WO2014018375A1

WO2014018375A1 - Cyp8b1 and uses thereof in therapeutic and diagnostic methods

Info

Publication number: WO2014018375A1
Application number: PCT/US2013/051124
Authority: WO
Inventors: Patrick Lorenzo Angelo FRANCHINI; Ian William TIETJEN; Michael Reuben HAYDEN; Roshini Rebecca SINGARAJA; Christopher Charles Alexander RADOMSKI; Michael David Percival; Maryanne Sharon EYERS; Richard Asher DEAN; Zhiwei XIE
Original assignee: Xenon Pharmaceuticals Inc.
Priority date: 2012-07-23
Filing date: 2013-07-18
Publication date: 2014-01-30

Abstract

The present invention embodiments relate to the diagnosis, prevention and/or treatment of diseases related to abnormal lipid metabolism. More particularly, disclosed herein is the discovery in human subjects having elevated HDL of mutated human CYP8B1 encoding genes and their encoded proteins, and exploitation of this discovery for use in compositions and methods for the diagnosis, treatment and prevention of cardiovascular diseases, such as dyslipidemia, atherosclerosis, low HDL diseases and related disorders, including methods for identifying agents that modulate CYP8B1 activity. Accordingly there are provided methods for detecting, diagnosing, prognosing or determining a predisposition to diseases related to abnormal lipid metabolism, as well as CYP8B1-directed screening assays, kits, antibodies, agents, nucleic acids, polypeptides, cells, vectors, transgenic animals and compositions.

Description

CYP8B1 AND USES THEREOF IN THERAPEUTIC

AND DIAGNOSTIC METHODS

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 980057_483WO_SEQUENCE_LISTING.txt. The text file is 374 KB, was created on July 18, 2013, and is being submitted electronically via EFS-Web.

BACKGROUND Technical Field

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.

Description of the Related Art

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 (CVD). In particular, 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.

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, however, suffers from a number of drawbacks that limit its applicability to the

understanding of human CVD processes. In 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.

For instance, 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.

122(2):558).

Yin et al. (2012 J. Lipid Res. 53:51 -65) characterized comprehensive lipid profiles in humans and in 24 mammalian models including five mouse strains, four nonhuman primates and six other nonprimate species, and found that in the majority of mouse models the lipid profiles did not mirror human dyslipidemia. In view of these and other profound differences between murine and human physiological regulation of lipid metabolism, evidence from murine model systems therefore cannot be predictive of the nature and degree of intervention needed for effective management of human CVD. Instead, demonstration of beneficial effects for improving CVD prognosis within a human metabolic and physiologic context would be desirable.

Despite advances in the field of CVD that have resulted in some improved treatment and prevention methods, CVD remains the number one cause of death in the U.S. Clearly there remains a need for additional methods for detecting and treating CVD and atherosclerosis, and in particular for increasing HDL levels in a patient without adversely affecting LDL levels. The compositions and methods described herein address these needs and offer other related advantages. BRIEF SUMMARY

According to certain embodiments of the present invention there are provided methods and compositions that are useful for determining the risk for or presence in a subject of a variety of cardiovascular diseases (CVD) and disorders, such as dyslipidemia, atherosclerosis, low HDL diseases and related disorders, including CVD 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, and that are also useful for increasing plasma HDL levels to treat and prevent such diseases and disorders.

In certain embodiments there is thus provided 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:

T at wildtype nucleotide position 483 of SEQ ID NO:2 which is substituted by C in said oligonucleotide,

C at wildtype nucleotide position 633 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 908 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

A at wildtype nucleotide position 1223 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 1348 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

G at wildtype nucleotide position 1371 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, and

G at wildtype nucleotide position 1545 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

or an oligonucleotide that is complementary thereto.

In certain embodiments there is provided 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:

C at wildtype nucleotide position 587 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

A at wildtype nucleotide position 1038 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

C at wildtype nucleotide position 1394 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 1439 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 1529 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, and

G at wildtype nucleotide position 1756 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

or an oligonucleotide that is complementary thereto.

In certain embodiments there is provided an isolated

C at wildtype nucleotide position 401 of SEQ ID NO:2 which is substituted by T in said oligonucleotide, C at wildtype nucleotide position 407 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

G at wildtype nucleotide position 474 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

C at wildtype nucleotide position 500 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

G at wildtype nucleotide position 563 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

C at wildtype nucleotide position 605 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 614 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

A at wildtype nucleotide position 71 1 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

G at wildtype nucleotide position 723 of SEQ ID NO:2 which is substituted by C in said oligonucleotide,

A at wildtype nucleotide position 759 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

C at wildtype nucleotide position 883 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 884 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 945 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

C at wildtype nucleotide position 1 185 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

A at wildtype nucleotide position 1334 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

C at wildtype nucleotide position 1351 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, C at wildtype nucleotide position 1482 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 1619 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

A at wildtype nucleotide position 1652 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

T at wildtype nucleotide position 1683 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

C at wildtype nucleotide position 1691 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

T at wildtype nucleotide position 1698 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

T at wildtype nucleotide position 1749 of SEQ ID NO:2 which is substituted by G in said oligonucleotide, ]

C at wildtype nucleotide position 1784 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 1793 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, and

G at wildtype nucleotide position 1806 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

or an oligonucleotide that is complementary thereto.

In certain embodiments 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,

A at wildtype amino acid sequence position 103 of SEQ ID NO:1 which is substituted by E in said polypeptide,

D at wildtype amino acid sequence position 195 of SEQ ID NO:1 which is substituted by N in said polypeptide,

K at wildtype amino acid sequence position 300 of SEQ ID NO:1 which is absent in said polypeptide and wherein L at wildtype amino acid sequence 299 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide,

D at wildtype amino acid sequence position 341 of SEQ ID NO:1 which is substituted by E in said polypeptide,

R at wildtype amino acid sequence position 349 of SEQ ID NO:1 which is substituted by Q in said polypeptide, and

R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which is substituted by H in said polypeptide,

but which does not significantly hybridize under moderately stringent conditions to a wild-type human CYP8B1 -encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:2,

or a wildtype amino acid position that is selected from:

(b) P at wildtype amino acid sequence position 88 of SEQ ID NO:1 which is substituted by S in said polypeptide,

K at wildtype amino acid sequence position 238 of SEQ ID NO:1 which is substituted by R in said polypeptide,

L at wildtype amino acid sequence position 357 of SEQ ID NO:1 which is substituted by F in said polypeptide,

Q at wildtype amino acid sequence position 372 of SEQ ID NO:1 which is substituted by K in said polypeptide,

V at wildtype amino acid sequence position 402 of SEQ ID NO:1 which is substituted by I in said polypeptide, and S at wildtype amino acid sequence position 488 of SEQ ID NO:1 which is substituted by N in said polypeptide,

or a wildtype amino acid position that is selected from:

(c) 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,

R at wildtype amino acid sequence position 50 of SEQ ID NO:1 which is substituted by Q in said polypeptide,

R at wildtype amino acid sequence position 59 of SEQ ID NO:1 which is substituted by C in said polypeptide,

V at wildtype amino acid sequence position 80 of SEQ ID NO:1 which is substituted by I in said polypeptide,

Q at wildtype amino acid sequence position 94 of SEQ ID NO:1 which is absent in said polypeptide and wherein T at wildtype amino acid sequence position 93 of SEQ ID NO:1 comprises a carboxyl terminus for said polypeptide,

L at wildtype amino acid sequence position 97 of SEQ ID NO:1 which is substituted by V in said polypeptide,

K at wildtype amino acid sequence position 129 of SEQ ID NO:1 which is substituted by M in said polypeptide,

G at wildtype amino acid sequence position 133 of SEQ ID NO:1 which is substituted by A in said polypeptide,

D at wildtype amino acid sequence position 145 of SEQ ID NO:1 which is substituted by Q in said polypeptide, F at wildtype amino acid sequence position 186 of SEQ ID NO:1 which is substituted by L in 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,

T at wildtype amino acid sequence position 287 of SEQ ID NO:1 which is substituted by M in said polypeptide,

T at wildtype amino acid sequence position 337 of SEQ ID NO:1 which is substituted by A in said polypeptide,

S at wildtype amino acid sequence position 342 of SEQ ID NO:1 which is substituted by R in said polypeptide,

P at wildtype amino acid sequence position 386 of SEQ ID NO:1 which is substituted by L in said polypeptide,

R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which is substituted by G in said polypeptide,

P at wildtype amino acid sequence position 432 of SEQ ID NO:1 which is substituted by S in said polypeptide,

R at wildtype amino acid sequence position 443 of SEQ ID NO:1 which is substituted by G in said polypeptide,

F at wildtype amino acid sequence position 453 of SEQ ID NO:1 which is substituted by C in said polypeptide,

L at wildtype amino acid sequence position 456 of SEQ ID NO:1 which is substituted by F in said polypeptide,

V at wildtype amino acid sequence position 458 of SEQ ID NO:1 which is substituted by Q in said polypeptide,

V at wildtype amino acid sequence position 475 of SEQ ID NO:1 which is substituted by G in said polypeptide,

P at wildtype amino acid sequence position 487 of SEQ ID NO:1 which is substituted by T in said polypeptide, D at wildtype amino acid sequence position 490 of SEQ ID NO:1 which is substituted by N in said polypeptide, and

R at wildtype amino acid sequence position 494 of SEQ ID NO:1 which is substituted by H in said polypeptide,

but which does not significantly hybridize under moderately stringent conditions to a wild-type human CYP8B1 -encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:2.

In certain embodiments there is provided 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:

M at wildtype amino acid sequence position 53 of SEQ ID NO:1 which is substituted by T in said polypeptide,

R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which is substituted by H in said polypeptide. In certain embodiments there is provided 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:

P at wildtype amino acid sequence position 88 of SEQ ID NO:1 which is substituted by S in said polypeptide,

V at wildtype amino acid sequence position 402 of SEQ ID NO:1 which is substituted by I in said polypeptide, and

S at wildtype amino acid sequence position 488 of SEQ ID NO:1 which is substituted by N in said polypeptide.

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,

D at wildtype amino acid sequence position 145 of SEQ ID NO:1 which is substituted by Q in said polypeptide,

F at wildtype amino acid sequence position 186 of SEQ ID NO:1 which is substituted by L in said polypeptide,

T at wildtype amino acid sequence position 337 of SEQ ID NO:1 which is substituted by A in said polypeptide, S at wildtype amino acid sequence position 342 of SEQ ID NO:1 which is substituted by R in said polypeptide,

P at wildtype amino acid sequence position 487 of SEQ ID NO:1 which is substituted by T in said polypeptide,

D at wildtype amino acid sequence position 490 of SEQ ID NO:1 which is substituted by N in said polypeptide, and

R at wildtype amino acid sequence position 494 of SEQ ID NO:1 which is substituted by H in said polypeptide.

In certain embodiments there is provided 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,

R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which is substituted by H in said polypeptide, or

(b) a wildtype amino acid position that is selected from: P at wildtype amino acid sequence position 88 of SEQ ID NO:1 which is substituted by S 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,

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,

R at wildtype amino acid sequence position 494 of SEQ ID NO:1 which is substituted by H in said polypeptide. In certain embodiments the antibody is a monoclonal antibody. In certain embodiments 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. In certain embodiments the antibody is a Fab or a Fab' fragment. In certain embodiments the antibody is a F(ab')2 fragment. In certain embodiments the antibody is a whole antibody.

In certain embodiments there is provided an antisense oligonucleotide that comprises any one of the above described polynucleotides. In certain embodiments there is provided a ribozyme that comprises any one of the above described polynucleotides. In certain embodiments there is provided a small interfering RNA that comprises any one of the above described polynucleotides.

Turning to another embodiment there is provided 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.

In another embodiment there is provided a method of stratifying a 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, 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. In certain further embodiments 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 O₂-binding domain, a CYP8B1 steroidogenic region and a CYP8B1 heme binding domain. In certain other further embodiments 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 O₂-binding domain, a CYP8B1 steroidogenic region and a CYP8B1 heme binding domain, and wherein the single nucleotide

polymorphism is a non-synonymous nucleotide substitution. In certain other further embodiments 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:

C at wildtype nucleotide position 633 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, G at wildtype nucleotide position 908 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 1371 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 1529 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 1545 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, and

G at wildtype nucleotide position 1756 of SEQ ID NO:2 which is substituted by A in said oligonucleotide.

In certain further embodiments 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:

G at wildtype nucleotide position 1545 of SEQ ID NO:2 which is substituted by A in said oligonucleotide.

In certain other further embodiments 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:

C at wildtype nucleotide position 401 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 407 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 614 of SEQ ID NO:2 which is substituted by G in said oligonucleotide, A at wildtype nucleotide position 71 1 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 1351 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

C at wildtype nucleotide position 1482 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

C at wildtype nucleotide position 1691 of SEQ ID NO:2 which is substituted by T in said oligonucleotide, T at wildtype nucleotide position 1698 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

T at wildtype nucleotide position 1749 of SEQ ID NO:2 which is substituted by G in said oligonucleotide,

G at wildtype nucleotide position 1806 of SEQ ID NO:2 which is substituted by A in said oligonucleotide.

In certain embodiments there is provided 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. In certain embodiments the agent is selected from:

(a) a com ound of formula I:

wherein:

m is 0, 1 , 2, 3, 4 or 5;

n is 1 , 2 or 3; X is -N- or -C(R⁶)-,

at each occurrence, R¹ is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, heterocyclylalkyl, or -OR⁷;

at each occurrence, R² is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cycloalkylalkyl, heterocyclylalkyl, or -OR⁷; or

R¹ and R² 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;

at each occurrence, R³ is the same or different and independently hydrogen, halogen, hydroxy, alkyl, alkoxy, aryl, cydoalkyi, heterocydyl, aralkyi, heteroaryl or heteroarylalkyi;

R⁴ and R⁵ is independently hydrogen or alkyl;

R⁶ is hydrogen or alkyl; and

each R⁷ 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;

(b) a com ound of formula II:

wherein:

— 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;

each R^14a and R^14b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^14a and R^14b together forms =0, =S, =C(R )₂; or =NR⁹;

R¹⁵ is hydrogen or alkyl;

each R^16a and R^16b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^16a and R^16b together forms =0, =S, =C(R⁹)₂; or =NR⁹;

each R^17a and R^17b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^17a and R^17b together forms =0, =S, =C(R⁹)₂; or =NR⁹;

R¹⁸ is hydrogen, hydroxy, alkoxy, or alkyl;

R¹⁹ is hydrogen or alkyl;

each R^20a and R^20b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^20a and R^20b together forms =0, =S, =C(R⁹)₂; or =NR⁹,

R²¹ is hydrogen or alkyl, or R²¹ and R^16a together form a bond; and

each R^22a and R^22b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^20a and R^20b together forms =0, =S, =C(R⁹)₂; or =NR⁹, or R^22b and R²¹ together form a bond,

(c) a compound of formula III:

wherein:

t is 0, 1 , 2, 3, 4 or 5;

each R⁹ is the same or different and independently hydrogen alkyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;

at each occurrence, R²⁷ is the same or different and independently hydrogen, alkyl, halogen, acyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, heterocyclylalkyl, -OR⁹, -N(R⁹)₂-, or -SR⁹; or two adjacent R²⁷, together with the carbons to which they attach, form a fused aryl, heteroaryl, heterocyclyl, or cycloalkyi ring;

each R^28a and R^28b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^28a and R^28b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

each R^29a and R^29b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^29a and R^29b together forms =O, =S, =C(R⁹)₂; or =NR⁹; or

R^28a and R^29a form a cycloalkyi or heterocyclyl ring; and

each R^30a and R^30b is the same or different and independently hydrogen, alkyl, acyl, aralkyl, or heteroarylalkyl,

(d) the antibody according to any one of claims 4-9;

(e) the antisense oligonucleotide of claim 10;

(f) the ribozyme of claim 1 1 ; and

(g) the small interfering RNA of claim 12.

In certain embodiments 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. In certain embodiments 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. In certain embodiments prior to the step of administering, 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 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 said agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject or said agent that is an inhibitor of human cytochrome P450-family 8-subfamily B- polypeptide 1 (CYP8B1 ) sterol 12-a-hydroxylase activity, wherein the mutation that results in decreased CYP8B1 activity comprises at least one substitution mutation of a human CYP8B1 polypeptide having the amino acid sequence set forth in SEQ ID NO:1 and said substitution is 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.

In certain embodiments 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 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 said agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject or said agent that is an inhibitor of human cytochrome P450-family 8- subfamily B-polypeptide 1 (CYP8B1 ) sterol 12-a-hydroxylase activity, wherein the mutation that results in decreased CYP8B1 activity comprises at least one substitution mutation of a human CYP8B1 polypeptide having the amino acid sequence set forth in SEQ ID NO:1 and said substitution is 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,

L at wildtype amino acid sequence position 97 of SEQ ID NO:1 which is substituted by V in said polypeptide, K at wildtype amino acid sequence position 129 of SEQ ID NO:1 which is substituted by M in said polypeptide,

L at wildtype amino acid sequence position 456 of SEQ ID NO:1 which is substituted by F in said polypeptide, V at wildtype amino acid sequence position 458 of SEQ ID NO:1 which is substituted by Q in said polypeptide,

In certain embodiments the cardiovascular disease or disorder is selected from dyslipidemia, atherosclerosis, low HDL diseases and related disorders. In certain embodiments the agent specifically binds to the CYP8B1 polypeptide.

In certain embodiments there is provided 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 agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder. In certain embodiments the method further comprises determining the base level of CYP8B1 polypeptide

expression and the test level of CYP8B1 polypeptide expression by quantifying CYP8B1 mRNA, and in certain other embodiments the method further comprises determining the base level of CYP8B1 polypeptide expression and the test level of CYP8B1 polypeptide expression by quantifying CYP8B1 protein. In certain embodiments there is provided 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. In certain

embodiments 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 .

In certain embodiments there is provided 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

determination that the test level of CYP8B1 activity is less than the base level of CYP8B1 activity indicates the candidate agent is an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder. In certain further embodiments the CYP8B1 activity is a sterol 12-a-hydroxylase activity. In certain other further embodiments the second CYP8B1 polypeptide is present in a cell when being contacted with the candidate agent. In certain embodiments the above-described method is performed in vitro. In certain embodiments each of the first and second CYP8B1 polypeptides, or fragment or variant thereof, comprises a CYP8B1 polypeptide catalytic domain. In certain embodiments each of the first and second 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.

In certain embodiments there is provided 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. In certain embodiments the above described agent inhibits a sterol 12-a-hydroxylase activity of the second CYP8B1 polypeptide. In certain further embodiments the agent specifically binds to the second CYP8B1 polypeptide. In certain further 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.

Turning to other embodiments there is provided 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. In certain embodiments there is provided amethod 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. In another embodiment there is provided 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, a V 475G mutation, a P487T mutation, a D490N mutation, and a R494H mutation.

In certain embodiments there is provided adiagnostic kit comprising as a first polynucleotide any one of the above-described

polynucleotides. In certain embodiments 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. In certain other embodiments 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, a P432S mutation, a R443G mutation, a F453C mutation, a L456F mutation, a V458Q mutation, a V 475G mutation, a P487T mutation, a D490N mutation, and a R494H mutation.

These and other aspects and embodiments of the herein described invention will be evident upon reference to the following detailed description and attached drawings. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference in their entirety, as if each was incorporated individually. Aspects and embodiments of the invention can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

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

CYP8B1 with high HDL in a family. Affected individuals (shaded black) were defined as having HDL >90^th percentile; half-shaded = potentially affected (HDL = 80^th- 89^th percentile); unshaded = unaffected. Values listed from top to bottom for each individual include: individual identifier code, date of birth, age at measure, total cholesterol, triglycerides, HDL [HDL percentile], and BMI. All lipid measurements were in mmol/L. Arrows indicate probands in which the CYP8B1 mutations were first identified. Mutated alleles in each individual are indicated by X.

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;

33.5%) or complete (CLOF, >90%) loss of function (A103E; 94.6%, K300X; 100%, R349Q; 100% and R407H; 98.5%).

Figure 5 is plot of wild type and complete 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 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.

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.

DETAILED DESCRIPTION

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. Surprisingly, mutations that impair CYP8B1 sterol 12-a-hydroxylase activity, and in some cases, mutations that do not result in apparent loss of sterol 12-a- hydroxylase activity, were found in human subjects exhibiting plasma HDL, LDL and triglyceride (TG) profiles and BMI that are associated with a cardiovascular disease risk that is lower than that of the general population. These and related unprecedented observations in humans also offer surprising advantages over prior understanding derived from murine models of dyslipidemia, which models differ from the human physiological context in part because in mice HDL is a significant cholesterol carrier. Compositions and methods that will find uses in the diagnosis and treatment of human patients having, or being at risk for developing, cardiovascular disease are therefore provided.

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.

As described herein there is a surprising and unexpected relationship between (i) unusually high HDL levels and/or subnormal LDL levels and/or subnormal triglyceride levels in human subjects, and (ii) the presence of loss-of-f unction mutations or of certain other herein described mutations in the CYP8B1 gene of these subjects. From this discovery, 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

cardiovascular diseases or disorders. As described in greater detail herein, "increased" or "decreased" levels of a given quantifiable parameter reflect statistically significant changes from a preestablished baseline level and/or from an art-accepted normal level of the parameter.

Also provided are 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 . As a brief background, CYP8B1 is involved in the biosynthesis of cholic acid from cholesterol. 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. Rev., 89:147-191 , 2009; Hylemon P.B., et al., J. Lip. Res., 50:1509-1520, 2009), where such increases and decreases occur in a statistically significant manner relative to normal ranges for these parameters that are present in control subjects who lack elevated cholic acid levels.

Without wishing to be bound by theory, it is believed that inhibition of 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. As chenodeoxycholic acid is more hydrophilic than cholic acid, 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 . 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. As part of the conversion of cholesterol to bile, 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

cardiovascular disorders associated with lipid metabolism (Bentivegna et al., PCT Published Application No. WO 2001/79224).

Dramatic differences in systemic cholesterol transport between mice and humans preclude the ability of the art to predict, from murine data, effective therapeutic strategies for cardiovascular disease in humans, despite studies in murine model systems that have considered CYP8B1 as a potential target for modulating HDL. For example, Li-Hawkins et al. showed that Cyp8b1 knockout mice had virtually no cholic acid and instead exhibited significantly elevated chenodeoxycholic acid, in addition to muricholic acid (2002 J. Clin. Invest., 1 10:1 191 -1200). Wang et al. described Cyp8b1 knockouts that also had elevated Abcai and Cyp7a1 gene expression, but no change in Scarbl or Apoal gene expression (2006 J. Lipid Res. 47:421 -430). These effects were exacerbated in mice fed a 0.5% cholesterol diet, suggesting that genes involved in the reverse cholesterol transport pathway (i.e., the transport of cholesterol from non-hepatic tissues to the liver) may be increased in the absence of Cyp8b1 (Wang J., et al., supra). Cyp8b1 knockout mice on a high cholesterol diet also had reduced hepatic cholesterol when compared to wild-type mice (Wang J., et al., supra). A recent genetic study showed that Cyp8b1 knockout mice crossed into an atherogenic ApoE knockout background had -50% reduction in aortic lesion area compared to control ApoE knockout mice (Slatis K., et al., J. Lipid Res., 51 :3289-3298, 2010). Despite these observations in the mouse, 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.

Thus, while the most common animal model in which to study atherosclerosis is the mouse, the murine model system fails to provide a predictive platform for human CVD therapy in view of several significant limitations, as also noted above. First, comparative studies indicate that the mouse uses high density lipoprotein (HDL) to transport cholesterol in the circulation almost exclusively, whereas in humans the majority of cholesterol is typically carried by low density lipoprotein (LDL) (Daugherty, 2002 Am J Med Sci. 323:3-10). This dramatic difference between murine and human atherosclerotic mechanisms results in part from the failure of mice to express cholesteryl ester transfer protein (CETP), a cholesterol-transfer protein that by contrast is normally present in humans (Plump et al, 1999 Arterioscler Thromb Vase Biol. 1999 19:1 105-1 1 10). As a result of this difference, genetic and/or dietary manipulation is necessary in murine experimental model systems to induce the development of atherosclerotic lesions. Use of such manipulations to model atherosclerosis in mice, however, frequently generates supraphysiological settings that limit the fidelity with which the art can translate experimental results from such murine studies to the human atherosclerotic condition. As also noted above, Yin et al. (2012 J. Lipid Res. 53:51 -65) characterized comprehensive lipid profiles in humans and in 24 mammalian models including five mouse strains, four nonhuman primates and six other nonprimate species, and found that in the majority of mouse models the lipid profiles did not mirror human dyslipidemia.

This lack of translatability from mice to humans is exemplified by the observations that few of the currently available classes of medication for the treatment of dislipidemia and atherosclerosis in humans are efficacious in commonly used mouse models. For instance, 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 ).

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. As described herein, there are provided for the first time the presently disclosed embodiments according to which such human genetic data may be usefully exploited.

A. CYP8B1 Polynucleotides and Polypeptides

Based on the identification of mutations in the human Cyp8b1 gene that are associated with increased levels of HDL, the present

embodiments provide novel CYP8B1 polynucleotide and polypeptide

sequences comprising one or more of these mutations, as described herein.

Portions of the human CYP8B1 -encoding DNA sequence of SEQ ID NO:2, and portions of a sample CYP8B1 -encoding DNA sequence derived from a biological source or subject as provided herein, are regarded as

"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 . (Genbank accession number NM_004391 .2) For instance, 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.

For example, 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. Ac. Res. 22:4673; CAP, www.no.embnet.org/clustalw.html; FASTA FASTP, Pearson, 1990 Proc. Nat. Acad. Sci. USA 85:2444; BLAST and BLAST 2.0 algorithms described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively).

In certain preferred embodiments, 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. In certain other preferred embodiments, 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

polymorphisms (SNPs) as shown in 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;

Sunyaev et al. 1999 Prot. Eng. 12:387). Certain SNPs in Table 1 may not result in detectable loss of function by these criteria and are referred to in Table 1 as "benign" mutations.

TABLE 1 . Human CYP8B1 SNPs

WT NT and MUTANT WT AA and Mutant AA Phenotype Position # NT Position #

in SEQ ID in SEQ ID

NO:2 NO:1

G1545 A R407 H LOF

C587 T P88 S benign

A1038 G K238 R benign

C1394 T L357 F benign

C1439 A Q372 K benign

G1529 A V402 I benign

G1756 A S488 N benign

C401 T R26 X LOF

C407 T R28 C LOF

G474 A R50 Q LOF

C500 T R59 C LOF

G563 A V80 I LOF

C605 T Q94 X LOF

C614 G L97 V LOF

A71 1 T K129 M LOF

G723 c G133 A LOF

A759 G D145 Q LOF

C883 A F186 L LOF

G884 A G187 S LOF

G945 A R207 H LOF

C1 185 T T287 M LOF

A1334 G T337 A LOF

C1351 A S342 R LOF

C1482 T P386 L LOF

G1545 A R407 G LOF

C1619 T P432 S LOF

A1652 G R443 G LOF WT NT and MUTANT WT AA and Mutant AA Phenotype Position # NT Position #

in SEQ ID in SEQ ID

NO:2 NO:1

T1683 G F453 C LOF

C1691 T L456 F LOF

T1698 G V458 G LOF

T1749 G V475 G LOF

C1784 A P487 T LOF

G1793 A D490 N LOF

G1806 A R494 H LOF

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 , and 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. With respect to the wildtype human CYP8B1 polynucleotide sequence, for example, these mutations are located at positions 1223, 483, 1348, and 1439,

respectively, of SEQ ID NO:2. For convenience, these polynucleotide mutations may be referred to herein by reference to their corresponding polypeptide substitutions. A summary of several non-limiting exemplary

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

It is to be understood that the terms "nucleotide,"

"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.

The term "polynucleotide" as referred to herein thus includes single-stranded and double-stranded nucleic acid polymers. In certain embodiments, 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,

phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,

phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term "polynucleotide" specifically includes single and double stranded forms of DNA.

The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate,

phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,

phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081 ; Stec et al., 1984, J. Am. Chem. Soc, 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et ai, 1991 , Anti-Cancer Drug Design, 6:539; Zon et al., 1991 , OLIGONUCLEOTIDES AND ANALOGUES: A

PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al., U.S. Pat. No. 5,151 ,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for any purpose. An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.

The term "vector" is used to refer to any molecule {e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "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.

The term "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. For example, 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.

The term "control sequence" as used herein 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. In particular embodiments, transcription control sequences for prokaryotes may include a promoter, ribosomal binding site, and transcription termination sequence. In other particular embodiments, 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. In certain

embodiments, "control sequences" can include leader sequences and/or fusion partner sequences.

As will be understood by those skilled in the art, 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.

As will also be recognized by the skilled artisan, 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.

As depicted in Figure 1 , 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. In particular embodiments, a full length CYP8B1

polynucleotide is the CYP8B1 gene sequence or the CYP8B1 cDNA sequence set forth in SEQ ID NO:2. In certain embodiments, 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.

In particular embodiments, 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. These may be readily determined by aligning CYP8B1 sequences from different species and identifying those polynucleotides and amino acids corresponding to the polynucleotides and amino acids modified in the human CYP8B1 mutant polynucleotides and proteins identified herein. In preferred embodiments, CYP8B1 polynucleotides and polypeptides are mammalian.

As described herein, polynucleotide compositions are provided that comprise a CYP8B1 polynucleotide sequence comprising one or more of the mutations described herein or encoding a mutant CYP8B1 polypeptide sequence described herein. In related embodiments, there are provided 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). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences, by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions. The term "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.

In additional embodiments, there are provided polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the CYP8B1 sequences disclosed herein. For example, 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. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 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. In particular embodiments, a polynucleotide fragment comprises one or more of the CYP8B1 mutations described herein. Accordingly, 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 .

In another embodiment, 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. For purposes of illustration, 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. One skilled in the art will understand that 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. For example, in another embodiment, 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. In particular embodiments, a polynucleotide that hybridizes to a CYP8B1 sequence comprises one or more of the mutations described herein.

The polynucleotides described herein or fragments thereof, regardless of the length of the coding sequence itself, 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. For example, polynucleotides of the present invention may be present in an expression vector. When comparing polynucleotide sequences, 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. Alternatively, 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

computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wl), or by inspection.

Certain preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which 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. Preferably, 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. 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.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can encode a CYP8B1 polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated within certain embodiments.

Additionally, alleles of the 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

substitutions of nucleotides. The resulting mRNA 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).

In general, polynucleotide sequences according to the herein described embodiments are isolated. "Isolated," as used herein, 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

manipulations such as those that are typical in recombinant biotechnology or other genetic engineering. As will be understood by those skilled in the art, the 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.

In certain embodiments, 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. For example, in particular embodiments, 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. Such 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. Thus, primers include CYP8B1 polynucleotide sequences that comprise any of the CYP8B1 polynucleotide mutations described herein, as well as complements thereof.

To permit hybridization under assay conditions, 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. Preferably, 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. In a particular embodiment, 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.

According to certain other embodiments of the present invention, there is provided 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

polypeptide comprising one of the mutations described herein. Also provided are variants of the CYP8B1 polypeptides described herein. 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

(determined as described herein), along its length, to a CYP8B1 polypeptide sequence set forth herein such as the polypeptide of SEQ ID NO:1 . In preferred embodiments, 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).

When comparing polypeptide sequences, 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. A

"comparison window" as used herein, 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

conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wl), using default parameters.

Alternatively, 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

Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which 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.

In one preferred approach, 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.

In general, polynucleotide and polypeptide compositions

(including fusion polypeptides) of the invention are isolated. An "isolated" polynucleotide or polypeptide is one that is removed from its original

environment. For example, a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. In certain embodiments, such polynucleotides and

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.

The CYP8B1 polynucleotides and polypeptides according to certain of the present invention embodiments 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). For example, short polynucleotide sequences may be synthetically produced, while longer polynucleotides may be produced from in vitro or in vivo expression systems. In addition, 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. In one illustrative example, 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. B. Methods of Modulating HDL and Triglycerides and Treating and Decreasing likelihood of occurrence of Cardiovascular Diseases and Disorders

Certain embodiments described herein relate in part to the surprising identification of high levels of plasma HDL in patients that lack a functioning CYP8B1 or that have a reduced functioning CYP8B1 , e.g., due to the presence of any one or more of the mutations identified herein in the gene encoding CYP8B1 (see, e.g., Table 1 ). 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).

Accordingly, in certain embodiments 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). In some embodiments, the agent reduces the expression and/or activity of CYP8B1 . In particular embodiments, 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. In one embodiment, 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. In particular embodiments, 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. In particular embodiments, 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.

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

Livingstone (Elsevier), London. See also, e.g., Smith, S.C. Jr, Benjamin, E.J., Bonow, R.O., Braun, L.T., Creager, M.A., Franklin, B.A., Gibbons, R.J., Grundy, S.M., Hiratzka, L.F., Jones, D.W., Lloyd-Jones, D.M., Minissian, M., Mosca, L., Peterson, E.D., Sacco, R.L., Spertus, J., Stein, J.H., Taubert, K.A. Circulation (201 1 ) 124(22):2458-73, AHA/ACCF Secondary Prevention and Risk Reduction Therapy for Patients with Coronary and other Atherosclerotic Vascular Disease: 201 1 Update: A Guideline from the American Heart Association and American College of Cardiology Foundation; Richmond, W. British Journal of Diabetes & Vascular Disease (2003) 3: 191 , When and how to measure lipids and their role in CHD risk prediction; Lear, S.A., Humphries, K.H., Kohli, S. and Birmingham, C.L. Obesity (2007) 15, 2817-2824, The Use of BMI and Waist Circumference as Surrogates of Body Fat Differs by Ethnicity; Littlea, R.R. and Sacks, D.B. Current Opinion in Endocrinology, Diabetes & Obesity (2009), 16:1 13-1 18, HbAl c: how do we measure it and what does it mean?).

As also noted above, "increased" or "decreased" levels of a given quantifiable parameter reflect statistically significant changes from a

preestablished baseline level and/or from an art-accepted normal level of the parameter. According to accepted principles and practices, those familiar with the relevant arts will recognize that 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. Medical Clinics North America (201 1 ) 95(5):919-37, Impact of obesity on cardiovascular disease: Degoma EM, Rader DJ. Nat Rev Cardiol. (201 1 ) 8(5):266-77, Protective role of HDL; Rutishauser J. Swiss Med Wkly (201 1 ) 21 ;141 :w13310, Benefits of LDL reduction; Syed IA, Khan WA. J Pak Med Assoc. (201 1 ) 61 (7):690-5, Glycated haemoglobin (HbA1 c)--a marker and predictor of cardiovascular disease.

According to certain preferred embodiments of the method for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder in a subject, 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. For example by way of illustration and not limitation, 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 assaying for CYP8B1 activity levels {e.g., by assaying for sterol 12-a-hydroxylase enzymatic activity) in a sample from a subject before and after exposure to the agent), or by assaying for CYP8B1 product levels {e.g., by assaying for 7,12-DiHCO) in a sample from a subject before and after exposure to the agent. In certain preferred embodiments, the agent that is an inhibitor of human cytochrome P450-family 8-subfamily B-polypeptide 1 (CYP8B1 ) sterol 12-a-hydroxylase activity may be selected from:

(a) a com ound of formula I:

wherein:

m is 0, 1 , 2, 3, 4 or 5;

n is 1 , 2 or 3;

X is -N- or -C(R⁶)-,

R⁴ and R⁵ is independently hydrogen or alkyl;

R⁶ is hydrogen or alkyl; and

each R⁷ 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;

(b) a com ound of formula II:

wherein:

— is independently a single or double bond;

each R⁹ is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, aralkyl, cycloalkyi, heterocyclyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;

each R^14a and R^14b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^14a and R^14b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

R¹⁵ is hydrogen or alkyl;

each R^16a and R^16b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^16a and R^16b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

each R^17a and R^17b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^17a and R^17b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

R¹⁸ is hydrogen, hydroxy, alkoxy, or alkyl;

R¹⁹ is hydrogen or alkyl;

each R^20a and R^20b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^20a and R^20b together forms =O, =S, =C(R⁹)₂; or =NR⁹, R²¹ is hydrogen or alkyl, or R²¹ and R^16a together form a bond; and

each R^22a and R^22b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyi, heterocyclyl, cycloalkylalkyi, or heterocyclylalkyl; or R^20a and R^20b together forms =O, =S, =C(R⁹)₂; or =NR⁹, or R^22b and R²¹ together form a bond,

as a stereoisomer, enantiomer, tautomer thereof or mixtures thereof, or a pharmaceutically acceptable salt; and

(c) a compound of formula III:

wherein:

t is 0, 1 , 2, 3, 4 or 5;

each R⁹ is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyi, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, or heterocyclylalkyl;

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,

as a stereoisomer, enantiomer, tautomer thereof or mixtures thereof, or a pharmaceutically acceptable salt.

"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,

isobutylenyl, 1 -pentenyl, 2-pentenyl, 3-methyl-1 -butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1 -butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 3-methyl-1 -butynyl, and the like.

"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, ~(CH₂)2 phenyl, ~(CH₂)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 ~CH₂ pyridinyl, - CH₂ 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. Thus, in addition to the heteroaryls listed 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 ~CH₂ 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.

The term "substituted" as used herein means any of the above groups {e.g., alkyl, alkenyl, alkynyl, homocyde) wherein at least one hydrogen atom is replaced with a substituent. In the case of a keto substituent ("-C(=0)- ") two hydrogen atoms are replaced. When substituted one or more of the above groups are substituted, "substituents" within the context of this invention include halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyi, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, as well as ~NR_aRb, ~NR_aC(=O)Rb -, NR_aC(=O)NR_aNRb, -NR_aC(=O)OR_b -NR_aSO₂Rb, -C(=O)R_a, -C(=O)OR_a, -- C(=O)NR_aR_b, -OC(=0) NR_aR_b, -OR_a, -SR_a, -SOR_a, -S(=O)₂R_a, -OS(=O)₂R_a and -S(=O)₂OR_a. In addition, the above 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.

In certain other preferred embodiments, 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.

From the disclosure herein for the first time of the relationship in humans between CYP8B1 activity and clinically significant circulating HDL levels, there are provided the present methods 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 to criteria described herein and/or as known in the art.

In a related embodiment, 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 . In particular embodiments, the agent reduces the expression and/or activity of CYP8B1 . In certain

embodiments, the cardiovascular disease or disorder is dyslipidemia, atherosclerosis, a low HDL disease, or a related disorder. In particular embodiments, 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

polynucleotide or mRNA.

As used herein, "subject" and "patient" are used interchangeably and refer to an individual having or at risk for having a particular disease or disorder. In certain embodiments the subject is an animal, and preferably a mammal, most preferably a human. In particular embodiments, 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.

Without intending to be bound by theory, 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 . In one embodiment, a CYP8B1 inhibitor may specifically inhibit or bind to a catalytic domain of a CYP8B1 protein. In particular embodiments, a CYP8B1 inhibitor may inhibit or bind a O2-binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide. In certain embodiments, a CYP8B1 inhibitor reduces or inhibits a hydroxylase activity of a CYP8B1 protein. In particular embodiments, 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 .

As used herein, unless the context makes clear otherwise,

"treatment," and similar words such as "treated," "treating" etc., 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.

As used herein, unless the context makes clear otherwise, "reducing the likelihood of occurrence," "prevention," and similar words such as "prevented," "preventing" etc., 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. Similarly, also included are 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. As used herein, "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 . As used herein, 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.

As used herein, 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 cholestasis, different eye disorders {e.g., Stargardt disease, autosomal recessive retinitis pigmentosa and cone-rode dystrophy), Tangier disease, and Zellweger syndrome. In certain embodiments, the CVD is atherosclerosis, coronary artery disease, or hypercholesterolemia. In certain embodiments, the CVD or related disorder is atherosclerosis.

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. The present discovery, that CYP8B1 functions to regulate HDL levels, links CYP8B1 with the foregoing processes.

In various embodiments of the present methods for treating or reducing the likelihood of occurrence of CVD, 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%. In certain embodiments, the target tissue where the expression or activity of CYP8B1 is measured or monitored is the liver. In other embodiments, the expression or activity of CYP8B1 is inhibited by not more than 50%, 40%, 30%, or 10%. Certain CYP8B1 loss-of-function mutation carriers are heterozygotes, whereby they only have one mutant copy of CYP8B1 . This suggests according to non-limiting theory that any

therapeutics that increase plasma HDL for treating cardiovascular diseases would only be required to inhibit CYP8B1 activity by a maximum of 50% in order to be effective. In some cases, it is possible that 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.

In certain embodiments, 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. In particular embodiments, 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.

According to certain embodiments of the herein described methods for treating or reducing the likelihood of occurrence of a cardiovascular disease or disorder in a 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.

In these and related embodiments, 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-family 8-subfamily B- polypeptide 1 (CYP8B1 ) sterol 12-a-hydroxylase activity, wherein the mutation that results in decreased CYP8B1 activity comprises at least one substitution mutation of a human CYP8B1 polypeptide having the amino acid sequence set forth in SEQ ID NO:1 and the substitution is at an amino acid position that corresponds to a wildtype amino acid position that is selected from those set forth in Table 1 . In preferred embodiments the mutation is a loss-of-function mutation.

Certain embodiments of the present invention further include pharmaceutical compositions comprising a CYP8B1 modulating agent, e.g., a CYP8B1 inhibitor or antagonist, and a pharmaceutically acceptable carrier, diluent or excipient. "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.

Pharmaceutical 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. The term 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. Furthermore, pharmaceutical compositions comprising an agent that is capable of decreasing a level of CYP8B1 expression or

CYP8B1 activity in the subject, or 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, may include one or more additional active agents, or may be administered in conjunction with one or more additional active agents.

C. Methods of Identifying CYP8B1 Mutations and Related Kits

According to certain embodiments of the present invention there are also provided methods for detecting 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.

As used herein, 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. For example, 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. In particular embodiments, 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.

These and related embodiments will find uses, for example, in pharmacogenomics, for the classification and/or stratification of a subject or patient population. In one embodiment, for example, such stratification may be achieved by identification in a subject or patient population of one or more distinct profiles of at least one 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. In another embodiment, 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.

By using the combination of the methods for determining at least one SNP that, as disclosed herein, is associated with a decreased risk of CVD, and methods known in the art for determining one or more of elevated HDL, decreased LDL, decreased plasma triglycerides, decreased BMI, and

decreased blood level of HbA1 c, an enhanced ability to detect the relative risk for CVD is provided by the present disclosure, along with other related advantages.

As described herein, 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. For instance, 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, may thereby permit stratifying the population according to cardiovascular disease risk.

Accordingly, in another preferred embodiment of the invention, 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. For example, 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. For example, detection in the 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. 77:2437, 1989), transcriptional amplification systems (TAS), strand displacement amplification (SDA), self-sustained sequence replication (3SR, Ghosh et al, in Molecular Methods for Virus Detection, 1995 Academic Press, NY, pp. 287-314), ligase chain reaction (LCR), single stranded conformational polymorphism analysis, Q-beta replicase assay, restriction fragment length polymorphism (RFLP, Botstein et al., Am. J. Hum. Gen.

32 2> A, 1980) analysis, cycled probe technology (CPT), oligonucleotide primer extension, and others known to persons skilled in the art.

To permit hybridization under assay conditions, 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. Preferably, 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. In a preferred embodiment, 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.

Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 57:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989). One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as a blood sample, and is reverse transcribed to produce cDNA molecules. 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. Alternatively, 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.

Related embodiments of the present invention include polynucleotide sequences 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 ). As such, it is contemplated that 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. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 1500, 1501 , 1502, 1503, 1504, 1505, 1506 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.

In particular embodiments, 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

substitution at a nucleotide position that corresponds to a wildtype nucleotide position that is selected from those set forth in Table 1 . The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. In one exemplary embodiment, 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.,

GeneChip™) to identify CYP8B1 proteins, orthologs, alleles, conservatively modified variants, and polymorphic variants. The present disclosure also provides, according to certain embodiments, kits relating to any of the CYP8B1 modulating agents and/or methods described herein. 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.

In particular embodiments, 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. In particular embodiments, 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 . In particular embodiments, 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.

In certain embodiments, a kit 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.

Furthermore, a kit may also be designed to operate through the use of an apparatus, instrument or device, including a computer.

D. Methods of Identifying Agents that Modulate CYP8B1 and Agents that Modulate CYP8B1

According to certain embodiments 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. In particular embodiments, 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. For example, 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.

Chem., 267:21319-21323).

In certain embodiments, an inhibitor or antagonist of CYP8B1 expression prevents or reduces the expression of CYP8B1 mRNA or protein. To determine if a test agent inhibits the expression of CYP8B1 mRNA, 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). 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. In particular embodiments, biological samples include cells that express CYP8B1 .

Similarly, 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). Certain embodiments thus contemplate 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 expression of CYP8B1 by a cell, thereby determining a base level of CYP8B1 expression; contacting the cell with a test agent;

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.

It is understood that 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. Typically, 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. One of skill in the art will appreciate that 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. Typically, the cells are the same type and express a comparable amount of CYP8B1 when grown under comparable conditions.

In certain embodiments, 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. In one embodiment, 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.

In certain related embodiments there is provided 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 an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder. The base and test levels of CYP8B1 expression may be determined, in accordance with the present disclosure, by quantifying CYP8B1 mRNA and/or by quantifying

CYP8B1 protein, using, respectively, methodologies for quantifying a specific mRNA or a specific protein that are well within the knowledge of the art.

In certain other related embodiments there is provided 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 determination that the test level of CYP8B1 activity is less than the base level of CYP8B1 activity indicates the candidate agent is an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder. In certain preferred embodiments the CYP8B1 activity is a sterol 12-a-hydroxylase activity that can be determined using established methodologies, such as the enzyme activity assay described in the Examples. The effect of the candidate agent on CYP8B1 activity may be assessed while the CYP8B1 polypeptide is present in a cell; the method may also be practiced in vitro. Preferably and in certain embodiments, each of the first and second CYP8B1 polypeptides (or a fragment or variant thereof as provided herein) comprises a CYP8B1 catalytic domain.

In certain other embodiments, each of the first and second

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).

The crystal structures of two P450 enzymes having a high degree of primary sequence identity to CYP8B1 have been solved; CYP8A1 (42% sequence identity to CYP8B1 , Protein Data Base (PDB) entry 3B6H) and CYP7A1 (36% sequence identity to CYP8B1 , PDB entry 3SN5; see also PDB entry 3DAX). These structures permit the construction of a 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. (Lopez-de-Brifias et al., J Gomput Aided Moi Des. 1 1 :395- 408, 1997; Lertkiatmongkol ei a!., BMC Res Notes, 4:321 , 201 1 ; Kanth et al. Comput Biol Chem. 34:226-31 , 2010.)

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. (Jefcoate, Methods Enzymol. 52:258-79, 1978; Yano et al., J Med Chem. 49:6987-7001 , 2006). 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. Hence, according to certain embodiments 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. 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 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. In a related embodiment, the agent specifically binds to a polynucleotide sequence encoding CYP8B1 or a complement thereof. In particular embodiments, the agent comprises a siRNA or an antisense oligonucleotide. In specific embodiments, the agent is a small molecule or an antibody.

In another embodiment, 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. In one embodiment, the CYP8B1 activity is conversion of a substrate.

In one embodiment, 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. Typically, 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. In a particular embodiment, the agent inhibits the conversion of a CYP8B1 substrate by CYP8B1 . In one embodiment, the agent specifically binds to CYP8B1 . In particular embodiments, 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;

measuring the activity of the CYP8B1 polypeptide after being contacted with the test agent, 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 treat or decrease likelihood of occurrence of a cardiovascular disease or disorder. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, the agent specifically binds to CYP8B1 . In certain embodiments, the agent is an antibody that binds specifically to CYP8B1 . In other

embodiments, the agent is a small molecule. In particular embodiments, 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. In certain embodiments, the agent binds to an O2-binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide. In certain embodiments, the CYP8B1 protein is a human CYP8B1 protein, and in one embodiment, the CYP8B1 protein is a wild-type human CYP8B1 protein.

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. As used herein, 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. In various embodiments, 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. In particular embodiments, 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 .

In various embodiments, 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.

In particular embodiments, the term "CYP8B1 inhibitor" or

"CYP8B1 antagonist" as used herein 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. In alternative embodiments, 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.

Polypeptides

In certain embodiments, the present invention contemplates the use of polypeptide inhibitors of 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.

According to certain presently contemplated embodiments there are provided 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. For example, in certain embodiments, 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. In one illustrative example, 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.

Antibodies

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.

The term "antibody" (Ab) 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. The term "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. In preferred embodiments, 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.

An "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. Examples of 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).

The antibodies described for use herein may be polyclonal or monoclonal antibodies. In particular embodiments, they are monoclonal.

Methods of producing polyclonal and monoclonal antibodies are known in the art and described generally, e.g., in U.S. Patent No. 6,824,780 and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, 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. To increase immunogenicity, an immunogen may be linked to, for example, glutaraldehyde or keyhole limpet hemocyanin (KLH). Following injection, the animals are bled periodically to obtain post-immune serum containing polyclonal anti-CYP8B1 antibodies. 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.

For certain embodiments, 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. For example, 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. For example, to recover monoclonal antibodies from mouse-derived hybridomas, 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.

In certain embodiments, antibodies may be produced recombinantly, using vectors and methods available in the art, as described further below. For example, 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.

In particular embodiments, antibodies are fully human antibodies. Human antibodies may be generated by in vitro activated B cells (see U.S.

Patent Nos. 5,567,610 and 5,229,275). In addition, 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

immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of

endogenous antibody production. Transfer of the human germ-line

immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. (See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806; 5,545,807; 5,569,825; 5,591 ,669; 5,770,429; 6,596,541 and 7,049,426; and PCT Patent Application Publication No. WO 97/17852.) Such animals may be genetically engineered to produce human antibodies that specifically recognize CYP8B1 polypeptides including mutant CYP8B1 polypeptides described herein.

In certain embodiments, antibodies are chimeric antibodies that comprise sequences derived from both human and non-human sources. In particular embodiments, these chimeric antibodies are humanized or

Primatized™. In practice, 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

corresponding sequences from a non-human animal. It is important that 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. To achieve this goal, according to one method, 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. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and imported sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

In one embodiment, a specific anti-CYP8B1 antibody that has activity as an inhibitor of CYP8B1 ("a CYP8B1 antibody inhibitor") may specifically inhibit or bind to a catalytic domain of a CYP8B1 protein, or a fragment or variant thereof. In particular embodiments, a CYP8B1 antibody inhibitor may inhibit or bind an O₂-binding domain, a steroidogenic region, or a heme binding domain of the CYP8B1 polypeptide. In certain embodiments, a CYP8B1 antibody inhibitor reduces or inhibits a hydroxylase activity of a CYP8B1 polypeptide, or of a fragment or variant thereof. In particular embodiments, 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 .

Polynucleotides

In certain embodiments, a CYP8B1 antagonist may be a polynucleotide. The term "polynucleotide" refers to a DNA or RNA (or mixed) molecule that has been isolated free of total genomic DNA of a particular species. As will be also recognized by the skilled artisan, 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

endogenous sequence) or may comprise a sequence that encodes a variant or derivative of such a sequence. Polynucleotides may consist of natural and/or non-natural bases.

In additional embodiments, polynucleotide fragments may comprise various lengths of contiguous stretches of sequence identical to or complementary to a CYP8B1 -encoding polynucleotide sequence. For example, 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. It will be readily understood that "intermediate lengths", in this context, 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.

In another embodiment 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

complementary sequence thereto. In particular embodiments, the

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. In particular embodiments, 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.

4,683,202 (incorporated herein by reference), by introducing selected

sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

In certain embodiments, 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;

5,789,573; and 5,610,288). 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).

Therefore, in certain embodiments, 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. In one embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In another embodiment, the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone. In another embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In another embodiment, 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). In each case, 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.

In certain embodiments, methods of the present invention are practiced using interfering RNA, e.g., siRNA, that targets a CYP8B1 mRNA, thereby reducing expression of CYP8B1 . The term "RNA interference" or "RNAi" 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). Duplex RNA can activate the RNA-induced silencing complex (RISC) to degrade target mRNA. The term "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

complementary to a target mRNA sequence. 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. For a review, see Gerwitz, J. Clin. Invest. 1 17(12):3612-3614 (2007) and Obbard et al., Phil. Trans. Roy. B. 364:99-1 15 (2008).

Interfering RNA includes, but is not limited to, "small-interfering

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). In some embodiments, the siRNA lacks a terminal phosphate. In some embodiments, the siRNA or miRNA duplex lacks 3' overhangs, i.e., has "blunt-ends".

Examples of siRNA 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.

2002/0086356); a double-stranded polynucleotide molecule assembled from a single oligonucleotide, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker (see, e.g., U.S. Patent Application Publication No. 2009/0005332 and PCT 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. Oligonucleotides, 17:223-236, 2007); and a circular single- stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double- stranded siRNA molecule. As used herein, "viral RNA" or "viRNA" 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. Upon interaction with RISC, 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. USA 99: 14236 (2002); Byrom et ai, Ambion TechNotes 10(1 ): 4-6 (2003); Kawasaki et ai, Nucleic Acids Res. 31 : 981 -7 (2003); Knight and Bass, Science 293: 2269-71 (2001 ); Robertson et ai, J. Biol. Chem. 243: 82 (1968); and U.S. Patent Application Publication No. 2005/0244858). Preferably, 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.

The phrase "inhibiting 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 . To examine the extent of gene silencing, 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) may be contacted with 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%.

As described above, an interfering RNA can be provided in several forms. For example, 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.

In preferred embodiments, the interfering RNA is a siRNA molecule that is capable of silencing expression of a target gene (i.e.,

CYP8B1 ). 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. In some embodiments, the siRNA lacks terminal phosphates. In some embodiments, the siRNA duplex lacks 3' overhangs, i.e., have blunt-ends.

In some embodiments, the antisense oligonucleotides or interfering RNA molecules described herein comprise at least one region of mismatch with its target sequence. As used herein, the term "region of mismatch" refers to a region of a siRNA that does not have 100%

complementarity to its target sequence. For example, 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. For example, 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). Typically, 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. Typically, 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. For example, 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. Typically, siRNA target sites are spaced at different postitions along the length of the target gene. To further enhance silencing efficiency of the siRNA sequences, 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.

Once a potential siRNA sequence has been identified, the sequence can be analyzed using a variety of criteria known in the art. For example, to enhance silencing efficiency, 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. One of skill in the art will appreciate that sequences with one or more of the foregoing characteristics may be selected for further analysis and testing as potential siRNA sequences. siRNA

sequences complementary to the siRNA target sites may also be designed.

Additionally, potential 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. However, one of skill in the art will appreciate that sequences with one or more of the foregoing characteristics may still be selected for further analysis and testing as potential siRNA sequences.

Once a potential siRNA sequence has been identified, 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

immunostimulatory, it can then be modified to decrease its immunostimulatory properties. As a non-limiting example, 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

immunostimulatory or a non-immunostimulatory siRNA. 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-β, IFN-α, IFN-γ, IL-6, IL-12, or a combination thereof. An 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

immunostimulatory properties have been reduced or abrogated.

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);

monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the "Western blot" method of Gordon et al. (U.S. Patent No.

4,452,901 ); immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem. 255:4980-4983 (1980)); enzyme-linked immunosorbent assays (ELISA) as described, for example, by Raines et al., J. Biol. Chem. 257:5154-5160 (1982); immunocytochemical techniques, including the use of fluorochromes (Brooks et al., Clin. Exp. Immunol. 39:477 (1980)); and neutralization of activity (Bowen- Pope et al., Proc. Natl. Acad. Sci. USA 81 :2396-2400 (1984)). In addition to the immunoassays described above, a number of other immunoassays are available, including those described in U.S. Patent Nos. 3,817,827; 3,850,752; 3,901 ,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.

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

intravenous injection in the lateral tail vein; (2) blood can be collected by cardiac puncture about 6 hours after administration and processed as plasma for cytokine analysis; and (3) 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-γ (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

transcribed from a transcriptional cassette in a DNA plasmid. 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).

An 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.

To form a long dsRNA, for synthetic RNAs, the complement is also transcribed in vitro and hybridized to form a dsRNA. If a naturally occuring RNA population is used, 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.

Alternatively, 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. 20:500 (2002); Miyagishi, et ai, Nat. Biotech. 20:497 (2002); Paul, et ai, Nat. Biotech. 20:505 (2002); and Sui, et ai, Proc. Natl Acad. Sci. USA 99:5515 (2002)). Typically, 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. The

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.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene 25:263-269 (1983);

Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see, U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those of skill in the art. Additional basic texts disclosing the general methods of use in this invention include Sambrook et ai, Molecular Cloning, A

Laboratory Manual {2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

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). The synthesis of

oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidites at the 3'- end. As a non-limiting example, small scale syntheses can be conducted on an Applied Biosystems synthesizer using a 0.2 μιτιοΙ scale protocol with a 2.5 min. coupling step for 2'-O-methylated nucleotides. Alternatively, synthesis at the 0.2 μιτιοΙ scale can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA). However, 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.

Alternatively, 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. For example, each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection. In certain other instances, the modified siRNA molecule can be synthesized as a single continuous

oligonucleotide fragment, wherein the self-complementary sense and antisense regions hybridize to form an siRNA duplex having hairpin secondary structure. In certain embodiments, the 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).

The 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

Application Publication Nos. 2006/0035254, 2007/0054873, and

2008/0085999). Similarly, the antisene oligonucleotides described herein can comprise at least one modified nucleotide. Examples of 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. (1984), are also suitable for use in the siRNA molecules of the present invention. Such 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. In certain instances, the siRNA molecule includes one or more G-clamp

nucleotides. 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)). In addition, 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.

In certain embodiments, the siRNA molecule can further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like. Examples of terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl

modifications, 4',5'-methylene nucleotides, l-( -D-erythrofuranosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1 ,5-anhydrohexitol nucleotides, L- nucleotides, a-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl

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.

Patent No. 5,998,203; Beaucage et al., Tetrahedron 49:1925, 1993). Non- limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate,

phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331 -417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994); and U.S. Patent Application Publication No. 2006/088490). Such chemical modifications can occur at the 5'-end and/or 3'- end of the sense strand, antisense strand, or both strands of the siRNA.

In some embodiments, 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.

2007/0265220). Additional examples of modified nucleotides and types of chemical modifications that can be introduced into the modified siRNA molecule are described, e.g., in UK Patent No. GB 2,397,818 B.

The modified siRNA molecules described herein can optionally comprise one or more non-nucleotides in one or both strands of the siRNA duplex. As used herein, the term "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.

In other embodiments, 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). In certain instances, the conjugate is a molecule that facilitates the delivery of the chemically-modified siRNA into a cell. Examples of 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. Patent Application Publication Nos. 2003/0130186 and 2004/01 10296; U.S. Patent Nos. 6,753,423 and 7,491 ,805). 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.

2004/0167090. Further examples include the conjugate molecules described in U.S. Patent Application Publication No. 2005/0239739. The type of conjugate used and the extent of conjugation to the chemically-modified siRNA molecule can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the siRNA. As such, one skilled in the art can screen chemically- modified siRNA molecules having various conjugates attached thereto to identify ones having improved properties using any of a variety of well-known in vitro cell culture or in vivo animal models.

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). In addition to lipid and polymer-based compositions, 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

Application Publication No. WO 2003/040399).

According to another embodiment of the invention, 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

Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):21 1 -20). The specificity of ribozymes has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozymes may be designed as described in PCT Patent

Application Publication Nos. WO 93/23569 and WO 94/02595 (each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for activity and delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary.

In another embodiment of the invention, peptide nucleic acids (PNAs) that target CYP8B1 are provided. 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

complementary to one or more portions of the CYP8B1 mRNA sequence, and such 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

6;254(5037):1497-500; Hanvey et al., Science 1992 Nov 27;258(5087):1481 -5; Hyrup and Nielsen, Bioorg. Med. Chem. 1996 Jan;4(1 ):5-23). This chemistry has three important consequences: firstly, in contrast to DNA or

phosphorothioate oligonucleotides, 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. Alternatively, 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. Once synthesized, 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.

Other applications of PNAs that have been described and will be apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like. Small Molecules

Inhibitory agents of the present invention further include large or small inorganic or organic molecules. In certain embodiments, 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.

In certain embodiments, a modulator includes a protecting group. The term "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.

Any of the 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.

In a particular embodiment, a small molecule inhibitor binds to CYP8B1 . In one embodiment, 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 , including small organic compounds, 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. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or

"comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; and so forth. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

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

Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H.

Margulies, Ethan M. Shevach, Warren Strober, 2001 and 2010, John Wiley & Sons, NY, NY); or other relevant Current Protocol publications and other like references. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

EXAMPLES

EXAMPLE 1

METHODOLOGIES FOR IDENTIFYING NOVEL MUTATIONS IN MULTIPLE HHDL

INDIVIDUALS In this and the following examples, study subjects with high HDL

(HHDL) from the Lipid Clinic Network and Vascular Research Network

(Amsterdam, Netherlands), The Centre for Molecular Medicine and

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). For the measurement of lipoprotein lipids, 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.

In order to sequence CYP8B1 in patients, 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. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386; http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi, MIT, Cambridge, MA). PCR products were amplified from genomic DNA, purified using the AMPure kit (Agencourt, Beverly, MA), and sequenced bidirectionally by fluorescent dye-terminator chemistry (Seqwright, Houston TX). Known and novel single nucleotide polymorphisms (SNPs) in the CYP8B1 gene were identified from manual sequence analysis and confirmed in dbSNP build 129 (National Center for Biotechnology Information, Bethesda, MD,

http://www.ncbi.nlm.nih.gov). Potential deleterious functional and splicing effects were estimated using the online Polymorphism Phenotyping-2 tool Polyphen version 2.0 (Adzhubei et al., 2010 Nat. Meth. 7(4):248;

http://genetics.dot.bwh.dot.harvard.dot.edu/pph/) and/or the Sorting Intolerant from Tolerant (SIFT) tool (Kumar et al., Nat Protoc. 2009; 4(7):1073-81 ;

http://sift.dot.jcvi.dot.org/).

EXAMPLE 2

NOVEL MUTATIONS IDENTIFIED IN MULTIPLE HHDL INDIVIDUALS

In order to identify mutations associated with high HDL, cohorts of 647 unrelated probands with high HDL (HHDL) and 398 unrelated probands with low HDL (LHDL) consisting of Dutch, Canadian, South African, Pakistani and Singaporean ancestry were assembled. HHDL was defined as HDL levels of at least 90^th percentile of the individual cohort while 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

Canadian probands. Lipid measures and medical histories were ascertained for the 174 HHDL probands with Dutch or Canadian ancestry (Table 3). Table 3: Lipid profiles of Dutch or Canadian ancestry HHDL individuals.

647 unrelated probands with HHDL and 398 unrelated probands with LHDL were sequenced for CYP8B1 . Following sequence analysis, 12 mutations were identified in HHDL and LHDL probands as summarized in Table 4. Seven mutations were novel and five mutations were rare and common SNPs previously identified in on-line databases. A further cohort of 133 probands with HDL levels of between the 70^th and 89^th percentiles was also sequenced for CYP8B1 and a single additional novel mutation (M53T) was identified.

Table 4: CYP8B1 mutations identified in HHDL and LHDL individuals.

The predicted damaging CYP8B1 mutations were found almost exclusively in probands with high HDL. Only a single CYP8B1 mutation carrier (R349Q) was found in the low HDL probands (< 10^th percentile) and this individual also had multiple mutations in the ABCA1 gene, indicating that reverse cholesterol transport was disrupted, leading to an abnormally low level of HDL. K300X was a loss-of-f unction mutation that resulted in the premature truncation of the protein at amino acid 300, which was situated approximately two-thirds of the way through the wild-type protein sequence (progressing in the N- to C- direction). 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.

Several mutations were found in multiple probands; for example, D341 E was found in two probands, and K238R was found in three probands. These mutations found in multiple probands were therefore potential

"Goldilocks alleles" that were likely to confer deleterious effects on protein function but that are common enough in humans to afford assessment of, for non-limiting example, lipid levels, cardiovascular disease (CVD) risk, and other clinical measures in large populations with sufficient statistical power.

EXAMPLE 3

CYP8B1 MUTATIONS SIGNIFICANTLY SEGREGATE WITH HHDL IN FAMILIES In order to validate that the CYP8B1 mutations were causative for

HHDL, the prevalence of HHDL in each of the proband's family members was investigated. When available, DNA from individuals from each family was genotyped, and it was found that individuals with the CYP8B1 mutations tended to segregate with higher HDL. For example, 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. EXAMPLE 4

EFFECT OF CYP8B1 MUTATIONS ON STEROL 12-a HYDROXYLASE ACTIVITY

In this test, an in vitro cell assay model was used to elucidate the effects of CYP8B1 mutations on expression and function. This test can also be used for high-throughput drug screening. This example describes the characterization of the enzymatic activity and protein stability properties of the following CYP8B1 mutants: M53T, A103E, D195N, K300X, D341 E, R349Q, R407H, P88S, K238R, L357F, Q372K, V402I, and S488N (see Tables 1 and 3), which were identified in individuals with HDLc > 90^th percentile.

HEK-293 cells were seeded into 6-well plates at 6.5 x 10⁵ 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. Mammalian expression vectors (pcDNA 3.1 (Invitrogen

Technolgies, Carlsbad, CA)) encoding wildtype (WT) human CYP8B1 (501 amino acids, SEQ ID NO:1 ) or of one of the CYP8B1 mutants (correspondingly mutated SEQ ID NO:1 in which the wildtype amino acid indicated by the single- letter symbol preceding the amino acid sequence position number was replaced with the amino acid indicated by the single-letter symbol following the sequence position number, with the exception of "X" residues in which the codon encoding the wildtype amino acid at the indicated amino acid sequence position number was replaced with a stop codon) were transfected into the cells using FuGENE® HD (Roche Diagnostics) at a DNA:FuGENE ratio of 2 μg:5 μΙ_, according to manufacturer's protocol. After a further 24 hours culturing, the cell medium was removed, cells were washed with 2 mL/well DMEM only, and the medium was replaced with 3 ml_ incubation media containing a final

concentration of 10 μΜ of 7a-hydroxy-4-cholesten-3-one (7-HCO) (Toronto Research Chemicals). The cells were cultured for a further 4 hours, after which 0.5 ml_ of cell-conditioned medium was collected from each well.

Media were centrifuged at 500 x g for 5 minutes at room

temperature, to remove residual cells, and 0.3 ml_ of supernatant was transferred to a 1 .5 ml_ Eppendorf® tube for liquid-liquid extraction using 3 volumes of ice-cold HPLC grade acetonitrile (ACN) containing a final

concentration of 0.35 μΜ D7-7a-hydroxy-4-cholesten-3-one (Toronto Research Chemicals). 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% ddH₂O:10% trifluoroacetic acid (TFA). 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). 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 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.

Table 5. CYP8B1 Activity in PLOF and CLOF Mutants

D195N 81 .9 (6.3)

D341 E 66.5 (5.5)

M53T 62.9 (7.2)

A103E 5.4 (0.6)

K300X 0

R349Q 0

R407H 1 .5 (1 .1 )

EXAMPLE 5

EFFECT OF CYP8B1 MUTATIONS ON CYP8B1 PROTEIN STABILITY AND STEROL 12-a

HYDROXYLASE ACTIVITY

In order to determine whether decreased CYP8B1 enzymatic activity in CYP8B1 mutants resulted from structural changes in the enzyme that decreased stability of the mutant CYP8B1 proteins relative to the wildtype human CYP8B1 , time-course experiments were performed in which cultured HEK-293 cells that had been transfected in vitro with recombinant expression vectors directing the expression of wildtype or mutant CYP8B1 were treated with cycloheximide (CHX) at various time points to halt protein expression.

HEK-293 cells were seeded into 6-well plates at 6.5x10⁵ 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. Mammalian expression vectors (pcDNA 3.1 (Invitrogen

Technolgies, Inc.)) encoding human CYP8B1 WT or CYP8B1 mutants were transfected into the cells using FuGENE® HD (Roche Diagnostics) at a

DNA:FuGENE ratio of 2 μg:5 μί 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 μg/mL cycloheximide (Sigma, St. Louis, MO). At 0, 1 , 2, 4, and 8 hours after addition of CHX, cell media were removed from separate wells and 0.2 mL ice-cold extraction buffer (0.025 M NH₄CI, pH 8.2, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.4 mg/mL sodium dodecyl sulfate (SDS), and 8 mg/mL TritonX-100) was added to the cells and the plates were frozen at -80 °C overnight. Plates were then thawed on ice, cells scraped from wells using a cell scraper (Corning, Inc., Tewksbury, MA), transferred to an ice-cold Eppendorf tube and cell lysates were sonicated for 30 seconds. After clearing cell lysates by centrifugation at 20,000 x g for 10 minutes at 4 °C, the protein concentration of lysates was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce

Chemicals, Inc., Rockford, IL) according to the manufacturer's protocol.

For western immunoblot analysis, cell lysates (20 μg) 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). 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. 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 . Eight hours post-termination of protein expression by CHX, the levels of the D195N mutant and the D341 E mutant were, respectively, approximately 80% and 85% of the levels of the wildtype CYP8B1 .

Results for benign CYP8B1 mutants are shown in Figure 7. The

P88S, K238R, L357F, Q372K and S488N mutations each did not cause detectable loss of stability for the respective mutant CYP8B1 proteins.

In order to determine whether decreased CYP8B1 enzymatic activity in CYP8B1 mutants resulted from structural changes in the enzyme that altered the catalytic activity, microsomal fractions of HEK-293 CYP8B1 WT and mutant transfectants were assayed for sterol-12-a-hydroxylase activity and the specificity constant (Vmax/Km) for each mutant calculated.

HEK-293 cells were seeded into 15 cm² sterile petri dishes (Corning) at 1 .1 x10⁷ 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 μg:91 μΙ_ 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. 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.

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 μΙ_ dispensed into each well of a 96-well plate. The plate was mixed on a microtitre plate shaker for 30 s. 70 μΙ_ of stop solution (acetonitrile, containing 0.75 μΜ D7-7a-hydroxy-4-cholesten-3-one (Toronto Research Chemicals) and 1 % formic acid) was dispensed into the negative control wells (time 0). A titration (final assay concentration 0.069-50 μΜ) of the CYP8B1 substrate 7a- hydroxy-4-cholesten-3-one (7-HCO, Toronto Research Chemicals) in 0.1 M potassium phosphate buffer, pH7.4, containing 4 mM NADPH (Sigma), was added to the wells in 17.5 μΙ_ and reactions incubated at room temperature for 20 and 40 minutes prior to addition of 70 μΙ_ of stop solution. Following mixing, and centrifugation at 3,000 x g for 20 minutes at 4 °C, 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.

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 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. 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. Of the CYP8B1 PLOF mutants, D195N and D341 E had specificity constants that were comparable to or greater than that of wildtype CYP8B1 , while the M53T mutant had a specificity constant value of about 55% that of the wildtype CYP8B1 . Thus, although 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 .

EXAMPLE 6

CYP8B1 MUTATION CARRIERS HAVE AN ATHEROPROTECTIVE PLASMA LIPID PROFILE Additional lipid profile measures were also compared between the

CYP8B1 carriers with experimentally confirmed loss of function mutations and control individuals. The lipid profiles for a cohort of 305 unrelated individuals, after adjusting for age, were obtained (Table 6). When compared to these 305 unrelated population controls with largely normal HDLc, a total of 23 individuals carrying heterozygous functionally damaging CYP8B1 mutations (both partial and complete loss of function) were observed to have a 27.0% elevated HDLc. This HDLc rise was highly significant (p=0.001 ).

In addition to an increase in HDL in CYP8B1 loss of function mutation carriers, a 28.5% (p=0.038) reduction in triglycerides (TG), a 6.7% decrease in LDLc (not significant) and a 5.4% (not significant) reduction in body mass index (BMI) were observed vs. 305 population controls. These data suggested that the effect of the CYP8B1 mutations was not specific to HDL modulation alone, but instead offered additional benefits through modulation of TG, LDLc and BMI.

Moreover, a gene dosage relationship was also observed, as the heterozygote complete loss of function carriers showed a greater beneficial effect on each parameter (HDLc, LDLc, TG and BMI, Figures 9, 10, 1 1 , 12).

Table 6: Lipid profiles of population controls.

For each measurement, the average (SD) is shown.

EXAMPLE 7

CYP8B1 MUTATIONS ARE ATHEROPROTECTIVE

To determine whether HDL-raising CYP8B1 mutations were atheroprotective in families, medical histories from all 4 complete loss of function CYP8B1 mutation carriers over age 30, in addition to 212 population control individuals over age 30, were obtained. 25 of 212 control individuals (1 1 .8 %) reported having CVD. In contrast, no complete loss of function CYP8B1 mutation carriers reported any CVD events or adverse events (e.g. diabetes, hypertension, stroke or gallstones). Although these observations were not statistically significant due to the relatively low number of CYP8B1 mutation carriers identified, they were consistent with a trend toward reduced CVD as HDL rose.

The data show for the first time that loss-of-function CYP8B1 mutations specifically raise HDLc and reduce triglycerides, LDLc and BMI in human families and populations without any deleterious effects on CVD or other health measures tested. Therefore, the identification and validation of HDL- raising mutations in CYP8B1 have an immediate application toward the development of diagnostics and therapeutics to decrease the likelihood of occurrence {e.g., prevent) of and/or for the treatment of CVD and CVD-related disorders.

EXAMPLE 8

IN VIVO STUDIES FOR ANTI-ATHEROSCLEROSIS

In this example, mouse studies are used to elucidate the effect of CYP8B1 on atherosclerosis.

a) Over-expression studies: In this test, expression vectors are constructed with either the wild-type CYP8B1 or mutant CYP8B1 and packaged into viruses (e.g., adenoviruses). ApoE -/- and Ldlr -/- atherogenic mice are injected with either the recombinant wild-type or mutant CYP8B1 adenoviruses and maintained on a high fat diet. Plasma HDL and lipid profiles are measured to observe the progression/prevention of atherosclerosis.

b) Down regulation studies: In this test, adenovirus-mediated siRNA-based silencing of CYP8B1 is used to observe the effect of down regulation of CYP8B1 on the progression/prevention of atherosclerosis.

Injection of Cyp8b1 siRNA-containing virus (e.g., adenovirus) into 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.

c) Cyp8b1 knockout studies: In this test, Cyp8b1 knockout mice are generated in order to assess the effects of CYP8B1 on lipid

homeostasis. 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.

EXAMPLE 9

IDENTIFICATION OF ADDITIONAL CYP8B1 MUTATIONS

An additional 27 human CYP8B1 mutations were found by querying the NHBLI Go Exome Sequencing Project (ESP) database (offered by the National Heart, Blood and Lung Institute of the National Institutes of Health (NHBLI), Bethesda, MD, through the University of Washington, Seattle, WA; http: //evs.gs.washington.edu/EVS/) and the 1000 Genomes database (Xue, Altshuler et al., 2010 Nature 467:1016) for mutations in the intronless human CYP8B1 genomic sequences.

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). As also presented above in Table 1 , there were 27 human CYP8B1 mutations identified by these queries, as follows: R26X, R28C, R50Q, R59C, V80I, Q94X, L97V, K129M, G133A, D145Q, F186L, G187S, R207H, T287M, T337A, S342R, P386L, R407G, P432S, R443G, F453C, L456F, V458Q, V475G, P487T, D490N, R494H.

EXAMPLE 10

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 .

Human liver microsomes (Xenotech) were diluted to 0.14 mg/ml using 0.1 M potassium phosphate buffer pH 7.4 and 25 μΙ_ was dispensed into each well of a 96 well plate. 0.35 μΙ_ of DMSO was dispensed into wells for positive and negative controls and 0.35 μΙ_ 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 μΙ_ 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

temperature, 10 μΙ_ of 3.5 μΜ 7a-hydroxy-4-cholesten-3-one (Toronto Research Chemicals), 7 mM NADPH (Sigma) in 0.1 M potassium phosphate buffer, pH 7.4 was dispensed into all wells. Following shaking, the plate was incubated at room temperature for 20 min, before 70 μΙ_ of acetonitrile containing 200 nM D7-7a-hydroxy-4-cholesten-3-one and 1 % formic acid was added into each well, except those for the negative controls. Following mixing, 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 μΜ. The antifungal agents ketoconazole and econazole were potent inhibitors of microsomal CYP8B1 activity with IC50 values of 2.0 and 0.21 μΜ

respectively. Table 7. Agents that modulate CYP8B1 enzyme activity

EXAMPLE 1 1

CYP8B1 ALTERNATE ISOFORM

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

(EnsembI 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.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent

applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

U.S. provisional patent application Serial No. 61/674,721 filed July 23, 2012 is incorporated herein by reference, in its entirety.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

What is claimed is:

1 . 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:

or an oligonucleotide that is complementary thereto.

2. An isolated polynucleotide comprising an oligonucleotide of at least 10 contiguous nucleotides and not more than 1506, 1505, 1504, 1503, 1502,

or an oligonucleotide that is complementary thereto.

3. 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:

C at wildtype nucleotide position 407 of SEQ ID NO:2 which is substituted by T in said oligonucleotide, G at wildtype nucleotide position 474 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

C at wildtype nucleotide position 1482 of SEQ ID NO:2 which is substituted by T in said oligonucleotide, G at wildtype nucleotide position 1545 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

or an oligonucleotide that is complementary thereto.

4. The isolated polynucleotide of any one of claims 1 -3 which 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, A at wildtype amino acid sequence position 103 of SEQ ID NO:1 which is substituted by E in said polypeptide,

or a wildtype amino acid position that is selected from:

S at wildtype amino acid sequence position 488 of SEQ ID NO:1 which is substituted by N in said polypeptide, but which does not significantly hybridize under moderately stringent conditions to a wild-type human CYP8B1 -encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:2,

or a wildtype amino acid position that is selected from:

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 by H in said polypeptide,

T at wildtype amino acid sequence position 287 of SEQ ID NO:1 which by M in said polypeptide,

T at wildtype amino acid sequence position 337 of SEQ ID NO:1 which by A in said polypeptide,

S at wildtype amino acid sequence position 342 of SEQ ID NO:1 which by R in said polypeptide,

P at wildtype amino acid sequence position 386 of SEQ ID NO:1 which by L in said polypeptide,

R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which by G in said polypeptide,

P at wildtype amino acid sequence position 432 of SEQ ID NO:1 which by S in said polypeptide,

R at wildtype amino acid sequence position 443 of SEQ ID NO:1 which by G in said polypeptide,

F at wildtype amino acid sequence position 453 of SEQ ID NO:1 which by C in said polypeptide,

L at wildtype amino acid sequence position 456 of SEQ ID NO:1 which by F in said polypeptide,

V at wildtype amino acid sequence position 458 of SEQ ID NO:1 which by Q in said polypeptide,

V at wildtype amino acid sequence position 475 of SEQ ID NO:1 which by G in said polypeptide,

P at wildtype amino acid sequence position 487 of SEQ ID NO:1 which by T in said polypeptide,

D at wildtype amino acid sequence position 490 of SEQ ID NO:1 which by N in said polypeptide, and

R at wildtype amino acid sequence position 494 of SEQ ID NO:1 which by H in said polypeptide, but which does not significantly hybridize under moderately stringent conditions to a wild-type human CYP8B1 -encoding polynucleotide having the nucleotide sequence set forth in SEQ ID NO:2.

5. 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:

6. 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:

7. 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 59 of SEQ ID NO:1 which is substituted by C in said polypeptide, V at wildtype amino acid sequence position 80 of SEQ ID NO:1 which is substituted by I in said polypeptide,

R at wildtype amino acid sequence position 407 of SEQ ID NO:1 which is substituted by G in said polypeptide, P at wildtype amino acid sequence position 432 of SEQ ID NO:1 which is substituted by S in said polypeptide,

8. 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:

A at wildtype amino acid sequence position 103 of SEQ ID NO:1 which is substituted by E in said polypeptide, D at wildtype amino acid sequence position 195 of SEQ ID NO:1 which is substituted by N in said polypeptide,

(b) a wildtype amino acid position that is selected from:

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 28 of SEQ ID NO:1 which is substituted by C in said polypeptide, R at wildtype amino acid sequence position 50 of SEQ ID NO:1 which is substituted by Q in said polypeptide,

S at wildtype amino acid sequence position 342 of SEQ ID NO:1 which is substituted by R in said polypeptide, P at wildtype amino acid sequence position 386 of SEQ ID NO:1 which is substituted by L in said polypeptide,

9. The antibody of claim 8 which is a monoclonal antibody.

10. The isolated antibody, or an antigen-binding fragment thereof, of claim 8 wherein the antibody is selected from the group consisting of a single chain antibody, a ScFv, a univalent antibody lacking a hinge region, and a minibody.

1 1 . The isolated antibody, or an antigen-binding fragment thereof, of claim 8 wherein the antibody is a Fab or a Fab' fragment.

12. The isolated antibody, or an antigen-binding fragment thereof, of claim 8 wherein the antibody is a F(ab')₂ fragment.

13. The isolated antibody, or an antigen-binding fragment thereof, of claim 8 wherein the antibody is a whole antibody.

14. An antisense oligonucleotide that comprises the polynucleotide of any one of claims 1 -3.

15. An antisense oligonucleotide that comprises the polynucleotide of claim 4.

16. A ribozyme that comprises the polynucleotide of any one of claims 1 -3.

17. A ribozyme that comprises the polynucleotide of claim 4.

18. A small interfering RNA that comprises the polynucleotide of any one of claims 1 -3.

19. A small interfering RNA that comprises the polynucleotide of claim 4.

20. 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.

21 . A method of stratifying a 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, 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.

22. The method of either claim 20 or claim 21 wherein 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 the group consisting of a CYP8B1 catalytic domain, a CYP8B1 O₂-binding domain, a CYP8B1 steroidogenic region and a CYP8B1 heme binding domain.

23. The method of either claim 20 or claim 21 wherein 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 the group consisting of a CYP8B1 O2-binding domain, a CYP8B1 steroidogenic region and a CYP8B1 heme binding domain, and wherein the single nucleotide polymorphism is a non-synonymous nucleotide substitution.

24. The method of either claim 20 or claim 21 wherein 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:

G at wildtype nucleotide position 1371 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, C at wildtype nucleotide position 1394 of SEQ ID NO:2 which is substituted by T in said oligonucleotide,

25. The method of either claim 20 or claim 21 wherein 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:

26. The method of either claim 20 or claim 21 wherein 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:

G at wildtype nucleotide position 884 of SEQ ID NO:2 which is substituted by A in said oligonucleotide, G at wildtype nucleotide position 945 of SEQ ID NO:2 which is substituted by A in said oligonucleotide,

G at wildtype nucleotide position 1806 of SEQ ID NO:2 which is substituted by A in said oligonucleotide. 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.

28. The method of claim 27 wherein the agent is selected from the group consisting of:

(a) a compound of formula I:

wherein:

m is 0, 1 , 2, 3, 4 or 5;

n is 1 , 2 or 3;

X is -N- or -C(R⁶)- at each occurrence, R is the same or different and

independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, or -OR⁷;

at each occurrence, R² is the same or different and

independently hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyl, heterocyclylalkyl, or -OR⁷; or R¹ and R² connected to the same carbon form a spiro ring, which can be optionally substituted with alkyi, aryl, heteroaryl, cydoalkyi,

heterocydyl, aralkyi, heteroarylalkyi, cydoalkylalkyi, or heterocyclylalkyl;

at each occurrence, R³ is the same or different and

independently hydrogen, halogen, hydroxy, alkyi, alkoxy, aryl, cydoalkyi,

heterocydyl, aralkyi, heteroaryl or heteroarylalkyi;

R⁴ and R⁵ is independently hydrogen or alkyi;

R⁶ is hydrogen or alkyi; and

each R⁷ is the same or different and independently hydrogen, alkyi, aryl, cydoalkyi, heterocydyl, heteroaryl, aralkyi, heteroarylalkyi, cydoalkylalkyi, or heterocyclylalkyl,

(b) a compound of formula II:

wherein:

— is independently a single or double bond;

each R⁹ is the same or different and independently hydrogen, alkyi, aryl, heteroaryl, aralkyi, cydoalkyi, heterocydyl, heteroarylalkyi, cydoalkylalkyi, or heterocyclylalkyl;

each R^14a and R^14b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyi, cydoalkyi, heterocydyl, cydoalkylalkyi, or heterocyclylalkyl; or R^14a and R^14b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

R¹⁵ is hydrogen or alkyi; each R^16a and R^16b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cydoalkyi, heterocydyl, cydoalkylalkyi, or heterocyclylalkyl; or R^16a and R^16b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

each R^17a and R^17b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cydoalkyi, heterocydyl, cydoalkylalkyi, or heterocyclylalkyl; or R^17a and R^17b together forms =O, =S, =C(R⁹)₂; or =NR⁹;

R¹⁸ is hydrogen, hydroxy, alkoxy, or alkyl;

R¹⁹ is hydrogen or alkyl;

each R^20a and R^20b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cydoalkyi, heterocydyl, cydoalkylalkyi, or heterocyclylalkyl; or R^20a and R^20b together forms =O, =S, =C(R⁹)₂; or =NR⁹,

R²¹ is hydrogen or alkyl, or R²¹ and R^16a together form a bond; and

each R^22a and R^22b is the same or different and independently hydrogen, hydroxy, alkoxy, alkyl, cydoalkyi, heterocydyl, cydoalkylalkyi, or heterocyclylalkyl; or R^20a and R^20b together forms =O, =S, =C(R⁹)₂; or =NR⁹, or R²² and R²¹ together form a bond,

(c) a compound of formula III:

wherein:

t is 0, 1 , 2, 3, 4 or 5;

each R⁹ is the same or different and independently hydrogen, alkyl, aryl, heteroaryl, cydoalkyi, heterocydyl, aralkyi, heteroarylalkyi, cydoalkylalkyi, or heterocyclylalkyl; at each occurrence, R is the same or different and independently hydrogen, alkyl, halogen, acyl, aryl, heteroaryl, cycloalkyi,

heterocyclyl, aralkyl, heteroarylalkyl, cycloalkylalkyi, heterocyclylalkyl, -OR⁹, -N(R⁹)₂-, or -SR⁹; or two adjacent R²⁷, 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^30a and R^30b is the same or different and independently hydrogen, alkyl, acyl, aralkyl, or heteroarylalkyl,

(d) the antibody according to any one of claims 4-9;

(e) the antisense oligonucleotide of claim 10;

(f) the ribozyme of claim 1 1 ; and

(g) the small interfering RNA of claim 12.

29. The method of either claim 27 or claim 28 wherein the

cardiovascular disease or disorder is selected from the group consisting of dyslipidemia, atherosclerosis, low HDL diseases and related disorders.

30. The method of either claim 27 or claim 28, wherein 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.

31 . The method of either claim 27 or claim 28 wherein the agent specifically binds to a CYP8B1 polypeptide catalytic domain.

32. The method of claim 31 wherein 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.

33. The method of claim 27 which 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 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 said agent that is capable of decreasing a level of CYP8B1 expression or CYP8B1 activity in the subject or said agent that is an inhibitor of human cytochrome P450-family 8-subfamily B- polypeptide 1 (CYP8B1 ) sterol 12-a-hydroxylase activity,

wherein the mutation that results in decreased CYP8B1 activity comprises at least one substitution mutation of a human CYP8B1 polypeptide having the amino acid sequence set forth in SEQ ID NO:1 and said substitution is at an amino acid position that corresponds to a wildtype amino acid position that is selected from:

34. The method of claim 27 which 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:

35. The method of either claim 33 or claim 34, wherein the cardiovascular disease or disorder is selected from the group consisting of dyslipidemia, atherosclerosis, low HDL diseases and related disorders.

36. The method of any one of claims 27, 33 and 34 wherein the agent specifically binds to the CYP8B1 polypeptide.

37. 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 agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder.

38. The method of claim 37 which comprises determining the base level of CYP8B1 polypeptide expression and the test level of CYP8B1 polypeptide expression by quantifying CYP8B1 mRNA.

39. The method of claim 37 which comprises determining the base level of CYP8B1 polypeptide expression and the test level of CYP8B1 polypeptide expression by quantifying CYP8B1 protein.

40. An agent for treating or decreasing likelihood of occurrence of of a cardiovascular disease or disorder that is identified according to the method of claim 37.

41 . The agent of claim 40 which 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 .

42. 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 determination that the test level of CYP8B1 activity is less than the base level of CYP8B1 activity indicates the candidate agent is an agent for treating or decreasing likelihood of occurrence of a cardiovascular disease or disorder.

43. The method of claim 42 wherein the CYP8B1 activity is a sterol 12-a-hydroxylase activity.

44. The method of claim 42 wherein the second CYP8B1 polypeptide is present in a cell when being contacted with the candidate agent.

45. The method of claim 42 which is performed in vitro.

46. The method of claim 42 wherein each of the first and second CYP8B1 polypeptides, or fragment or variant thereof, comprises a CYP8B1 polypeptide catalytic domain.

47. The method of claim 42 wherein each of the first and second 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.

48. An agent for treating or decreasing likelihood of occurrence of of a cardiovascular disease or disorder that is identified according to the method of claim 42.

49. The agent of claim 48 which inhibits a sterol 12-a-hydroxylase activity of the second CYP8B1 polypeptide.

50. The agent of claim 49 which specifically binds to the second CYP8B1 polypeptide.

51 . The agent of claim 50 which 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.

52. 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.

53. 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.

54. 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, a V 475G mutation, a P487T mutation, a D490N mutation, and a R494H mutation.

55. A diagnostic kit comprising as a first polynucleotide the polynucleotide of either claim 1 or claim 2.

56. The diagnostic kit of claim 55, further comprising 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.

57. The diagnostic kit of claim 55, further comprising 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, a P432S mutation, a R443G mutation, a F453C mutation, a L456F mutation, a V458Q mutation, a V 475G mutation, a P487T mutation, a D490N mutation, and a R494H mutation.