDP and StAR, cholesterol catabolism was significantly increased. Specifically, the catabolic product pg / cell ratios were as follows: empty + empty, 2.83E-03; CDP + empty, 1.26E-01; and CDP + StARStAR, 1.07E+00. These results correspond to a 44.52-fold increase in the presence of CDP and a further 8.49-fold increase in the presence of StARStAR protein.

[0064] Claim 1. A coding sequence for a steroid production acute regulatory (StAR) protein; and A nucleic acid composition comprising a coding sequence for a cholesterol-degrading protein (CDP) containing one or more of the following: cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0065] Claim 2. The nucleic acid of Claim 1, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO: 7.

[0066] Claim 3. The nucleic acid of claim 1 or 2, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO: 7.

[0067] Claim 4. The nucleic acid of any one of claims 1 to 3, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

[0068] Claim 5. The nucleic acid of any one of claims 1 to 4, wherein the CDP comprises P450.

[0069] Claim 6. The nucleic acid of any one of claims 1 to 5, wherein the CDP comprises a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0070] Claim 7. The nucleic acid of any one of claims 1 to 6, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

[0071] Claim 8. The nucleic acid according to any one of claims 1 to 7, wherein the nucleic acid further comprises a cell-specific promoter sequence.

[0072] Claim 9. The nucleic acid according to any one of claims 1 to 8, wherein the nucleic acid is contained in liponoparticles.

[0073] Claim 10. The nucleic acid according to any one of claims 1 to 8, wherein the nucleic acid is contained in a viral vector.

[0074] Claim 11. A nucleic acid according to any one of claims 1 to 10 for use in the treatment of a subject with high cholesterol.

[0075] Claim 12. A nucleic acid according to any one of claims 1 to 11 for use in the transformation of mammalian cells.

[0076] Claim 13. The nucleic acid of claim 12, wherein mammalian cells are transformed in vitro.

[0077] Claim 14. The nucleic acid of claim 12, wherein mammalian cells are transformed in vivo.

[0078] Claim 15. The nucleic acid according to any one of claims 1 to 14, wherein the nucleic acid is contained in liponoparticles.

[0079] Claim 16. The nucleic acid according to any one of claims 1 to 15, wherein the nucleic acid is contained in cells of a subject that have high cholesterol or are at risk of developing it.

[0080] Claim 17. A nucleic acid according to any one of claims 1 to 16 for use in reducing cholesterol in a subject with high cholesterol.

[0081] Claim 18. A coding sequence of an engineered steroid-producing acute regulatory (StAR) protein or a portion thereof; and Mammalian cells containing coding sequences for cholesterol-degrading proteins (CDPs), including one or more of the following: cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0082] Claim 19. The mammalian cell of claim 18, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO: 7.

[0083] Claim 20. The nucleic acid of claim 18 or 19, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO: 7.

[0084] Claim 21. A mammalian cell according to any one of claims 18 to 20, wherein the StAR protein comprises at least a portion of the TOMM20 protein sequence.

[0085] Claim 22. A mammalian cell according to any one of claims 18 to 21, wherein the CDP comprises P450.

[0086] Claim 23. A mammalian cell according to any one of claims 18 to 22, wherein the CDP comprises a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0087] Claim 24. A mammalian cell according to any one of claims 18 to 23, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

[0088] Claim 25. A mammalian cell according to any one of claims 18 to 24, wherein the coding sequence of StAR or CDP further comprises a cell-specific promoter sequence.

[0089] Claim 26. A mammalian cell according to any one of claims 18 to 25, wherein a coding sequence for StAR or CDP is incorporated into the genome of the mammalian cell.

[0090] Claim 27. A mammalian cell according to any one of claims 18 to 25, wherein the coding sequence of StAR or CDP is not incorporated into the genome of the mammalian cell.

[0091] Claim 28. Mammalian cells of any one of claims 18 to 27 for use in the treatment of subjects suffering from or at risk of developing high cholesterol.

[0092] Claim 29. Mammalian cells according to any one of claims 18 to 28, for use in reducing cholesterol in a subject.

[0093] Claim 30. Mammalian cells according to any one of claims 18 to 29, wherein the mammalian cells are selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells.

[0094] Claim 31. An engineered acute steroid production regulatory (StAR) protein or a portion thereof; and Mammalian cells containing cholesterol-degrading proteins (CDPs) including one or more of the following: cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolism enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0095] Claim 32. The mammalian cell of claim 31, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO: 7.

[0096] Claim 33. A mammalian cell according to claim 31 or 32, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO: 7.

[0097] Claim 34. A mammalian cell according to any one of claims 31 to 33, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

[0098] Claim 35. A mammalian cell according to any one of claims 31 to 34, wherein the CDP contains P450.

[0099] Claim 36. A mammalian cell according to any one of claims 31 to 35, wherein the CDP comprises a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0100] Claim 37. A mammalian cell according to any one of claims 31 to 36, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

[0101] Claim 38. A mammalian cell according to any one of claims 31 to 37, wherein at least 10% of the total amount of StAR protein and CDP in the cell is located in the mitochondrial membrane.

[0102] Claim 39. A mammalian cell according to any one of claims 31 to 38, wherein the StAR protein or CDP is encoded by a gene integrated into the genome of the mammalian cell.

[0103] Claim 40. A mammalian cell according to any one of claims 31 to 39, wherein the StAR protein or CDP is encoded by an extragenomic gene.

[0104] Claim 41. Mammalian cells according to any of claims 31 to 39, for use in the treatment of subjects suffering from or at risk of developing high cholesterol.

[0105] Claim 42. Mammalian cells according to any one of claims 31 to 40 for use in reducing cholesterol in a subject.

[0106] Claim 43. Mammalian cells according to any one of claims 18 to 29, wherein the mammalian cells are selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells.

[0107] Claim 44. Expressing a non-naturally engineered acute steroid production regulatory (StAR) protein or a portion thereof, and This involves expressing one or more cholesterol-degrading proteins (CDPs), including cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx). A method to improve cholesterol breakdown within mammalian cells.

[0108] Claim 45. The method of claim 44, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO: 7.

[0109] Claim 46. The method of claim 44 or 45, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO: 7.

[0110] Claim 47. Any method of claims 44 to 46, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

[0111] Claim 48. Any method of claims 44 to 47, wherein the CDP includes P450.

[0112] Claim 49. The method according to any one of claims 44 to 48, wherein the CDP comprises a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0113] Claim 50. Any method of claims 44 to 49, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

[0114] Claim 51. A method of any one of claims 44 to 50, wherein a StAR protein and CDP are expressed from a cell-specific promoter sequence.

[0115] Claim 52. Any method of claims 44 to 51 for use in treating a person who has or is at risk of developing high cholesterol.

[0116] Claim 53. Any method of claims 44 to 52 for use in reducing cholesterol in a subject that is high in cholesterol.

[0117] Claim 54. The method according to any one of claims 44 to 53, wherein at least about 10% of the total amount of intracellular StAR proteins and CDPs is located in the mitochondrial membrane.

[0118] Claim 55. A method of any one of claims 44 to 54, wherein a StAR protein or CDP is expressed from a gene integrated into the genome of a mammalian cell.

[0119] Claim 56. A mammalian cell according to any one of claims 44 to 54, wherein the StAR protein or CDP is of extragenomic origin.

[0120] Claim 57. Any method from Claims 44 to 56 for use in treating a person who has or is at risk of developing high cholesterol.

[0121] Claim 58. Any method from Claims 44 to 57 for use in reducing cholesterol in a subject.

[0122] Claim 59. The method according to any one of claims 44 to 58, wherein the cells are selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells.

[0123] Claim 60. Any method of claims 44 to 59, wherein the cholesterol is LDL.

[0124] Claim 61. The method according to any one of claims 44 to 59, wherein the cholesterol is 7 kC.

[0125] Claim 62. A method for treating a subject at risk of developing or suffering from high cholesterol, comprising administering at least one modified cell to the patient, Modified cells contain non-native, engineered acute steroid production regulatory (StAR) proteins or parts thereof, A method comprising one or more cholesterol-degrading proteins (CDPs), including cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol-metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0126] Claim 63. The method of claim 62, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO: 7.

[0127] Claim 64. The method of claim 62 or 63, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO: 7.

[0128] Claim 65. Any method of claims 62 to 64, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

[0129] Claim 66. Any method of claims 62 to 65, wherein the CDP includes P450.

[0130] Claim 67. The method according to any one of claims 62 to 66, wherein the CDP comprises a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0131] Claim 68. Any method of claims 62 to 67, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

[0132] Claim 69. A method of expressing a StAR protein and a CDP from a cell-specific promoter sequence, as described in any of claims 62 to 68.

[0133] Claim 70. Any method from Claims 62 to 76 for use in reducing cholesterol in a subject that is high in cholesterol.

[0134] Claim 71. The method according to any one of claims 62 to 71, wherein at least about 10% of the total amount of intracellular StAR proteins and CDPs is located in the mitochondrial membrane.

[0135] Claim 72. A mammalian cell according to any one of claims 62 to 72, wherein the StAR protein or CDP is expressed from a gene integrated into the genome of the mammalian cell.

[0136] Claim 73. Any method of claims 62 to 72, wherein the StAR protein or CDP is derived from an extragenomic gene.

[0137] Claim 74. The method according to any one of claims 62 to 73, wherein the patient's total cholesterol is higher than 200 mg / dl.

[0138] Claim 75. The method according to any one of claims 62 to 74, wherein the patient has non-alcoholic fatty liver disease.

[0139] Claim 76. The method according to any one of claims 62 to 74, wherein the patient has non-alcoholic steatohepatitis.

[0140] Claim 77. The method of any one of claims 62 to 76, wherein the modified cells are derived from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells.

[0141] Claim 78. A method of any one of claims 62 to 77 for reducing the total cholesterol concentration in a patient's blood by more than 5%.

[0142] Claim 79. A method for modifying a mammalian mitochondria, comprising inserting at least one unnaturally engineered acute regulatory (StAR) protein or a portion thereof into the outer mitochondrial membrane of a mammal, A method wherein the mitochondria contain one or more cholesterol-degrading proteins (CDPs), including cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolism enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), and P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0143] Claim 80. The method of claim 79, wherein the StAR protein comprises residues 63 to 188 of SEQ ID NO: 7.

[0144] Claim 81. The method of claim 79 or 80, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO: 7.

[0145] Claim 82. Any method of claims 79 to 81, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

[0146] Claim 83. Any method of claims 79 to 82, wherein the CDP includes P450.

[0147] Claim 84. The method according to any one of claims 79 to 83, wherein the CDP comprises a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

[0148] Claim 85. Any method of claims 79 to 84, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

[0149] Claim 86. A method of expressing a StAR protein and a CDP from a cell-specific promoter sequence, as described in any of claims 79 to 85.

[0150] Claim 87. Any method of claims 79 to 86 for use in treating a person who has or is at risk of developing high cholesterol.

[0151] Claim 88. Any method from Claims 79 to 87 for use in reducing cholesterol in a subject that is high in cholesterol.

[0152] Claim 89. The method according to any one of claims 79 to 88, wherein at least about 10% of the total amount of intracellular StAR proteins and CDPs is located in the mitochondrial membrane.

[0153] Claim 90. A mammalian cell according to any one of claims 79 to 90, wherein the StAR protein or CDP is expressed from a gene integrated into the genome of the mammalian cell.

[0154] Claim 91. Any method of claims 79 to 89, wherein the StAR protein or CDP is derived from an extragenomic gene.

[0155] Claim 92. Any method of claims 79 to 91 for use in treating a person who has or is at risk of developing high cholesterol.

[0156] Claim 93. Any method from Claims 79 to 92 for use in reducing cholesterol in a subject that is high in cholesterol.

[0157] Claim 94. The method according to any one of claims 79 to 93, wherein the cells are selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, and their stem cells or progenitor cells.

[0158] While several embodiments have been disclosed, it is likely that further embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. If so, the present invention can be modified in various obvious aspects without departing in any way from the spirit and scope of the invention. Therefore, the detailed description should be considered to be essentially for the purpose of aiding understanding and not limiting.

[0159] All references disclosed herein, whether patent or non-patent, are incorporated herein by reference as if they were included in their entirety at the time of their citation. In the event of any conflict between a reference and the specification, the specification, including its definitions, shall prevail.

[0160] Although this disclosure is described in some detail, it is understood that the disclosure is made through examples, and any changes to the details or structure may be made without departing from the spirit of the disclosure as defined in the attached claims.

Claims

1. A nucleic acid composition for use in reducing cholesterol in a subject with high cholesterol, or in treating a subject who has high cholesterol or is at risk of developing it, The coding sequence of a steroid production acute regulatory (StAR) protein containing at least one copy of residues 63 to 188 of SEQ ID NO: 7; and A nucleic acid composition containing the coding sequence of a cholesterol-degrading protein (CDP) which is a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx).

2. The nucleic acid composition according to claim 1, wherein the StAR protein contains duplicate residues 63 to 188 of SEQ ID NO:

7.

3. The nucleic acid composition according to claim 1 or 2, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

4. A nucleic acid composition according to any one of claims 1 to 3, wherein the coding sequence of CDP further codes for one or more of the following: cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolizing enzyme B (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), or 3β-hydroxysteroid dehydrogenase 2 (HSD2).

5. A nucleic acid composition according to any one of claims 1 to 4, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

6. A nucleic acid composition further comprising a liver-specific promoter sequence, any one of claims 1 to 5.

7. A nucleic acid composition according to any one of claims 1 to 6, contained in lipid nanoparticles.

8. A nucleic acid composition according to any one of claims 1 to 7 for use in the transformation of mammalian liver cells.

9. A nucleic acid composition for use in reducing cholesterol in a subject with high cholesterol, as described in any of claims 1 to 8.

10. A nucleic acid composition according to any one of claims 1 to 8, for use in the treatment of subjects at risk of developing or suffering from high cholesterol.

11. A nucleic acid composition according to any one of claims 1 to 10, wherein P450-FdxR-Fdx comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with SEQ ID NO:

1.

12. A composition comprising mammalian cells for use in reducing cholesterol in a subject with high cholesterol, or in treating a subject who has high cholesterol or is at risk of developing it, Mammalian cells that have been modified to contain at least one copy of residues 63 to 188 of SEQ ID NO: 7, or a portion thereof, of a modified steroid-producing acute regulatory (StAR) protein; and It contains a cholesterol-degrading protein (CDP) which is a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), A composition in which mammalian cells are not human fertilized eggs, human embryos, or human germ cells.

13. The composition of claim 12, wherein the StAR protein comprises a duplication of residues 63 to 188 of SEQ ID NO:

7.

14. The composition of claim 12 or 13, wherein the StAR protein comprises at least a portion of the TOMM20 protein.

15. A composition comprising any one of claims 12 to 14, wherein the StAR protein and CDP comprise at least one mitochondrial signaling protein.

16. A composition according to any one of claims 12 to 15, wherein the StAR protein or CDP is encoded by a gene integrated into the genome of a mammalian cell.

17. The composition according to any one of claims 12 to 16, wherein the mammalian cells are selected from hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, hematopoietic cells, or stem cells or progenitor cells of hepatocytes, nerve cells, osteocytes, muscle cells, endothelial cells, epithelial cells, immune cells, or hematopoietic cells.

18. A composition according to any one of claims 12 to 17, wherein the cells are mammalian liver cells.

19. A composition according to any one of claims 12 to 18, wherein P450-FdxR-Fdx comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with SEQ ID NO:

1.

20. A coding sequence for a steroid production acute regulatory (StAR) protein comprising at least one copy of residues 63 to 188 of SEQ ID NO: 7; and A coding sequence for a cholesterol-degrading protein (CDP) containing a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), wherein P450-FdxR-Fdx is a coding sequence containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with SEQ ID NO:

1. A nucleic acid composition containing the following:

21. An engineered steroid production acute regulatory (StAR) protein or a portion thereof comprising at least one copy of residues 63 to 188 of SEQ ID NO: 7; and A cholesterol-degrading protein (CDP) comprising a P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), wherein P450-FdxR-Fdx comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with SEQ ID NO:

1. Mammalian cells that include human fertilized eggs, human embryos, or human germ cells.

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