Reduction of excess lipids through targeted expression of microbial cholesterol catabolism genes

Abstract

The present invention provides a composition for reducing cholesterol and lipid accumulation in mammals. [Solution] A composition is provided for reducing cholesterol and lipid accumulation in a mammalian subject, comprising mammalian cells containing at least one humanized cholesterol-degrading enzyme and at least one suicide gene.

Description

[Technical Field]

[0001] field The processes, methods, and systems described herein are directed toward the treatment of cholesterol-related diseases by inducing microbial catabolic genes and enzymes through cell therapy and / or gene therapy.

[0002] Cross-reference of related applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 983102, filed February 28, 2020, entitled "Reduction of excess lipids in the liver by targeted expression of microbial cholesterol catabolic genes," and U.S. Provisional Patent Application No. 63 / 094075, filed October 20, 2020, entitled "Reduction of excess lipids in the liver by targeted expression of microbial cholesterol catabolic genes." These applications are incorporated herein by reference in their entirety.

[0003] Sequence List This application is submitted electronically in ASCII format and includes a sequence listing which is incorporated in its entirety by reference. The ASCII version, created on February 25, 2021, is named P286540_WO_01-507078_00010_SL.txt and has a size of 82265 bytes. [Background technology]

[0004] background High intracellular cholesterol levels are characteristic of numerous conditions, from atherosclerosis to fatty liver disease. Treatment for this lipid and / or cholesterol overgrowth typically involves altering lipoprotein metabolism, as this reduces the human cells' ability to break down cholesterol.

[0005] Cholesterol derivatives can be toxic in some cases. For example, the accumulation of 7-ketocholesterol (7KC), a pro-inflammatory, pro-oxidative, pro-apoptotic, and fibrinolytic oxysterol, is associated with various cardiovascular, ocular, and neurological diseases.

[0006] There is a need for compositions and methods to lower lipid and cholesterol levels in individuals suffering from excessive lipids and cholesterol. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 is a scatter plot showing serum triglycerides on day 0 and day 14 (d14); liver triglycerides on d14; serum and liver fatty acids on d14; and serum cholesterol esters on d14 for mice in groups 1-3. The bars at the top of the figure show the relationship of p-values ​​between the groups. [Figure 2] Figure 2 is a bar graph showing free cholesterol in the liver and serum on day 14, and total serum cholesterol on day 14, for mice in groups 1-3. [Figure 3] Figure 3 is a Western blot showing protein expression in the liver. [Figure 4] Figure 4 shows the results of treating mice prone to atherosclerosis with one aspect of the compositions, cells, treatment methods and / or methods of the present disclosure. [Figure 5] Figure 5 shows the anatomy of the heart (top) and the aorta (bottom). [Figure 6] Figure 6 shows an exemplary nucleotide sequence disclosed in the present invention. [Figure 7] Figure 7 shows the amino acid and nucleotide sequences disclosed in the present invention. [Figure 8] Figure 8 shows the amino acid sequence disclosed in the present invention. [Figure 9] Figure 9 shows the amino acid sequence disclosed in the present invention. [Figure 10-1]Figure 10 shows the nucleotide sequence disclosed in the present invention. [Figure 10-2] Same as above [Figure 10-3] Same as above [Overview of the project]

[0008] Detailed explanation High levels of serum cholesterol are associated with atherosclerosis and coronary artery disease. In atherosclerosis, lipid plaques are deposited within the walls of blood vessels, particularly in the heart. Atherosclerosis is a cause of cardiovascular disease (CVD), myocardial infarction, stroke, and peripheral vascular disease, all of which are among the leading causes of death in the United States. Genetic abnormalities in many different aspects of lipoprotein metabolism, as well as various side effects of malnutrition, a sedentary lifestyle, and other diseases (particularly diabetes, hypothyroidism, and kidney disease), can accelerate the development and progression of atherosclerosis.

[0009] High cholesterol leads to fat accumulation in various tissues, including the liver. Overweight or obese individuals, or those with high triglyceride levels, diabetes, and / or high cholesterol, may have excess fat in their liver. Hepatic fat accumulation exceeding approximately 5-10% of total liver weight may indicate the presence of a more serious lesion and / or the development of non-alcoholic fatty liver disease (NAFLD). NAFLD can occur in lean / non-obese individuals and is known to be caused by impaired cholesterol signaling and pathways. NAFLD is a collective term for liver diseases ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH) and cirrhosis (irreversible, progressive scarring of the liver). While these diseases are associated with fat accumulation in cells (hepatocytes), NASH encompasses inflammation (hepatitis) and / or scarring (fibrosis) of the liver. NASH can ultimately lead to hepatocyte destruction and hepatic necrosis. Free hepatic cholesterol is a major lipotoxic molecule that may play a significant role in the development of NASH.

[0010] Current treatments for excessive cholesterol and / or lipid levels generally aim to alter lipoprotein metabolism pathways and / or signaling, and there are few treatments that effectively target already accumulated lipids and cholesterol.

[0011] Applicants disclose herein compositions, methods and systems for delivering cholesterol metabolism transgenes to mammalian cells or tissues to reduce and / or prevent the accumulation of excessive cholesterol. In some embodiments, the compositions and methods of the present disclosure may be useful for reducing cholesterol and lipoprotein accumulation in various tissues, including the arterial wall.

[0012] A method is disclosed herein that includes administering to a mammalian subject one or more proteins involved in cholesterol metabolism. In some embodiments, the one or more proteins can be bacterial-derived enzymes involved in cholesterol catabolism or breakdown. In many embodiments, administration of one or more of the proteins and / or enzymes of the present disclosure can provide for the breakdown of various lipids and / or cholesterol in the cells of one or more subjects.

[0013] Cholesterol-degrading enzyme Various cholesterol and cholesterol-related genes and proteins are disclosed herein. In various embodiments, the genes and proteins of the present disclosure can be enzymes or transport proteins. In some embodiments, the genes and proteins of the present disclosure can serve in cholesterol catabolism / breakdown or the transport of cholesterol across membranes or lipid bilayers. As used herein, the term "cholesterol" refers to the formula C 27 H 46Cholesterol, or cholesteryl alcohol, is a sterol of O whose IUPAC name is cholesta-5-en-3β-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. In this book, the term "cholesterol" refers to oxidized cholesterol, C 27 H 46 This also includes cholesterol derivatives, such as O2, oxycholesterol, or 5,6-epoxycholesterol, 7-ketocholesterol (7KC), cholestane-3β,5α,6β-triol, and 7-α / β-hydroxycholesterol. In this text, cholesterol-related and cholesterol catabolic or degrading proteins may refer to proteins that interact with, bind to, or act on cholesterol, cholesterol derivatives, and compounds within the cholesterol metabolic pathway.

[0014] The cholesterol-related genes and proteins of the present disclosure can be selected from cholesterol dehydrogenase (CholD), 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), anaerobic cholesterol metabolism B enzyme (acmB), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), ATP-binding cassette subfamily A, member 1 (ABCA1; ncbi.nlm.nih.gov / nuccore / NM_005502.4 accession number NM_005502), ATP-binding cassette, subfamily G, member 2 variant (ABCG2; ncbi.nlm.nih.gov / nuccore / NM_004827.3 accession number NM_004827.3), and combinations thereof. In some embodiments, the proteins of the present disclosure comprise an amino acid sequence having an identity greater than about 80% with any one or more of the proteins encoded by SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or SEQ ID NOs: 1 and 13. In many embodiments, the currently defined identity can span sequences longer than about 100, 150, 200, 250, 300, 350, or 400 amino acids and shorter than about 500, 450, 400, 350, 300, 250, 200, or 150 amino acids. In many embodiments, the sequence identity exceeds about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and can be less than about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, or 85%.

[0015] In many embodiments, the cholesterol-related genes and proteins of this disclosure may be involved in the transport of cholesterol across the cell membrane. In various embodiments, the ABCA1 (SEQ ID NO: 11) and ABCG2 (SEQ ID NO: 12) genes and proteins may be useful in lowering cholesterol in a subject. ABCA1 may also be referred to as CERP or cholesterol efflux regulatory protein. The ABCG2 protein is present in the cell membrane of mammalian cells and is involved in the transport of various compounds from the cell. In the case of ABCA1, cholesterol is transported to apoA1 and apoE. ABCG2 may be present in the bile canal membrane of hepatocytes and may be involved in the efflux of compounds into bile. ABCG2 is known to require high levels of membrane cholesterol to exhibit maximum activity, and the inventors recently demonstrated, by examining pure ABCG2 reconstituted in proteoliposomes, that cholesterol is an essential activator when bile acids significantly regulate the activity of this protein.

[0016] One or more cholesterol-related genes, proteins, and enzymes of this disclosure may be packaged in one or more vectors, constructs, or cassettes. In various embodiments, a cassette containing one or more cholesterol-related proteins or cholesterol-degrading enzymes may be referred to as a cholesterol catabolic cassette (CCC). In some embodiments, the cassette may be a polynucleotide construct and may contain a nucleic acid sequence encoding a protein having identity with the protein encoded by any one of SEQ ID NOs: 1 or 13. In some embodiments, the cassette may be a construct having a protein sequence having approximately 80% or more identity with one or more protein sequences of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the cassette may be a ribonucleic acid encoding one or more proteins of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0017] Compositions, vectors, methods, and protocols useful for lowering cholesterol levels in at least one cell of a target mammalian subject are disclosed herein. In many embodiments, the subject is a human being who has or is at risk of developing one or more conditions associated with high cholesterol levels.

[0018] Vectors, cassettes, and constructs Various vehicles may be used to deliver vectors, cassettes, or constructs to target cells. In some embodiments, the vector may be a chemical vector, a viral vector, and / or a non-viral vector. In some embodiments, the vector may be selected from viruses, nanoparticles, liposomes, cell membrane-permeable peptides, etc. If the vector is a viral vector, the virus may be a mammalian virus, a non-mammalian virus, or a synthetic virus. In some embodiments, the vector may be an adenovirus, a retrovirus, etc. If the vector is a nanoparticle or liposome, the construct may be RNA or DNA, e.g., mRNA. In many embodiments, lipid nanoparticles (LNPs) may be used to deliver polynucleotides encoding one or more cholesterol-related proteins or cholesterol catabolic enzymes, e.g., mRNA or other treatment nucleic acids. Often, the use of LNPs may enable the delivery of more RNAs and / or DNAs than can be delivered by other means. In some embodiments, the LNPs and / or LNP systems of this disclosure may comprise one or more variations of four components that can optimize their integrity and organ specificity, particularly organ specificity for the liver. In some embodiments, the LNP variation may comprise one or more ionizable cationic lipids, phospholipids (typically phosphatidylcholine), cholesterol, and PEG-lipids. Furthermore, some LNP formulations may comprise one or more fusion-associated small transmembrane proteins that may contribute to improved cell / tissue-specific delivery of the LNP, as well as to reliable fusion, particularly highly efficient fusion, and direct intracellular delivery of the nucleic acid payload for treatment into the cytoplasm via the endocytosis pathway.

[0019] The vectors, cassettes, and constructs of this disclosure may target specific types of cells or tissues and / or the expression of genes encoded by the vectors, cassettes, and constructs, and the constructs may be limited and / or optimized for specific cells and cell types. In some embodiments, particles comprising the vectors, cassettes, and constructs of this disclosure, such as lipid nanoparticles, may comprise a membrane containing one or more transmembrane proteins having affinity for receptors or outer membrane proteins on cells of the target cell or target tissue. In many embodiments, promoter sequences may be used for gene expression of the disclosure, which may include continuous or discontinuous sequences and / or 5' untranslated regions, where the promoter exhibits limited or no activity in non-target cells or tissues. In some embodiments, the promoters of this disclosure may be resistant to silencing, which may result in decreased expression over time. In some embodiments, for example, target cells may be liver cells, such as cells present in liver tissue. In such embodiments, the vector, cassette, and construct may include one or more promoters that can be targeted to hepatocytes by particles containing membrane proteins having affinity for hepatocyte receptors or hepatocyte membrane proteins, and / or that are specific to the liver and show little or no activity in other types of cells and tissues.Some examples of promoters in this disclosure are CMV, Ef1a, ABCA1 (ncbi.nlm.nih.gov / nuccore / AF275948.1?report=genbank&to=149034), or promoters in Sequence ID Nos. 1 and 13, e.g., approximately 100bp to 2kbp, e.g., 200bp to 1.5kbp, and in some embodiments, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1.0kb, 1.2kbp, 1.3kbp, 1.4kb. It may have more than 80% identity with respect to continuous or discontinuous portions greater than p, 1.5kbp, 1.6kbp, 1.7kbp, 1.8kbp, or 1.9kbp, and less than approximately 2.5kbp, 2.4kbp, 2.3kbp, 2.2kbp, 2.1kbp, 2.0kbp, 1.9kbp, 1.8kbp, 1.7kbp, 1.6kbp, 1.5kbp, 1.4kbp, 1.3kbp, 1.2kbp, 1.1kbp, 1.0kbp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, or 300bp.

[0020] gene therapy Cholesterol-related proteins, such as cholesterol-degrading enzymes, can be targeted to mammalian cells, for example, at a target where desired. In some embodiments, such proteins are targeted to cells in one or more vectors. In some embodiments, the vector may comprise one or more enzymes, proteins, peptides, nucleic acids, or combinations thereof. For example, in one embodiment where the vector comprises nucleic acids, the vector further comprises one or more mammalian regulatory sequences comprising nucleic acid sequences that regulate the expression of one or more cholesterol-related proteins or cholesterol-degrading enzymes (which may comprise one or more bacterial cholesterol catabolic enzymes). In some embodiments, the vector may be directly or indirectly targeted to a variety of cells and tissues. In some embodiments, the vector may be delivered to liver tissue, hepatocytes, blood vessels, arterial endothelial cells, muscle cells, epithelial cells, macrophages, hepatocytes, hepatic stellate cells, Kupffer cells, hepatic sinusoidal endothelial cells, or any other cells that may contain or be associated with excess cholesterol or that may act to reduce cholesterol levels in a target. In some embodiments, the vectors of this disclosure may be preferentially taken up by hepatocytes, endothelial cells, and / or macrophages.

[0021] The vectors, constructs, enzymes, and methods of this disclosure may be useful for reducing the concentration of at least one lipid in target cells treated by the vectors, constructs, enzymes, or methods. In many embodiments, the at least one lipid is cholesterol. In many embodiments, the concentration of lipids may be reduced in one or more mammalian cells before a reduction in lipids is detected in the serum. In some embodiments, the cells may be hepatocytes or other mammalian cells.

[0022] cell therapy Cholesterol-degrading enzymes can be targeted to mammalian cells in a subject. In some embodiments, these cells are modified in vitro to contain one or more cholesterol-related genes or proteins and then administered to the subject. In some embodiments, the delivery of cholesterol-degrading genes, proteins, and enzymes may involve a cell therapy approach. In one embodiment, one or more cells may be isolated from a subject requiring treatment. In several other embodiments, one or more cells may be obtained from a related or unrelated donor to the subject. In many embodiments, the cells may be stem cells or induced pluripotent stem cells. The cells may be obtained from various sources, such as tissue, blood, bone marrow, umbilical cord blood, etc., taken from the subject or donor. The cells may be modified to express one or more cholesterol-related genes, proteins, and / or enzymes. The modified cells may then be administered to the subject.

[0023] Pluripotent stem cells (PSCs) can be modified to express one or more cholesterol-related genes or proteins, such as one or more cholesterol-degrading enzymes. In some embodiments, pluripotent stem cells can be induced pluripotent stem cells (iPSCs). iPSCs can be derived from a subject (self) or from a related or unrelated donor (xenogeneic or allogeneic). In some embodiments, iPSCs can be modified to reduce immunogenicity, i.e., to reduce rejection or attack by the subject's immune system. In some embodiments, cells can be modified to prevent or suppress the expression of one or more genes, proteins, or receptors related to immunogenicity, such as major histocompatibility (MHC) genes, such as MHC class I and MHC class II. In some embodiments, such genes can be deleted in iPSCs before administration to a subject. For some cell types, deletion of MHC class I expression can lead to recognition and attack (e.g., lysis) by natural killer (NK) cells. To overcome this "missing self" reaction, genes can be introduced into cells before administration. This may be called gene knock-in. In some embodiments, a single heavy chain of a non-polymorphic HLA gene (e.g., HLAE) can be knocked in.

[0024] In some embodiments, it may be useful to remove the administered cells after the subject has been treated. In such embodiments, the cells of the Disclosure may be modified before administration to the subject to contain one or more genes that can help remove, excrete, destroy or kill the administered cells, for example, after administration to the subject. In some embodiments, the genes are those that, when expressed, make the cells sensitive to one or more compounds and / or kill the cells. In some embodiments, these may be referred to as “suicide genes.” In many embodiments, the genes are related to cytochrome P4502 B1, human enteric carboxylesterase, and cytosine deaminase, which can convert cyclophosphamide, irinotecan, and fluorocytosine into active metabolites, respectively. In some embodiments, the genes are thymidine kinases (TKs), such as herpes simplex virus-derived TKs (HSV-TKs). In many embodiments, the expression of suicide genes may make the modified cells more susceptible to damage by compounds or molecules that do not affect (or have minimal effect on) cells that do not possess suicide genes. In many embodiments, for example, when the suicide gene is HSV-TK, the compound may be a prodrug, such as ganciclovir (GCV). In such embodiments, HSV-TK modified cells can be killed with low doses of GCV, but cells lacking the HSV-TK gene cannot.

[0025] Cells expressing one or more cholesterol-related genes or proteins, such as cholesterol-degrading enzymes, can be differentiated. In many embodiments, cells can be treated to promote differentiation into selected cell types. In such embodiments, cells can be transformed, transfected, stimulated and / or treated with one or more factors, hormones, peptides, proteins, compounds, or molecules to promote differentiation. In many embodiments, PSCs of this disclosure can be treated to differentiate into immune cells, such as monocytes, macrophages, and dendritic cells. In several other embodiments, cells can be differentiated into a target tissue or a similar cell type.

[0026] In short, human iPSCs can be differentiated into monocytes as follows: Human iPSCs can be seeded and cultured using methods well known in this field. Cells can be cultured for approximately two days in bone morphogenetic protein 4 (BMP4), activin A, and CHIR99021 (GSK-3 inhibitor) to induce mesoderm formation. Next, cells are cultured for three days in vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), SB431542 (TGF-βR inhibitor), and stem cell factor (SCF) to differentiate into hematopoietic endothelial cells (HE). HE cells are CD144 + / CD34 + / CD73 - And this is further CD43 + These cells can be differentiated into HPCs. In many cases, differentiation efficiency can be determined by FACS analysis scoring for mesoderm (CD140a+) and HE (CD144+CD34+CD73-) cells on days 2 and 5, respectively. From cells on days 5 to 9, hematopoietic cells are induced by growth with VEGF, FGF2, SCF, interleukin-3 (IL-3), interleukin-6 (IL-6), and thrombopoietin (TPO). Here again, differentiation efficiency can be assessed based on the number of round HPCs in the population, or the efficiency can be quantified by FACS analysis scoring of the HPC-specific marker CD43 (e.g., on day 9). On day 9, round HPCs are initially harvested before adherent cell dissociation, using TrypLE and Accutase sequentially to minimize cellular stress. Finally, monocytes are induced from the HPCs by growth in suspension culture in a medium containing IL-3, IL-6, and macrophage colony-stimulating factor (M-CSF). Monocyte induction from HPC takes 5-6 days.

[0027] Modified monocytes and / or macrophages containing one or more genes selected from cholesterol-degrading enzymes, suicide genes, etc., can migrate to sites of inflammation, such as blood vessels with atherosclerotic lesions and / or liver tissue containing hepatocytes with fat, after administration to a subject. In many embodiments, modified monocytes and / or macrophages can enter sites of inflammation, such as blood vessels with inflamed atherosclerotic lesions, and promote phagocytosis and / or degradation of cholesterol. This may help reduce atherosclerotic plaque size, total atheroma volume, cholesterol concentration, and / or local inflammation.

[0028] The compositions, cells, methods, and treatments of this disclosure are useful for the treatment of atherosclerosis and hypercholesterolemia. In many embodiments, the treatment approaches and compositions of this disclosure may be useful for the treatment or prevention of type I to VI atherosclerotic lesions, such as existing type VI or associated lesions with signs of thrombosis, fissures, and hepatocellular carcinoma. In many cases, the compositions, cells, methods, and treatments of this disclosure are useful for treating and preventing the onset / formation of such lesions, including complex lesions.

[0029] Cholesterol-related diseases and disorders The compositions and methods of this disclosure can treat or prevent a variety of diseases and conditions. In many embodiments, the diseases or conditions treated by the compositions and methods of this disclosure are cholesterol-related diseases and conditions. Some examples of diseases and conditions that can be treated by the compositions and methods of this disclosure are disclosed below.

[0030] Excessive native LDL and a high LDL:HDL ratio have been shown to be strongly associated with cardiovascular disease, atherosclerosis, stroke, and coronary heart disease and heart attacks. Acetylated LDL is a chemically modified form of LDL that does not exist in vivo. Both acetylated LDL and oxidized LDL are taken up by macrophages, converting those cells into foam cells. Often, all components of LDL are sensitive to oxidation, producing an oxidized form of LDL (oxLDL). Uptake of oxLDL by arterial macrophages is strongly involved in plaque formation. Unlike unmodified LDL, oxLDL is taken up by arterial wall macrophages in an unregulated manner via LDL scavenger receptors. Oxysterols are 10 to 100 times more reactive than native cholesterol, with 7-ketocholesterol (7KC) being the most toxic, and it is also the most abundant in oxLDL.

[0031] Studies have shown that high circulating 7KC levels are associated with a higher risk of future cardiovascular events and a higher overall mortality rate. 7KC is a pro-inflammatory, pro-oxidative, pro-apoptotic, and fibrogenic molecule that alters endothelial cell function by impairing cell membranes and ion transport pathways crucial for vasodilation.

[0032] In patients with hypercholesterolemia, 7KC can account for approximately 57% of plasma oxysterols. The next most abundant is 7-α / β-hydroxycholesterol, the direct product of 7KC metabolism (21% of plasma oxysterols). In arterial plaque, 55% of oxysterols are reported to be 7KC, with cholestane-3β,5α,6β-triol and 7-α / β-hydroxycholesterol being the second and third most abundant, respectively, at 13% and 12%.

[0033] As previously stated, NASH (non-alcoholic steatohepatitis) is another cholesterol-related disease that can be treated with the compositions and methods of this disclosure. Changes in cholesterol homeostasis and transport lead to the accumulation of free cholesterol in the liver, which results in NAFLD (non-alcoholic fatty liver disease) through hepatocyte damage and non-parenchymal cell activation. In particular, free cholesterol overload in and around mitochondria leads to mitochondrial dysfunction, promoting inflammation, fibrosis, and hepatocyte death.

[0034] Other cholesterol-related diseases and conditions that can be treated or prevented by the compositions and methods of this disclosure include alveolar proteinosis (PAP), eye diseases, neurodegenerative diseases, Niemann-Pick disease type C (NPC), and lysosomal acid lipase (LAL) deficiency. In animals with lungs, cholesterol affects the regulation of surfactant fluidity and function, and since this fluidity can undergo abrupt changes, especially under extreme temperatures, reduced cholesterol clearance is a major disorder that promotes the development of PAP. Regarding eye diseases, oxysterols and especially 7KC cause degeneration of retinal cells. Therefore, high oxysterol levels may be involved in a variety of eye diseases, including macular degeneration (AMD), choroidal neovascularization (CNV), glaucoma, and cataracts.

[0035] High oxysterol levels also lead to alterations in cerebral cholesterol metabolism. Cholesterol metabolism can be a significant part of several brain disorders, including the progression of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and dementia. Various oxysterols, including 7KC, induced by the auto-oxidation of cholesterol, have been identified in the postmortem brains of Alzheimer's patients. Many of these etiologies may also be common to chronic epilepsy. In particular, a link between epilepsy and atherosclerosis has been suggested. Therefore, treatments for 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) ​​and accumulate in brain tissue, ultimately leading to neurodegeneration.

[0036] Various other diseases may be associated with high cholesterol levels and can be treated with the compositions and methods of this disclosure. For example, patients with Niemann-Pick disease type C (NPC) are unable to excrete cholesterol, leading to the accumulation of cholesterol and oxysterols, primarily in the liver, spleen, and brain. A positive correlation between the 7KC profile and disease severity has been reported. Furthermore, in patients with lysosomal acid lipase (LAL) deficiency, cholesterol esters and triglycerides accumulate in lysosomes, potentially leading to hypercholesterolemia, hyperlipidemia, and / or atherosclerosis. In such patients, plasma levels of oxysterols, including 7KC, are also very high. Increased oxysterol production further enhances oxidative stress and worsens the condition.

[0037] The compositions and methods of this disclosure are useful for treating diseases or conditions associated with excess cholesterol and / or lipid accumulation 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 oxidized cholesterol species, such as 7-ketocholesterol. In some embodiments, the disease or condition may be one or more of the following: 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.

[0038] The compositions, cells, methods, and treatments of this disclosure may be useful in treating, alleviating, or improving various diseases, disorders, or conditions associated with excess cholesterol. In one embodiment, the disease, disorder, or condition may be one or more of the following: early type II lesions (i.e., macrophage foam cell formation), type III lesions or pre-atheromas (i.e., having a small extracellular lipid pool), type IV lesions or atheromas (i.e., having an extracellular lipid core), type V lesions or fibrous atheromas (i.e., atheromas with fibrous thickening).

[0039] treatment The cell therapies of this disclosure may be useful in reducing atheroma or atherosclerotic plaque. In some embodiments, the therapies of this disclosure may reduce atheroma by about 5% to about 100%, for example, about 70% to about 90%, and by more than about 30%, 40%, 50%, 60%, or more. For example, the treatment methods of this disclosure may reduce atheroma volume in a patient population requiring such treatment, where volume is based on imaging by one or more of the following: invasive intravascular ultrasound (IVUS), newer non-invasive imaging methods, for example, B-mode ultrasound, computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI). In many embodiments, the atheroma volume in the population may be reduced by more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and by 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%, compared to a control population not treated with the compositions, cells, methods, or treatments of the Disclosure. In many cases, the period of decline is more than about one month after treatment or about 24 months after treatment, for example, about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 2 It is longer than 1 month, 22 months, 23 months, and 24 months, and shorter than approximately 36 months, 30 months, 25 months, 24 months, 23 months, 22 months, 21 months, 20 months, 19 months, 18 months, 17 months, 16 months, 15 months, 14 months, 13 months, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month.In some embodiments, for example, when measurements are made in subjects based on normalized plaque fractions, the treated population (e.g., with 2 or more subjects) may show a mean decrease of approximately 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and less than approximately 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, a decrease of approximately 42%.

[0040] The vectors, constructs, enzymes, and methods of this disclosure may be useful for reducing the amount of at least one cholesterol in cells. In many embodiments, the vectors, constructs, enzymes, and methods of this disclosure may reduce cellular cholesterol levels before acting on systemic cholesterol levels, for example, serum cholesterol levels of the subject. In one embodiment, a reduction in cholesterol in at least one cell of the subject may result in a reduction in systemic cholesterol levels. In many embodiments, the level of total or free cholesterol in tissues may be reduced by more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. In many embodiments, the vectors, constructs, enzymes and methods of this disclosure may be useful in the treatment or prevention of atherosclerosis, cardiovascular disease (CVD), myocardial infarction, stroke, peripheral vascular disease, diabetes mellitus, hypothyroidism, renal disease, hepatic disease, fatty liver, non-alcoholic fatty liver disease, NAFLD, obesity, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis and hepatic fibrosis. In many embodiments, the vectors, constructs, enzymes and methods of this disclosure may be useful in the treatment or prevention of cirrhosis, steatohepatitis, inflammation or hepatic fibrosis. [Example 1]

[0041] Administration to mammals Three groups of five mice (C57BL / 6J; Jackson Lab #000664) were injected with an empty vector (Group 1), a mammalian expression vector containing the sequence of bacterial cholesterol catabolic enzyme (Groups 2 and 3). The injection was performed via the tail vein, and approximately 10 12 copies of the vector, cassette, or construct were administered to each mouse.

[0042] After injection, the mice were fed a high-fat diet (Envigo TD 88137). Food and water were provided ad libitum, and the body weight of each mouse was measured daily and the data were recorded.

[0043] On day 14, the mice were sacrificed. Serum and liver tissues were collected for analysis.

Example 2

[0044] Serum and liver lipid concentrations As described above, serum samples were collected prior to sacrifice and liver samples were collected after sacrifice. Triglycerides in serum at day 0 (d0) and day 14 (d14) and in d14 liver homogenates were quantified using a triglyceride colorimetric assay kit (Item No. 10010303) from Cayman Chemical (Ann Arbor, MI). Briefly, serum was treated with a mixture of lipoprotein lipase, glycerol kinase, glycerol phosphate oxidase, peroxidase, 4-aminoantipyrine (4-AAP), and N-ethyl-N-(3-sulfopropyl)-m-anisidine (ESPA). Thereby, glycerol is released from triglycerides, phosphorylated, oxidized, hydrogen peroxide is released, which reacts with 4-AAP and ESPA to exhibit purple color, which is measured by absorbance at 530 - 550 nm.

[0045] Triglyceride levels in serum and liver samples were quantified, and the results are shown in the graph of Figure 1. The graph shows that serum triglyceride levels at d0 and d14 were similar in all three groups. Liver tissue triglyceride levels were also similar in the three groups.

[0046] Serum and liver free fatty acid levels on day 14 were tested by a colorimetric / fluorescence assay (Abcam; ab65341 Free Fatty Acid Quantitative Assay Kit) according to the manufacturer's instructions. Briefly, fatty acids were converted to CoA derivatives, oxidized to produce color or fluorescence, and quantified by colorimetric measurement (spectrophotometric method at λ=570 nm) or fluorescence measurement (Ex / Em=535 / 587 nm). Serum samples were tested directly. Fatty acids in tissue samples were extracted and dried according to the manufacturer's protocol. Briefly, samples were first washed with cold PBS and then homogenized in the presence of Triton X-100 and chloroform. Samples were centrifuged to separate the organic phase, which was then taken, dried, and subjected to processing.

[0047] Free fatty acid levels in serum and liver were quantified on day 14. The results are shown in Figure 1. The results showed little difference in serum or liver fatty acid levels among the three groups.

[0048] Cholesterol ester, total cholesterol, and free cholesterol levels were examined by colorimetric / fluorescence assays (Abcam; ab65359, Cholesterol / Cholesteryl Ester Quantitative Assay Kit) according to the manufacturer's protocol. Briefly, for the analysis of total cholesterol, samples were treated with cholesterol esterase to convert cholesterol esters to cholesterol. The cholesterol was then treated with cholesterol oxidase to produce peroxides, which were quantified by colorimetric assay (λmax=570nm) or fluorescence assay (Ex / Em=535 / 587nm). Cholesterol ester levels were determined by subtracting the free cholesterol measured before cholesterol esterase treatment. Serum samples were tested directly according to the manufacturer's protocol. Tissue samples were first homogenized in a mixture of chloroform, isopropanol, and NP-40 before analysis, centrifuged, the organic phase was separated, and dried.

[0049] Cholesterol ester levels in mouse serum were quantified on day 14. The results are shown in Figure 1. The results showed little difference in serum cholesterol ester levels among the three groups. [Example 3]

[0050] Analysis of free and total cholesterol Total cholesterol and free cholesterol levels in serum and liver tissue were analyzed and compared. Samples were tested as described above.

[0051] On day 14, serum free and total cholesterol levels were similar in all three groups, but free cholesterol levels in liver tissue were higher in the control mice than in any of the test groups. Specifically, as shown in Figure 2, total cholesterol levels in liver tissue were substantially the same in G2 and G3 (average free cholesterol approximately 30 mg / dl). On the other hand, the total cholesterol amount in the liver of the control group (G1) was approximately 50% higher, and the free cholesterol was approximately 45 mg / dl.

[0052] These results demonstrate that the construct of this disclosure, targeted to liver cells, is effective in reducing fatty liver in mice fed a high-fat diet. As described above, serum cholesterol levels at 2 weeks were similar to those in the treatment group in the control mice. Furthermore, no adverse effects were observed at the time of sacrifice. [Example 4]

[0053] Recombinant protein expression in liver tissue The results of the above test were confirmed by a protein assay. Specifically, the expression of recombinant protein in liver tissue was confirmed by Western blotting. Briefly, liver tissue was collected two weeks after injection. The tissue was homogenized in lysis buffer, the lysate was loaded onto an SDS-polyacrylamide gel, separated by electrophoresis, and the proteins were transferred to a suitable membrane for Western blotting. The membrane was subjected to Western blotting using FLAG (see figure above) antibody to detect recombinant protein tagged with FLAG. The results are shown in Figure 3.

[0054] As shown in Figure 3 (upper panel), the expression of recombinant protein (approximately 120 kD) was specific to liver tissue of mice treated with the construct / cassette of this disclosure. In contrast, samples from control mice (empty vector; lanes 1, 7, and 8) did not react with the FLAG antibody. Loading was examined by probing with anti-GAPDH and anti-tubulin antibodies. These results are shown in the two lower panels of Figure 3.

[0055] The data as a whole demonstrate that the vector, including the construct / cassette of this disclosure, results in the expression of cholesterol catabolic recombinant protein in the liver tissue of treated mice.

[0056] Legend: G1, Group 1 - Empty vector as a control; G2, Group 2 - AAV1-HR; G3, Group 3 - AAV6-HR. M: Mouse. [Example 5]

[0057] Cell therapy treatment for atherosclerosis ApoE-null mice, which are prone to atherosclerosis, were treated with in vitro modified cells expressing one or more cholesterol-degrading enzymes. Briefly, stem cells were modified with a control expression vector or an expression vector containing one or more coding regions of the cholesterol metabolism genes described herein.

[0058] After treatment, mice were sacrificed, and tissue sections were prepared of the aortic root, aortic arch (including the brachiocephalic artery, right subclavian artery, right common carotid artery, left common carotid artery, and left subclavian artery), and aortic tree (descending / thoracic aorta and abdominal aorta). The sections were analyzed for changes compared to untreated control mice. Specifically, the sections were analyzed to measure the plaque region / vascular wall region and the plaque region / vascular lumen region.

[0059] To evaluate any changes in atherosclerotic lesions, such as the volume, size, or composition of plaques, one or more of the following non-invasive contrast-enhancing techniques may be used: non-invasive carotid ultrasound, carotid magnetic resonance imaging, aortic computed tomography, coronary magnetic resonance imaging, and positron emission tomography (PET). A PET tracer is used to aid in the evaluation. In one embodiment, the PET tracer is 18F-fluorodeoxyglucose (18F-FDG). The PET tracer can be taken up by macrophages due to its high metabolic activity, thereby allowing macrophage cells to be identified relative to less active surrounding cells. For this reason, the PET tracer can be used as a surrogate for inflammation. This allows for an evaluation of inflammatory plaque activity across multiple vascular beds. The approach described herein was used in the Cardiovascular Inflammation Reduction Trial (CIRT) - Imaging Study (NCT02576067). [Example 6]

[0060] Genetic therapy for atherosclerosis

[0061] Animal care, handling, and drug administration Twenty male ApoE-null mice (jax.org / strain / 002052) were randomly assigned to three test groups of 10, 5, and 5 mice. On day 0 of the study, all mice were implanted with a subcutaneous osmotic minipump (alzet.com / products / alzet_pumps / ) to enable continuous release of angiotensin II (0.7 mg / kg / day for 4 weeks), and were fed a high-fat diet (insights.envigo.com / hubfs / resources / data-sheets / 88137.pdf) for 4 weeks. The mice were then returned to a normal diet and divided into three groups, each receiving one of the following intravenous doses via the tail vein: Group 1 (n=10 mice) - 5 × 10 13 vg / kg AAV6-Empty (2x10 13 vg / mL); Group 2 – 5×10 13 vg / kg AAV6-CDP (cholesterol-degrading protein, 2 × 10⁻¹⁰ 13 vg / mL); Group 2 – 5×10 13 vg / kg AAV6-CDP (2×10 13 (vg / mL)

[0062] To investigate whether there was any variation between batches of AAV6-CDP, groups 2 and 3 were injected with different batches of AAV6-CDP.

[0063] Euthanasia of animals, tissue collection and processing: Mice were euthanized on day 28 after treatment by final euthanasia via CO2 narcosis. Approximately 0.5 ml of blood was collected from the mandibular vein of the mice, processed, and used as serum for later analysis. The mice were then slowly perfused through the left ventricle with 10 ml of PBS + 0.5 ml of MEDTA, followed by 10 ml of PBS. Continuous perfusion effusion from the right atrium and decolorization of the liver were carefully monitored for macroscopic assessment of effective systemic perfusion. The liver, lungs, spleen, kidneys, and brain were collected, placed in ice-cold PBS, and processed.

[0064] Following systemic PBS+EDTA / PBS perfusion and excision of specific organs, the cardiac / aortic tree structure was perfused through the left ventricle with 5 ml of 10% neutral buffered formalin (NBF). The structure was then carefully excised from the animals and immersed in 10% NBF overnight at 4°C. The tissue was then placed in 15% sucrose and left at 4°C for 6–12 hours (i.e., until the tissue sank), and then transferred to 30% sucrose and left overnight at 4°C until the tissue sank. The sucrose solution is hypertonic and can dehydrate the tissue. The tissue sank to the bottom of the container once it reached equilibrium with the 15% and 30% sucrose solutions. Thorough dehydration of the tissue before freezing in the OCT embedding medium is important to prevent freeze damage due to ice crystal expansion.

[0065] The heart and aortic tree are cut into five sections: (1) heart-aortic root (HR-AR); (2) chest-I or T1; (3) chest-II or T2; (4) abdomen-I or A1; and (5) abdomen-II or A2. The preparation methods for these samples are briefly described below.

[0066] Heart-Aortic Root (HR-AR): The heart and aortic root were separated from the remaining aortic tree. The heart was cut along the red line shown in Figure 5 using a scalpel blade. Approximately 70% of the ventricle (from the apex to 3 mm from the base, i.e., the lower portion) was removed. The remaining HR-AR (upper portion) was placed in a tissue mold and embedded in the OCT, ensuring that the aortic root was perpendicular to the bottom surface of the tissue mold. The mold was rapidly frozen in isopentane cooled with dry ice for 3-5 minutes until the tissue block solidified and turned white. The tissue block was kept frozen on dry ice for 30 minutes and stored in a -80°C freezer until frozen sections were prepared.

[0067] Next, the remainder of the aortic block, including the aortic tree, was fragmented as shown in Figure 5 (below). Briefly, the thoracic-I (T1) structure includes the aortic arch, including the innominate artery, right subclavian artery, right common carotid artery, left carotid artery, and left subclavian artery; the thoracic-II (T2) structure includes the aorta from the 7th rib to the diaphragm, including the intercostal arteries; the abdominal-I (A1) structure includes the aorta from below the diaphragm to the middle of the abdominal aorta, including the celiac artery, superior mesenteric artery, and right / left renal arteries; and the abdominal-II (A2) structure includes the aorta from the middle of the abdominal aorta to below the level of the iliac artery bifurcation, including the inferior mesenteric artery and the common iliac artery at the bifurcation of the iliac artery.

[0068] The T1, T2, A1, and A2 structures were immersed to the same depth in a common freezing mold containing OCT embedding medium, in the appropriate orientation as shown in the illustration. The mold was rapidly frozen in isopentane cooled with dry ice for 3–5 minutes until the tissue blocks solidified and turned white. The tissue blocks were kept frozen on dry ice for 30 minutes and stored in a -80°C freezer until frozen sections were prepared. The tissue blocks were sectioned as follows: Heart - AR Block - The ventricular tissue was incised and removed until the aortic sinus was reached. This was confirmed by checking under a microscope until the three aortic valves were visible. Once all the aortic valves were visible, 10 μm sections were cut out and mounted on slides as 10 μm serial sections. The slides were then stained with Oil Red O / Meyer hematoxylin. Thoracic I, thoracic II, abdominal I, and abdominal II blocks (i.e., four aortic blocks) - For Oil Red O / Meyer hematoxylin, use 10 μm thick serial sections spaced 100 μm apart for every 10 serial sections. Based on the Oil Red O / H&E data, select serial sections (e.g., sections 12, 13, 14, and 15) are stained for Flag, F4 / 80, CD45, and α-SMC actin, respectively.

[0069] The oil red O staining protocol is as follows:An ORO stock was prepared by adding 2.5 g of ORO to 400 ml of 99% (vol / vol) isopropanol and mixing with a magnetic stirrer at room temperature (RT) for 2 hours. To prepare the ORO working solution, 1.5 parts of the ORO stock solution were added to 1 part of distilled water. The solution was left at 4°C for 10 minutes and filtered through a 45 μm filter. Frozen sections were equilibrated at room temperature (RT) for 10 minutes, washed with 60% isopropanol, and incubated with the ORO working solution at room temperature (RT) for 15 minutes. The sections were washed with 60% isopropanol, counterstained with Meyer hematoxylin, washed with running tap water, and covered with coverslips.

[0070] Plaque volume was evaluated as follows: The plaque area and vascular lumen area were determined, and the level of plaque volume was quantified by calculating the ratio of the total plaque area to the total vascular lumen area. The ratio was determined for both control-treated mice and CDP-treated mice.

[0071] While several embodiments have been disclosed, further embodiments of the present invention may become apparent to those skilled in the art from the detailed description. As will be apparent, the present invention can be modified in various obvious ways without departing from the spirit and scope of the invention. Therefore, the detailed description should be understood as essentially descriptive and not limiting.

[0072] References included herein, both patent and non-patent documents, are incorporated into this text in their entirety by reference, as if they were cited in their respective sections. In the event of any inconsistency between a reference and the specification, the provisions of this specification, including definitions, shall prevail.

[0073] While this disclosure has been described with a certain degree of specificity, the description is for illustrative purposes only, and it should be understood that changes in detail or structure may be made without deviating from the spirit of the invention as defined in the claims.

Claims

1. A method for reducing the concentration of lipids in at least one cell of an object requiring treatment for a disease or condition, A step of administering a vector containing the construct to the target; A step of causing the construct to enter the at least one cell and produce a modified cell; The step of causing the modified cells to degrade the lipids. Methods that include...

2. The method according to claim 1, wherein the construct comprises a nucleic acid sequence encoding one or more of the following, or a protein having homology to one or more of the following: Proteins encoded by 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), SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

3. The method according to claim 1 or 2, wherein the construct comprises a nucleic acid sequence encoding one or more of the following, or a protein having homology to one or more of the following: Proteins encoded by humanized 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

4. The method according to any one of claims 1 to 3, wherein the construct comprises at least humanized 3-ketosteroid 9α-hydroxylase (KshAB), and the humanized KshAB comprises a mitochondrial targeting sequence in the KshAB coding sequence.

5. The method according to any one of claims 1 to 4, wherein the construct is a nucleic acid containing a eukaryotic promoter sequence of a CMV promoter sequence.

6. The method according to any one of claims 1 to 5, wherein the construct is a nucleic acid of more than 200 nucleotides derived from sequences selected from sequence numbers 1 and 13.

7. The method according to any one of claims 1 to 5, wherein the construct is a protein selected from one or more proteins encoded by SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and SEQ ID NOs: 1 and 13.

8. The method according to any one of claims 1 to 7, wherein the cells are hepatocytes.

9. The method according to any one of claims 1 to 8, wherein the subject suffers from a disease or condition associated with high cholesterol levels, selected from one or more of the following: atherosclerosis, cardiovascular disease (CVD), myocardial infarction, stroke, peripheral vascular disease, diabetes mellitus, hypothyroidism, kidney disease, liver disease, fatty liver, non-alcoholic fatty liver disease, NAFLD, obesity, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, and hepatic fibrosis.

10. The method according to any one of claims 1 to 9, wherein the subject suffers from fatty liver or NAFLD.

11. A method for treating a subject who requires treatment for a cholesterol-related disease or condition, A step of creating modified cells by administering a vector containing one or more cholesterol-degrading genes to mammalian cells; A step of administering the modified cells to the subject; A step of causing the modified cells to break down cholesterol; thereby Steps to treat the aforementioned target Methods that include...

12. The method according to claim 11, wherein one or more cholesterol-degrading genes encode a protein having homology to the protein encoded by 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), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

13. The method according to claim 11 or 12, wherein one or more cholesterol-degrading genes encode a protein having homology to the proteins encoded by humanized 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

14. The method according to any one of claims 11 to 13, wherein one or more cholesterol-degrading genes encode a protein having homology to humanized 3-ketosteroid 9α-hydroxylase (KshAB), and the humanized KshAB includes a mitochondrial targeting sequence in its KshAB coding sequence.

15. The method according to any one of claims 11 to 14, wherein the vector comprises a eukaryotic promoter sequence of a CMV promoter sequence.

16. The method according to any one of claims 11 to 15, wherein the vector comprises nucleic acids of more than approximately 200 nucleotides of one or more sequences of sequence number 1 or 13.

17. The method according to any one of claims 11 to 15, wherein the one or more cholesterol-degrading genes encode one or more proteins of the proteins encoded by SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

18. The method according to any one of claims 11 to 17, wherein the cells are stem cells.

19. The method according to any one of claims 11 to 18, wherein the modified cells are differentiated before administration to a target.

20. The method according to any one of claims 11 to 19, wherein the modified cells administered to the subject are selected from immune cells, monocytes, and macrophages.

21. The method according to any one of claims 11 to 20, wherein the modified cells administered to the subject are macrophages.

22. The method according to any one of claims 11 to 21, wherein the modified cells administered to the subject are heterogeneous or homogeneous cells.

23. The method according to any one of claims 11 to 21, wherein the modified cells administered to the subject are autologous cells.

24. The method according to any one of claims 11 to 22, wherein the modified cells administered to the subject are modified at one or more loci involved in autoimmunity, and the loci are one or more MHC loci.

25. The method according to any one of claims 11 to 24, wherein the modified cells administered to the subject contain a suicide gene.

26. The method according to any one of claims 11 to 25, wherein the subject suffers from a disease or condition associated with high cholesterol levels or toxic cholesterol derivatives selected from one or more of the following: atherosclerosis, cardiovascular disease (CVD), myocardial infarction, stroke, peripheral vascular disease, diabetes mellitus, hypothyroidism, kidney disease, liver disease, fatty liver, non-alcoholic fatty liver disease, NAFLD, obesity, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, hepatic fibrosis, eye disease, lung disease, neurological disease, and neurodegenerative disease.

27. The method according to any one of claims 11 to 26, wherein the subject is suffering from atherosclerosis.

28. The method according to any one of claims 11 to 27, wherein, after treatment, at least one atheroma in the subject is reduced in volume by more than 50%.

29. At least one humanized cholesterol-degrading enzyme; and At least one suicide gene A composition for cell therapy containing mammalian cells.

30. The composition according to claim 29, wherein the cells further comprise at least one modified MHC gene locus.

31. The composition according to claim 29 or 30, wherein one or more cholesterol-degrading genes encode a protein having homology to the proteins encoded by 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), SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

32. The composition according to any one of claims 29 to 31, wherein one or more cholesterol-degrading genes encode a protein having homology to the proteins encoded by humanized 3-ketosteroid Δ1-dehydrogenase (Δ1-KstD), 3-ketosteroid 9α-hydroxylase (KshAB), 3β-hydroxysteroid dehydrogenase 2 (HSD2), P450-ferredoxin reductase-ferredoxin fusion protein (P450-FdxR-Fdx), SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

33. The composition according to any one of claims 29 to 32, wherein one or more cholesterol-degrading genes encode a protein having homology to humanized 3-ketosteroid 9α-hydroxylase (KshAB), and the humanized KshAB includes a mitochondrial targeting sequence in the KshAB coding sequence.

35. The composition according to any one of claims 29 to 34, wherein the cells contain nucleic acids of one or more sequences of sequence number 1 or 13.

36. The composition according to any one of claims 29 to 35, wherein one or more cholesterol-degrading genes encode one or more proteins of the proteins encoded by SEQ ID NOs: 2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and SEQ ID NOs: 1 and 13.

37. The composition according to any one of claims 29 to 36, wherein the mammalian cells are stem cells.

38. The composition according to any one of claims 29 to 37, wherein the mammalian cells are induced pluripotent stem cells.

39. The composition according to any one of claims 29 to 38, wherein the mammalian cells are differentiated mammalian cells.

40. The composition according to any one of claims 29 to 39, wherein the mammalian cells are selected from immune cells, monocytes, and macrophages.

41. The composition according to any one of claims 29 to 40, wherein the mammalian cell is a macrophage.

42. The composition according to any one of claims 29 to 41, wherein the mammalian cell comprises at least one modified MHC gene locus.

43. The composition according to any one of claims 29 to 42, for administration to subjects suffering from a disease or condition associated with high cholesterol levels, selected from one or more of the following: atherosclerosis, cardiovascular disease (CVD), myocardial infarction, stroke, peripheral vascular disease, diabetes mellitus, hypothyroidism, renal disease, hepatic disease, fatty liver, non-alcoholic fatty liver disease, NAFLD, obesity, non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, and hepatic fibrosis.

44. The composition according to claim 43, wherein the subject is suffering from atherosclerosis.

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