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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/136—Screening for pharmacological compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention is directed in certain embodiments to compositions and methods for treating, preventing and/or diagnosing cardiovascular diseases (CVD), such as dyslipidemia, atherosclerosis, low HDL diseases or related disorders. More specifically, the present embodiments relate to identification of mutations in the human gene encoding CYP8B1 as diagnostic targets for cardiovascular diseases, and to modulation of the activity or expression of CYP8B1 for the prevention and treatment of cardiovascular diseases.
• CVD cardiovascular diseases
• Heart disease is the leading cause of death in the United States, and more than one in four deaths each year are associated with a
• cardiovascular disease or disorder cardiovascular disease or disorder
• Coronary artery disease is the most common form of cardiovascular disease.
• Coronary artery disease is caused by the hardening and narrowing of arteries due to the formation of atherosclerotic plaques, i.e., atherosclerosis.
• An atherosclerotic plaque, or an atheroma is an accumulation of lipids, cholesterol and white blood cells, particularly macrophages, deposited on a blood vessel wall.
• High density lipoprotein (HDL) has been shown to have cardioprotective and particularly antiatherogenic effects that have been linked to its role in reverse cholesterol transport (i.e., the transport of cholesterol from non-hepatic tissues to the liver), and a low level of HDL is considered to be a risk factor for CVD.
• mouse model Among currently used animal models for CVD and in particular atherosclerosis, rodent models and specifically the mouse model have proven popular in view of the large number of available genetically defined mouse strains, murine cell lines, isolated murine genes, antibody-defined gene products, ease of manipulation, and other factors.
• the mouse model suffers from a number of drawbacks that limit its applicability to the
• mice for example, cholesterol transport is mediated primarily by HDL, while in humans it is low density lipoprotein (LDL) that is responsible for cholesterol transport. Additionally, mice fail to express cholesteryl ester transfer protein (CETP), a cholesterol-transfer protein that is typically present in humans (Plump et al, 1999 Arterioscler Thromb Vase Biol. 1999 19:1 105-1 1 10). Hence, comparatively severe departures from typical physiological conditions are required in mice in order to replicate certain CVD manifestations such as those seen in atherosclerosis, calling into question whether other, undetermined effects undermine the fidelity with which the murine system models human disease.
• CETP cholesteryl ester transfer protein
• statins the most successful and widely used class of therapeutics for human dislipidemia and atherosclerosis, fail to provide comparable effects in mice (Zadelaar et al, 2007 Arterioscler Thromb Vase Biol. 27:1706-21 ). It was recently reported, in a murine in vivo system that was experimentally manipulated to exhibit elevated levels of serum cholesterol and of oxidized LDL, that simvastatin failed to lower cholesterol levels even though oxidized LDL levels were lowered (Owens et al., 2012 J. Clin. Invest.
• CVD remains the number one cause of death in the U.S.
• the compositions and methods described herein address these needs and offer other related advantages.
• CVD cardiovascular diseases
• disorders such as dyslipidemia, atherosclerosis, low HDL diseases and related disorders
• CVD cardiovascular diseases
• HDL high density lipoprotein
• LDL low density lipoprotein
• TG plasma triglyceride
• BMI body-mass index
• an isolated polynucleotide comprising an oligonucleotide of at least 10 contiguous nucleotides and not more than 1506, 1505, 1504, 1503, 1502, 1501 , 1500, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 40, 30, 20 or 15 contiguous nucleotides of a human CYB8B1 -encoding sequence as set forth in SEQ ID NO:2 which encodes a human CYP8B1 polypeptide as set forth in SEQ ID NO:1 , wherein the oligonucleotide comprises at least one nucleotide substitution at a nucleotide position that corresponds to a wildtype nucleotide position that is selected from:
• polynucleotide comprising an oligonucleotide of at least 10 contiguous nucleotides and not more than 1506, 1505, 1504, 1503, 1502, 1501 , 1500, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 40, 30, 20 or 15 contiguous nucleotides of a human CYB8B1 -encoding sequence as set forth in SEQ ID NO:2 which encodes a human CYP8B1 polypeptide as set forth in SEQ ID NO:1 , wherein the oligonucleotide comprises at least one nucleotide substitution at a nucleotide position that corresponds to a wildtype nucleotide position that is selected from:
• polynucleotide comprising an oligonucleotide of at least 10 contiguous nucleotides and not more than 1506, 1505, 1504, 1503, 1502, 1501 , 1500, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 40, 30, 20 or 15 contiguous nucleotides of a human CYB8B1 -encoding sequence as set forth in SEQ ID NO:2 which encodes a human CYP8B1 polypeptide as set forth in SEQ ID NO:1 , wherein the oligonucleotide comprises at least one nucleotide substitution at a nucleotide position that corresponds to a wildtype nucleotide position that is selected from:
• any one of the isolated polynucleotides just described hybridizes under moderately stringent conditions to a mutant human CYP8B1 -encoding polynucleotide that encodes a mutant human CYP8B1 polypeptide which differs in amino acid sequence from the amino acid sequence set forth in SEQ ID NO:1 by at least one amino acid substitution that is present at an amino acid position that corresponds to a wildtype amino acid position that is selected from: (a) M at wildtype amino acid sequence position 53 of SEQ ID NO:1 which is substituted by T in said polypeptide,
• T at wildtype amino acid sequence position 93 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide
• an isolated polypeptide comprising at least 10 and no more than 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 1 1 contiguous amino acids of a human CYP8B1 protein having the amino acid sequence set forth in SEQ ID NO:1 , wherein the polypeptide comprises at least one amino acid substitution at an amino acid position that corresponds to a wildtype amino acid position that is selected from:
• R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which is substituted by H in said polypeptide.
• an isolated polypeptide comprising at least 10 and no more than 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 1 1 contiguous amino acids of a human CYP8B1 protein having the amino acid sequence set forth in SEQ ID NO:1 , wherein the polypeptide comprises at least one amino acid substitution at an amino acid position that corresponds to a wildtype amino acid position that is selected from:
• an isolated polypeptide comprising at least 10 and no more than 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 1 1 contiguous amino acids of a human CYP8B1 protein having the amino acid sequence set forth in SEQ ID NO:1 , wherein the polypeptide comprises at least one amino acid substitution at an amino acid position that corresponds to a wildtype amino acid position that is selected from:
• R at wildtype amino acid sequence position 26 of SEQ ID NO:1 which is absent in said polypeptide and wherein L at wildtype amino acid sequence position 25 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide, R at wildtype amino acid sequence position 28 of SEQ ID NO:1 which is substituted by C in said polypeptide,
• T at wildtype amino acid sequence position 93 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide
• an isolated antibody or an antigen-binding fragment thereof, that specifically binds to an isolated polypeptide that comprises at least 10 contiguous amino acids of a human CYP8B1 protein having the amino acid sequence set forth in SEQ ID NO:1 , wherein the polypeptide comprises at least one amino acid substitution at an amino acid position that corresponds to a wildtype amino acid position (a) that is selected from:M at wildtype amino acid sequence position 53 of SEQ ID NO:1 which is substituted by T in said polypeptide,
• S at wildtype amino acid sequence position 488 of SEQ ID NO:1 which is substituted by N in said polypeptide or (c) a wildtype amino acid position that is selected from: R at wildtype amino acid sequence position 26 of SEQ ID NO:1 which is absent in said polypeptide and wherein L at wildtype amino acid sequence position 25 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide,
• T at wildtype amino acid sequence position 93 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide
• G at wildtype amino acid sequence position 187 of SEQ ID NO:1 which is substituted by S in said polypeptide
• R at wildtype amino acid sequence position 207 of SEQ ID NO:1 which is substituted by H in said polypeptide
• the antibody is a monoclonal antibody.
• the isolated antibody, or an antigen-binding fragment thereof is selected from the group consisting of a single chain antibody, a ScFv, a univalent antibody lacking a hinge region, and a minibody.
• the antibody is a Fab or a Fab' fragment.
• the antibody is a F(ab')2 fragment.
• the antibody is a whole antibody.
• an antisense oligonucleotide that comprises any one of the above described polynucleotides.
• a ribozyme that comprises any one of the above described polynucleotides.
• a small interfering RNA that comprises any one of the above described polynucleotides.
• a method for determining the risk for or presence in a subject of a cardiovascular disease that would be ameliorated by one or more of (i) an increased level of plasma high density lipoprotein (HDL) in the subject, (ii) a decreased level of plasma low density lipoprotein (LDL) in the subject, (iii) a decreased level of plasma triglyceride (TG) in the subject, (iv) a decreased body-mass index (BMI) in the subject, and (v) a decreased blood level of hemoglobin A1 c in the subject, the method comprising: determining the presence, in CYP8B1 -encoding DNA in a biological sample from the subject, of at least one single nucleotide
• polymorphism that is associated with a decreased risk of cardiovascular disease.
• cardiovascular disease that would be ameliorated by one or more of (i) an increased level of plasma high density lipoprotein (HDL) in one or more of the subjects, (ii) a decreased level of plasma low density lipoprotein (LDL) in one or more of the subjects, (iii) a decreased level of plasma triglyceride (TG) in one or more of the subjects, (iv) a decreased body-mass index (BMI) in one or more of the subjects, and (v) a decreased blood level of hemoglobin A1 c in the subject, the method comprising: determining absence or presence, in CYP8B1 - encoding DNA in a biological sample from each subject, of at least one single nucleotide polymorphism that is associated with decreased risk for the cardiovascular disease, wherein presence of said at least one polymorphism indicates decreased risk for the cardiovascular disease, and therefrom stratifying the population according to cardiovascular disease risk.
• HDL plasma high density lipoprotein
• LDL low density lipoprotein
• TG plasma t
• At least one single nucleotide polymorphism that is associated with the decreased risk of cardiovascular disease is present in a CYP8B1 -encoding DNA region that encodes a CYP8B1 region that is selected from a CYP8B1 catalytic domain, a CYP8B1 O 2 -binding domain, a CYP8B1 steroidogenic region and a CYP8B1 heme binding domain.
• At least one single nucleotide polymorphism that is associated with the decreased risk of cardiovascular disease is present in a CYP8B1 -encoding DNA region that encodes a CYP8B1 region that is selected from a CYP8B1 O 2 -binding domain, a CYP8B1 steroidogenic region and a CYP8B1 heme binding domain, and wherein the single nucleotide
• polymorphism is a non-synonymous nucleotide substitution.
• at least one single nucleotide polymorphism that is associated with the decreased risk of cardiovascular disease is a single nucleotide polymorphism located at a nucleotide that corresponds to a wildtype nucleotide position of SEQ ID NO:2 that is selected from the group consisting of:
• At least one single nucleotide polymorphism that is associated with the decreased risk of cardiovascular disease is a single nucleotide polymorphism located at a nucleotide that corresponds to a wildtype nucleotide position of SEQ ID NO:2 that is selected from the group consisting of:
• At least one single nucleotide polymorphism that is associated with the decreased risk of cardiovascular disease is a single nucleotide polymorphism located at a nucleotide that corresponds to a wildtype nucleotide position of SEQ ID NO:2 that is selected from the group consisting of:
• a method for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI) in the subject, and (v) a decreased blood level of hemoglobin A1 c in the subject, the method comprising administering to the subject an agent that is selected from (a) an agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject, and (b) an agent that is an inhibitor of human cytochrome P450-family 8- subfamily B-polypeptide 1 (CYP8B1 ) sterol 12-a-hydroxylase activity in the subject.
• the agent is selected from:
• n 0, 1 , 2, 3, 4 or 5;
• n 1 , 2 or 3;
• X is -N- or -C(R 6 )-,
• R 1 is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, heterocyclylalkyl, or -OR 7 ;
• R 2 is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, heterocyclylalkyl, or -OR 7 ; or
• R 1 and R 2 connected to the same carbon form a spiro ring, which can be optionally substituted with alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, or heterocyclylalkyl;
• R 3 is the same or different and independently hydrogen, halogen, hydroxy, alkyl, alkoxy, aryl, cydoalkyi, heterocydyl, aralkyi, heteroaryl or heteroarylalkyi;
• R 4 and R 5 is independently hydrogen or alkyl
• R 6 is hydrogen or alkyl
• each R 7 is the same or different and independently hydrogen, alkyl, aryl, cydoalkyi, heterocydyl, heteroaryl, aralkyi, heteroarylalkyi, cycloalkylalkyl, or heterocyclylalkyl,
• each R is independently a single or double bond; each R is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, aralkyl, cycloalkyi, heterocyclyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;
• R 15 is hydrogen or alkyl
• R 18 is hydrogen, hydroxy, alkoxy, or alkyl
• R 19 is hydrogen or alkyl
• R 21 is hydrogen or alkyl, or R 21 and R 16a together form a bond
• t 0, 1 , 2, 3, 4 or 5;
• each R 9 is the same or different and independently hydrogen alkyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;
• R 27 is the same or different and independently hydrogen, alkyl, halogen, acyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, heterocyclylalkyl, -OR 9 , -N(R 9 ) 2 -, or -SR 9 ; or two adjacent R 27 , together with the carbons to which they attach, form a fused aryl, heteroaryl, heterocyclyl, or cycloalkyi ring;
• R 28a and R 29a form a cycloalkyi or heterocyclyl ring
• each R 30a and R 30b is the same or different and independently hydrogen, alkyl, acyl, aralkyl, or heteroarylalkyl,
• the cardiovascular disease or disorder is selected from dyslipidemia, atherosclerosis, low HDL diseases and related disorders. In certain embodiments at least one of: (i) administering the agent increases plasma HDL levels in the subject; (ii) administering the agent decreases plasma LDL levels in the subject; and (iii) administering the agent decreases plasma triglyceride levels in the subject.
• the agent specifically binds to a CYP8B1 polypeptide catalytic domain. In certain embodiments the agent specifically binds to a substrate access channel, a steroidogenic region or product egress channel, or a heme prosthetic group interface domain of the CYP8B1 polypeptide catalytic domain.
• the method comprises a method for identifying said human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, the method comprising the steps of: (a) determining whether a candidate human subject has a reduced level of CYP8B1 activity relative to a control subject known to have a normal level of CYP8B1 activity, by testing a biological sample obtained from the candidate subject for presence of a mutant CYP8B1 polypeptide which comprises a mutation that results in decreased CYP8B1 activity, or for presence of a polynucleotide encoding said mutant CYP8B1 polypeptide, wherein the presence of
• the method comprises, prior to the step of administering, a method for identifying said human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, the method comprising the steps of: (a) determining whether a candidate human subject has a reduced level of CYP8B1 activity relative to a control subject known to have a normal level of CYP8B1 activity, by testing a biological sample obtained from the candidate subject for presence of a mutant CYP8B1 polypeptide which comprises a mutation that results in decreased CYP8B1 activity, or for presence of a polynucleotide encoding said mutant CYP8B1 polypeptide, wherein the presence
• T at wildtype amino acid sequence position 93 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide
• the cardiovascular disease or disorder is selected from dyslipidemia, atherosclerosis, low HDL diseases and related disorders.
• the agent specifically binds to the CYP8B1 polypeptide.
• a method for identifying an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, comprising: comparing (i) a base level of CYP8B1 polypeptide expression by a first cell that has not been contacted with a candidate agent, to (ii) a test level of the CYP8B1 polypeptide expression by a second cell that has been contacted with the candidate agent, wherein a determination that the test level of CYP8B1 polypeptide expression is less than the base level of CYP8B1 polypeptide expression indicates the candidate agent is an increased level of plasma high density
• the method further comprises determining the base level of CYP8B1 polypeptide expression and the test level of CYP8B1 polypeptide expression by quantifying CYP8B1 protein.
• an agent for treating or decreasing likelihood of occurrence of of a cardiovascular disease or disorder that is identified according to the above described method.
• the agent specifically binds to a polynucleotide sequence encoding the CYP8B1 polypeptide, said CYP8B1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 .
• a method for identifying an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, comprising comparing (i) a base level of CYP8B1 activity by a first CYP8B1 polypeptide, or a fragment or variant thereof, that has not been contacted with a candidate agent, to (ii) a test level of the CYP8B1 activity by a second CYP8B1 polypeptide, or a fragment or variant thereof, that has been contacted with the candidate agent, wherein a
• each of the first and second CYP8B1 polypeptides, or fragment or variant thereof comprises a CYP8B1 polypeptide catalytic domain.
• each of the first and second CYP8B1 polypeptides, or fragment or variant thereof comprises a substrate access channel, a
• an agent for treating or decreasing likelihood of occurrence of of a cardiovascular disease or disorder that is identified according to the above described method.
• the above described agent inhibits a sterol 12-a-hydroxylase activity of the second CYP8B1 polypeptide.
• the agent specifically binds to the second CYP8B1 polypeptide.
• the agent specifically binds to a substrate access channel, a steroidogenic region or product egress channel, or a heme prosthetic group interface domain of the CYP8B1 polypeptide.
• a method for identifying a human subject having reduced CYP8B1 activity comprising determining if a polynucleotide sequence of a CYP8B1 gene in a biological sample obtained from said subject encodes a CYP8B1 sequence comprising at least one mutation selected from the group consisting of: a M53T mutation, a P88S mutation, an A103E mutation, a D195N mutation, a K238R mutation, a K300X mutation, a D341 E mutation, an R349Q mutation, an L357F mutation, a Q372K mutation, a V402I mutation, an R407H mutation, and an S488N mutation, and thereby determining that the subject has reduced CYP8B1 activity.
• a method for identifying a human subject having reduced CYP8B1 activity comprising determining if a polynucleotide sequence of a CYP8B1 gene in a biological sample obtained from said subject encodes a CYP8B1 sequence comprising at least one mutation selected from the group consisting of a M53T mutation, an A103E mutation, a D195N mutation, a K300X mutation, a D341 E mutation, an R349Q mutation, and an R407H mutation, and thereby determining that the subject has reduced CYP8B1 activity.
• a method for identifying a human subject having reduced CYP8B1 activity comprising determining if a polynucleotide sequence of a CYP8B1 gene in a biological sample obtained from said subject encodes a CYP8B1 sequence comprising at least one mutation selected from the group consisting of a R26X mutation, a R28C mutation, a R50Q mutation, a R59C mutation, a V80I mutation, a Q94X mutation, a L97V mutation, a K129M mutation, a G133A mutation, a D145Q mutation, a F186L mutation, a G187S mutation, a R207H mutation, a T287M mutation, a T337A mutation, a S342R mutation, a P386L mutation, a R407G mutation, a P432S mutation, a R443G mutation, a F453C mutation, a L456F mutation, a V458Q mutation
• the kit further comprises a second polynucleotide that hybridizes under moderately stringent conditions to a wild- type CYP8B1 polynucleotide, such that the first and second polynucleotides are capable of amplifying, in a polymerase chain reaction (PCR), a CYP8B1 - encoding polynucleotide which encodes a mutant CYP8B1 that comprises at least one mutation selected from the group consisting of: a M53T mutation, a P88S mutation, an A103E mutation, a D195N mutation, a K238R mutation, a K300X mutation, a D341 E mutation, a R349Q, a L357F mutation, a Q372K mutation, a V402I mutation, a R407H mutation, and a S488N mutation.
• PCR polymerase chain reaction
• the diagnostic kit further comprises a second polynucleotide that hybridizes under moderately stringent conditions to a wild- type CYP8B1 polynucleotide, such that the first and second polynucleotides are capable of amplifying, in a polymerase chain reaction (PCR), a CYP8B1 - encoding polynucleotide which encodes a mutant CYP8B1 that comprises at least one mutation selected from the group consisting of: R26X mutation, a R28C mutation, a R50Q mutation, a R59C mutation, a V80I mutation, a Q94X mutation, a L97V mutation, a K129M mutation, a G133A mutation, a D145Q mutation, a F186L mutation, a G187S mutation, a R207H mutation, a T287M mutation, a T337A mutation, a S342R mutation, a P386L mutation, a R407G mutation
• Figure 1 is a schematic representation of human CYP8B1 protein structure including relative locations of 13 mutations identified in high HDL individuals.
• the M53T, A103E, K300X, R349Q, Q372K, R407H and S488N mutations were identified in single probands; whereas the P88S, D195N, K238R, D341 E, L357F and V402I mutations were identified in multiple probands.
• Figure 2 is a schematic representation of human CYP8B1 protein structure including relative locations of 27 predicted damaging CYP8B1 mutations identified in NHLBI Grand Opportunity Exome Sequencing Project (ESP) and 1000 Genomes Databases. The functional predictions were made using Polyphen2 and assuming truncation mutations result in inactive enzyme.
• Figure 3 depicts the segregation of the K300X mutation of
• Figure 4 is a bar graph that shows the relative catalytic activities of empty vector control (pcDNA3.1 ), wild type (WT) and mutant CYP8B1 enzymes expressed heterologously in HEK-293 cells. CYP8B1 activities were determined by measuring the conversion of exogenous substrate (7a-hydroxy- 4-cholesten-3-one) to product (7a,12a-dihydroxy-4-cholesten-3-one) in the cell media.
• CYP8B1 mutations either (i) have no significant effect (benign) on enzyme activity (Q372K, K238R, V402I, L357F, S488N, P88S), or (ii) cause partial (PLOF) loss of function (M53T; 37.1 %, D195N; 18.1 %, and D341 E;
• Figure 5 is plot of wild type and complete loss of function mutant
• the K300X mutation resulted in total loss of CYP8B1 protein expression, whereas R349Q and R407H mutations decreased CYP8B1 protein stability compared to wild-type enzyme and may thereby have reduced protein expression.
• the A103E mutation did not affect CYP8B1 protein stability compared to wild-type enzyme.
• Figure 6 is plot of wild type and partial loss-of-function mutant CYP8B1 protein levels in HEK-293 cells at different times following the cessation of protein synthesis resulting from the addition of cycloheximide.
• the western blot below shows representative data from a single experiment.
• the D195N and D341 E mutations destabilized CYP8B1 protein, whereas the M53T mutation did not affect protein stability.
• Figure 7 is plot of wild type and benign mutant CYP8B1 protein levels in HEK-293 cells at different times following the cessation of protein synthesis resulting from the addition of cycloheximide.
• the western blot below shows representative data from a single experiment.
• Benign CYP8B1 mutants (P88S, K238R, L357F, Q372K, S488N) had the same protein stability as wild- type enzyme.
• Figure 8 is a bar graph showing the specificity constants
• Vmax/Km for the sterol-12-a-hydroxylase activities of microsomal preparations of the wild type CYP8B1 and each of the loss-of-function mutants.
• A103E, K300X, R349Q, and R407H complete loss-of-function mutants had no CYP8B1 activity, whereas M53T partial loss-of -function mutant had a specificity constant (Vmax/Km) value that was 55% that of wild-type CYP8B1 .
• the specificity constants of the other mutants were similar to that of wild type enzyme.
• Figure 9 is a dot plot depicting plasma HDLc levels measured in individuals identified as being heterozygous for a partial and complete loss-of- function (LOF) CYP8B1 mutation described herein, and population control individuals.
• LEF loss-of- function
• Figure 10 is a dot plot that shows the relative triglyceride concentrations of partial (pLOF) and complete (cLOF) loss-of-function CYP8B1 mutation carriers and population control individuals.
• Figure 1 1 is a dot plot that shows the relative LDLc concentrations of partial and complete LOF CYP8B1 mutation carriers and population control individuals.
• Figure 12 is a dot plot that shows the relative body mass index (BMI) of partial and complete LOF CYP8B1 mutation carriers and population control individuals.
• BMI body mass index
• the presently disclosed invention embodiments are based in part on the unexpected discovery that in humans, mutations in the gene encoding CYP8B1 (cytochrome P450, family 8, subfamily B, polypeptide 1 also known as sterol 12-a-hydroxy!ase), including mutations responsible for partial or complete impairment of the sterol 12-a-hydroxylase enzymatic activity of CYP8B1 , can result in beneficially elevated plasma high density lipoprotein (HDL) levels relative to the HDL levels detected in humans having normal CYP8B1 activity.
• HDL plasma high density lipoprotein
• Described herein is the identification of novel mutations, including loss-of-f unction and reduced function mutations, in the CYP8B1 gene of human subjects having unusually high levels of high density lipoprotein cholesterol (HDLc).
• Certain embodiments are thus based on the discovery of previously unknown mutations in the human CYP8B1 gene and its CYP8B1 protein product, and certain embodiments derive from exploiting the association disclosed herein for the first time between one or more herein described CYP8B1 mutations in a human subject and one or more of (i) an increased level of plasma high density lipoprotein (HDL) in the human subject, (ii) a decreased level of plasma low density lipoprotein (LDL) in the human subject, (iii) a decreased level of plasma triglyceride (TG) in the human subject, (iv) a decreased body-mass index (BMI) in the human subject, and (v) a decreased blood level of hemoglobin A1 c in the human subject.
• the present invention provides, in certain embodiments, methods and compositions to modulate CYP8B1 , e.g., to reduce the expression and/or activity of CYP8B1 , resulting in increased plasma HDL levels and/or decreased plasma LDL levels and/or reduced triglyceride (TG) levels and/or decreased body-mass index (BMI) and/or decreased blood levels of hemoglobin A1 c, which will find uses in treating, diagnosing and/or decreasing likelihood of occurrence of
• CVD cardiovascular disease
• methods for determining cardiovascular disease (CVD) risk in a subject and for stratifying a population of subjects according to CVD risk based on the determination of mutations in the human CYP8B1 polypeptide as described herein, including loss-of-function mutations described herein, and/or based on the determination of single nucleotide polymorphisms (SNPs) that occur as oligonucleotide substitutions in the CYP8B1 -encoding polynucleotide sequence relative to the normal wildtype CYP8B1 -encoding gene sequence as described herein. These substitutions include SNPs responsible for causing loss-of-function mutations in CYP8B1 .
• SNPs single nucleotide polymorphisms
• Cholic acid is a hydrophobic bile acid that promotes intestinal cholesterol absorption. Elevated cholic acid levels are implicated in increased levels of intestinal cholesterol absorption, VLDL production, hepatic cholesterol esters and APOB-containing particles, more concentrated and hydrophobic bile acid (potentially leading also to increased gallstone risk), and decreased levels of bile acid synthesis and hepatic ABCA1 expression (Norlin and Wikvall, Curr. Mol. Med., 7:199-218, 2007; Lefebvre P., et ai, Physiol.
• CYP8B1 which results in reduced cholic acid biosynthesis and instead directs bile acid production predominantly to the chenodeoxycholic acid component, leads to an increase in the HDL plasma level of a patient.
• this effect of CYP8B1 inhibition would lead to reduced cholesterol absorption from the gut, reduced hepatic cholesterol esters and APOB-containing particles, elevated bile acid synthesis, reduced VLDL production, and increased ABCA1 expression, resulting in elevated HDLc, reduced LDLc and/or triglycerides, reduced gallstone risk, and/or reduced atherosclerosis.
• CYP8B1 (Cytochrome P450, family 8, subfamily B, polypeptide 1 ), also known as sterol 12-alpha-hydroxylase, is an enzyme that is part of the neutral bile acid synthesis pathway.
• the human CYP8B1 cDNA encodes a 501 amino acid protein having the amino acid sequence set forth in SEQ ID NO:1 and that has, respectively, 42%, 35% and 36% amino acid similarity to human CYP8A1 , CYP7A1 and CYP7B1 .
• human CYP8B1 When CYP8B1 amino acid sequences are compared across mammalian species, human CYP8B1 has 99% amino acid sequence similarity to chimpanzee CYP8B1 , 82% amino acid sequence similarity to pig CYP8B1 , 81 % amino acid sequence similarity to dog CYP8B1 , 78% amino acid sequence similarity to rabbit CYP8B1 , 75% amino acid sequence similarity to mouse and rat CYP8B1 , and 54% amino acid sequence similarity to chicken CYP8B1 .
• On-line databases such as BioGPS report that CYP8B1 is expressed exclusively in the liver.
• CYP8B1 is required for biosynthesis of cholic acid (CA), a major component of bile, and a product of cholesterol metabolism.
• CA cholic acid
• the intermediate metabolite 7a-hydroxy-4- cholesten-3-one (7-HCO) is converted by CYP8B1 to 7a,12a-dihydroxy-4- cholesten-3-one (7,12-DiHCO), eventually leading through a series of downstream steps to the production of cholic acid.
• 7-HCO can also be converted to chenodeoxycholic acid (CDCA), which occurs via an alternate metabolic pathway that does not involve CYP8B1 .
• CYP8B1 determines the ratio of cholic acid to chenodeoxycholic acid, which in turn determines the hydrophobicity of bile acids. Both cholesterol levels and hydrophobicity of bile acids down-regulate the activity of CYP8B1 , and thus changes in the levels of cholesterol affect the activity of CYP8B1 , which could be linked to
• CYP8B1 As a target for elevating HDL levels prior to the present disclosure, there is no human validation of CYP8B1 as a target for elevating HDL levels prior to the present disclosure. Further, previous reports failed to indicate the ultrastructural fine specificity (e.g., which CYP8B1 portion, region, domain, conformational structure or other structural feature) by which an agent (e.g., a chemical compound), should desirably antagonize the CYP8B1 protein in humans. As also noted below, CYP inhibitors may include certain agents that function by coordinating with heme groups and certain other agents that are substrate analogues, such as non-catalyzable substrate mimetics.
• the murine model system fails to provide a predictive platform for human CVD therapy in view of several significant limitations, as also noted above.
• HDL high density lipoprotein
• LDL low density lipoprotein
• CETP cholesteryl ester transfer protein
• Human genetic data demonstrating that a mutation in a particular gene is associated with an improvement in plasma lipoprotein profile ⁇ e.g., raised HDL cholesterol and/or lowered LDL cholesterol) are therefore considerably more predictive of relevance to the human condition than rodent data.
• plasma lipoprotein profile e.g., raised HDL cholesterol and/or lowered LDL cholesterol
• inventions provide novel CYP8B1 polynucleotide and polypeptide
• corresponding nucleic acid sequences, regions, fragments or the like based on the convention for numbering CYP8B1 nucleic acid positions according to SEQ ID NO:2 in which nucleotides 326-1831 encode the CYP8B1 polypeptide having the amino acid sequence set forth in SEQ ID NO:1 .
• a portion of a CYP8B1 - encoding polynucleotide sequence may correspond to the CYP8B1 -encoding sequence of SEQ ID NO:2 when a sample CYP8B1 -encoding DNA sequence is aligned with the human CYP8B1 -encoding DNA sequence of SEQ ID NO:2 such that at least 70%, preferably at least 80% and more preferably at least 90% of the nucleotides in a given sequence of at least 20 consecutive nucleotides of a sequence are identical.
• a portion of the CYP8B1 -encoding DNA sequence in a biological sample containing DNA from a subject suspected of having or being at risk for having cardiovascular disease or, as another example, a portion of the CYP8B1 -encoding DNA sequence in CYP8B1 -encoding DNA containing at least one single nucleotide polymorphism ⁇ e.g., mutated CYP8B1 DNA) that is associated with a decreased risk or decreased likelihood of occurrence of cardiovascular disease (e.g., decreased in a statistically significant manner relative to a randomly seleted population sample) as provided herein, may be aligned with a corresponding portion of the CYP8B1 - encoding DNA sequence of SEQ ID NO:2 using any of a number of alignment procedures and/or tools with which those having ordinary skill in the art will be familiar [e.g., CLUSTAL W, Thompson et al., 1994 Nucl.
• a sample CYP8B1 -encoding DNA sequence is greater than 95%, 96%, 97%, 98% or 99% identical to a corresponding CYP8B1 -encoding DNA sequence of SEQ ID NO:2.
• a sample CYP8B1 -encoding DNA sequence is identical to a corresponding CYP8B1 -encoding DNA sequence of SEQ ID NO:2.
• Those oligonucleotide probes having sequences that are identical in corresponding regions of the DNA sequence of SEQ ID NO:2 and sample DNA may be identified and selected following hybridization target DNA sequence analysis, to verify the absence of mutations. Mutations disclosed herein in the DNA sequence encoding human CYP8B1 include single nucleotide
• SNPs polymorphisms
• Table 1 which also shows amino acid substitutions that are caused by the indicated mutations or premature stop codons ("X") that result in truncated CYP8B1 polypeptide products.
• Certain SNPs in Table 1 may result in complete or partial loss of function (LOF) for the resulting CYP8B1 polypeptide product, as determined by assaying CYP8B1 mutants for sterol 12-a-hydroxylase activity or by in silico modeling using Polyphen2 (polymorphism phenotyping) software (Adzhubei et al., 2010 Nature Meths. 7(4):248; see also Ramensky et al. 2002 Nucl. Ac. Res. 30:3894;
• Human CYP8B1 SNPs are presented in Table 1 . Polypeptide mutations are identifed by indicating the wild type amino acid, followed by its position number within the full lenth human CYP8B1 polypeptide, followed by the amino acid replacement for the wild type amino acid. X indicates a stop codon. For example, M53T indicates that a threonine residue has replaced the wild-type methionine residue at position 53 of this human CYP8B1 polypeptide mutant.
• a K300X mutation results in a truncated CYP8B1 polypeptide, whereas other mutations result in the following single amino acid substitutions as compared to the wild-type human CYP8B1 polypeptide sequence: a M53T mutation, a D341 E mutation, and a Q372K mutation.
• the full length wild-type human CYP8B1 protein sequence is provided in SEQ ID NO:1
• the wild-type human cDNA that encodes the human CYP8B1 protein is provided in SEQ ID NO:2.
• Nucleotides 326-1831 of SEQ ID NO:2 are the coding sequence that encodes the CYP8B1 protein of SEQ ID NO:1 .
• Exemplary polynucleotide sequences e.g., codons, encoding the above-identified amino acid substitutions are as follows: Chr3:42891415 T>A (K300X), Chr3:42892155 T>C (M53T), Chr3: 42891290 OG (D341 E), and Chr3:42891 199 C>A (Q372K), based on hg18 human genome release.
• these mutations are located at positions 1223, 483, 1348, and 1439,
• CYP8B1 polynucleotide mutations and corresponding amino acid mutations, including seven mutations that result in CYP8B1 loss-of-function (LOF) mutants, is provided in Table 2.
• SNP nucleotide substitions are referred to as, for example, A>T, which indicates that the wild type A is replaced by a T in the mutant CYP8B1 polynucleotide.
• Table 2 Exemplary Novel CYP8B1 mutations identified in HHDL individuals
• oligonucleotide and “polynucleotide” as used herein encompass DNA, RNA, or combinations thereof, unless otherwise indicated. Further, the terms DNA and RNA should be understood to include not only naturally occurring nucleic acids, but also sequences containing nucleotide analogs or modified
• nucleotides such as those that have been chemically or enzymatically modified, for example DNA phosphorothioates, RNA phosphorothioates, and 2'- O-methyl ribonucleotides.
• polynucleotide as referred to herein thus includes single-stranded and double-stranded nucleic acid polymers.
• the nucleotides comprising the polynucleotide can be
• ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide Modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose and
• internucleotide linkage modifications such as phosphorothioate
• polynucleotide specifically includes single and double stranded forms of DNA.
• nucleotides includes deoxyribonucleotides and ribonucleotides.
• modified nucleotides includes nucleotides with modified or substituted sugar groups and the like.
• oligonucleotide linkages includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate,
• An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.
• vector is used to refer to any molecule ⁇ e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.
• expression vector refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
• operably linked means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
• a transcription control sequence "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.
• control sequence refers to polynucleotide sequences that can affect expression, processing or intracellular localization of coding sequences to which they are ligated or operably linked. The nature of such control sequences may depend upon the host organism.
• transcription control sequences for prokaryotes may include a promoter, ribosomal binding site, and transcription termination sequence.
• transcription control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences and polyadenylation sequences.
• control sequences can include leader sequences and/or fusion partner sequences.
• polynucleotides may include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the skilled person.
• polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
• RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide according to the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
• Polynucleotides may comprise a native sequence or may comprise a sequence that encodes a variant or derivative of such a sequence.
• each of the CYP8B1 mutations described herein results in a single amino acid change to, or a truncation of, the CYP8B1 protein.
• CYP8B1 polynucleotide and polypeptide sequences comprising one or more of the mutations identified herein may correspond to a full length CYP8B1 polynucleotide or polypeptide sequence, or they may be fragments or variants thereof.
• a CYP8B1 polynucleotide is the CYP8B1 gene sequence or the CYP8B1 cDNA sequence set forth in SEQ ID NO:2.
• a CYP8B1 polynucleotide comprises or consists of the coding region of the CYP8B1 cDNA sequence set forth in SEQ ID NO:2, and mutants thereof further comprise one or more of the mutations described herein.
• CYP8B1 polynucleotide sequences are double-stranded or single-stranded, and may include either or both sense and antisense strands. While the mutant CYP8B1 polynucleotide and polypeptides of certain preferred embodiments are provided herein with reference to the human CYP8B1 sequences, it is understood that other embodiments also contemplate non-human CYP8B1 polynucleotides and polypeptides comprising corresponding mutations, respectively.
• CYP8B1 polynucleotides and polypeptides are mammalian.
• polynucleotide compositions comprise a CYP8B1 polynucleotide sequence comprising one or more of the mutations described herein or encoding a mutant CYP8B1 polypeptide sequence described herein.
• polynucleotide variants having substantial identity to CYP8B1 sequences comprising one or more of the mutations described herein, for example, those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a herein disclosed polynucleotide sequence identified using the methods described herein ⁇ e.g., BLAST analysis using standard parameters, as described below).
• BLAST analysis using standard parameters, as described below.
• polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions.
• variants should also be understood to encompasses homologous genes of xenogenic origin. In particular embodiments, a
• polynucleotide variant comprises one or more of the mutations described herein.
• polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the CYP8B1 sequences disclosed herein.
• polynucleotides may comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 1500, 1501 , 1502, 1503, 1504, 1505, 1506 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between.
• a polynucleotide fragment comprises one or more of the CYP8B1 mutations described herein.
• certain preferred embodiments are directed to an isolated polynucleotide comprising an oligonucleotide of at least 10 contiguous nucleotides and not more than 1506, 1505, 1504, 1503, 1502, 1501 , 1500, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 40, 30, 20 or 15 contiguous nucleotides of a human CYB8B1 -encoding sequence as set forth in SEQ ID NO:2 which encodes a human CYP8B1 polypeptide as set forth in SEQ ID NO:1 , wherein the oligonucleotide comprises at least one nucleotide substitution at a nucleotide position that corresponds to a wildtype nucleotide position that is selected from those set forth in Table 1 .
• polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof.
• Hybridization techniques are well known in the art of molecular biology.
• suitable moderately stringent conditions for testing the hybridization of a polynucleotide to other polynucleotides may include prewashing in a solution of 5 X SSC, 0.5% SDS, 1 .0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS.
• the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed.
• suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65°C or 65-70°C.
• a polynucleotide that hybridizes to a CYP8B1 sequence comprises one or more of the mutations described herein.
• polynucleotides described herein or fragments thereof may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
• polynucleotides of the present invention may be present in an expression vector.
• two sequences are said to be "identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below.
• Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
• a “comparison window” as used herein refers to a segment of at least about 20 contiguous nucleotide positions, usually 30 to about 75, or 40 to about 50, in which a nucleotide sequence may be compared to a reference sequence of the same number of contiguous nucleotide positions after the two sequences are optimally aligned.
• Optimal alignment of sequences for comparison may be conducted, for instance, using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wl), using default parameters.
• optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981 ) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by
• BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
• BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides of the invention.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
• the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• additions or deletions i.e., gaps
• the percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
• genes comprising the polynucleotide sequences provided herein are also regarded as being within the scope of certain herein disclosed embodiments. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or
• RNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
• isolated means that a polynucleotide is substantially physically apart and away from the physical environment in which it occurs naturally, such as other coding sequences, and that the DNA molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. This use of “isolated” refers to the DNA molecule as originally obtained and removed from a natural source, and does not exclude genes or coding regions later added to the DNA segment by human
• polynucleotide compositions described herein may include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or may be modified synthetically by human intervention.
• polynucleotides may be single-stranded oligonucleotide primers, e.g., that bind specifically to a region of a CYP8B1 encoding polynucleotide comprising a mutation described herein.
• oligonucleotide primers bind to a CYP8B1 encoding polynucleotide comprising a mutation described herein, under moderately stringent hybridization conditions, but do not bind to a wild-type CYP8B1 polynucleotide under the same conditions.
• primers may be used, e.g., to detect the presence of a CYP8B1 polynucleotide mutation described herein.
• Primers may hybridize to either the coding or non-coding strand of a CYP8B1 DNA or to a CYP8B1 mRNA or cDNA sequence. Accordingly, primers may include sequences that correspond to either the coding or non-coding strand of a CYP8B1 DNA sequence.
• primers include CYP8B1 polynucleotide sequences that comprise any of the CYP8B1 polynucleotide mutations described herein, as well as complements thereof.
• oligonucleotide primers and probes may comprise an oligonucleotide sequence that is at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, and preferably at least 20 nucleotides, in length.
• oligonucleotide primers and/or probes hybridize to a polynucleotide comprising a CYP8B1 mutant sequence described herein under moderately stringent conditions, as defined above.
• Oligonucleotide primers and/or probes which may be usefully employed in diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
• the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a CYP8B1 polynucleotide sequence and include a mutation as disclosed herein.
• an isolated polypeptide comprising at least 10 and no more than 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 1 1 contiguous amino acids of a human CYP8B1 protein having the amino acid sequence set forth in SEQ ID NO:1 , wherein the polypeptide comprises at least one amino acid substitution at an amino acid position that corresponds to a wildtype amino acid position that is selected from those set forth in Table 1 .
• Certain such embodiments may provide polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a CYP8B1
• a CYP8B1 polypeptide variant typically exhibits at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity
• CYP8B1 polypeptide sequence set forth herein such as the polypeptide of SEQ ID NO:1 .
• the presently provided polypeptide fragments and variants comprise one or more of the mutations described herein such as the CYP8B1 mutations listed in Table 1 .
• a polypeptide "variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the herein disclosed polypeptide sequences. In many instances, a variant will contain conservative substitutions. A "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Conservative substitutions are known in the art. In one embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer (i.e., by five, four, three or two amino acids, or by one amino acid).
• two sequences are said to be “identical” if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below.
• Comparisons between two polypeptide sequences are typically performed by comparing the amino acid sequences of the polypeptides over a comparison window to identify and compare local regions of sequence similarity.
• comparison window refers to a segment of at least about 20 contiguous amino acid positions, usually 30 to about 75, or 40 to about 50 amino acids, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• optimal alignment of sequences for comparison may be
• BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
• BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polypeptides described herein.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score.
• the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 amino acid positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
• isolated polynucleotide or polypeptide is one that is removed from its original
• a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system.
• polypeptides are also purified, e.g., are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
• CYP8B1 polynucleotides and polypeptides may be readily produced using conventional molecular biology techniques (see, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other like references).
• short polynucleotide sequences may be synthetically produced, while longer polynucleotides may be produced from in vitro or in vivo expression systems.
• a wild-type CYP8B1 polynucleotide may be readily cloned and altered, e.g., by site-directed mutagenesis to produce a CYP8B1 polynucleotide comprising one or more of the mutations described herein.
• Polypeptides, fragments and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art.
• such polypeptides are synthesized using any of the commercially available solid- phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963.)
• Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
• Longer polypeptides may be recombinantly expressed using any of a large number of expression systems known and available in the art.
• HDL is one of the five major types of lipoproteins present in the blood that function to transport hydrophilic molecules including cholesterol and triglycerides. HDL is the densest type of lipoprotein due to its high protein content.
• HDL-associated proteins include, for example, Apolipoprotein A-l (ApoA-l), ApoA-ll, ApoC3, ATP binding cassette transporter A1 (ABCA1 ), and lecithin cholesterol acyltransferase (LCAT).
• the invention relates to a method for increasing the plasma HDL in a subject in need thereof, comprising providing an agent to the subject, wherein the agent inhibits CYP8B1 ⁇ e.g., completely, or substantially and in a statistically significant manner, impairs a CYP8B1 activity or expression).
• the agent reduces the expression and/or activity of CYP8B1 .
• a reduction in the expression of CYP8B1 means a reduced amount or level of CYP8B1 polypeptide in the subject or a biological sample (e.g., blood or plasma) obtained from the subject.
• the agent does not modulate the amount of plasma LDL and/or triglycerides. In another embodiment, the agent decreases the amount of plasma LDL and/or triglycerides.
• the agent is a small molecule that inhibits a biological activity of CYP8B1 , an antibody that specifically binds and inhibits CYP8B1 , or an antisense oligonucleotide, ribozyme or siRNA comprising a sequence that specifically binds to a CYP8B1 encoding polynucleotide or to CYP8B1 mRNA in a manner that suppresses ⁇ e.g., decreases with statistical significance) or abolishes CYP8B1 expression.
• the antisense or siRNA inhibits expression of the CYP8B1 polypeptide.
• Certain embodiments that are expressly contemplated herein are therefore directed to a method for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL) in the human subject, (ii) a decreased level of plasma low density lipoprotein (LDL) in the human subject, (iii) a decreased level of plasma triglyceride (TG) in the human subject, (iv) a decreased body-mass index (BMI) in the human subject, and (v) a decreased blood level of hemoglobin A1 c (HbAl c) in the human subject.
• HDL plasma high density lipoprotein
• LDL low density lipoprotein
• TG plasma triglyceride
• BMI body-mass index
• HbAl c hemoglobin A1 c
• Criteria for determining HDL, LDL, TG, BMI and HbA1 c are well known in the art, including established reference ranges and methodologies for determining baseline levels in a subject ⁇ e.g., Marshall, W.J. and Bangert, S.K., Clinical Biochemistry: Metabolic and Clinical Aspects (2008), Churchill
• a clinical benefit may result from increasing HDL and/or from decreasing LDL, TG, BMI and HbAl c, or at least that a clinical benefit may correlate with, respectively, such increases and/or decreases, in situations where a definitive cause-effect relationship has not been established. See, e.g., Talayero BG, Sacks FM, Curr Cardiol Rep. (201 1 ) 13(6):544-52, The role of triglycerides in atherosclerosis; Zalesin KC et al.
• the method comprises administering to the subject an agent that may be (a) an agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject, and/or (b) an agent that is an inhibitor of human cytochrome P450-family 8-subfamily B-polypeptide 1
• CYP8B1 sterol 12-a-hydroxylase activity in the subject.
• Methodologies for determining whether an agent decreases CYP8B1 expression levels and/or CYP8B1 activity levels are described herein and known in the art.
• expression levels of the CYP8B1 polypeptide may be determined by assaying for CYP8B1 polypeptides in a sample from a subject before and after exposure to the agent (e.g., by biochemical characterization of the sample for CYP8B1 polypeptides therein, or by immunochemical testing of the sample using specific anti-CYP8B1 antibodies), or by assaying for CYP8B1 -encoding mRNA levels in a sample from a subject before and after exposure to the agent [e.g., by northern blot hybridization using a CYP8B1 -specific probe, or by reverse transcription-PCR using CYP8B1 -specific oligonucleotide primers), or by
• n 0, 1 , 2, 3, 4 or 5;
• n 1 , 2 or 3;
• X is -N- or -C(R 6 )-
• R 1 is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, heterocyclylalkyl, or -OR 7 ;
• R 2 is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, heterocyclylalkyl, or -OR 7 ; or
• R 1 and R 2 connected to the same carbon form a spiro ring, which can be optionally substituted with alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, or heterocyclylalkyl;
• R 3 is the same or different and independently hydrogen, halogen, hydroxy, alkyl, alkoxy, aryl, cydoalkyi, heterocydyl, aralkyi, heteroaryl or heteroarylalkyi;
• R 4 and R 5 is independently hydrogen or alkyl
• R 6 is hydrogen or alkyl
• each R 7 is the same or different and independently hydrogen alkyl, aryl, cydoalkyi, heterocydyl, heteroaryl, aralkyi, heteroarylalkyi, cycloalkylalkyl, or heterocyclylalkyl, as a stereoisomer, enantiomer, tautomer thereof or mixtures thereof, or a pharmaceutically acceptable salt;
• — is independently a single or double bond
• each R 9 is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, aralkyl, cycloalkyi, heterocyclyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;
• R 15 is hydrogen or alkyl
• R 18 is hydrogen, hydroxy, alkoxy, or alkyl
• R 19 is hydrogen or alkyl
• t 0, 1 , 2, 3, 4 or 5;
• each R 9 is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;
• R 27 is the same or different and independently hydrogen, alkyl, halogen, acyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, heterocyclylalkyl, -OR 9 , -N(R 9 ) 2 -, or -SR 9 ; or two adjacent R 27 , together with the carbons to which they attach, form a fused aryl, heteroaryl, heterocyclyl, or cycloalkyi ring;
• R 28a and R 29a form a cycloalkyi or heterocyclyl ring; and each R and R is the same or different and independently hydrogen, alkyl, acyl, aralkyl, or heteroarylalkyl,
• Alkyl means a straight chain or branched, noncyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon containing from 1 to 10 carbon atoms.
• Representative saturated straight chain alkyls include methyl, ethyl, n- propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
• Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
• Cyclic alkyls are also referred to herein as “homocycles” or “homocyclic rings.”
• Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl” or “alkynyl", respectively).
• Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1 -butenyl, 2-butenyl,
• Alkoxy means an alkyl moiety attached through an oxygen bridge (i.e.,—O— alkyl) such as methoxy, ethoxy, and the like.
• Alkylthio means an alkyl moiety attached through a sulfur bridge (i.e., -S-alkyl) such as methylthio, ethylthio, and the like.
• Alkylsulfonyl means an alkyl moiety attached through a sulfonyl bridge (i.e., -SO2 -alkyl) such as methylsulfonyl, ethylsulfonyl, and the like.
• Alkylamino and dialkylamino mean one or two alkyl moieties attached through a nitrogen bridge (i.e., — N-alkyl) such as methylamino, ethylamino, dimethylamino, diethylamino, and the like.
• Aryl means an aromatic carbocyclic moiety such as phenyl or naphthyl.
• Arylalkyl means an alkyl having at least one alkyl hydrogen atom replaced with an aryl moiety, such as benzyl, ⁇ (CH 2 )2 phenyl, ⁇ (CH 2 )3 phenyl, - -CH(phenyl) 2 , and the like.
• Heteroaryl means an aromatic heterocycle ring of 5- to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and bicyclic ring systems.
• Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.
• Heteroarylalkyl means an alkyl having at least one alkyl hydrogen atom replaced with a heteroaryl moeity, such as ⁇ CH 2 pyridinyl, - CH 2 pyrimidinyl, and the like.
• Halogen means fluoro, chloro, bromo and iodo.
• Haloalkyl means an alkyl having at least one hydrogen atom replaced with halogen, such as trifluoromethyl and the like.
• Heterocycle (also referred to as a “heterocyclic ring”) means a 4- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring.
• the heterocycle may be attached via any heteroatom or carbon atom.
• Heterocycles include heteroaryls as defined above.
• heterocycles also include morpholinyl, pyrrol id inonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
• Heterocyclealkyl means an alkyl having at least one alkyl hydrogen atom replaced with a heterocycle, such as ⁇ CH 2 morpholinyl, and the like.
• Homocyde (also referred to herein as “homocyclic ring”) means a saturated or unsaturated (but not aromatic) carbocyclic ring containing from 3- 7 carbon atoms, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene, and the like.
• substituted means any of the above groups ⁇ e.g., alkyl, alkenyl, alkynyl, homocyde) wherein at least one hydrogen atom is replaced with a substituent.
• substituents may be further substituted with one or more of the above substituents, such that the substituent is substituted alkyl, substituted aryl, substituted arylalkyl, substituted heterocycle or substituted heterocyclealkyl.
• R a and R b in this context may be the same or different and independently hydrogen, alkyl, haloalkyi, substituted aryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl or substituted heterocyclealkyl.
• the agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject may be an antibody that specifically binds to a human CYP8B1 polypeptide as provided herein, or an antisense oligonucleotide, ribozyme or siRNA as provided herein that specifically interferes with human CYP8B1 expression.
• cardiovascular disease or disorder for treating or decreasing the likelihood of occurrence of a cardiovascular disease or disorder in a subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL) in the human subject, (ii) a decreased level of plasma low density lipoprotein (LDL) in the human subject, (iii) a decreased level of plasma triglyceride (TG) in the human subject, (iv) a decreased body-mass index (BMI) in the human subject, and (v) a decreased blood level of hemoglobin A1 c (HbA1 c) in the human subject, wherein at least one of (i)-(v) results from administering the subject agent.
• the cardiovascular disease or disorder may in certain embodiments be dyslipidemia, atherosclerosis, a disease characterized by low HDL levels, or another related disorder, the absence or presence of which may be determined according
• the present invention includes a method for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a subject in need thereof, comprising providing an agent to the subject, wherein the agent inhibits CYP8B1 .
• the agent reduces the expression and/or activity of CYP8B1 .
• the cardiovascular disease or disorder is dyslipidemia, atherosclerosis, a low HDL disease, or a related disorder.
• the agent is a small molecule that inhibits a biological activity of CYP8B1 , an antibody that specifically binds and inhibits CYP8B1 , or an antisense or siRNA comprising a sequence that binds a CYP8B1
• subject and “patient” are used interchangeably and refer to an individual having or at risk for having a particular disease or disorder.
• the subject is an animal, and preferably a mammal, most preferably a human.
• the subject's plasma HDL level is less than 10 mg/dl, less than 20 mg/dl, less than 30 mg/dl, less than 40 mg/dl, or less than 60 mg/dl prior to administration of the agent the reduces the expression and/or activity of CYP8B1 to the subject.
• a CYP8B1 inhibitor or antagonist may act by either preventing or reducing the expression of CYP8B1 or by preventing or reducing one or more CYP8B1 activities.
• a CYP8B1 inhibitor that reduces the expression of CYP8B1 may act to reduce expression at the mRNA level or the protein level, resulting in reduced amounts of CYP8P1 polypeptides.
• a CYP8B1 inhibitor that reduces an activity of CYP8B1 protein may bind to CYP8B1 .
• a CYP8B1 inhibitor may specifically inhibit or bind to a catalytic domain of a CYP8B1 protein.
• a CYP8B1 inhibitor may inhibit or bind a O2-binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide.
• a CYP8B1 inhibitor reduces or inhibits a hydroxylase activity of a CYP8B1 protein.
• the CYP8B1 inhibitor reduces or inhibits the conversion of 7a-hydroxy-4-cholesten-3-one (7-HCO) to 7a,12a-dihydroxy-4-cholesten-3-one (7,12-DiHCO) by CYP8B1 .
• treatment indicates an approach for obtaining beneficial or desired results, including and preferably clinically desirable results.
• Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.
• reducing the likelihood of occurrence include approaches for preventing, inhibiting, or decreasing the likelihood of the onset or recurrence of a disease or condition, in a manner that exhibits statistical significance, for example, when compared to the results obtained when the indicated method steps are omitted.
• preventing, inhibiting, or decreasing the likelihood of the occurrence or recurrence of the symptoms of a disease or condition or optionally delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition.
• prevention and similar words also include reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
• Methods according to these and related embodiments may be practiced using an effective amount or a therapeutically effective amount of an agent that inhibits CYP8B1 .
• an "effective amount” or a “therapeutically effective amount” of an agent or substance is that amount sufficient to affect a desired biological effect, such as beneficial results, including clinical results.
• the terms “disease” and “disorder” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms has been identified by clinicians.
• Cardiovascular diseases and disorders that may be treated or for which the likelihood of occurrence may be decreased ⁇ e.g., reduced in a statistically significant manner relative to control conditions in which the present embodiments are not practiced) according to the methods of the present invention include, but are not limited to, adrenoleukodystrophy, atherosclerosis, stroke, heart failure, Alzheimer's disease, angina, cardiovascular disease, cerebrovascular disease, congestive heart failure, coronary artery disease (or coronary heart disease), coronary microvascular disease, coronary restenosis, cystic fibrosis, diabetes, dyslipidemias, HDL-, familial HDL deficiency (FHA), hypercholesterolemia, hypertension, ischemic heart disease, metabolic syndrome, myocardial infarction, obesity, lipid disorders, low LDL diseases and related disorders (e.g., abetalipoproteinemia (ABL) and familial hypobetalipoproteinemia (FHBL)), peripheral arterial disease, peripheral vascular disease, progressive familial intrahepatic
• HDL has been implicated in many other biological processes, including but not limited to: prevention or reduction in the likelihood of occurrence of lipoprotein oxidation, absorption of endotoxins, protection against Trypanosoma brucei infection, modulation of endothelial cells and prevention or reduction in the likelihood of occurrence of platelet aggregation.
• Agents that modulate HDL levels by inhibiting CYP8B1 may also be used in modulating one or more of the foregoing processes.
• CYP8B1 functions to regulate HDL levels, links CYP8B1 with the foregoing processes.
• the expression or activity of CYP8B1 in the subject is reduced by not less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
• the target tissue where the expression or activity of CYP8B1 is measured or monitored is the liver.
• the expression or activity of CYP8B1 is inhibited by not more than 50%, 40%, 30%, or 10%.
• cardiovascular diseases would only be required to inhibit CYP8B1 activity by a maximum of 50% in order to be effective.
• the identified mutations are mild in their effects yet still correlate with increased HDL, and thus even a 40%, 30%, 20% or 10% inhibition of CYP8B1 function may be enough to raise plasma HDL or to treat or decrease likelihood of occurrence of cardiovascular diseases.
• the subject's plasma HDL level may be increased (with statistical significance) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to the HDL level prior to treatment.
• the subject's plasma HDL level is greater than 40 mg/dl or greater than 60 mg/dl at some time following administration of the agent that inhibits CYP8B1 ⁇ e.g., the agent that reduces the expression and/or activity of CYP8B1 ) to the subject.
• the method may comprises, prior to the step of administering the agent that is capable of decreasing CYP8B1 expression or activity in the subject, a method for identifying the human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject.
• HDL plasma high density lipoprotein
• LDL decreased level of plasma low density lipoprotein
• TG plasma triglyceride
• BMI body-mass index
• a decreased blood level of hemoglobin A1 c in the subject a decreased blood level of hemoglobin A1 c in the subject.
• the method further comprises the steps of (a) determining whether a candidate human subject has a reduced level of CYP8B1 activity relative to a control subject known to have a normal level of CYP8B1 activity, by testing a biological sample obtained from the candidate subject for presence of a mutant CYP8B1 polypeptide which comprises a mutation that results in decreased CYP8B1 activity, or for presence of a polynucleotide encoding said mutant CYP8B1 polypeptide, wherein the presence of said mutant CYP8B1 polypeptide or mutant CYP8B1 polypeptide-encoding polynucleotide indicates a reduced level of CYP8B1 activity; and (b) where the candidate subject does not exhibit a reduced level of CYP8B1 activity, administering to the subject the agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject or the agent that is an inhibitor of human cytochrome P450
• compositions comprising a CYP8B1 modulating agent, e.g., a CYP8B1 inhibitor or antagonist, and a pharmaceutically acceptable carrier, diluent or excipient.
• a pharmaceutically acceptable carrier, diluent or excipient includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration, Health Canada or the European Medicines Agency as being acceptable for use in humans or domestic animals.
• compositions may be administered in vivo to increase plasma HDL or for treating or decreasing likelihood of occurrence of (e.g., preventing) of a cardiovascular disease or disorder.
• Typical routes of administering the pharmaceutical compositions of the invention include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
• Pharmaceutical compositions of the invention are formulated so as to allow the agent contained therein to be bioavailable upon administration of the composition to a human.
• Agents are provided to a subject in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of subject to which the agent is administered; the mode and time of administration; the rate of excretion; the drug combination; and the type or severity of the condition to be treated.
• pharmaceutical compositions comprising an agent that is capable of decreasing a level of CYP8B1 expression or
• CYP8B1 activity in the subject may include one or more additional active agents, or may be administered in conjunction with one or more additional active agents.
• a CYP8B1 mutation as provided herein e.g., a reduced function or loss-of-function mutation such as those disclosed in Table 1 , in a CYP8B1 gene of a subject.
• These and related methods may be practiced advantageously, for example, to identify a subject as having a higher than average level of plasma HDL, or to determine the risk for or presence in a subject of CVD.
• a "loss-of-function mutation” refers to a mutation, either naturally occurring or synthesized, that results in either a lack of normal expression of the encoded polypeptide, or that results in a polypeptide that does not possess a functional characteristic of the non-mutated polypeptide.
• a loss-of-function mutation in CYP8B1 may result in little or no expression of the CYP8B1 polypeptide, or it may result in the expression of a CYP8B1 polypeptide that has little or no enzymatic activity.
• a subject is identified as having a mutation in the CYP8B1 gene by deternnining that the subject has at least one CYP8B1 gene (DNA) sequence that encodes a CYP8B1 polypeptide (amino acid) sequence which comprises one of the mutations presented in Table 1 .
• Certain embodiments described herein therefore provide a method for identifying a human subject having reduced CYP8B1 activity, comprising determining if a polynucleotide sequence of a CYP8B1 gene in a biological sample obtained from said subject encodes a CYP8B1 sequence comprising at least one mutation selected from those set forth in Table 1 , and in preferred embodiments those characterized in Table 1 as loss-of-function mutations.
• CYP8B1 SNP or the resultant mutated CYP8B1 polypeptide as disclosed herein (e.g., Table 1 ), including determination for each SNP of its influence on or correlation with a relevant phenotype ⁇ e.g., complete LOF, partial LOF, association with one or more of elevated HDL, decreased LDL, decreased plasma triglycerides, decreased BMI, decreased blood level of HbA1 c).
• Such profiles may define parameters indicative of a subject's predisposition to develop a cardiovascular disease or related disorder, and may further be useful in the identification and definition of novel subtypes of such disorders.
• correlation of one or more phenotypic traits in a subject with at least one of the CYP8B1 mutations set forth in Table 1 may be used to gauge the subject's responsiveness to, or the efficacy of, a particular therapeutic treatment.
• determination of the presence of one or more of the SNPs presented in Table 1 may therefore also be used to stratify a patient population of human subjects according to risk for or presence of a cardiovascular disease that would be ameliorated by one or more of (i) an increased level of plasma high density lipoprotein (HDL) in one or more of the subjects, (ii) a decreased level of plasma low density lipoprotein (LDL) in one or more of the subjects, (iii) a decreased level of plasma triglyceride (TG) in one or more of the subjects, (iv) a decreased body-mass index (BMI) in one or more of the subjects, and (v) a decreased blood level of hemoglobin A1 c in the subject.
• HDL plasma high density lipoprotein
• LDL decreased level of plasma low density lipoprotein
• TG plasma triglyceride
• BMI body-mass index
• polymorphism that is associated with decreased risk for the cardiovascular disease wherein presence of said at least one polymorphism indicates decreased risk for the cardiovascular disease, may thereby permit stratifying the population according to cardiovascular disease risk.
• determination of levels of at least one CYP8B1 SNP (or the resultant mutated CYP8B1 polypeptide) in a biological sample from a subject may provide a useful correlative indicator for that subject.
• a subject so classified based on the presence of at least one CYP8B1 mutation may be monitored using art- accepted CVD clinical parameters referred to herein, such that correlation between a particular CYP8B1 mutation (and/or the level of CYP8B1 expression and/or activity in each subject) and any particular clinical score used to evaluate CVD or a related disorder may be monitored.
• stratification of a CVD patient population according to incidence of one or more of the CYP8B1 mutations disclosed herein ⁇ e.g., Table 1 ) may provide useful markers by which to correlate the relative efficacy of any candidate therapeutic agent being used in CVD patients.
• a CYP8B1 mutation may be detected by determining the polynucleotide or amino acid sequence of a CYP8B1 gene or mRNA or protein and comparing it with a wild type CYP8B1 gene or mRNA or protein sequence.
• CYP8B1 polynucleotide sequence obtained from a biological sample of a nucleotide sequence encoding a M53T mutation, a K300X mutation, a D341 E mutation, a Q372K mutation, or any of the other CYP8B1 mutations set forth in Table 1 indicates presence of a CYP8B1 mutation in the subject from which the sample was derived. Additional functional mutations can be identified by comparing the sample CYP8B1 sequence with the wild type CYP8B1 sequence and further evaluating the expression and activity of the CYP8B1 from the sample.
• the presence of a gene allele or mRNA comprising a CYP8B1 mutation described herein ⁇ e.g., Table 1 ) in a subject, or in a biological sample obtained from a subject, may be determined using a variety of techniques, including hybridization-based assays employing a polynucleotide primer that specifically binds to a CYP8B1 polynucleotide sequence comprising a mutation described herein and that does not bind to a wild-type CYP8B1 polynucleotide sequence. In other techniques such as SNP detection techniques,
• oligonucleotide primers may be complementary to wildtype sequence regions adjacent to a SNP, which is then identified by extension of the primer using the SNP-containing polynucleotide as a template, followed by amplification and sequencing or other sequence-dependent characterization of the extended sequence to reveal the presence of the SNP (i.e., as a deviation from the wildtype sequence).
• exemplary and non-limiting methodologies for mutation detection in a polynucleotide include polymerase chain reaction (PCR, Gibbs et al., Nucl. Ac. Res.
• TAS transcriptional amplification systems
• SDA strand displacement amplification
• SR self-sustained sequence replication
• LCR ligase chain reaction
• RFLP restriction fragment length polymorphism
• oligonucleotide primers and probes should comprise an oligonucleotide sequence that is at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, and preferably at least 20 nucleotides, in length.
• oligonucleotide primers and/or probes hybridize to a polynucleotide comprising a CYP8B1 SNP sequence described herein under moderately stringent conditions, as defined above.
• Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
• the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a CYP8B1 polynucleotide sequence having a mutation as disclosed herein.
• PCR amplification using at least one specific primer that hybridizes to a mRNA sequence comprising a CYP8B1 mutation described herein generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis.
• a detectable label may be incorporated into the generated cDNA, and the presence of a CYP8B1 sequence comprising a mutation described herein detected based upon detection of the label, e.g., a fluorescent label.
• nucleic acid segments that can be advantageously used as probes or primers for nucleic acid hybridization, e.g., in diagnostic assays to determine if a subject has one of the CYP8B1 mutations described herein ⁇ e.g., mutations set forth in Table 1 ).
• nucleic acid segments that comprise a sequence region of at least about 12 or 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 12 to 15 nucleotide long contiguous sequence disclosed herein will find particular utility.
• an oligonucleotide primer comprises a mutated CYP8B1 gene sequence described herein, such as an isolated polynucleotide comprising an oligonucleotide of at least 10 contiguous nucleotides and not more than 1506, 1505, 1504, 1503, 1502, 1501 , 1500, 1000, 500, 400, 300, 200, 150, 100, 75, 50, 40, 30, 20 or 15 contiguous nucleotides of a human CYB8B1 -encoding sequence as set forth in SEQ ID NO:2 which encodes a human CYP8B1 polypeptide as set forth in SEQ ID NO:1 , wherein the oligonucleotide comprises at least one nucleotide
• oligonucleotide primers comprising a sequence corresponding to a region of a CYP8B1 sequence that encodes a mutation presented in Table 1 , such as a M53T mutation, a K300X mutation, a D341 E mutation, or a Q372K mutation, or a complement thereof, can be used in high density oligonucleotide array technology ⁇ e.g.,
• kits of the present invention may be used for diagnostic or treatment methods.
• a kit of the present invention may further provide instructions for use of a composition or agent and packaging.
• a kit comprises one or more polynucleotide primers that may be used to amplify a wild-type or mutant CYP8B1 gene from a biological sample obtained from a subject.
• one of the primers encodes a CYP8B1 sequence which comprises a M53T mutation, a K300X mutation, a D341 E mutation, or a Q372K mutation, or any of the other CYP8B1 mutations set forth in Table 1 .
• a diagnostic kit comprises both a first primer that comprises a sequence encoding a CYP8B1 mutation that appears in Table 1 (e.g., a K300X mutation, a D341 E mutation, or a Q372K mutation) and a second primer that comprises a sequence encoding a wild-type CYP8B1 sequence, such that the first and second primer may be used together to amplify, e.g., by PCR, a CYP8B1 polynucleotide comprising the mutation present in the first primer.
• Diagnostic kits useful in identifying wild-type or CYP8B1 mutations in a subject may further comprise additional agents useful in performing PCR, such as a Taq polymerase and polynucleotide mixture.
• kits comprises one or more agents capable of reducing the expression or activity of a CYP8B1 .
• a kit may optionally also include devices, reagents, containers or other components.
• kit may also be designed to operate through the use of an apparatus, instrument or device, including a computer.
• the present invention provides methods of identifying an agent for treating or reducing the likelihood of occurrence of a cardiovascular disease or disorder in a human subject, such as an agent that is capable of inhibiting CYP8B1 expression or activity.
• an inhibitor of CYP8B1 activity prevents or reduces or otherwise substantially impairs ⁇ e.g., decreases in a statistically significant manner relative to the result that pertains when the agent is not present) the capability of CYP8B1 to convert CYP8B1 substrate to a downstream product, e.g., 7,12-diHCO or cholic acid.
• CYP8B1 can be contacted with a test agent in the presence of a substrate of CYP8B1 , and an inhibitor of
• CYP8B1 will prevent or reduce the conversion of the substrate in comparison to the conversion of the substrate by CYP8B1 in the absence of the inhibitor.
• Conversion of the substrate can be determined using methods known in the art (Ahlberg, J. et al., J. Lip. Res., 20:107-1 15, 1979; Ishada, H. et al., J. Biol.
• an inhibitor or antagonist of CYP8B1 expression prevents or reduces the expression of CYP8B1 mRNA or protein.
• CYP8B1 mRNA levels in biological samples that either have been contacted with the test agent, or that have not been so contacted, can be measured, for example, by reverse transcriptase polymerase chain reaction (RT-PCR).
• RT-PCR reverse transcriptase polymerase chain reaction
• a lower CYP8B1 mRNA level in the sample that has been contacted with the test agent in comparison to the untreated sample indicates that the test agent is an inhibitor of CYP8B1 mRNA expression.
• biological samples include cells that express CYP8B1 .
• the amount of CYP8B1 protein produced in a biological sample contacted with a test agent can be compared to the amount of CYP8B1 protein produced in an untreated biological sample to determine if the test agent is an inhibitor of CYP8B1 protein expression.
• the amount of CYP8B1 protein may be measured by, for example, an enzyme linked immunosorbant assay (ELISA).
• ELISA enzyme linked immunosorbant assay
• determining the level of expression of CYP8B1 thereby determining a test level of CYP8B1 expression; and comparing the base level of CYP8B1 expression and the test level of CYP8B1 expression, wherein a test level that is less than the base level of CYP8B1 expression indicates the agent may be used to treat or decrease likelihood of occurrence of a cardiovascular disease or disorder.
• methods that recite measuring CYP8B1 expression or activity in a cell include measuring CYP8B1 expression levels or activity in one or more populations of cells. For example, expression or activity may be measured in a first population of cells in the absence of a test agent to determine a base level, and expression or activity may be measured in a second population of cells contacted with the test agent to determine a test level.
• the two populations of cells are the same cell type and/or are obtained from the same source, e.g., a cell culture may be divided to produce both the first and second population of cells.
• methods used to determine expression levels or activity of a polynucleotide or protein may require more than one cell. In addition, the methods may result in the descruction of the cells, such that a different cell or cell population must be used for comparative purposes.
• a method for identifying an agent for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder may comprise the steps of determining the level of expression of CYP8B1 by a cell that is not contacted with the agent, determining the level of expression of CYP8B1 by a cell that is contacted with the agent; and comparing the determined levels, wherein a lower level of expression by the cell that has been contacted with the agent indicates the agent may be used to increase HDL or to treat or decrease likelihood of occurrence of a cardiovascular disease or disorder.
• the cells are the same type and express a comparable amount of CYP8B1 when grown under comparable conditions.
• screening methods are practiced using a population of cells, wherein certain cells of the population are contacted with a test agent, and other cells of the population are not.
• determining the level of expression of CYP8B1 comprises measuring the amount of CYP8B1 mRNA. In another embodiment, determining the level of expression of CYP8B1 comprises measuring the amount of CYP8B1 protein.
• a method for identifying an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, which method comprises comparing (i) a base level of CYP8B1 polypeptide expression by a first cell that has not been contacted with a candidate agent, to (ii) a test level of the CYP8B1 polypeptide expression by a second cell that has been contacted with the candidate agent, wherein a determination that the test level of CYP8B1 polypeptide expression is less than the base level of CYP8B1 polypeptide expression indicates the candidate agent is
• CYP8B1 protein using, respectively, methodologies for quantifying a specific mRNA or a specific protein that are well within the knowledge of the art.
• a method for identifying an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, comprising comparing (i) a base level of CYP8B1 activity by a first CYP8B1 polypeptide, or a fragment or variant thereof, that has not been contacted with a candidate agent, to (ii) a test level of the CYP8B1 activity by a second CYP8B1
• each of the first and second CYP8B1 polypeptides comprises a CYP8B1 catalytic domain.
• CYP8B1 polypeptides or fragment or variant thereof, comprises a substrate access channel, a steroidogenic region or product egress channel, or a heme prosthetic group interface domain of the respective CYP8B1 polypeptide.
• Protein X-ray crystal structures have demonstrated that mammalian cytochrome P450 enzymes all have a common fold, or tertiary structure, and contain two domains: (i) a short N-terminal membrane binding domain of approximately 25 amino acids, and (ii) the remainder of the protein containing the amino acids involved in the binding of the essential heme cofactor.
• This second region is involved in forming an interface with the redox partners, NADPH-P450 reductase and cytochrome b5, the substrate binding site, and a channel or channels that allow the access and egress of substrates and products, respectively, from the enzyme active site (Otyepka et al., Biochim Biophys Acta. 1770:376-89, 2007; Cojocaru et al., Biochim Biophys Acta, 1770:390-401 , 2007; Denisov et al., J Inorg Biochem, 108:150-8, 2012).
• CYP8A1 (42% sequence identity to CYP8B1 , Protein Data Base (PDB) entry 3B6H)
• CYP7A1 (36% sequence identity to CYP8B1 , PDB entry 3SN5; see also PDB entry 3DAX).
• CYP8B1 homology model by which can be identified the specific amino acids that are involved in the interaction of CYP8B1 with its heme cofactor ⁇ e.g., the heme prosthetic group interface domain), and by which can also be identified the specific amino acids of CYP8B1 that form the substrate binding site, the substrate access and product egress channel(s) ⁇ e.g., steroidogenic region), and the interface with the redox partners.
• Cytochrome P450 crystal structures have shown that specific compounds can interact with the enzyme by coordinating, via a nitrogen- containing moiety, to the heme iron ⁇ e.g., ritonavir binding CYP3A4, PDB entry 3NXU), or by binding in the CYP substrate-binding site ⁇ e.g., S-warfarin binding CYP3A4, PDB entry 3NXU and Schoch et a ⁇ ., J Biol Chem, 283:17227-37, 2008). Interactions of inhibitors with either the heme iron or substrate binding site can be identified by visible spectroscopy.
• Ligands that coordinate to the heme iron cause a shift in the heme Soret absorption band resulting in a Type II difference spectrum, whereas those interacting with the substrate binding site result in a distinct Type I difference spectrum.
• Inhibitors that do not cause an appreciable Soret band shift may be expected to bind in either the access/egress channel(s) or at the interface with the redox partners.
• an agent for treating, or decreasing likelihood of occurrence of, a cardiovascular disease or disorder in a human subject who would benefit from one or more of (i) an increased level of plasma high density lipoprotein (HDL), (ii) a decreased level of plasma low density lipoprotein (LDL), (iii) a decreased level of plasma triglyceride (TG), (iv) a decreased body-mass index (BMI), and (v) a decreased blood level of hemoglobin A1 c in the subject, comprises an agent that inhibits a sterol 12-a- hydroxylase activity.
• HDL plasma high density lipoprotein
• LDL decreased level of plasma low density lipoprotein
• TG plasma triglyceride
• BMI body-mass index
• a decreased blood level of hemoglobin A1 c in the subject comprises an agent that inhibits a sterol 12-a- hydroxylase activity.
• the agent may specifically bind to a CYP8B1 polypeptide, and may in certain embodiments specifically bind to a CYP8B1 substrate access channel, or to a CYP8B1 steroidogenic region or product egress channel, or to a heme prosthetic group interface domain of the CYP8B1 polypeptide.
• An agent that specifically binds to a CYP8B1 steroidogenic region or product egress channel may include any agent that specifically binds to the CYP8B1 sterol 12-a-hydroxylase enzyme active site, and preferably inhibits enzyme activity in a statistically significant manner, which may include complete, substantial or partial inhibition of sterol 12-a-hydroxylase enzyme activity.
• An agent that specifically binds to a CYP8B1 heme prosthetic group interface domain includes an agent that inhibits sterol 12-a-hydroxylase activity and that interferes with heme binding to the CYP8B1 polypeptide and which may be readily detected on this basis.
• An agent that specifically binds to a CYP8B1 heme prosthetic group interface domain includes an agent that inhibits sterol 12-a-hydroxylase activity and that interferes with heme binding to the CYP8B1 polypeptide and which may be readily detected on this basis.
• CYP8B1 access channel includes an agent that inhibits sterol 12-a-hydroxylase enzyme activity in statistically significant manner, including complete, substantial or partial inhibition of sterol 12-a-hydroxylase enzyme activity, but which does so without detectably binding to the CYP8B1 enzymatic active site and also without detectably binding to CYP8B1 heme prosthetic group, nor to the CYP8B1 heme prosthetic group interface domain (i.e., the specific CYP8B1 amino acids that are involved in the interaction of CYP8B1 with its heme cofactor).
• Another embodiment provides an agent useful for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, wherein the agent is identified according to a method described above.
• the agent specifically binds to a polynucleotide sequence encoding CYP8B1 or a complement thereof.
• the agent comprises a siRNA or an antisense oligonucleotide.
• the agent is a small molecule or an antibody.
• the present invention provides a method for identifying an agent for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, comprising the steps of: measuring an activity of CYP8B1 , thereby determining a base level of activity; contacting CYP8B1 with a test agent; measuring the activity of
• CYP8B1 thereby determining a test level of activity; and comparing the base level and the test level of activity, wherein a test level that is less than the base level of CYP8B1 activity indicates the agent may be used to increase plasma HDL or to treat or decrease likelihood of occurrence of a cardiovascular disease or disorder.
• the CYP8B1 activity is conversion of a substrate.
• the present invention includes a method for identifying an agent for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, comprising the steps of: determining the level of activity of CYP8B1 by a cell that has not been contacted with the agent, determining the level of activity of CYP8B1 by a cell that has been contacted with the agent; and comparing the determined levels, wherein a lower level of activity by the cell that has been contacted with the agent indicates the agent may be used to increase HDL or to treat or decrease likelihood of occurrence of a cardiovascular disease or disorder.
• the cells are the same type and exhibit a comparable amount of CYP8B1 activity when grown under comparable conditions.
• Another embodiment provides an agent useful for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, wherein the agent is identified according to a herein-described method.
• the agent inhibits the conversion of a CYP8B1 substrate by CYP8B1 .
• the agent specifically binds to CYP8B1 .
• the agent is a small organic molecule or an antibody that binds specifically to CYP8B1 .
• Another embodiment of the invention provides a method for identifying an agent for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, comprising the steps of: measuring an activity of a CYP8B1 polypeptide comprising a catalytic domain of CYP8B1 , or a variant or a fragment thereof, thereby determining a base level of activity; contacting the CYP8B1 polypeptide comprising the catalytic domain, or the variant or a fragment thereof, with a test agent;
• the CYP8B1 polypeptide comprises a substrate access channel, a steroidogenic region or product egress channel, or a heme prosthetic group interface domain ⁇ e.g., a heme binding domain) of the CYP8B1 polypeptide.
• a related embodiment provides a method for identifying an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, comprising the steps of: measuring an activity of a CYP8B1 polypeptide comprising a catalytic domain of CYP8B1 in the absence of the test agent; measuring the activity of the CYP8B1 polypeptide in the presence of the test agent; and comparing the two levels of activity measured, wherein a lower measured activity in the presence of the test agent indicates that the agent may be used to treat or decrease likelihood of occurrence of a cardiovascular disease or disorder.
• the CYP8B1 polypeptide comprises an O2-binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide.
• Another embodiment provides a method for identifying an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, comprising the steps of: contacting a CYP8B1 polypeptide comprising a catalytic domain of CYP8B1 , or a variant or a fragment thereof, with a test agent, and determining whether the test agent binds to the CYP8B1 polypeptide, wherein binding of the test agent to the polypeptide identifies the test agent as an agent useful for increasing plasma HDL or for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder.
• the CYP8B1 polypeptide comprises a substrate access channel, a steroidogenic region or product egress channel, or a heme prosthetic group interface domain ⁇ e.g., a heme binding domain) of the CYP8B1 polypeptide.
• Another embodiment provides an agent useful in treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder, wherein the agent is identified according to one of the above methods.
• the agent specifically binds to CYP8B1 .
• the agent is an antibody that binds specifically to CYP8B1 .
• the agent is a small molecule.
• an agent identified according to a method described herein or used according to a method described herein specifically binds to a catalytic domain of a CYP8B1 protein.
• the agent binds to an O2-binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide.
• the CYP8B1 protein is a human CYP8B1 protein, and in one embodiment, the CYP8B1 protein is a wild-type human CYP8B1 protein.
• CYP8B1 Any agent that inhibits or reduces the expression level or activity of CYP8B1 may be used to practice the herein described methods, i.e., any CYP8B1 inhibitor or antagonist.
• a CYP8B1 inhibitor may be an antagonist of a CYP8B1 functional activity or expression level.
• a CYP8B1 inhibitor may also include an agent that specifically binds to a catalytic domain of CYP8B1 or variants or fragments thereof.
• an agent is considered to specifically bind to a polypeptide, e.g., a catalytic domain of CYP8B1 , if it binds to the polypeptide with at least two-fold, three-fold, five-fold, or ten-fold higher affinity than the affinity with which it binds to a structurally unrelated control polypeptide.
• a polypeptide e.g., a catalytic domain of CYP8B1
• polypeptides and polynucleotides are human CYP8B1 polypeptides and polynucleotides, and in related embodiments, the herein described methods and agents reduce or inhibit the expression and/or activity of human CYP8B1 .
• CYP8B1 inhibitors include, but are not limited to, small molecules ⁇ e.g., small organic molecules, such as a drug or prodrug); antibodies or fragments thereof; proteins, polypeptides and peptide fragments; and polynucleotides, including, e.g., expression vectors, siRNA, antisense oligonucleotides; and the like.
• CYP8B1 inhibitor or
• CYP8B1 antagonist refers to agents or compounds that inhibit the expression ⁇ e.g., level) or an activity of a CYP8B1 polypeptide by at least or at least about 10%, at least or at least about 15%, at least or at least about 20%, at least or at least about 25%, at least or at least about 30%, at least or at least about 35%, at least or at least about 40%, at least or at least about 45%, at least or at least about 50%, at least or at least about 55%, at least or at least about 60%, at least or at least about 65%, at least or at least about 70%, at least or at least about 75%, at least or at least about 80%, at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, at least or at least about 99%, or 100% as compared with a control or reference sample or compound.
• the inhibition may be over two-fold, or over five-fold, or over 10-fold, or over 100-fold, or over 300-fold, or over 500-fold or over 1000-fold, when compared with a control or reference sample or compound.
• CYP8B1 antagonists include competitive antagonists (i.e., antagonists that compete with an agonist for binding to CYP8B1 ) and noncompetitive antagonists.
• CYP8B1 antagonists include antibodies. The antibodies may be monoclonal. They may be human or humanized antibodies.
• CYP8B1 antagonists also include polypeptides and nucleic acids that bind to CYP8B1 polypeptides or polynucleotides and inhibit CYP8B1 activity or expression. The CYP8B1 antagonists may be selective or mixed CYP8B1 antagonists.
• the present invention contemplates the use of polypeptide inhibitors of CYP8B1 .
• the term CYP8B1 As used herein, the term
• polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
• the polypeptides are not limited to a specific length; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
• This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
• a polypeptide may be an entire protein, or a partial sequence thereof. Particular polypeptides of interest are modulators of CYP8B1 activity or expression levels.
• polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a CYP8B1 polypeptide.
• an inhibitor of CYP8B1 is a polypeptide comprising or consisting of a fragment of a CYP8B1 polypeptide.
• Such a polypeptide may act as a dominant-negative inhibitor of a CYP8B1 activity.
• Polypeptides may be prepared using any of a variety of well known synthetic and/or recombinant techniques, the latter of which are further described below.
• Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those skilled in the art.
• such polypeptides are synthesized using any of the commercially available solid- phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc, 85:2149-2146, 1963.)
• Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.
• Certain embodiments contemplate the use of antibodies that specifically bind to a CYP8B1 protein, or variants or fragments thereof, as CYP8B1 antagonists. Accordingly, the present invention provides such antibodies, and variants or fragments thereof, as well as the methods and reagents used to produce them. As will be understood by the skilled artisan, general description of antibodies herein and methods of preparing and using the same also apply to individual antibody polypeptide constituents and antibody fragments.
• antibody as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity, e.g., specifically bind to CYP8B1 and inhibit or antagonize CYP8B1 function.
• immunoglobulin Ig is used interchangeably with “antibody” herein.
• an "isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• the antibody is purified: (1 ) to greater than 95% by weight of antibody as determined by the Bradford method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, silver stain.
• Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
• antibody fragment is a polypeptide comprising or consisting of a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
• antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641 ,870; Zapata et al., 1995 Protein Eng. 8(10): 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
• immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
• the antibodies described for use herein may be polyclonal or monoclonal antibodies. In particular embodiments, they are monoclonal.
• an immunogen comprising a CYP8B1 polypeptide or antigenic portion thereof is initially injected into a suitable animal ⁇ e.g., mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule incorporating one or more booster immunizations.
• a suitable animal e.g., mice, rats, rabbits, sheep and goats
• an immunogen may be linked to, for example, glutaraldehyde or keyhole limpet hemocyanin (KLH).
• polyclonal antibodies may then be purified from such antisera by, for example, affinity chromatography using a CYP8B1 polypeptide or antigenic portion thereof coupled to a suitable solid support.
• Such polyclonal antibodies may be used directly, e.g., for screening purposes and Western blots.
• monoclonal antibodies may be desired.
• Monoclonal antibodies may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:51 1 -519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines ⁇ i.e., hybridomas) capable of producing antibodies having the desired specificity ⁇ i.e., reactivity with the polypeptide of interest).
• Hybridoma cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal.
• the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
• a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
• Antigen-specific repertoires can be recovered from immunized animals by hybridoma technology as described above, single-cell RT-PCR for selected B cells, antibody display technologies, and other methods known in the art.
• antibodies would be secreted into the culture supernatant and can be isolated by means known in the art such as ammonium sulfate precipitation and column chromatography using protein A, protein G, etc.
• Such isolated antibody can be used for further testing and characterization of the antibody to determine potency in vitro and in vivo, affinity, etc.
• antibodies may be produced recombinantly, using vectors and methods available in the art, as described further below.
• the variable regions of a monoclonal antibody can be recovered and sequenced by standard molecular biology methods, such as RT-PCR.
• the polynucleotide sequences encoding the H and L chains can be cloned into a suitable expression vector known in the art and transfected into a suitable host cell ⁇ e.g., mammalian cells, yeast cells, bacteria) to secrete antibody into the culture supernatant.
• Other methods of production include generating ascites by injecting hybridoma cells into the peritoneal cavity of an animal ⁇ e.g., mice), transgenic animals that secrete the antibody into milk or eggs, and transgenic plants that make antibody in the fruit, roots or leaves.
• the recombinant antibody can be isolated by various methods such as affinity chromatography.
• antibodies are fully human antibodies.
• Human antibodies may be generated by in vitro activated B cells (see U.S.).
• human antibodies may also be produced in transgenic animals ⁇ e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous
• Such animals may be genetically engineered to produce human antibodies that specifically recognize CYP8B1 polypeptides including mutant CYP8B1 polypeptides described herein.
• antibodies are chimeric antibodies that comprise sequences derived from both human and non-human sources. In particular embodiments, these chimeric antibodies are humanized or
• humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some
• immunoglobulin framework (FR) residues are substituted by residues from analogous sites in rodent antibodies.
• Chimeric antibodies for use as described herein may also include fully human antibodies wherein the human hypervariable region or one or more complementarity determining regions (CDRs) are retained, but one or more other regions of immunoglobulin sequence have been replaced by
• chimeric antibodies retain high binding affinity for the desired antigen ⁇ e.g., a CYP8B1 polypeptide or fragment or variant thereof as provided herein) and other favorable biological properties.
• chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using three- dimensional models of the parental human and non-human sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
• a specific anti-CYP8B1 antibody that has activity as an inhibitor of CYP8B1 may specifically inhibit or bind to a catalytic domain of a CYP8B1 protein, or a fragment or variant thereof.
• a CYP8B1 antibody inhibitor may inhibit or bind an O 2 -binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide.
• a CYP8B1 antibody inhibitor reduces or inhibits a hydroxylase activity of a CYP8B1 polypeptide, or of a fragment or variant thereof.
• the CYP8B1 antibody inhibitor reduces or inhibits the conversion of 7a-hydroxy-4-cholesten-3-one (7-HCO) to 7a,12a-dihydroxy-4-cholesten-3- one (7,12-DiHCO) by CYP8D1 .
• a CYP8B1 antagonist may be a polynucleotide.
• polynucleotide refers to a DNA or RNA (or mixed) molecule that has been isolated free of total genomic DNA of a particular species.
• polynucleotides may be single-stranded ⁇ e.g., coding or antisense) or double-stranded (or include both single- and double-stranded regions), and may be DNA ⁇ e.g., genomic, cDNA or synthetic) or RNA molecules (or include regions of both DNA and RNA).
• RNA molecules may include, but are not limited to, HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns, and fragments and variants thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. Polynucleotides may comprise a native sequence (i.e., an
• Polynucleotides may consist of natural and/or non-natural bases.
• polynucleotide fragments may comprise various lengths of contiguous stretches of sequence identical to or complementary to a CYP8B1 -encoding polynucleotide sequence.
• polynucleotides are provided that comprise at least about 10, 12, 15, 18, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 1500, 1501 , 1502, 1503, 1504, 1505, 1506 or more contiguous nucleotides of one or more of the sequences disclosed herein (e.g., SEQ ID NO:2 or mutated versions of SEQ ID NO:2 that contain one or more of the SNPs set forth in Table 1 ) as well as all intermediate lengths therebetween.
• intermediate lengths means any length between the quoted values, such as 1 1 , 12, 13, 14, etc.; 16, 17, 18, 19, etc.; 21 , 22, 23, etc.; 30, 31 , 32, etc.; 50, 51 , 52, 53, etc.; 100, 101 , 102, 103, etc.; 150, 151 , 152, 153, etc.; including all integers through 200-500; 500-1 ,000, and the like.
• polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a CYP8B1 gene or mRNA sequence, or to a fragment thereof, or to a
• polynucleotide composition may hybridize to a CYP8B1 gene or mRNA sequence comprising a CYP8B1 mutation as presently disclosed (e.g., a mutation presented in Table 1 ) to a greater extent than the extent to which it hybridizes to a wild-type CYP8B1 gene or mRNA.
• the polynucleotide composition selectively hybridizes to a mutant CYP8B1 gene or mRNA sequence that comprises at least one of the mutations disclosed in Table 1 but does not hybridize to a wild-type CYP8B1 gene or mRNA.
• Hybridization techniques are well known in the art of molecular biology and are also described herein.
• Small polynucleotide segments or fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent No.
• methods of the present invention are practiced using antisense polynucleotides that target a CYP8B1 mRNA, thereby reducing expression of CYP8B1 .
• Antisense oligonucleotides have been demonstrated to be effective and specifically targetable inhibitors of protein synthesis, and, consequently, provide a therapeutic approach by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease.
• the efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of
• polygalacturonase and the muscarinic type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Patent Nos. 5,739,1 19 and 5,759,829). Further, examples of antisense inhibition have been demonstrated with the multiple drug resistance gene (MDG1 ), ICAM-1 , and human EGF (U.S. Patent Nos. 5,801 ,154;
• Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g., cancer (U.S. Patent Nos. 5,747,470 and 5,783,683).
• the present invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to a CYP8B1 gene or mRNA, or a complement thereof.
• the antisense oligonucleotides comprise DNA or derivatives thereof.
• the oligonucleotides comprise RNA or derivatives thereof.
• the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone.
• the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof.
• the antisense oligonucleotide compositions may comprise one DNA strand and one RNA strand in a duplex, wherein either the DNA strand or the RNA strand may be the antisense sequence (see, e.g., U.S. Patent Application Publication No. 2008/0085999).
• preferred compositions comprise a sequence region that is complementary, and more preferably substantially complementary, and even more preferably, completely complementary to a CYP8B1 gene or mRNA.
• RNA interference refers to the mechanism by which short single-stranded RNA (ssRNA) binds to a complementary mRNA sequence to form double-stranded RNA (i.e., duplex RNA or dsRNA) and mediates the degradation and/or inhibits the translation of the specific mRNA (see, e.g., U.S. Patent Application Publication No. 2008/0221054).
• ssRNA short single-stranded RNA
• dsRNA double-stranded RNA
• Duplex RNA can activate the RNA-induced silencing complex (RISC) to degrade target mRNA.
• RISC RNA-induced silencing complex
• interfering RNA or "interfering RNA sequence” as used herein refers to RNA that targets (i.e., silences, reduces, or inhibits) expression of a target gene (i.e., by mediating the degradation of mRNAs which are complementary to the antisense sequence of the interfering RNA) when the interfering RNA is in the same cell as the target gene.
• Interfering RNA thus refers to the double stranded RNA formed by two complementary strands or by a single, self-complementary strand.
• Interfering RNA further refers to ssRNA that is derived from duplex RNA and is
• Interfering RNA typically has substantial or complete sequence identity to all or a portion of the target gene.
• the sequence of the interfering RNA can correspond to the full length target gene, or a subsequence thereof.
• Interfering RNA includes, but is not limited to, "small-interfering RNA
• RNA or “siRNA,” “short hairpin RNA” or “shRNA,” and “microRNA” or “miRNA,” i.e., interfering RNA of about 15-60, 15-50, 15-50, or 15-40 nucleotides in length, more typically about, 15-30, 15-25 or 19-25 nucleotides in length, and is often about 20-24 or about 21 -22 or 21 -23 nucleotides in length (e.g., each complementary sequence of an siRNA or miRNA duplex is 15-60, 15-50, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides in length, often about 20-24 or about 21 -22 or 21 -23 nucleotides in length, and the double stranded siRNA is about 15-60, 15-50, 15-50, 15-40, 15-30, 15-25 or 19-25 often about 20-24 or about 21 -22 or 21 -23 base pairs in length).
• siRNA and miRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides, preferably of about 2 to about 3 nucleotides and 5' phosphate termini (see, e.g., U.S. Patent Nos. 7,056,704 and 7,078,196).
• the siRNA lacks a terminal phosphate.
• the siRNA or miRNA duplex lacks 3' overhangs, i.e., has "blunt-ends".
• siRNA examples include, without limitation, a double-stranded polynucleotide molecule assembled from two separate oligonucleotides, wherein one strand is the sense strand and the other is the complementary antisense strand (see, e.g., U.S. Patent Application Publication No.
• WO 2006/074108 a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions (e.g., shRNA, see, Wang et al., Molecular Therapy, 12(3):562-568, 2005; lives et al., Ann. N. Y. Acad. Sci., 1082:52-55, 2006; and Vlassov et al.
• RNA refers to siRNA derived from a virus.
• miRNA typically refers to naturally occurring, i.e., endogenous, non-coding RNA that induces RNAi (see, U.S. Patent Nos. 7,387,896 and 7,459,547).
• miRNA is derived from "pre-microRNA” or "pre-miRNA” that typically has a hairpin structure having self-complementary sense and antisense regions or a single-stranded stem-loop structure having self- complementary sense and antisense regions.
• Pre-miRNA can be cleaved by Dicer into an miRNA duplex.
• the sense and antisense strands of the miRNA duplex unwind.
• the antisense strand is complementary to and interacts with the target mRNA to drive its degradation, e.g., by an argonaute protein of the RISC.
• siRNA can be chemically synthesized or may be encoded by a plasmid ⁇ e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops). siRNA can also be generated by cleavage of longer dsRNA ⁇ e.g., dsRNA greater than about 25 nucleotides in length) with the E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA duplexes (see, e.g., Yang et al., Proc. Natl Acad. Sci. USA 99: 9942-7 (2002); Calegari et al., Proc. Natl Acad. Sci.
• dsRNA are at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length.
• a dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer.
• the dsRNA can encode an entire gene transcript or a partial gene transcript.
• inhibitors expression of a target gene refers to the ability of an antisense oligonucleotide, siRNA or miRNA of the invention to silence, reduce, or inhibit expression of a target gene, e.g., CYP8B1 .
• a test sample ⁇ e.g., a biological sample from organism of interest expressing the target gene or a sample of cells in culture expressing the target gene
• an siRNA or miRNA that silences, reduces, or inhibits expression of the target gene.
• Expression of the target gene in the test sample is compared to expression of the target gene in a control sample ⁇ e.g., a biological sample from an organism of interest expressing the target gene or a sample of cells in culture expressing the target gene) that is not contacted with the siRNA or miRNA.
• Control samples ⁇ i.e., samples expressing the target gene) are assigned a value of 100%.
• Silencing, inhibition, or reduction of expression of a target gene is achieved when the value of the test sample relative to the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10%.
• Suitable assays include, e.g., examination of protein or mRNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, protein assays ⁇ e.g., the Bradford protein assay), as well as phenotypic assays known to those of skill in the art.
• an “effective amount” or “therapeutically effective amount” of an antisense oligonucleotide, siRNA or miRNA is an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence, e.g., of a CYP8B1 -encoding sequence, in comparison to the normal expression level detected in the absence of the antisense oligonucleotide, siRNA or miRNA. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with the antisense oligonucleotide, siRNA or miRNA relative to the control is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
• an interfering RNA can be provided in several forms.
• an interfering RNA can be provided as one or more isolated siRNA duplexes, longer double-stranded RNA (dsRNA), pre- miRNA, miRNA, or as siRNA or dsRNA transcribed from a transcriptional cassette in a DNA plasmid.
• the interfering RNA may also be chemically synthesized.
• the interfering RNA is a siRNA molecule that is capable of silencing expression of a target gene (i.e.,
• the siRNA is typically from about 15 to about 30 nucleotides in length.
• the synthesized or transcribed siRNA can have 3' overhangs of about 1 -4 nucleotides, preferably of about 2-3 nucleotides, and 5' phosphate termini.
• the siRNA lacks terminal phosphates.
• the siRNA duplex lacks 3' overhangs, i.e., have blunt-ends.
• the antisense oligonucleotides or interfering RNA molecules described herein comprise at least one region of mismatch with its target sequence.
• region of mismatch refers to a region of a siRNA that does not have 100%
• a siRNA may have at least one, two, or three regions of mismatch.
• the regions of mismatch may be contiguous or may be separated by one or more nucleotides.
• the regions of mismatch may comprise a single nucleotide or may comprise two, three, four, or more nucleotides.
• a single nucleotide substitution may be made to introduce a G:U wobble base pair as described in U.S. Patent No. 7,459,547.
• Suitable siRNA sequences that target CYP8B1 can be identified using any means known in the art. Typically, the methods described in Elbashir et al., Nature 41 1 :494-498 (2001 ) and Elbashir et al., EMBO J. 20: 6877-6888 (2001 ) are combined with rational design rules set forth in Reynolds et al., Nature Biotech. 22:326-330 (2004).
• the sequence within about 50 to about 100 nucleotide 3' of the AUG start codon of a transcript from the target gene of interest is scanned for dinucleotide sequences (e.g., AA, CC, GG, or UU) (see, e.g., Elbashir, et al., supra).
• the nucleotides immediately 3' to the dinucleotide sequences are identified as potential siRNA target sequences.
• the 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, or more nucleotides immediately 3' to the dinucleotide sequences are identified as potential siRNA target sites.
• the dinucleotide sequence is an AA sequence and the 19 nucleotides immediately 3' to the AA dinucleotide are identified as a potential siRNA target site.
• siRNA target sites are spaced at different postitions along the length of the target gene.
• potential siRNA target sites may be further analyzed to identify sites that do not contain regions of homology to other coding sequences. For example, a suitable siRNA target site of about 21 base pairs typically will not have more than 16-17 contiguous base pairs of homology to other coding sequences. If the siRNA sequences are to be expressed from an RNA Pol III promoter, siRNA target sequences lacking more than 4 contiguous A's or T's are selected.
• the sequence can be analyzed using a variety of criteria known in the art.
• the siRNA sequences may be analyzed by a rational design algorithm to identify sequences that have one or more of the following features: (1 ) G/C content of about 25% to about 60% G/C; (2) at least 3 A/Us at positions 15-19 of the sense strand; (3) no internal repeats; (4) an A at position 19 of the sense strand; (5) an A at position 3 of the sense strand; (6) a U at position 10 of the sense strand; (7) no G/C at position 19 of the sense strand; and (8) no G at position 13 of the sense strand.
• siRNA design tools that incorporate algorithms that assign suitable values of each of these features and are useful for selection of siRNA can be found at, e.g., http://boz094.ust.hk/RNAi/siRNA.
• sequences with one or more of the foregoing characteristics may be selected for further analysis and testing as potential siRNA sequences.
• sequences complementary to the siRNA target sites may also be designed.
• siRNA target sequences with one or more of the following criteria can often be eliminated as siRNA: (1 ) sequences comprising a stretch of 4 or more of the same base in a row; (2) sequences comprising homopolymers of Gs (i.e., to reduce possible non-specific effects due to structural characteristics of these polymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in a row; and (5) sequence comprising direct repeats of 4 or more bases within the candidates resulting in internal fold-back structures.
• sequences with one or more of the foregoing characteristics may still be selected for further analysis and testing as potential siRNA sequences.
• the sequence can be analyzed for the presence of any immunostimulatory properties, e.g., using an in vitro cytokine assay or an in vivo animal model. Motifs in the sense and/or antisense strand of the siRNA sequence such as GU-rich motifs can also provide an indication of whether the sequence may be immunostimulatory. Once an siRNA molecule is found to be
• a siRNA sequence can be contacted with a mammalian responder cell under conditions such that the cell produces a detectable immune response to determine whether the siRNA is an
• the mammalian responder cell may be from a na ' fve mammal (i.e., a mammal that has not previously been in contact with the gene product of the siRNA sequence).
• the mammalian responder cell may be, e.g., a peripheral blood mononuclear cell (PBMC), a macrophage, and the like.
• the detectable immune response may comprise production of a cytokine or growth factor such as, e.g., TNF-a, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-6, IL-12, or a combination thereof.
• siRNA molecule identified as being immunostimulatory can then be modified to decrease its immunostimulatory properties by replacing at least one of the nucleotides on the sense and/or antisense strand with modified nucleotides such as 2'OMe nucleotides ⁇ e.g., 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-cytosine, and/or 2'OMe-adenosine).
• the modified siRNA can then be contacted with a mammalian responder cell as described above to confirm that its
• Suitable in vitro assays for detecting an immune response include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Patent No. 4,376,1 10);
• a non-limiting example of an in vivo model for detecting an immune response includes an in vivo mouse cytokine induction assay that can be performed as follows: (1 ) siRNA can be administered by standard
• cytokines can be quantified using sandwich ELISA kits according to the manufacturers' instructions ⁇ e.g., mouse and human IFN-a (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF-a (eBioscience; San Diego, CA); and mouse IL-6, TNF-a, and IFN- ⁇ (BD Biosciences; San Diego, CA)).
• sandwich ELISA kits according to the manufacturers' instructions ⁇ e.g., mouse and human IFN-a (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF-a (eBioscience; San Diego, CA); and mouse IL-6, TNF-a, and IFN- ⁇ (BD Biosciences; San Diego, CA)).
• Monoclonal antibodies that specifically bind cytokines and growth factors are commercially available from multiple sources and can be generated using methods known in the art (see, e.g., Kohler and Milstein, Nature 256: 495-497, 1975, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York (1999)). Generation of monoclonal antibodies has been previously described and can be accomplished by any means known in the art (Buhring et ai, Hybridoma, 10:1 , 77-78, 1991 ). In some methods, the monoclonal antibody is labeled ⁇ e.g., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) to facilitate detection.
• siRNA can be provided in several forms including, e.g., as one or more isolated siRNA duplexes, longer dsRNA, ssRNA having self- complementary sense and antisense regions, or as siRNA or dsRNA
• siRNA may also be chemically synthesized.
• the siRNA sequences may have overhangs ⁇ e.g., 3' or 5' overhangs as described in Elbashir et ai, Genes Dev. 15:188 (2001 ), Nykanen et ai., Cell 107:309 (2001 ), and U.S. Patent Application Publication No. 2007/0275465), or may lack overhangs, i.e., have blunt ends (see, e.g., U.S. Patent No. 7,452,987).
• RNA population can be used to provide long precursor RNAs, or long precursor RNAs that have substantial or complete identity to a selected target sequence can be used to make the siRNA.
• the RNAs can be isolated from cells or tissue, synthesized, and/or cloned according to methods well known to those of skill in the art.
• the RNA can be a mixed population (obtained from cells or tissue, transcribed from cDNA, subtracted, selected etc.), or can represent a single target sequence.
• RNA can be naturally occurring, ⁇ e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or a cloned cDNA), or chemically synthesized.
• the complement is also transcribed in vitro and hybridized to form a dsRNA.
• the RNA complements are also provided ⁇ e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerases.
• the precursor RNAs are then hybridized to form double stranded RNAs for digestion.
• the dsRNAs can be directly administered to a subject or can be digested in vitro prior to administration.
• one or more DNA plasmids encoding one or more siRNA or antisense oligonucleoide templates are used to provide siRNA.
• siRNA can be transcribed as single-stranded sequences that automatically fold into duplexes with hairpin loops from DNA templates in plasmids having RNA polymerase III transcriptional units, for example, based on the naturally occurring transcription units for small nuclear RNA U6 or human RNase P RNA H1 (see, Brummelkamp, et ai, Science 296:550 (2002); Donze, et al., Nucleic Acids Res. 30:e46 (2002); Paddison, et al., Genes Dev. 16:948 (2002); Yu, et ai, Proc. Natl Acad. Sci. USA 99:6047 (2002); Lee, et al., Nat. Biotech.
• a transcriptional unit or cassette will contain an RNA transcript promoter sequence, such as an H1 -RNA or a U6 promoter, operably linked to a template for transcription of a desired siRNA sequence and a termination sequence, comprised of 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp, Science, supra).
• the selected promoter can provide for constitutive or inducible transcription.
• compositions and methods for DNA-directed transcription of RNA interference molecules are described in detail in U.S. Patent No. 6,573,099.
• transcriptional unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. Patent Nos. 5,962,428 and 5,910,488.
• the selected plasmid can provide for transient or stable delivery of a target cell. It will be apparent to those of skill in the art that plasmids originally designed to express desired gene sequences can be modified to contain a transcriptional unit cassette for transcription of siRNA.
• the siRNA can also be chemically synthesized.
• the oligonucleotides that comprise the siRNA molecule can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc. 109:7845 (1987); Scaringe et al., Nucl. Acids Res. 18:5433 (1990); Wincott et al., Nucl. Acids Res. 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio. 74:59 (1997).
• oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidites at the 3'- end.
• small scale syntheses can be conducted on an Applied Biosystems synthesizer using a 0.2 ⁇ scale protocol with a 2.5 min. coupling step for 2'-O-methylated nucleotides.
• synthesis at the 0.2 ⁇ scale can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA).
• Protogene Protogene
• a larger or smaller scale of synthesis is also within the scope of the present invention.
• Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.
• siRNA molecules can also be synthesized via a tandem synthesis technique, wherein both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize to form the siRNA duplex.
• the linker can be a polynucleotide linker or a non- nucleotide linker.
• the tandem synthesis of siRNA can be readily adapted to both multiwell/multiplate synthesis platforms as well as large scale synthesis platforms employing batch reactors, synthesis columns, and the like.
• the siRNA molecule can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA.
• each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
• the modified siRNA molecule can be synthesized as a single continuous
• siRNA duplex is joined by a chemical linkage formed by chemical linkage groups such as polyethylene glycol chains, purine analogs, and methylene blue (see, e.g., U.S. Patent Application Publication No. 2004/0053875).
• siRNA molecules described herein can comprise at least one modified nucleotide in the sense and/or antisense strand (see, e.g., U.S. Patent Nos. 5,898,031 ; 6,107,094; 7,432,250; and 7,452,987 and U.S. Patent
• the antisene oligonucleotides described herein can comprise at least one modified nucleotide.
• modified nucleotides suitable for use in the present invention include, but are not limited to, ribonucleotides having a 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro, 2'-deoxy, 5-C- methyl, 2'-methoxyethyl, 4'-thio, 2'-amino, or 2'-C-allyl group.
• Modified nucleotides having a Northern conformation such as those described in, e.g., Saenger, Principles of Nucleic Acid Structure, Springer- Verlag Ed.
• modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides ⁇ e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'- methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'- fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, and 2'-azido nucleotides.
• LNA locked nucleic acid
• MOE methoxyethoxy
• the siRNA molecule includes one or more G-clamp
• a G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. Soc. 120:8531 -8532 (1998)).
• nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6- nitroindole (see, e.g., Loakes, Nucl. Acids Res. 29:2437-2447 (2001 )) can be incorporated into the siRNA molecule.
• a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6- nitroindole (see, e.g., Loakes, Nucl. Acids Res. 29:2437-2447 (2001 )
• the siRNA molecule can further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like.
• terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl
• nucleotides acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'- inverted abasic moieties, 5'-5'-inverted nucleotide moieties, 5'-5'-inverted abasic moieties, 3'-5'-inverted deoxy abasic moieties, 5'-amino-alkyl phosphate, 1 ,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1 ,2-aminododecyl phosphate, hydroxypropyl phosphate, 1 ,4- butanediol phosphate, 3'-phosphoramidate, 5'-phosphoramidate,
• hexylphosphate aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'- phosphorothioate, 5'-phosphorothioate, phosphorodithioate, and bridging or non-bridging methylphosphonate or 5'-mercapto moieties (see, e.g., U.S.
• Non- limiting examples of phosphate backbone modifications include phosphorothioate,
• the sense and/or antisense strand can further comprise a 3'-terminal overhang having about 1 to about 4 (e.g., 1 , 2, 3, or 4) 2'-deoxy ribonucleotides and/or any combination of modified and unmodified nucleotides (see, e.g., U.S. Patent Application Publication No.
• the modified siRNA molecules described herein can optionally comprise one or more non-nucleotides in one or both strands of the siRNA duplex.
• non-nucleotide refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity.
• the group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1 '-position.
• chemical modification of the siRNA comprises attaching a conjugate to the chemically-modified siRNA molecule.
• the conjugate can be attached at the 5' and/or 3'-end of the sense and/or antisense strand of the chemically-modified siRNA via a covalent attachment such as, e.g., a biodegradable linker.
• the conjugate can also be attached to the chemically-modified siRNA, e.g., through a carbamate group or other linking group (see, e.g., U.S. Patent Nos. 6,803,198; 7,122,649; and 7,125,975).
• the conjugate is a molecule that facilitates the delivery of the chemically-modified siRNA into a cell.
• conjugate molecules suitable for attachment to a chemically-modified siRNA include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S.
• steroids such as cholesterol
• glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives
• Other examples include the lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleaving agent, and cross-linking agent conjugate molecules described in U.S. Patent Application Publication Nos. 2005/01 19470 and 2005/0107325.
• Yet other examples include the 2'-O-alkyl amine, 2'-O-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in U.S. Patent Application Publication No. 2005/0153337. Additional examples include the hydrophobic group, membrane active compound, cell penetrating compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in U.S. Patent Application Publication No.
• Polynucleotide compositions including siRNA may be delivered systemically using a variety of lipid-based delivery agents known in the art. For example, see, PCT Patent Application Publication Nos. WO 2005/105152, WO 2006/069782, WO 2007/121947, and WO 2008/042973.
• a number of hydrophilic polymer-based delivery systems that utilize hydrophilic polymers, such as polyoxazoline and HPMA-polyamine, are known in the art (see, e.g., PCT Patent Application Publication Nos. WO 2003/066054, WO 2003/066068, and WO 2003/066069).
• peptide compositions may also be used for the delivery of siRNA (see, e.g., PCT Patent Application Publication No. WO 2008/036929).
• Polynucleotides including siRNA may also be delivered using a viral vector deliver (see, e.g., U.S. Patent Application Publication No. 2007/02191 18 and PCT Patent
• the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of CYP8B1 .
• Ribozymes are RNA-protein complexes that cleave nucleic acids in a site- specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc. Natl. Acad. Sci. USA. 1987
• Ribozymes may be designed as described in PCT Patent
• PNAs peptide nucleic acids that target CYP8B1
• PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug Dev. 7(4) 431 -37, 1997).
• PNA can be utilized in a number of methods that traditionally have used RNA or DNA.
• a review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol. 1997 Jun; 15(6):224-9). As such, in certain embodiments, one may prepare PNA sequences that are
• PNA compositions may be used to regulate, alter, decrease, or reduce the translation of CYP8B1 -specific mRNA, and thereby alter the level of CYP8B1 activity in a host cell to which such PNA compositions have been administered.
• PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et ai, Science 1991 Dec
• PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.
• PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et ai, Bioorg. Med. Chem. 1995 Apr;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.
• Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N- terminal amine.
• PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements.
• the identity of PNAs and their derivatives can be confirmed by mass spectrometry.
• U.S. Patent No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.
• PNAs small Molecules
• Inhibitory agents of the present invention further include large or small inorganic or organic molecules.
• modulators are small organic molecules, or derivatives or analogs thereof. Non-limiting examples of such small molecules are described above as the compounds of formulae I, II, and III.
• a modulator includes a protecting group.
• protecting group refers to chemical moieties that block at least some reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed (or “cleaved”). Examples of blocking/protecting groups are described, e.g., in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999.
• modulators may possess one or more chiral centers and each center may exist in the R or S configuration.
• Modulators of the present invention include all diastereomeric, enantiomeric, and epimeric forms as well as mixtures thereof. Stereoisomers may be obtained, if desired, by methods known in the art as, for example, the separation of stereoisomers by chiral chromatographic columns. Modulators further include of /V-oxides, crystalline forms (also known as polymorphs), and pharmaceutically acceptable salts, as well as active metabolites of any inhibitor. All tautomers are included within the scope of the modulators presented herein. In addition, the
• modulators described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
• the solvated forms of the modulators presented herein are also included within the present invention.
• a small molecule inhibitor binds to CYP8B1 .
• a small molecule binds to a catalytic region of CYP8B1 and interferes or reduces a CYP8B1 activity or CYP8B1 binding to a subtrate.
• Inhibitors of CYP8B1 may be identified using methods described herein and/or by routine screening procedures available in the art, e.g., using commercially available libraries of such compounds or according to established CYP8B1 enzyme activity assays such as assays of CYP8B1 sterol 12-a-hydroxylase activity.
• Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation ⁇ e.g., electroporation, lipofection).
• Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et ai, 2001 , MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in
• Therapeutics (Vancouver, Canada), The University of Capetown, South Africa and The National University of Singapore were selected.
• the main criterion was an HDL cholesterol level >90 th percentile or ⁇ 10 th percentile in the proband.
• Blood was drawn in EDTA-containing tubes for plasma lipoprotein cholesterol, and triglyceride analyses and stored at -80°C. Leukocytes were isolated from the buffy coat for DNA extraction.
• Lipoprotein measurement was performed on fresh plasma as described (Rogler et a/., Arterioscler. Thromb. Base. Biol. 15(5):683-90, 1995).
• total cholesterol and triglyceride levels were determined in total plasma, whereas HDL cholesterol was measured in plasma separated at density d ⁇ 1 .006 g/mL after preparative ultracentrifugation, before and after precipitation with dextran manganese.
• DNA primers were designed to overlap the CYP8B1 open reading frame coding sequence, as well as an upstream exon and adjacent untranslated and intronic region boundaries, as described in the UCSC Genome Bioinformatics Human Genome Browser Gateway March 2006 release (http://genome.ucsc.edu/, Univ. of California- Santa Cruz, Santa Cruz, CA; Hinrichs et al., 2006 Nucl. Ac. Res. 34(Database issue):D590-8). Primer sequences were designed using standard algorithms (Primer 3; Rozen and Skaletsky (2000), Primer3 on the WWW for general users and for biologist programmers.
• HHDL 647 unrelated probands with high HDL
• LHDL 398 unrelated probands with low HDL
• HHDL was defined as HDL levels of at least 90 th percentile of the individual cohort
• LHDL was defined as HDL levels of less than 10 th percentile of the individual cohort.
• Pedigrees and family member DNA samples were available for 208 of 258 Dutch and
• Table 4 CYP8B1 mutations identified in HHDL and LHDL individuals.
• the other predicted damaging mutations introduced non- conservative amino acid substitutions which likely disrupted protein domains, secondary structures or small "hinge” regions between the secondary structures.
• Most damaging mutations encoded amino acid residues that were highly conserved across vertebrates. For example, the amino acid D341 was completely conserved across all species in which the sequence has been determined and M53 was conserved in all mammals except opossum.
• HHDL the prevalence of HHDL in each of the proband's family members was investigated.
• DNA from individuals from each family was genotyped, and it was found that individuals with the CYP8B1 mutations tended to segregate with higher HDL.
• the three K300X mutation carriers in the pedigree shown in Figure 3 all had HDL cholesterol greater than the 85 th percentile, whereas their first degree relative non-carriers had HDL cholesterol less than the 70 th percentile.
• Expansion of the families of the K300X and D341 E mutation carrier probands resulted in the identification of an additional 16 CYP8B1 mutation carriers.
• HEK-293 cells were seeded into 6-well plates at 6.5 x 10 5 cells/2 mL/well in incubation media (Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-glutaimine) and cultured for 24 hours at 37 °C, 5% CO2.
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• 2 mM L-glutaimine Mammalian expression vectors
• Extractions were vortexed for 10 seconds, centrifuged at 20,000 x g for 10 minutes at 4 °C and 1 ml_ of supernatant was then transferred to a 96- deep well plate for drying under vacuum at 60 °C. Samples were reconstituted with 0.125 ml_ of 50% methanol:40% ddH 2 O:10% trifluoroacetic acid (TFA).
• TFA trifluoroacetic acid
• Ionization was performed by electrospray in positive ion mode and detection was carried out by multiple reaction monitoring the following transitions: 401 .50 > 176.70 m/z for 7a-hydroxy-4-cholesten-3-one; 408.50 > 177.10 m/z for D7- 7a-hydroxy-4-cholesten-3-one and 417.60 > 381 .50 m/z for 7a, 12a-dihydroxy- 4-cholesten-3-one).
• CYP8B1 sterol-12-a-hydroxylase activity was determined based on the amount of 7,12-diHCO product formed normalized to the internal standard, and results normalized to the activity of wildtype human CYP8B1 .
• Figure 4 shows that human CYP8B1 mutant "benign" forms (P88S, K238R, L357F, Q372K, V402I, S488N) could be classified as those mutants having no significant difference ( ⁇ 10%) in activity from wildtype CYP8B1 , while partial loss-of-function (PLOF) mutants (M53T, D195N, D341 E) exhibited 15-50% loss of activity relative to wildtype human CYP8B1 , and complete loss-of-function (CLOF) mutants (A103E, K300X, R349Q and R407H) exhibited >90% loss of enzyme activity in the sterol-12-a-hydroxylase assay. Activity data are summarized in Table 5.
• HEK-293 cells were seeded into 6-well plates at 6.5x10 5 cells/2 mL/well in incubation media (Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-glutaimine) and cultured for 24 hours at 37 °C, 5% CO2.
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• 2 mM L-glutaimine Mammalian expression vectors
• DNA:FuGENE ratio of 2 ⁇ g:5 ⁇ according to manufactures protocol After a further 24 hours culturing, the cell media was removed and replaced with 2 mL incubation media containing a final concentration of 20 ⁇ g/mL cycloheximide (Sigma, St. Louis, MO).
• cell lysates (20 ⁇ g) were prepared and electrophoresed in SDS-PAGE gels (NuPAGE® 4-12% Bis-Tris (Novex, San Diego, CA)) at 200 V for 50 min, followed by 60 min electroblot- transfer at 30 V to solid phase polyvinylidene difluoride (PVDF) membranes (BIO-RAD Laboratories, Hercules, CA).
• PVDF polyvinylidene difluoride
• the membrane was blocked for 1 hour at room temperature in blocking buffer (1 x tris-buffered saline (TBS), 0.1 % Tween-20 with 5% w/v nonfat dry milk), probed overnight at 4 °C with a polyclonal anti-CYP8B1 antibody in blocking buffer (1 :200 dilution of Abgent AP8787b), washed 3 x 5 minutes in TBS-T (1x TBS, 0.1 % Tween-20), probed for 1 hour at room temperature with a goat anti-rabbit IgG (H+L)-HRP conjugate secondary antibody in blocking buffer (1 :3,000 dilution of Cat#170-6515 BIO- RAD) and washed 3 x 5 or 3 x 10 minutes in TBS-T.
• blocking buffer (1 x tris-buffered saline (TBS), 0.1 % Tween-20 with 5% w/v nonfat dry milk
• TBS-T tris-buffered saline
• Chemiluminescent substrate (SuperSignal West Pico (Pierce)) was applied for 1 minute, blots were exposed to Blue x-ray film and densitometry of autoradiograms was performed using an Alpha Imager 1220 (Alpha Innotech Corp., San Leandro, CA)
• results for CLOF CYP8B1 mutants are shown in Figure 5.
• the K300X mutation resulted in the complete loss of detectable CYP8B1 protein expression compared to wildtype CYP8B1 .
• the A013E mutant by contrast, exhibited stability that was comparable to that of wildtype CYP8B1 .
• the R349Q and R407H mutants exhibited decreased levels of protein expression, and decreased stability, relative to the wildtype CYP8B1 protein. Eight hours post- termination of protein expression by CHX, the levels of the R349Q mutant and the R407H mutant were, respectively, approximately 5% and 1 % of the levels of the wildtype CYP8B1 .
• results for PLOF CYP8B1 mutants are shown in Figure 6.
• the M53T mutation did not cause detectable loss of stability for the mutant CYP8B1 protein.
• the D195N and D341 E mutations by contrast, exhibited reduced stability relative to that of wildtype CYP8B1 .
• HEK-293 cells were seeded into 15 cm 2 sterile petri dishes (Corning) at 1 .1 x10 7 cells/20 mL/dish in incubation media (Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 2 mM L-glutaimine) and cultured for 24 hours at 37 °C, 5% CO2.
• Mammalian expression vectors pcDNA 3.1 (Invitrogen technologies)) encoding human CYP8B1 WT or
• CYP8B1 mutants were transfected into the cells using FuGENE® HD (Roche Diagnostics) at a DNA:FuGENE ratio of 36 ⁇ g:91 ⁇ _ according to manufactures protocol. After a further 48 hours culturing, the cells where trypsinized, washed in ice-cold phosphate buffered saline solution (PBS), pelleted at 500 x g for 5 minutes at 4 °C and frozen overnight at -80 °C. The cell pellet was thawed on ice, resuspended in 1 ml_ ice-cold 5 mM hepes, pH7.4 containing protease inhibitors cocktail (Roche) and incubated on ice for 15 minutes.
• FuGENE® HD Roche Diagnostics
• the cell suspension was homogenized in a 2 mL ice-cold dounce (KONTES Glass Company), with 20 strokes before being adjusted to a final concentration of 0.25 M sucrose and centrifuged at 6,000 x g for 10 minutes at 4 °C to remove cell debris. The supernatant was centrifuged at 15,000 x g for 20 minutes at 4 °C to remove the mitochondrial fraction, followed by 105,000 x g for 60 minutes at 4 °C to isolate the microsomal fraction. The cell pellet was washed with wash buffer (0.15 M KCI, pH 7.5, 0.01 M EDTA) and centrifuged at 105,000 x g for 60 minutes at 4 °C. The remaining microsomes were resuspended in 0.25 M sucrose and protein concentration determined using a bicinchoninic acid (BCA) protein assay kit (Pierce) according to the manufacturer's protocol.
• BCA bicinchoninic acid
• the prepared CYP8B1 WT and mutant microsomes were diluted to 0.2 mg/mL using 0.1 M potassium phosphate buffer pH 7.4 and 17.5 ⁇ _ dispensed into each well of a 96-well plate.
• the plate was mixed on a microtitre plate shaker for 30 s.
• 70 ⁇ _ of stop solution acetonitrile, containing 0.75 ⁇ D7-7a-hydroxy-4-cholesten-3-one (Toronto Research Chemicals) and 1 % formic acid
• the CYP8B1 enzymatic reaction product, 7a, 12a-dihydroxy-4-cholesten-3-one (7,12-diHCO), and the internal standard D7-7a-hydroxy-4-cholesten-3-one were quantified by C18 ultra performance liquid chromatography/ electrospray ionization/ tandem mass spectrometry (UPLC-ESI-MS/MS) after dilution into 1 :1 acetonitrile/water containing 1 % formic acid.
• UPLC-ESI-MS/MS ultra performance liquid chromatography/ electrospray ionization/ tandem mass spectrometry
• the CYP8B1 enzymatic rate was determined for each substrate concentration and the Vmax and Km calculated for CYP8B1 WT and mutant microsomes using GraphPad Prism 5.0 (GraphPad Software). The effects of the various CYP8B1 mutations on sterol- 12-a-hydroxylase activity were calculated as the specificity constant (Vmax/Km) for each mutant. As shown in Figure 8, the CYP8B1 CLOF mutants (A103E, K300X, R349Q, R407H) had essentially no enzyme activity.
• D195N and D341 E had specificity constants that were comparable to or greater than that of wildtype CYP8B1
• the M53T mutant had a specificity constant value of about 55% that of the wildtype CYP8B1 .
• the A103E (Fig. 5) and M53T (Fig. 6) mutant protein products were stable, the A103E mutation essentially abrogated all enzyme activity and the M53T mutation caused catalytic efficiency that was markedly lower than that of wildtype CYP8B1 .
• CYP8B1 MUTATION CARRIERS HAVE AN ATHEROPROTECTIVE PLASMA LIPID PROFILE Additional lipid profile measures were also compared between the
• Table 6 Lipid profiles of population controls.
• mice studies are used to elucidate the effect of CYP8B1 on atherosclerosis.
• Cyp8b1 siRNA-containing virus e.g., adenovirus
• ApoE -/- and Ldlr -/- atherogenic mice maintained on a high fat diet.
• Plasma HDL and lipid profiles are measured to assess the progress/prevention of atherosclerosis.
• Cyp8b1 knockout mice are generated in order to assess the effects of CYP8B1 on lipid
• Cyp8b1 -I- mice are crossed with ApoE -/- or Ldlr -/- mice.
• the resultant strain is maintained on a high fat diet and the turnover rate of the radiolabelled HDLc is measured in order to determine if HDL rising is mediated through increased synthesis or increased turnover.
• Mutations responsible for causing disruptions to the CYP8B1 structure in a manner that significantly decreases sterol-12-a-hydroxylase activity were identified using the Polyphen2 (Polymorphism Phenotyping version 2) software tool (Adzhubei et al., 2010 Nature Meths. 7(4):248; see also Ramensky et al. 2002 Nucl. Ac. Res. 30:3894; Sunyaev et al. 1999 Prot. Eng. 12:387).
• AGENTS THAT INHIBIT CYP8B1 ACTIVITY This example describes agents that were capable of decreasing wildtype CYP8B1 sterol 12-a-hydroxylase activity to levels at or below the enzyme activity levels observed for CYP8B1 partial-loss-of-function (PLOF) mutants described herein, including CYP8B1 PLOF mutants identified in human subjects presenting with plasma HDL levels that were significantly increased relative to plasma HDL levels in normal subjects expressing wildtype CYP8B1 .
• PLOF partial-loss-of-function
• Human liver microsomes (Xenotech) were diluted to 0.14 mg/ml using 0.1 M potassium phosphate buffer pH 7.4 and 25 ⁇ _ was dispensed into each well of a 96 well plate. 0.35 ⁇ _ of DMSO was dispensed into wells for positive and negative controls and 0.35 ⁇ _ of test compound dissolved in DMSO was dispensed into wells for the titrations. The plate was mixed on a microtitre plate shaker for 30 s. 70 ⁇ _ of acetonitrile, containing 200 nM D7-7a- hydroxy-4-cholesten-3-one (Toronto Research Chemicals) and 1 % formic acid was dispensed into the negative control wells. After 10 min at room
• the CYP8B1 product, 7a, 12a-dihydroxy-4-cholesten-3-one and the internal standard D7-7a- hydroxy-4-cholesten-3-one were quantified by C18 UPLC-ESI-MS/MS after dilution into 1 :1 acetonitrile/water containing 1 % formic acid. Elution was performed with a gradient of 5% to 95% acetonitrile, 0.1 % formic acid.
• Ionization was performed by electrospray in positive ion mode and detection was carried out by multiple reaction motoring the following transitions; 401 .50 > 176.70 m/z for 7a-hydroxy-4-cholesten-3-one; 408.50 > 177.10 m/z for D7-7a- hydroxy-4-cholesten-3-one and 417.60 > 381 .50 m/z for 7a, 12a-dihydroxy-4- cholesten-3-one).
• Exemplary agents shown in Table 7 exhibited IC50 values ⁇ 10 ⁇ .
• the antifungal agents ketoconazole and econazole were potent inhibitors of microsomal CYP8B1 activity with IC50 values of 2.0 and 0.21 ⁇
• CYP8B1 transcripts Two CYP8B1 transcripts were identified; the first transcript (EnsembI nucleotide ID ENST00000316161 ), represented a single exon transcript of 3,950 bps translating into a 501 amino acid residue protein
• EndombI protein ID ENSP00000318867 and a second (EnsembI nucleotide ID ENST00000437102) represented a 2 exon transcript of 1 ,896 bps translating into a 496 amino acid residue protein (EnsembI protein ID ENSP00000404499). Only the 501 amino acid form of CYP8B1 has been identified thus far in vivo. The putative second isoform of CYP8B1 contained the same 449 N-terminal amino acids as the first isoform with a distinct 47 amino acid C-terminus. The protein and nucleotide sequences are presented in the Sequence Listing as SEQ ID NO:83 and SEQ ID NO:84 respectively. Variants of the second transcript were also identified in high HDL individuals using the same CYP8B1 DNA primers as described in Example 1 . In these cases the program SIFT was used to identify variants predicted to be damaging to protein activity.

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