JP2003520026A - Novel ABC transporter and its use - Google Patents

Abstract

(57) Abstract The present invention provides an isolated nucleic acid molecule encoding a novel ABC transporter family member, designated ABCB12 transporter nucleic acid molecule. The present invention also provides an antisense nucleic acid molecule, a recombinant expression vector containing the ABCB12 transporter nucleic acid molecule, a host cell into which the expression vector has been introduced, and a non-human transgenic animal into which the ABCB12 transporter gene has been introduced or disrupted. I do. The invention further provides screening assays for isolated ABCB12 transporter proteins, fusion proteins, antigenic peptides, anti-ABCB12 transporter antibodies, and ABCB12 transporter modulators. Diagnostic and therapeutic methods using the compositions of the present invention are also provided.

Description

Detailed Description of the Invention

RELATED APPLICATION This application was filed on Aug. 20, 1999 and Aug. 30, 1999, the entire contents of which are hereby incorporated by reference, both entitled “
Novel ABC Transporter and Its Utilization, "which claims priority under US provisional applications 60 / 161,724 and 60 / 151,473. The entire contents of all patents, patent applications, and references cited throughout this specification are hereby incorporated by reference.

BACKGROUND OF THE INVENTION The ABC transporter protein is a large protein superfamily whose characteristics are conserved in both prokaryotes and eukaryotes. The ABC transporter catalyzes ATP-dependent transport of endogenous and exogenous substrates across biological membranes in Borst, P .. (1997) Seminar.
in Cancer Biology 8: 131-213) and / or alters the function of heterologous proteins allosterically (Higgins CF, 1995, Cell 82: 693-696). Several ABC transporters are multidrug resistant (Ambudkar SV et al., (1999), Ann, just to name a few).
u. Rev. Toxicol., 39: 361-398), cystic fibrosis (Riordin JR et al., (1989)
Science 245: 1066-1073), Atherosclerosis (Brooks-Wilson A et al., (19
99) Nature Genetics 22: 336-345), hyperinsulinemic hypoglycemia (Thomas PM et
al., (1995) Science 268: 46-429), macular degeneration (Allikmets R et al., (1997)
) Science 277: 1805-1807), and adrenoleukodystrophy (Mosser J et al.,
(1993) Nature 361: 726-730) and has been associated with clinically relevant phenotypes.

Although genes associated with these disease phenotypes have been identified to some extent, there are numerous putative ABC transporters in the human genome, as evidenced by the partial sequences found in various EST databases. (Allikmets et al., (
1996) Hum Mol Genet 5: 1649-1655). However, the usefulness of such information is limited due to the absence of the full-length nucleotide sequence of the coding region of the relevant gene or its post-translational protein product.

[0004]   The present invention relates, at least in part, to the ABCB12 transporter nucleic acid and protein components herein.
Offspring (widely expressed mammalian ABC half-transporter (original:ubiquitouslyexpressed m ammalianABC half-transporter) (UMAT) followed by a rat homologue)
A new ATP-binding cassette (ABC) transporter family called human UMAT)
It is based on the discovery of Lee members. The ABCB12 transporter molecules of the present invention have various
Cellular processes, especially cell membranes of neurotoxic molecules such as β-amyloid peptides,
Is a group of researchers to develop modulators that regulate transport across the blood-brain barrier (BBB) and elsewhere.
Useful as a target. Neurotoxic molecules such as β-amyloid peptides are
Involved in neurological disorders such as Luzheimer's disease (eg Goate et al. (1991)
Nature 349: 704; Games et al. (1995) Nature 373: 523; and Suzuki et al. (19
94) Science 264: 1336). Other nerves involved in toxic polypeptides
Examples of diseases include prion disease, Huntington's chorea, Parkinson's disease, etc.
(For a review, see Hardy et al. (1998) Science 282: 1075-1079).
.

Therefore, it can be expected that if the modulator of the human ABCB12 transporter can be used to modulate the export of amyloid β protein, it will be possible to modulate amyloid deposits and thus Alzheimer's disease.

In addition, the ABCB12 transporter molecules of the present invention are useful as targets for developing multidrug resistance modulators. Furthermore, the molecules of the invention are useful as diagnostic and therapeutic tools.

Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule encoding an ABCB12 transporter protein or a biologically active portion thereof, as well as a primer or hybridization probe for detecting a nucleic acid encoding ABCB12. And a nucleic acid fragment suitable as.

In one embodiment, the ABCB12 transporter nucleic acid molecule of the present invention is directed to the nucleotide sequence set forth in SEQ ID NO: 1 or 3 (eg, for the entire length of this nucleotide sequence) or its complement. , At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%
, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more identical.

In one preferred embodiment, the isolated nucleic acid molecule of interest is SEQ ID NO: 1 or 3
The nucleotide sequence shown in 1 above or its complementary sequence is included. In another example, the nucleic acid molecule of interest comprises SEQ ID NO: 3 and nucleotides 1-164 of SEQ ID NO: 1. In another embodiment, the nucleic acid molecule of interest comprises SEQ ID NO: 3 and nucleotides of SEQ ID NO: 1.
Including 2697-2893.

In another example, the ABCB12 transporter nucleic acid molecule comprises a nucleotide sequence that encodes a protein having an amino acid sequence that is sufficiently homologous to the amino acid sequence of SEQ ID NO: 2. In certain preferred embodiments, the ABCB12 transporter nucleic acid molecule has at least 80%, 81%, 82%, 83%, 84%, 85%, 86% of the full length amino acid sequence of SEQ ID NO: 2. 87%, 88%
, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, including a nucleotide sequence encoding a protein having a homologous amino acid sequence.

In another preferred embodiment, the isolated nucleic acid molecule is one that encodes the amino acid sequence of the human ABCB12 transporter having the amino acid sequence of SEQ ID NO: 2. In yet another preferred embodiment, the nucleic acid molecule is at least 2696 nucleotides long. In a further preferred embodiment, the nucleic acid molecule is at least 2574 nucleotides in length and encodes a single protein having ABCB12 transporter activity (as described herein).

Another embodiment of the invention specifically detects an ABCB12 transporter nucleic acid molecule relative to a nucleic acid molecule, preferably a nucleic acid molecule encoding a non-ABCB12 transporter protein.
An ABCB12 transporter nucleic acid molecule. For example, in one embodiment, such nucleic acid molecule comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 3
00, 400, 500, 500-1000, 1000-1500, 1500-2000, 2000-2500, or 2500-3000 or more nucleotides in length and / or under stringent conditions SEQ ID NO: 1
It hybridizes to a nucleic acid molecule containing the nucleotide sequence shown in or its complementary sequence. Here, the nucleic acid molecule has a length within a range in which the lower limit is one of the numbers listed above and the upper limit is another number with respect to the number of nucleotides contained in the entire length. Well, it should be understood that it may be, for example, a molecule that is 60-80, 300-1000, or 150-400 nucleotides in length.

In a preferred embodiment, the nucleic acid molecule is at least 15 (eg, contiguous) nucleotides in length and hybridizes under stringent conditions to nucleotides 165-2696 of SEQ ID NO: 1. In another preferred embodiment, the nucleic acid molecule comprises nucleotides 165-2696 of SEQ ID NO: 1.

In another preferred embodiment, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, provided that the nucleic acid molecule is Hybridizes under stringent conditions to a nucleic acid molecule comprising SEQ ID NO: 1 or 3.

Another embodiment of the invention is the use of the ABCB12 transporter nucleic acid molecule, such as the coding strand of the ABCB12 transporter.
Provided is an isolated nucleic acid molecule that is antisense to a transporter nucleic acid molecule.

Another aspect of the invention provides a vector comprising an ABCB12 transporter nucleic acid molecule. In some embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing the vector of the invention.
In another embodiment, the invention provides a host cell containing the nucleic acid molecule of the invention. The invention further provides a protein, preferably the ABCB12 transporter protein,
To produce the protein by culturing a host cell according to the present invention containing a recombinant expression vector, such as a mammalian host cell such as a non-human mammalian cell, in a suitable medium. It is provided.

Another aspect of the invention features isolated or recombinant ABCB12 transporter proteins and polypeptides. In a preferred embodiment, the protein, preferably the ABCB12 transporter protein, comprises at least one transmembrane domain, SEQ ID
At least about 80%, 81%, 82%, 83%, 84%, 85%, 8 based on the amino acid sequence of NO: 2
6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more homologous amino acid sequences. In yet another preferred embodiment, the protein, preferably the ABCB12 transporter protein, comprises at least one transmembrane domain and hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1. Encoded by a nucleic acid molecule having a soybean nucleotide sequence.

In another embodiment, the invention features a fragment of a protein having the amino acid sequence of SEQ ID NO: 2, wherein the fragment is at least 15 amino acid sequences of SEQ ID NO: 2. An amino acid (for example, a continuous amino acid). In another example, the protein, preferably the ABCB12 transporter protein, has the amino acid sequence of SEQ ID NO: 2.

In another embodiment, the invention provides a nucleotide sequence of SEQ ID NO: 1 or 3 or its complementary sequence of at least 80%, 85%, 86%, 87%, 88%, 89%, 90%. , 91%, 92%, 93%
, 94%, 95%, 96%, 97%, 98% or more, isolated protein, preferably ABCB12 transporter protein, encoded by a nucleic acid molecule consisting of homologous nucleotide sequences. Furthermore, the present invention provides an isolated protein encoded by a nucleic acid molecule consisting of a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3 or its complementary sequence, Preferably it is characterized by the ABCB12 transporter protein.

The protein of the present invention or a portion thereof, such as a biologically active portion thereof,
A non-ABCB12 transporter polypeptide (eg, a heterologous amino acid sequence) can be operably linked to form a fusion protein. The invention further features an antibody, such as a monoclonal or polyclonal antibody, that specifically binds to a protein of the invention, preferably the ABCB12 transporter protein. In addition, the ABCB12 transporter protein, a biologically active portion thereof, or an expressible nucleic acid encoding them is
It may be incorporated into a pharmaceutical composition and the pharmaceutical composition may optionally include a pharmaceutically acceptable carrier.

In another aspect, the invention provides an agent capable of detecting an ABCB12 transporter nucleic acid molecule, protein or polypeptide, such that the presence of an ABCB12 transporter nucleic acid molecule, protein or polypeptide is detected in a biological sample. The present invention provides a method for detecting the presence of an ABCB12 transporter nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample.

In another aspect, the invention provides for the ABCB12 transporter by contacting the biological sample with an agent capable of detecting an indicator of ABCB12 transporter activity such that the presence of ABCB12 transporter activity is detected in the biological sample. Provided is a method of detecting the presence of transporter activity in a biological sample.

In another aspect, the invention provides that ABCB12 transporter activity in a cell is modulated such that ABCB12 transporter activity is modulated.
Provided is a method of modulating ABCB12 transporter activity, comprising the step of contacting a cell capable of expressing ABCB12 transporter with an agent that modulates 12 transporter activity. In one embodiment, the agent inhibits ABCB12 transporter activity. In another embodiment, the agent stimulates ABCB12 transporter activity. In one example, the agent is an antibody that specifically binds to the ABCB12 transporter protein. In another embodiment, the agent modulates the ability of the ABCB12 transporter to allosterically alter the function of other membrane proteins. In another embodiment, the agent modulates ABCB12 transporter expression by modulating transcription of the ABCB12 transporter gene or translation of ABCB12 transporter mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the ABCB12 transporter mRNA or the coding strand of the ABCB12 transporter gene.

[0024] In one example, a subject having an abnormality characterized by aberrant or unwanted ABCB12 transporter protein or nucleic acid expression or activity is utilized in the subject method.
Treatment is by administration of an agent that is an ABCB12 transporter modulator. In one example, the ABCB12 transporter modulator is an ABCB12 transporter protein. In another embodiment, the ABCB12 transporter modulator is an ABCB12 transporter nucleic acid molecule. In yet another embodiment, the ABCB12 transporter modulator is, for example, a carbohydrate-based, lipid-based, nucleic acid-based, natural organic-based, or artificially-synthesized organic. Based molecules, such as peptides, peptidomimetics,
Is a small molecule such as.

The invention further provides (i) an abnormal modification or mutation of the gene encoding the ABCB12 transporter protein, (ii) misregulation of said gene, and (iii) an abnormal post-translational modification of the ABCB12 transporter protein, To provide a diagnostic assay for determining the presence or absence of a genetic alteration characterized by at least one of the above, wherein the wild type of said gene encodes a protein with ABCB12 transporter activity. is doing.

In another aspect, the invention provides a step of providing an indicator composition comprising an ABCB12 transporter protein having ABCB12 transporter activity, contacting said indicator composition with a test compound, said test compound comprising: By examining the effect on ABCB12 transporter activity in the indicator composition, identifying a compound that modulates the activity of ABCB12 transporter protein (eg, ABCB12 transporter protein for membranes), by binding to ABCB12 transporter protein. Or to modulate the activity of the ABCB12 transporter protein.

[0027] Detailed Description of the Invention The present invention, at least in part, here ABCB12 transporter nucleic acid and protein molecules ( "widely expressed mammalian ABC half - transporter (Language: u biquitously e xpresse
d m ammalian A BC half- t ransporter ) "(following the rat homologue called UMAT)) based on the discovery of novel human ATP-binding cassette (ABC) transporter family members called the human UMAT also to mention) is there. The ABC transporter molecule is a transmembrane protein that catalyzes ATP-dependent transport of endogenous and exogenous substrates across biological membranes. The ABC transporter has been implicated in the transport of polypeptides, such as neurotoxic polypeptides such as β-amyloid involved in Alzheimer's disease. Other neurological disorders caused by neurotoxic polypeptides include prion disease, Parkinson's chorea, Huntington's disease, etc. (Review Hardy et al. (1998) Science 282:
See 1075-1079). In particular, ABC transporters are involved in the transport of substrates across the blood-brain barrier. Thus, transport of substances in brain cells and across the blood-brain barrier is controlled, at least in part, by ABC transporter molecules.

In addition, the ABC transporter is associated with multidrug resistance found in cells, especially cells that are resistant to cytotoxic anti-cancer agents (Borst, P. (1997) Sem. Cancer Bio
. 8: 131-134).

Accordingly, the ABCB12 transporter molecule of the present invention can be used to develop novel diagnostic targets and therapeutic agents that control intracellular transport in brain cells (eg, nerve cells) and transport across the blood-brain barrier. A good target. Furthermore, the ABCB12 transporter molecule is a suitable target for developing diagnostic targets and therapeutic agents for detecting and / or treating cells or tissues having multidrug resistance such as cancer.

In particular, the novel human ABCB12 transporter molecules described herein will have one or more of the following functions and / or uses:

Regarding the ABC transporter expressed in the brain, it has been suggested that the ABC transporter is involved in the transport of the substrate through the blood-brain barrier (Schinkel AH, et al, (1994) Cell, 77, 491). The identification of the sequence of the human ABCB12 transporter described here allows the development of new strategies to alter the function of the blood-brain barrier. Many of the drugs that appear to be of utility in treating brain disorders have been abandoned due to their inability to enter the brain at therapeutically relevant concentrations, but the present invention provides drug delivery to the brain. It is possible to develop strategies to support

The ABC transporter expressed in the brain has its deposition on senile plaques (as described in US patent application Ser. Nos. 08 / 847,616 and PCT / US98 / 08463, the contents of which are incorporated herein by reference). Is a potential transporter of a peptide, a β-amyloid peptide, which is a basic feature of Alzheimer's disease. Therefore, identifying novel transporters that regulate β-amyloid deposition is important in developing therapeutics for Alzheimer's disease.

Drosophila melanogaster white
) It has been reported that a human homologue of the gene is associated with mood disorders and panic disorders (Nakamura M et al., (1999) Mol. Psychiatry, 4, 155-162). Is a member of the super family. Since the human ABCB12 transporter of the present invention is expressed in the brain, it may be involved in mood disorder and panic disorder. The identification of the sequence of the human ABCB12 transporter described here allows the development of new treatments for mood disorders and panic disorders.

It has been shown that the ABC transporter is involved in the phenomenon of multidrug resistance (Ling, V., (
1997) Cancer Chemother Pharmacol 40: S3-S8; Naomi, M., et al., (1998) Can
cer Research, 58: 1332-1337). According to the present invention, the ability of ABCB12 to contribute to the multidrug resistance phenotype was determined, and the theory of Boer, R. et al. (Boer, R., et al. ((1996) Eu.
It would be possible to design drugs that could reduce multidrug resistance using techniques similar to those of the ropean Journal of Cancer, 32A: 857-861).

The term “family”, as used herein for proteins and nucleic acid molecules of the present invention, refers to two or more as defined herein having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology. It is intended to mean a protein or nucleic acid molecule of. Such family members may be naturally occurring or non-naturally occurring and may be from the same or different species. For example, in one family, the first protein of human origin,
It may contain different proteins of human origin, or it may contain homologues of non-human origin. Members of a family will have more common functional characteristics.

For example, the family of ABC transporter proteins comprises at least one “transmembrane domain”, and preferably two transmembrane domains. The term “transmembrane domain” as used herein includes amino acid sequences that extend through the cell membrane and are about 18 amino acid residues in length. More preferably, the transmembrane domain is at least about 1.
It is preferable that it contains 8, 20, 25, 30, 35, 40, or 45 or more residues and extends through the cell membrane. Transmembrane domains are, for example, incorporated herein by reference, Zagotta WN et al, (1996) Annual Rev. Ne.
See uronsci. 19: 235-63. Amino acid residue 27 of the human ABCB12 transporter protein
-50, 262-285, 304-326, 383-405, 503-521, and 530-548 comprise transmembrane domains. One or more of these transmembrane domains may associate to form a single domain that extends across the membrane.

The isolated protein of the invention, preferably the ABCB12 transporter protein, is SEQ ID NO:
Has an amino acid sequence sufficiently homologous to the amino acid sequence of ID NO: 2, or SEQ ID NO:
: 1 or 3 is encoded by a nucleotide sequence sufficiently homologous. As used herein, the term "sufficiently homologous" means that the first amino acid or nucleotide sequence has a sufficient or minimal number of identical or equivalent (eg, similar flanking) residues to the second amino acid or nucleotide sequence. Containing amino acid residues or nucleotides (such as chain-bearing amino acid residues) such that these first and second amino acid or nucleotide sequences have a common structural domain or motif and / or a common functional activity. Say there is. For example, an amino acid or nucleotide sequence that has a common structural domain has at least 60% homology across the amino acid sequences of these domains,
More preferably it has 70-80% homology, and even more preferably 90-95% homology, contains at least one, and preferably two structural domains or motifs and is sufficiently homologous. As defined here. Furthermore, an amino acid or nucleotide sequence having at least 60%, more preferably 70-80%, or 90-95% homology and having a common functional activity is also said to be sufficiently homologous herein. Define it.

“ABCB12 transporter activity” and “ABCB12 transporter biological activity” used interchangeably herein
Alternatively, "functional activity of the ABCB12 transporter" means that the ABCB12 transporter protein, polypeptide or nucleic acid molecule, when examined in vitro or in vitro by standard techniques
CB12 transporter Responsive cell or ABCB12 transporter protein substrate.
Preferably, ABCB12 transporter activity is capable of acting as an energy-dependent (ATP) molecular pump.

In one example, ABCB12 activity is a direct activity, such as, for example, association with a membrane associated protein and / or transport of an endogenous or exogenous substrate across a biological membrane. In another example, ABCB12 activity is the ability of the polypeptide to allosterically alter the function of other membrane proteins. For example, in specific cells, modulation of p-glycoprotein by ABCB12 transporter modulators has been shown to change the magnitude of the volume-dependent chloride current (Higgins, CF Volume-activa).
ted chloride currents associated with the multidrug resistance P-glycopr
otein, J. Physiol. 482: 31S-36S (1995)). Thus, in this model, p-glycoprotein and other ABC transporters have multiple functions, one of which is to alter the function of other membrane proteins allosterically. For example, the present invention provides a model in which changes in the transport of substrates such as β-amyloid, cytotoxic drugs, or other small molecules involve the ABCB12 transporter or the like altering other membrane proteins allosterically. It matches. Thus, ABCB12 activity is at least one or more of the following activities: (i) activation of ABCB12-dependent signaling pathways; (ii) transmembrane transmembrane substrates (eg, cytotoxic agents, β-amyloid) Modulation of transport; (iii) ABCB12 protein interaction with non-ABC2 membrane associated molecules; (iv) ABCB
Modulation of development or differentiation of 12-expressing cells; (v) Modulation of development or differentiation of non-ABCB12-expressing cells; (vi) Modulation of homeostasis of ABCB12-expressing cells; and (vii) Modulation of homeostasis of non-ABCB12-expressing cells.

Accordingly, another embodiment of the present invention is an isolated ABCB12 having ABCB12 transporter activity.
It is characterized by transporter proteins and polypeptides. A preferred protein is an ABCB12 transporter protein having at least one transmembrane domain, preferably two transmembrane domains, and preferably ABCB12 transporter activity.

Nucleotide sequence of the isolated human ABCB12 transporter protein cDNA and human AB
The deduced amino acid sequence of the CB12 transporter polypeptide is shown in Figure 5 and SEQ ID NO: 1, respectively.
And 2 are shown.

The human ABCB12 transporter gene, which is approximately 2893 nucleotides long, has a molecular weight of approximately 94 kDa.
Encodes a protein of about 843 amino acid residues in length.

[0043]   Various aspects of the invention are detailed in the following subsections.

I. Isolated Nucleic Acid Molecules One aspect of the invention is an isolated nucleic acid molecule encoding an ABCB12 transporter protein or a biologically active portion thereof, or a nucleic acid molecule encoding ABCB12 (eg, ABCB12 transporter mR
Nucleic acid fragment sufficient as a hybridization probe for identifying (e.g., NA) and a fragment that can be used as a PCR primer for amplification or mutation of an ABCB12 transporter nucleic acid molecule. As used herein, the term “nucleic acid molecule” includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA), and analogs of DNA or RNA produced using nucleotide analogs, Is intended. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, when referring to genomic DNA, the term "isolated" includes nucleic acid molecules that have been separated from the previous chromosome to which the genomic DNA was naturally associated. Preferably, the "isolated" nucleic acid is the genomic DNA of the organism from which the nucleic acid was derived and is a sequence that naturally flanks the nucleic acid (ie, at the 5'and 3'ends of the nucleic acid. Array) should not be included. For example, in various examples, an isolated ABCB12 transporter nucleic acid molecule is provided with about 5 kb, 4 kb, 3 kb, 1 kb that naturally flanks the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid was obtained. , 0.
It may contain a nucleotide sequence of less than 5 kb or 0.1 kb. Furthermore, for example, cD
An "isolated" nucleic acid molecule, such as an NA molecule, is substantially free of other cellular material or media when produced recombinantly, or when chemically synthesized,
It may be substantially free of chemical precursors or other chemicals.

Nucleic acid molecules of the present invention, such as nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1 or 3, or portions thereof, may be prepared using standard molecular biology techniques and sequence information provided herein. Can be used to isolate. Using all or part of the nucleic acid sequence of SEQ ID NO: 1 or 3 as a hybridization probe, the ABCB12 transporter nucleic acid molecule can be isolated using standard hybridization and cloning techniques ( Sambrook, J., Fritsh, EF, and Mania
tis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Ha
rbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
, NY, 1989). Furthermore, a nucleic acid molecule encompassing all or part of SEQ ID NO: 1 or 3 is a polymerase chain reaction (PCR) method using a synthetic oligonucleotide primer designed based on the sequence of SEQ ID NO: 1 or 3. However, it can be isolated.

The nucleic acids of the present invention can be amplified using standard PCR amplification techniques, using cDNA, mRNA, or optionally genomic DNA as a template and further suitable oligonucleotide primers. . The nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis. In addition, ABCB12
Oligonucleotides corresponding to transporter nucleotide sequences can be made by standard synthetic techniques, such as using an automated DNA synthesizer.

In one preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 corresponds to human ABCB12 cDNA. This cDNA includes a sequence encoding the human ABCB12 protein (ie, "coding region" at nucleotides 165-2696), a 5'-terminal untranslated sequence (nucleotide 1-164) and a 3'-terminal untranslated sequence ( Nucleotides 2697-2893). Optionally, the nucleic acid molecule has a coding region of SEQ ID NO: 1 (eg SEQ
It may contain only nucleotides 165-2696 corresponding to ID NO: 3).

In another preferred embodiment, the isolated nucleic acid molecule of the invention is the complement of the nucleotide sequence set forth in SEQ ID NO: 1 or 3, or a portion of any of these nucleotide sequences. A nucleic acid molecule that is a sequence. The nucleic acid molecule complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3 means that the nucleotide sequence shown in SEQ ID NO: 1 or 3 is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 3. It is capable of hybridizing to the indicated nucleotide sequence to form a stable duplex.

In yet another preferred embodiment, the isolated nucleic acid molecule of the invention is SEQ ID NO: 1.
Alternatively, at least about 70%, 75%, 80%, 85%, 86%, 87% relative to the total length of the nucleotide sequence shown in 3 or a portion of any of these nucleotide sequences.
, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, comprising homologous nucleotide sequences.

Furthermore, the nucleic acid molecule of the present invention may be, for example, a fragment that can be used as a probe or primer, or a fragment that encodes a portion of the ABCB12 transporter protein, such as a biologically active portion of the ABCB12 transporter protein. It may comprise only a part of the nucleic acid sequence of ID NO: 1 or 3. Designed to be used to identify and / or clone other ABCB12 transporter family members and ABCB12 transporter homologues from other species based on the nucleotide sequence determined by cloning the ABCB12 transporter gene Prepared probes and primers can be prepared. The probe / primer typically comprises substantially purified oligonucleotide. This oligonucleotide is typically the sense sequence of SEQ ID NO: 1 or 3, or SE
At least 12 or 15 of the antisense sequences of Q ID NO: 1 or 3 or naturally occurring allelic variants or variants of SEQ ID NO: 1 or 3, preferably about 20 or 25 More preferably about 30, 35, 40,
It comprises a region of nucleotide sequence that hybridizes under stringent conditions to 45, 50, 55, 60, 65, or 75 contiguous nucleotides. In certain exemplary embodiments, the nucleic acid molecules of the invention are 50, 60, 70, 80, 90, 100, 1
50, 200, 300, 400, 500-1000, 1000-1500, 1500-2000, or 2000-2500, or 25
It comprises a nucleotide sequence that is 00-3000 or more nucleotides in length and hybridizes under stringent hybridization conditions to the nucleic acid molecule set forth in SEQ ID NO: 1 or 3.

Probes based on the ABCB12 transporter nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In a preferred embodiment, the probe may further include a labeling group attached thereto, for example, the labeling group may be a radioisotope, a fluorescent compound or an enzyme. Alternatively, it may be an enzyme cofactor. Such a probe is called ABCB
It can be used as a part of a diagnostic test kit for identifying cells or tissues in which 12 transporter proteins are erroneously expressed.For example, by measuring the level of nucleic acid encoding ABCB12 in a cell sample taken from a subject. For example, ABCB12 transporter mRNA levels may be detected or it may be determined whether the genomic ABCB12 transporter gene is mutated or deleted.

A nucleic acid fragment encoding “the biologically active portion of the ABCB12 transporter protein” has SEQ ID
A portion of the nucleotide sequence of NO: 1 or 3 that encodes a polypeptide having ABCB12 transporter bioactivity (the biological activity of ABCB12 transporter protein is described herein) is isolated and isolated from the ABCB12 transporter protein. This can be made by expressing the encoded portion (eg, by recombinant expression in vivo) and assessing the activity of the encoded portion of the ABCB12 transporter protein.

Furthermore, the present invention differs from the nucleotide sequence shown in SEQ ID NO: 1 or 3 by virtue of the degeneracy of the genetic code, but the nucleotide sequence shown in SEQ ID NO: 1 or 3 is Also included are nucleic acid molecules which encode the same ABCB12 transporter protein as encoded. In another embodiment, the isolated nucleic acid molecule of the invention is SE
It has a nucleotide sequence that encodes a protein having the amino acid sequence shown in Q ID NO: 2.

Those skilled in the art will appreciate that in addition to the ABCB12 transporter nucleotide sequence shown in SEQ ID NO: 1 or 3, DNA sequence polymorphisms leading to alterations in the amino acid sequence of the ABCB12 transporter protein may be present in a population (eg, It will be appreciated that it will exist within a human population. Due to natural allelic variation, it is believed that such genetic polymorphisms in the ABCB12 transporter gene are among individuals within a population. The terms "gene" and "recombinant gene" as used herein refer to a nucleic acid molecule containing an open reading frame that encodes an ABCB12 transporter protein, preferably a mammalian ABCB12 transporter protein, but which is not a coding strand. May include sequences and introns.

Allelic variants of the human ABCB12 transporter include functional and non-functional ABCB12
Both transporter proteins are included. A functional allelic variant is ABCB12
It is a naturally occurring amino acid sequence variant of the human ABCB12 transporter that retains the ability to bind transporter ligands. Functional allelic variants typically include only conservative substitutions at one or more amino acids of SEQ ID NO: 2 or residues of nonessential residues in non-critical regions of this protein. Substitution, deletion or insertion may have occurred.

A non-functional allelic variant is a naturally occurring amino acid sequence variant of the human ABCB12 transporter protein that has no ability to bind an ABCB12 transporter ligand. A non-functional allelic variant typically refers to non-conservative substitutions, deletions or insertions or premature truncations in the amino acid sequence of SEQ ID NO: 2, or significant residues or important The region may have been replaced, inserted or deleted.

The invention further provides non-human orthologs of the human ABCB12 transporter protein.
The ortholog of human ABCB12 transporter protein is isolated from a non-human organism,
It is a protein having the ABCB12 transporter activity of the human ABCB12 transporter protein. Human
The orthologue of the ABCB12 protein contains an amino acid sequence which is abbreviated as SEQ ID NO: 2 and thus can be easily identified.

In addition, nucleic acid molecules that encode other ABC transporter family members and thus have a nucleotide sequence that differs from the ABC transporter sequence of SEQ ID NO: 1 or 3 are also within the scope of the invention. Is intended. For example, another ABC transporter cDNA can be identified based on the nucleotide sequence of the human ABCB12 transporter. further,
Encodes the ABCB12 transporter protein from different species such as mammals, and thus SE
Nucleic acid molecules having a nucleotide sequence that differs from the ABCB12 transporter sequence of Q ID NO: 1 or 3 are also intended to be included within the scope of the present invention. For example, a mouse ABCB12 transporter cDNA can be identified based on the nucleotide sequence of the human ABCB12 transporter.

Nucleic acid molecules corresponding to naturally occurring allelic variants and homologues of the ABCB12 transporter cDNA of the present invention may be any of the cDNAs disclosed herein, based on their homology to the ABCB12 transporter nucleic acids disclosed herein, or , A portion thereof can be identified by utilizing it as a hybridization probe based on standard hybridization techniques under stringent hybridization conditions. Further, the AB of the present invention
Nucleic acid molecules corresponding to naturally occurring allelic variants and homologues of the CB12 transporter cDNA can also be isolated by mapping to the same chromosome or locus as the ABCB12 transporter gene.

Thus, in another embodiment, the isolated nucleic acid molecule of the invention comprises at least 15,20
, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3. In other embodiments, the nucleic acid is at least 30, 50, 100, 150, 200, 300, 400, 500, 600, 7
The nucleotide length is 00, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2739 or more. As used herein, the term "hybridize under stringent conditions" refers to hybridization and washing conditions that allow nucleotide sequences that are at least 75% homologous to one another to typically remain hybridized to each other. Say, it's intended. Preferably, the conditions are at least about 80%, even more preferably at least about 85%, 86%, 87%, 88%, 89%, 90%, 9%.
Sequences that are 1%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to each other are typically conditions under which they will remain hybridized to each other. Such stringent conditions are known to those of skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & S
ons, NY (1989), 6.3.1-6.3.6. A suitable, but non-limiting example of stringent hybridization conditions is 6 times sodium chloride / sodium citrate (SSC) hybridization at about 45 ° C. followed by 0.2 times SSC, 0.
One or more washes in 50%, preferably 55 ° C, more preferably 60 ° C, and even more preferably 65 ° C in 1% SDS. Preferably, the isolated nucleic acid molecule of the invention which hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 or 3 may correspond to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule that has a naturally-occurring nucleotide sequence (eg, encodes a natural protein).

Those skilled in the art will appreciate that in addition to naturally occurring allelic variants of the ABCB12 transporter sequences that may be present in the population, mutations will occur in the nucleotide sequence of SEQ ID NO: 1 or 3. It will also be appreciated that, as a result, the functional capacity of the ABCB12 transporter protein does not change, but only the amino acid sequence of the encoded ABCB12 transporter protein may change. For example, nucleotide substitutions that result in amino acid substitutions at "non-critical" amino acid residues may occur in the sequence of SEQ ID NO: 1 or 3. An "insignificant" amino acid residue is an ABCB12 transporter (eg, SEQ ID N
O: 2 sequence, etc.) is a residue whose biological activity does not change even if it changes from the wild-type sequence, while the “important” amino acid residue is a residue necessary for biological activity. Is.

Accordingly, another aspect of the invention pertains to nucleic acid molecules that encode the ABCB12 transporter protein with alterations in amino acid residues that are not important for activity. Such an ABCB12 transporter protein differs in amino acid sequence from SEQ ID NO: 2, but retains biological activity. In one example, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, provided that the protein is at least about 75% SEQ ID NO: 2, 80 %, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more, comprising amino acid sequences that are homologous.

An isolated nucleic acid molecule encoding an ABCB12 transporter protein homologous to the protein of SEQ ID NO: 2 has a substitution, addition, or addition of one or more nucleotides to the nucleotide sequence of SEQ ID NO: 1 or 3. It is possible to make deletions by introducing one or more amino acid substitutions, additions or deletions into the encoded protein. To introduce mutations into SEQ ID NO: 1 or 3, for example, site-directed mutagenesis and
Standard techniques such as PCR-mediated mutagenesis can be used. Preferably, conservative amino acid substitutions are made at one or more predicted non-critical amino acid residues. A "conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (
For example, glycine, asparagine, glutamine, serine, threonine, tyrosine,
Cysteine), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan)
, Amino acids with beta branched side chains (eg threonine, valine, isoleucine,
) And amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue within the ABCB12 transporter protein is preferably replaced with another amino acid residue within the same side chain family. Instead, in another example, mutations are randomly introduced in all or part of the ABCB12 transporter coding sequence, such as by saturation mutagenesis, and the resulting mutants are screened for ABCB12 transporter biological activity The remaining mutant may be identified. After mutagenesis of SEQ ID NO: 1 or 3, the encoded protein may be recombinantly expressed and the activity of that protein may be examined.

In one preferred embodiment, mutant ABCB12 transporter proteins are assayed, for example ABCB12
The ability to interact with molecules that are not ABCB12 transporters, such as transporter ligands and polypeptides or small molecules, can also be investigated.

Another aspect of the invention relates to an isolated nucleic acid molecule that is antisense thereto, in addition to the above-described nucleic acid molecule that encodes the ABCB12 transporter protein.
An "antisense" nucleic acid refers to a "sense" nucleic acid that encodes a protein, such as complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. It comprises a complementary nucleotide sequence. Thus, the antisense nucleic acid can hydrogen bond to the sense nucleic acid. The antisense nucleic acid may be complementary to the entire ABCB12 transporter coding strand, or only to a portion thereof. In one example, the antisense nucleic acid molecule is antisense to the "codon region" of the coding strand of the nucleotide sequence encoding ABCB12. The term "codon region" refers to a region of a nucleotide sequence that comprises codons translated into amino acid residues (eg, the codon region of human ABCB12 corresponds to SEQ ID NO: 3). In another example, the antisense nucleic acid molecule is antisense to the "non-codon region" of the coding strand of the nucleotide sequence encoding ABCB12. The term "non-codon region" refers to the 5'and 3'terminal sequences that flank the codon region that are not translated into amino acids (ie, also referred to as the 5'and 3'terminal untranslated regions).

With the disclosure herein of the coding strand sequence encoding the ABCB12 transporter (eg, SEQ ID NO: 3), antisense nucleic acids of the invention are designed based on the Watson-Crick base pairing law. be able to. The antisense nucleic acid molecule of interest is AB
It may be complementary to the entire codon region of CB12 transporter mRNA, but preferably,
The oligonucleotide may be antisense only to a part of the codon region or non-codon region of ABCB12 transporter mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation start site of ABCB12 transporter mRNA. Antisense oligonucleotides include, for example, about 5, 10, 15, 20, 25.
, 30, 35, 40, 45 or 50 nucleotides in length or longer. The antisense nucleic acid of the present invention can be constructed by utilizing a chemical synthesis method and an enzyme ligation method which are known methods in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) can be formed using naturally occurring nucleotides, or to enhance the biological stability of the molecule, or formed between antisense and sense nucleic acids. Can be chemically synthesized using a variety of modified nucleotides designed to enhance the physical stability of the duplex, such as phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides that can be used to make the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 -(Carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1-methylguanine , 1-methylinosine, 2
, 2-Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2- Thiouracil, beta-D-mannosylkeosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracil, keosine, 2 -Thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil , 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, using an expression vector in which the nucleic acid is subcloned in the antisense orientation (ie, the RNA transcribed from the inserted nucleic acid is in the antisense orientation with respect to the target nucleic acid of interest. Will be further explained in the subsection of),
The antisense nucleic acid can also be produced biologically.

The antisense nucleic acid molecules of the invention are typically provided by their hybridization or binding to intracellular mRNA and / or genomic DNA encoding the ABCB12 transporter protein, eg transcription and / or translation. Or administered in situ to inhibit expression of the protein, such as by
This hybridization can be based on conventional nucleotide complementation to form a stable duplex, or in the case of antisense nucleic acid molecules that bind to DNA duplexes, for example, in the groove of the double helix. It may be due to physical interaction. One example of the route of administration of the antisense nucleic acid molecules of the invention includes direct injection at the tissue site. Alternatively, the antisense nucleic acid molecule may be modified to target certain cells prior to systemic administration. For example, in the case of systemic administration, an antisense molecule may be modified so that it specifically binds to a receptor or antigen expressed on a given cell surface. For example, a peptide or antibody that binds to the cell surface receptor or antigen. Alternatively, the antisense nucleic acid molecule may be linked. In addition, the antisense nucleic acid molecule can be delivered to cells using the vectors described herein. In order to achieve a sufficient intracellular concentration of antisense molecule, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an alpha anomeric nucleic acid molecule. Alpha-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA, in which the strands are parallel to each other, in contrast to the normal β-unit (Gaultier et al. (1987). ) Nucleic Acids. Res. 15: 6625-6641). Further, the antisense nucleic acid molecule is a 2'-o-methylribonucleotide (Inoue et al.
(1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al.
al. (1987) FEBS Lett. 215: 327-330).

[0070] In yet another embodiment, the antisense nucleic acid of the invention is a ribozyme. A ribozyme is an RNA molecule having a ribonuclease activity and having a catalytic action, and can cleave a single-stranded nucleic acid when it has a region complementary to a single-stranded nucleic acid of a partner such as mRNA. Thus, using ribozymes (eg, Hammerhead Ribozyme (described in Haselhoff and Gerlach (1988) Nature 334: 585-591), the ABCB12 transporter mRNA transcripts are catalytically cleaved to produce the ABCB12 transporter mRNA.
It can inhibit the translation of NA. Ribozymes having specificity for a nucleic acid encoding ABCB12 can be designed based on the nucleotide sequence of the ABCB12 transporter cDNA disclosed herein (ie, SEQ ID NO: 1). For example, a derivative of Tetrahymena L-19 IVS RNA in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the mRNA encoding ABCB12 can be constructed. For example, Cech et al., U.S. Pat.No. 4,987,071 and Cec.
See h et al., US Pat. No. 5,116,742. Depending on the selection, the ABCB12 transporter mRNA can be used to screen a pool of RNA molecules for catalytic RNA with specific ribonuclease activity. For example, Bartel, D. adn Szostak, J. W
(1993) Science 261: 1411-1418.

Alternatively, a triple helix structure that targets a nucleotide sequence complementary to a regulatory region of the ABCB12 transporter (eg, ABCB12 transporter promoter and / or enhancer) in a target cell to prevent transcription of the ABCB12 transporter gene. To form AB
It can also inhibit the expression of the CB12 transporter gene. In general, Helene, C. (19
91) Anticancer Drug Des. 6 (6): 569-84; Helene, C. et al. (1992) Ann. N. Y.
Acad. Sci. 660: 27-36; and Maher, LJ (1992) Bioassays 14 (12): 807-15.
Please refer to.

In yet another embodiment, the ABCB12 transporter nucleic acid molecule of the present invention may be modified with a base moiety, an east moiety or a phosphate backbone moiety to enhance stability, hybridization or solubility, etc. of the molecule. it can. For example, the phosphate deoxyribose backbone of nucleic acid molecules can be modified to produce peptide nucleic acids (Hyrup B. et al. (1996) Bioor.
ganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the term "peptide nucleic acid" or "PNA" refers to DNA in which the backbone of deoxyribose phosphate has been replaced by a pseudopeptide backbone, leaving only four natural nucleobases.
Nucleic acid mimics such as mimics. It has been shown that the neutral backbone of PNA can specifically hybridize to DNA and RNA under conditions of low ionic strength. PNA
Oligomers are described above in Hyrup B. et al. (1996); Perry-O'Keefe et al. Proc. Na.
It can be synthesized using the standard solid phase peptide synthesis protocol described in tl. Acad. Sci. 93: 14670-675.

The PNA of the ABCB12 transporter nucleic acid molecule can be used in therapeutic and diagnostic applications. For example, PNA
When used as an antisense or antigene substance, gene expression can be modulated in a sequence-specific manner by, for example, inducing transcription or translation arrest, or inhibiting replication. The PNA of the ABCB12 transporter nucleic acid molecule can be further analyzed for single base pair mutations in a gene (for example, by PNA-specified PCR clamping).
; Used as an "artificial restriction enzyme" when used in combination with another enzyme (eg, S1 nuclease (Hyrup B. (1996) et al., Cited above)), or for DNA sequencing or hybridization Use as probe or primer (
(Hyrup B. (1996) et al., Supra; Perry-O'Keefe, supra).

In another example, modification of PNAs of the ABCB12 transporter nucleic acid molecule may be performed by attaching lipophilic or other auxiliary groups to the PNA (eg, to enhance their stability or uptake into cells). Or by forming a PNA-DNA chimera or using other drug delivery techniques known in the art such as the use of liposomes. For example, PNA-DNA chimeras of ABCB12 transporter nucleic acid molecules can be generated that would combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (such as RNAse H and DNA polymerase) to interact with the DNA moiety while allowing the PNA moiety to provide high binding affinity and specificity. PNA-D
For ligation of NA chimera, it is preferable to use a linker having an appropriate length selected in terms of stacking of bases, number of bonds between nucleic acid bases, and direction (Hyrup B. (1996) et al., Supra). The synthesis of PNA-DNA chimeras is described in Hyrup B. (1996) et al., Supra, and Finn PJ.
et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, DNA strands were synthesized on a solid support using standard phosphoramidite coupling chemistry, eg, 5 '-(4-methoxytrityl) amino-5'.
Modified nucleoside analogues, such as -deoxy-thymidine phosphoramidite, are available between the PNA and the 5'end of DNA (Mag, M. et al. (1989) Nu
cleic Acid Res. 17: 5973-88). Next, the PNA monomer is coupled stepwise to generate a chimeric molecule having a PNA segment at the 5'end and a DNA segment at the 3'end (Finn PJ (1996) et al.). . Alternatively, a DNA segment at the 5'end and 3
It is also possible to synthesize a chimeric molecule with a PNA segment at the end (Peterser, KH
et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

In another embodiment, the oligonucleotide is provided with a peptide (eg, to target a host cell receptor in vitro), or a cell membrane (eg, Letsinge).
r et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al.
(1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT publication No. WO088 / 09810) or blood-brain barrier (see PCT publication No. WO89 / 10134, for example). Other ancillary groups, such as agents that facilitate transport, may also be included. In addition, the oligonucleotide can be labeled with a cleavage agent that is triggered by hybridization (eg, Krol e).
(1988) Bio-Techniques 6: 958-976) or an intercalating agent (see, eg, Zon (1988) Pharm. Res. 5: 539-549). . For this purpose, the oligonucleotide may be conjugated to another molecule, such as a peptide, a hybridization-induced cross-linking agent, a transport agent, or a hybridization-induced cleavage agent.

II. Isolated ABCB12 Transporter Protein and Anti-ABCB12 Transporter Antibody One aspect of the invention is used as an isolated ABCB12 transporter protein and biologically active portions thereof and as immunogens that raise anti-ABCB12 transporter antibodies. To polypeptide fragments suitable for In one example, the native ABCB12 transporter protein can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another example, the ABCB12 transporter protein is recombined.
Generated by DNA technology. As an alternative to recombinant expression, the ABCB12 transporter protein or polypeptide may be chemically synthesized using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portion thereof, includes
ABCB12 transporter protein is substantially free of cellular material or other contaminating proteins from the original cell or tissue source, or, if chemically synthesized, contains chemical precursors or other chemicals. I can't. ABCB for the language "abbreviation of cellular material is absent"
The preparation of the 12 transporter protein includes that the protein is separated from the cellular components of the original cells produced by isolation or recombination. In one example, the language "substantially free of cellular material" includes less than about 30% (by dry weight) of non-ABCB12 transporter protein (herein "contaminated protein"). ), More preferably less than about 20% non-ABCB12 transporter protein, even more preferably less than about 10% non-ABCB12 transporter protein, and most preferably less than about 5% non-ABCB12 transporter protein. Is included.
When the ABCB12 transporter protein or a biologically active portion thereof is recombinantly produced, it is preferable that the culture medium is substantially absent, that is, the culture medium is less than about 20% of the volume of the protein preparation, more preferably about It should comprise less than 10%, and most preferably less than about 5%.

The language “substantially free of chemical precursors or other chemicals” refers to a preparation of the ABCB12 transporter protein in which the protein is a chemical precursor or other chemical involved in the synthesis of this protein. It includes being separated from the substance. In one example, the language "abbreviating chemical precursors or other chemicals" does not include ABCB12.
The transport protein preparation is less than about 30% (by dry weight) chemical precursor or non-ABCB12 transporter chemical, more preferably less than about 20% chemical precursor or non-ABC.
B12 transporter chemicals, even more preferably less than about 10% chemical precursors or non-ABCB
12 transporter chemicals, and most preferably less than about 5% chemical precursors or non-ABCB12
Includes having transporter chemicals.

As used herein, the “bioactive portion” of the ABCB12 transporter protein includes a fragment of the ABCB12 transporter protein that participates in the interaction between ABCB12 transporter molecules and non-ABCB12 transporter molecules. Be done. The biologically active part of the ABCB12 transporter protein is
For example, a peptide that is sufficiently homologous to the amino acid sequence of ABCB12 transporter protein, such as the amino acid sequence of SEQ ID NO: 2, or that contains an amino acid sequence derived from the same amino acid sequence, Contains a small number of amino acids,
Included are peptides that exhibit at least one of the activities of the ABCB12 transporter protein. Typically, the bioactive portion is one that comprises a domain or motif that has at least one of the activities of the ABCB12 transporter protein. The biologically active portion of the ABCB12 transporter protein can be, for example, 10, 25, 50, 100, 200,
It may be a polypeptide having an amino acid length of 300, 400, 500, 600, 700, 800 or more. The bioactive portion of the ABCB12 transporter protein can be used as a target for developing agents that modulate the ABCB12 transporter-mediated activity.

In one example, the bioactive portion of the ABCB12 transporter protein comprises at least one transmembrane domain. It will be appreciated that in the present invention, a suitable bioactive portion of the ABCB12 transporter protein will contain at least one transmembrane domain. Another suitable bioactive portion of the ABCB12 transporter protein is
It will contain at least two transmembrane domains. One or more of these transmembrane domains may associate to form a domain that extends across the membrane. Additionally or alternatively, the bioactive portion of the ABCB12 transporter protein may comprise Walkermain, eg, the Walker A and / or Walker B domain (see Figure 2; Patel et al. . (1998) Tren
ds Cell Biol 8: 65-71) It may be convenient to use any of the numerous art-recognized molecular modeling techniques described herein to identify these domains (see also Example 2). I want to be). Furthermore, another bioactive portion in which other regions of the protein have been deleted may be produced by a recombinant technique, and one or more of the functional activities of the natural ABCB12 transporter protein may be evaluated.

In one preferred embodiment, the ABCB12 transporter protein has the amino acid sequence set forth in SEQ ID NO: 2. In another example, the ABCB12 transporter protein is substantially homologous to SEQ ID NO: 2 and retains the functional activity of the protein of SEQ ID NO: 2, but is described in subsection I above. As mentioned, the amino acid sequences differ due to natural allelic variation or mutagenesis. Thus, in another example, the ABCB12 transporter protein has at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8% of SEQ ID NO: 2.
7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more,
It is a protein comprising homologous amino acid sequences.

To determine the percent identity between two amino acid sequences or between two nucleic acid sequences, the sequences are aligned to allow optimal comparison (eg, optimal alignment). May introduce gaps in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). In one preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 70%, preferably 80% of the length of the reference sequence,
90% or 100% (e.g., the second sequence with SEQ ID
At least 253 when aligned with the NO: 2 ABCB12 transporter amino acid sequence,
Preferably at least 337, more preferably at least 422, even more preferably at least 506 and even more preferably at least 590, 674, 759 or 843 amino acid residues may be aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If one position in the first sequence is occupied by the same amino acid residue or nucleotide as in the corresponding position in the second sequence, then the molecules are identical at that position (as used herein). The “identity” of an amino acid or nucleic acid is equivalent to the “homology” of an amino acid or nucleic acid.The percentage of identity between two sequences must be introduced to optimally align the two sequences. It is a function of the number of gaps and the number of identical positions between the sequences, taking into account the length of each gap.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one preferred embodiment, the percentage of identity between two amino acid sequences is determined using standard comparison software recognized in the art and utilizing standard parameter settings. For example, (http:
Needleman & Wanche (J. Mol. Biol. (48): 444-453 (19) incorporated into the GAP program of the GCG software package (available at www.gcg.com).
79)) algorithm using either a Blossom 62 matrix or a PAM250 matrix,
It can be used with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http://www.gcg.com), NWSgapdna.CMP.
It can be determined using the matrix and with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. In another example, the percentage of identity between two nucleotide or amino acid sequences is determined by the E. Meyers and
Using W. Miller's algorithm (CABIOS, 4: 11-17 (1989)), using the PAM120 weight residue table with a gap length penalty of 12 and a gap penalty of 4, it is possible to determine it can.

In addition, the nucleic acid and protein sequences of the present invention can be used as a “query sequence” to search public databases, eg, to identify other family members or related sequences. Such searches include Altschul,
et al. (1990) J. Mol. Biol. 215: 403-10 using the NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the ABCB12 transporter nucleic acid molecule of the invention. BLAST protein searches can be performed using the XBLAST program with score = 50, wordlength = 3
An amino acid sequence homologous to the ABCB12 transporter protein molecule can be obtained. To perform a gapped alignment for comparison, use Gapped BLAST, Altschul et
al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and Gapped BLAST programs, each program (e.g. X
BLAST and NBLAST) default parameters may be utilized. http: //www.ncbi.
See nlm.nih.gov.

The invention further provides ABCB12 transporter chimeras or fusion proteins. The ABCB12 transporter "chimeric protein" or "fusion protein" used here is
An ABCB12 transporter polypeptide is included that is operably linked to a non-ABCB12 transporter polypeptide. "ABCB12 transporter polypeptide" refers to a polypeptide having an amino acid sequence corresponding to ABCB12, while "non-ABCB12 transporter polypeptide"
The term, for example, refers to a polypeptide having an amino acid sequence corresponding to a protein which is different from the ABCB12 transporter protein and is not substantially homologous to the ABCB12 transporter protein, such as a protein derived from the same or a different organism. The ABCB12 transporter polypeptide within the ABCB12 transporter fusion protein may correspond to all or part of the ABCB12 transporter protein. In one preferred embodiment, the ABCB12 transporter fusion protein comprises at least one bioactive portion of the ABCB12 transporter protein. In another preferred embodiment, the ABCB12 transporter fusion protein comprises at least two bioactive portions of the ABCB12 transporter protein. Within this fusion protein, the term "operably linked" refers to the ABCB12 transporter polypeptide and non-AB
It is intended to refer to the CB12 transporter polypeptides fused to each other in frame. The non-ABCB12 transporter polypeptide may be fused to the N-terminus or the C-terminus of the ABCB12 transporter polypeptide.

For example, in one embodiment, the fusion protein is a GST-ABCB12 transporter fusion protein, provided that the ABCB12 transporter sequence is fused to the C-terminus of the GST sequence. Purification of recombinant ABCB12 is convenient using such a fusion protein.

In another embodiment, the fusion protein is the ABCB12 transporter protein containing a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian host cells), heterologous signal sequences can be utilized to increase expression and / or secretion of the ABCB12 transporter.

The ABCB12 transporter fusion protein of the invention may be incorporated into pharmaceutical compositions and administered to a subject in vitro. The ABCB12 transporter fusion protein can also be used to influence the bioavailability of the ABCB12 transporter substrate. Utilization of the ABCB12 transporter fusion protein can be performed, for example, by (i) aberrant modification or mutation of the gene encoding the ABCB12 transporter protein; (ii) misexpression of the ABCB12 transporter gene; Would be therapeutically useful in treating disorders caused by significant post-translational modifications.

In addition, the ABCB12 fusion proteins of the invention can also be used as immunogens to produce anti-ABCB12 transporter antibodies in a subject, purify ABCB12 transporter ligands, and to bind ABCB12 transporter substrates. It could also be used in a screening assay to identify molecules that inhibit the ABCB12 transporter interaction.

Preferably, the ABCB12 transporter chimera or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, in order to ligate DNA fragments encoding different polypeptide sequences in-frame to each other, it can be carried out based on a conventional technique. For example, a blunt end or a cohesive end is used for ligation. Appropriately add cohesive ends, which are digested with to make suitable ends. There are methods such as treatment with alkaline phosphatase to avoid unnecessary conjugation, and enzymatic ligation. In another example, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, the gene is PCR-amplified using an anchor primer that creates a complementary overhang between two consecutive gene fragments, which is then annealed and re-amplified to produce the chimeric gene. Sequences may be generated (eg, Current Protocols in Molecular Biology, eds. Ausubel et
al. John Wiley & Sons: 1992). Furthermore, many expression vectors that already encode a fusion moiety (eg, GST polypeptide etc.) are commercially available. The nucleic acid encoding the ABCB12 transporter may be cloned into such an expression vector and the fusion portion ligated in frame to the ABCB12 transporter protein.

The present invention also relates to variants of the ABCB12 transporter protein that act as either ABCB12 transporter agonists or ABCB12 transporter antagonists. Variants of the ABCB12 transporter protein include mutagenesis methods such as discrete point mutations,
Alternatively, it can be produced by cleaving the ABCB12 transporter protein. The agonist of the ABCB12 transporter protein may have the same biological activity as the biological activity of the naturally-occurring ABCB12 transporter protein, or may retain the biological activity less than that. An antagonist of the ABCB12 transporter protein can inhibit one of the activities of the naturally occurring ABCB12 transporter protein by, for example, competitively modulating the activity of the ABCB12 transporter protein. in this way,
Treatment with a variant with limited function can elicit a particular biological effect. In one example, treatment of a subject with a variant that has less biological activity than the naturally occurring protein has a naturally occurring ABCB12.
There are fewer side effects on the subject as compared to treatment with transporter proteins.

In one embodiment, the ABCB12 transporter agonist (mimetic) or ABCB12
Identification of variants of the ABCB12 transporter protein that act as either transporter antagonist can be accomplished by screening combinatorial libraries of variants of the ABCB12 transporter protein, such as truncation mutants, for ABCB12 transporter protein agonist or antagonist activity. ,It can be carried out. In one example, combinatorial mutagenesis at the nucleic acid level is performed to generate a variegated library of ABCB12 transporter variants and encoded by a variegated gene library. A variegated library of ABCB12 transporter variants includes, for example, a degenerate set of potential ABCB12 transporter sequences
Mixtures of synthetic oligonucleotides can be expressed so that they can be expressed as individual polypeptides or, alternatively, as a set of larger fusion proteins (for phage display) that internally contain a set of ABCB12 transporter sequences. It can be prepared by ligating with an enzyme to form a gene sequence. There are a variety of methods available to generate libraries of potential ABCB12 transporter variants from degenerate oligonucleotide sequences. Chemical synthesis of the degenerate gene sequence may be performed on an automated DNA synthesizer, which synthetic gene is then ligated into a suitable expression vector. Utilizing a degenerate set of genes, one mixture can provide all the sequences encoding the desired combination of potential ABCB12 transporter sequences. Methods of synthesizing degenerate oligonucleotides are known in the art (eg Narang, SA (1983) Tetrahedron 39: 3; Itakura et al.
. (1984) Annu. Rev. Biochem. 53: 323: Itakura et al. (1984) Science 198: 10.
56; Ike et al. (1983) Nucleic Acid Res. 11: 477).

Moreover, a library of fragments of sequences encoding the ABCB12 transporter protein is used to screen and screen variants of the ABCB12 transporter protein, AB
A variegated population of CB12 transporter fragments can be generated. In one embodiment, the double-stranded PCR fragment of the ABCB12 transporter coding sequence is nuclease treated to denature and restore the double-stranded DNA under conditions where nicking occurs only once per molecule. To form double-stranded DNA that is thought to contain sense / antisense pairs from products with different nicks, and the single-stranded portion is treated with S1 nuclease from the re-formed double strand. By removing and ligating the resulting fragment library into an expression vector, a library of coding sequence fragments can be prepared. According to this method, it is possible to obtain an expression library encoding N-terminal fragments, C-terminal fragments and internal fragments of various sizes of ABCB12 transporter protein.

Several techniques are known in the art for screening a gene product of a combinatorial library prepared by point mutation or truncation, and for screening a cDNA library for a gene product having a predetermined property. . Such techniques can be adapted for rapid screening of gene libraries generated by combinatorial mutagenesis of ABCB12 transporter proteins. The most widely used technique for screening large gene libraries and accustomed to high-throughput analysis is to clone the gene library into a replicable expression vector and select suitable cells in the resulting vector library. There is a technique in which the combinatorial gene is expressed under conditions such that the vector encoding the gene in which the product is detected can be easily isolated by transforming and detecting the desired activity. Recursive Ensemble Mutagenesis (RE
M) is a new technique that increases the frequency of functional variants in libraries.
This technique can also be used in combination with a screening assay to identify ABCB12 transporter variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.
USA 89: 7811-7815: Delgrave et al. (1993) Protein Engineering 6 (3): 327-33.
1).

Using the isolated ABCB12 transporter protein or a portion or fragment thereof as an immunogen, standard techniques for preparing polyclonal and monoclonal antibodies can be used to generate antibodies that bind to the ABCB12 transporter. . Full length ABCB12
Transporter proteins may be used, or alternatively, the invention uses ABCB as the immunogen.
We propose to utilize an antigenic peptide fragment of the 12 transporter. A preferable epitope included in the antigenic peptide is a region of ABCB12 transporter located on the surface of this protein, for example, a hydrophilic region or a highly antigenic region.

The ABCB12 transporter immunogen is typically used to raise antibodies by immunizing a suitable subject (eg, rabbit, goat, mouse or other mammal) with this immunogen. . Suitable immunogenic formulations are, for example, recombinantly expressed ABCB12.
It may contain a transporter protein or a chemically synthesized ABCB12 transporter polypeptide. The formulation may further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory substance. Immunogenic ABCB
Immunization of a suitable subject with a 12-transporter formulation induces a polyclonal anti-ABCB12 transporter antibody response.

Accordingly, another aspect of the invention pertains to anti-ABCB12 transporter antibodies. As used herein, the term "antibody" specifically binds (immunoreacts with) an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule, ie, an antigen such as the ABCB12 transporter. A molecule containing a site. Examples of immunologically active portions of immunoglobulins include F (ab) and F (ab ', which are obtained by treating the antibody with an enzyme such as pepsin.
) There are ₂ fragments. The present invention provides polyclonal and monoclonal antibodies that bind to the ABCB12 transporter. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a population of antibody molecules that contain only one antigen binding site capable of immunoreacting with a particular epitope of the ABCB12 transporter. Thus, a monoclonal antibody composition typically will be specific to the immunoreacted partner.
It exhibits a single binding affinity for the ABCB12 transporter protein.

Polyclonal anti-ABCB12 transporter antibodies can be used to transform suitable subjects as described above.
It can be prepared by immunizing with a CB12 transporter immunogen. The anti-ABCB12 transporter antibody titer of the immunized subject can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized ABCB12 transporters. If necessary, an antibody molecule targeting the ABCB12 transporter may be isolated from a mammal (for example, blood) and further purified by a known technique such as protein A chromatography to obtain an IgG fraction. After a suitable period of time from immunization, for example, when the anti-ABCB12 transporter antibody titer becomes the highest, antibody-producing cells are collected from the subject, and the cells are used to
The hybridoma technology first described by hler and Milstein (1975) Nature 256: 495-497 (also Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al.
(1980) J. Biol. Chem. 255: 4980-83; Yeh et al. (1976) Proc. Natl. Acad.
Sci. USA 76: 2927-31; and Yeh et al. (1982) Int. J. Cancer 29: 269-75), more recent human B cell hybridoma technology (Kozvor et al. (198).
3) Immunol Today 4:72), EBV hybridoma technology (Cole et al. (1985), Mo.
Noclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or the trioma technique, or a standard technique such as a trioma technique can be used to produce the monoclonal antibody.
Techniques for producing monoclonal antibody hybridomas are known (generally R.
H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Ana
lyses, Plenum Publishing Corp., New York, New York (1980); EA Lerner
(1981) Yale J. Biol. Med., 54: 387-402; ML Gefter et al. (1977) Somati
c Cell Genet. 3: 231-36). Briefly, an immortal cell line (typically myeloma) was fused to lymphocytes (typically splenocytes) taken from a mammal immunized with the ABCB12 transporter immunogen, as described above, The resulting culture supernatant of hybridoma cells is screened to identify hybridomas producing monoclonal antibodies that bind to the ABCB12 transporter.

Any of the many known protocols used to fuse lymphocytes and immortalized cell lines may be utilized to generate anti-ABCB12 transporter monoclonal antibodies (eg, G. Galfre et al. (1977) Nature 266: 55052; Gefter quoted above.
See et al. Somatic Cell Genet., Lerner, Yale J. Biol. Med., cited above, Kenneth, Monoclonal Antibodies, cited above). Furthermore, one of ordinary skill in the art will appreciate that there are convenient variations of such methods. Often, immortal cell lines (eg myeloma cell lines) are obtained from the same mammalian species from which the lymphocytes are derived. For example, a mouse hybridoma can be prepared by fusing lymphocytes taken from a mouse immunized with the immunogenic preparation of the present invention with an immortalized mouse cell line. Suitable immortal cell lines are mouse myeloma cell lines that are sensitive to medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”. Eg P3-NS1 / 1-Ag4-1, P3. Any of a number of myeloma cell lines, such as the -x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines, may be used as fusion partners based on standard techniques. Available from, typically HA
T-sensitive mouse myeloma cells were transformed into mouse splenocytes using polyethylene glycol (“PEG”).
) Is used for fusion. Next, the hybridoma cells formed by the fusion are selected using HAT medium that kills unfused cells or myeloma cells that have fused without proliferative ability (unfused splenocytes have not been transformed). To die after a few days). Hybridoma cells producing a monoclonal antibody of the invention are detected by looking for antibodies that bind to ABCB12 in the hybridoma culture supernatant, eg, using a standard ELISA assay.

Instead of producing hybridomas that secrete monoclonal antibodies, screening recombinant combinatorial immunoglobulin libraries (eg, antibody phage display libraries) with the ABCB12 transporter and isolating members of the immunoglobulin library that bind to ABCB12. However, monoclonal anti-ABCB12 transporter antibodies can be identified and isolated. A commercially available kit is available for preparing and screening a phage display library (for example, Pharmacia recombinant phage.
Antibody System, Catalog No. 27-9400-1; and Stratagene's SurfZAP ^™ Phage Display Kit, Catalog No. 240.
612). Further, examples of methods and reagents particularly suitable for use in making and screening antibody display libraries are found in, for example, Radner et al., US Pat. No. 5,223,40.
No. 9; Kang et al. PCT International Publication WO92 / 18619; Dower et al. PCT International Publication WO91 / 17271; Winter et al. PCT International Publication WO92 / 20791; Markland et al. PCT International Publication WO92.
/ 15679; Breitling et al., PCT International Publication WO93 / 01288; McFerty et al., P.
CT International Publication WO92 / 01047; Garrard et al. PCT International Publication WO92 / 09690; Radnor et al. PCT International Publication WO90 / 02809; Fuchs et al. (1991) Bio / Technology 9: 1370-137.
2; Hay et al. (1992) Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989
) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J 12: 725-734; Hawk
ins et al. (1992) J. Mol. Biol. 226: 889-896; Clarkson et al. (1991) Natu
re 352: 624-628; Gram et al. (1992) Proc. Na: tl. Acad. Sci. USA 89: 3576-3
580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al.
(1991) Nuc. Acid Res. 19: 4133-4137; Barbas et al. (1991) Proc. Natl. Aca
d. Sci. USA 88: 7978-7982; and McCafferty et al. Nature (1990) 348: 552-55.
Can be seen in 4.

Recombinant anti-ABCB12 transporter antibodies that also include both human and non-human portions, such as chimeric and humanized monoclonal antibodies, and that can be made using standard recombinant DNA are within the scope of the invention. By the way. Such chimeric and humanized monoclonal antibodies are described, for example, in Robinson et al., International Application PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi.M.
1,496; Morrison et al. European Patent Application No. 173,494; Neuberger et al. PCT.
International Publication WO86 / 01533; U.S. Pat. No. 4,816,567 to Cavily et al .; European Patent Application No. 125,023 to Cavily et al .; Better et al. (1988) Science 240: 1041-1.
043; Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et
al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad.
Sci. USA 84: 214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; W.
ood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Ca.
ncer Inst. 80: 1553-1559); Morrison, SL (1985) Science 229: 1202-1207;
Oi et al. (1986) Bio Techniques 4: 214; Winter, US Pat. No. 5,225,539;
Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science
239: 1534: and Beidler et al. (1988) J. Immunol. 141: 4053-4060 and the like, and can be produced by recombinant DNA technology known in the art.

Anti-ABCB12 transporter antibodies (eg, monoclonal antibodies) are eg
Standard techniques such as chromatography or immunoprecipitation can be used to isolate the ABCB12 transporter. Anti-ABCB12 transporter antibodies are useful in purifying native ABCB12 transporters from cells and recombinantly produced ABCB12 transporters expressed in host cells. In addition, anti-ABCB12 transporter antibodies can be used to detect ABCB12 transporter proteins (eg, in cell lysates or cell supernatants) to assess the abundance and expression pattern of ABCB12 transporter proteins. Anti-ABCB12 transporter antibodies can be used diagnostically to observe protein levels in tissues as part of a clinical laboratory procedure, for example to determine the effect of any given treatment regimen.
Detection can be easily accomplished by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes are horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin, and avidin / biotin. Examples of suitable fluorescent substances are umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. An example of a luminescent material is luminol. Examples of bioluminescent materials are luciferase, luciferin, and aequorin. Examples of suitable radioactive materials are ¹²⁵ I, ¹³¹ I, ³⁵ S, ³³ P, ³² P, or ³ H.

III. Recombinant expression vector and host cell Another aspect of the present invention relates to a vector, preferably an expression vector, containing a nucleic acid encoding the ABCB12 transporter protein (or part thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular, double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). Other vectors, such as non-episomal mammalian vectors, integrate into the host cell's genome upon introduction into the host cell and replicate with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Generally, a practical expression vector in recombinant DNA technology is often in the form of a plasmid. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to encompass other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for expression of this nucleic acid in a host cell, which recombinant expression vector comprises It means that one abnormal regulatory sequence selected on the basis of the host cell used for is contained in a state functionally linked to the nucleic acid sequence to be expressed. "Operably linked" in a recombinant expression vector means that the nucleotide sequence of interest is the expression of that nucleotide sequence (eg in an in vitro transcription / translation system or when the vector is introduced into a host cell). It is intended to mean linked to regulatory sequences in a possible manner (in the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements such as polyadenylation signals. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Acad.
There is an explanation in emic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells, and those that direct the expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). Be done. Those skilled in the art will appreciate that the design of the expression vector will depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, etc. By introducing the expression vector of the present invention into a host cell, a protein or peptide (for example, ABCB12 transporter protein, mutated AB or the like) encoded by the nucleic acid described herein, including the fusion protein or peptide is encoded.
CB12 transporter proteins, fusion proteins, etc.) can be produced.

Recombinant expression vectors of the invention can be designed to express the ABCB12 transporter protein in prokaryotic or eukaryotic cells. For example, the ABCB12 transporter protein is described in, for example, E. It can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are also Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, CA (1990) has explanation. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, such as by using T7 promoter regulatory sequences and T7 polymerase.

For expression of proteins in prokaryotes, E. coli was used exclusively with vectors containing constitutive or inducible promoters to direct the expression of either fusion or non-fusion proteins. It is done in Kori. Using a fusion vector, the number of amino acids in the protein encoded in it, often at the amino terminus of the recombinant protein,
You can increase. Such fusion vectors typically serve three purposes: 1) increase expression of the recombinant protein; 2) increase the solubility of the recombinant protein; and 3) act as a ligand in affinity purification. This helps in the purification of recombinant proteins. In the case of fusion expression vectors, a proteolytic cleavage site is often introduced at the junction between the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. . Such enzymes, and recognition sequences recognized by them, include Factor Xa,
There are thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia), which fuses glutathione-S-transferase (GST), maltose E-binding protein, or protein A, respectively, to a target recombinant protein.
Biotech; Smith, DB and Johnson, KS (1988) Gene 67: 31-40),
There are pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ).

The purified fusion protein can be used to assay ABCB12 transporter activity (such as the direct or competitive assays detailed below) or to generate antibodies specific to ABCB12 transporter protein. It can be used to do so. In one preferred embodiment, the ABCB12 transporter fusion protein expressed with the retroviral expression vector of the present invention is used to infect bone marrow cells, which are then transplanted into irradiated recipients. can do. The pathology of this recipient will be examined after sufficient time has elapsed (eg, six (6) weeks later).

Suitable inducible unfused E. Examples of E. coli expression vectors include pTrc (Amann et al., (1988
) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technolo
gy: Methods in Enzymology 185, Academic Press, San Diego, California (19
90) 60-89). Targeted gene expression from the pTrc vector depends on transcription from the hybrid trp-lac fusion promoter by the host RNA polymerase. pET
Targeted gene expression from the 11d vector relies on co-expressed viral RNA polymerase (T7 gn1) mediated transcription from the T7 gn10-lac fusion promoter. This viral polymerase can either host BL21 (DE3) or the T7 gn1 gene in lacUV
5 HMS174 (DE3) derived from a sedentary prophage, which is under the transcriptional control of a promoter, is provided.

E. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in host bacteria that have compromised the ability to proteolytically cleave the recombinant protein (Gottesman, S. et al. , Gene Expression Technol
ogy: Methods in Enzymology 185, Academic Press, San Diego, California (1
990) 119-128). Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector such that the individual codons corresponding to each amino acid are replaced by E. It is a method to be used preferentially in E. coli (Wada eta l., (1992) Nucleic Acids Res. 20: 2111
-2118). Such alterations of the nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another example, the ABCB12 transporter expression vector is a yeast expression vector. Yeast S
． An example of a vector for expression in cerevisiae is pYEPSec1 (Ba
ldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz,
(1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123.
), PYES2 (Invitrogen Corporation, San Diego, Calif.) And picZ (Invitrogen Corporation, San Diego, Calif.).

Alternatively, the ABCB12 transporter protein may be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors that can be used to express proteins in cultured insect cells (eg, Sf 9 cells) include pAc series (Smith e
t al. (1983) Mol. Cell. Biol. 3: 2156-2165) and pVL series (Lucklow and
Summers (1989) Virology 170: 31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature
329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulators. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Sa
mbrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laborat
ory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
See Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 16 and 17.

In another embodiment, the recombinant mammalian expression vector of interest is one that is capable of directing the expression of a nucleic acid preferentially in a particular cell type (eg, nucleic acid using tissue-specific regulators). Is expressed). Tissue-specific regulators are known in the art.
Non-limiting examples of suitable tissue-specific promoters include albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphocyte-specific promoter (Calame and Eaton (1988). Adv. Immunol. 43: 235-275), especially the T cell receptor promoter (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and the immunoglobulin promoter (Banerji et al. (1983) Cell 33). : 729-740; Queen a
nd Baltimore (1983) Cell 33: 741-748), a neuron-specific promoter (eg, neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad.
Sci. USA 86: 5473-5477), pancreas-specific promoter (Edlund et al. (1985) Sc
ience 230: 912-916), and mammary gland-specific promoters (eg, milk whey promoter; US Pat. No. 4,873,316 and European Patent Publication 264,166). Developmentally regulated promoters such as the mousehox promoter (Kessel and Gru
ss (1990) Science 249: 374-379) and α-fetoprotein promoter (Campes
and Tilghman (1989) Genes Dev. 3: 537-546).

Furthermore, the present invention relates to the present invention cloned into an expression vector in the antisense orientation.
A recombinant expression vector comprising a DNA molecule is provided. That is, this D
The NA molecule is operably linked to regulatory sequences in a manner that allows expression (by transcription of this DNA molecule) of an RNA molecule that is antisense to the ABCB12 transporter mRNA.
The regulatory sequences operably linked to the nucleic acid cloned in the antisense orientation should be selected such that they direct the continuous expression of this antisense RNA molecule in various cell types, eg viral promoters and / or enhancers. You can Alternatively, regulatory sequences that direct constitutive, tissue-specific or cell-type specific expression of antisense RNA may be selected. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus, provided that the antisense nucleic acid is produced under the control of a highly efficient regulatory region, Can be determined by the cell type into which the vector is introduced. For methods of regulating gene expression with antisense genes, see Weintraub, H. et al., Antisense RNA as
a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vo
See l. 1 (1) 1986.

Another aspect of the invention includes, for example, an ABCB12 transporter nucleic acid molecule in a recombinant expression vector or a sequence that enables homologous recombination into a particular site of the host cell's genome. And a host cell into which the ABCB12 transporter nucleic acid molecule of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that such terms refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Such progeny may, in fact, not be identical to the parental cell, since certain alterations may occur in later generations due to mutations or environmental influences, but still here. Within the scope of this term to be used.

The host cell may be prokaryotic or eukaryotic. For example, the ABCB12 transporter protein was transformed into E. It can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells. Other suitable host cells are known to those of skill in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "
“Transfection” is known in the art for introducing foreign nucleic acids (eg, DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Is intended to mean the technology of. Suitable methods for transforming or transfecting host cells are described in Sambrook, et al. (Molecular Cloning: A Labor
atory Manual.2nd.ed., Cold Spring Harbor Laboratory, Cold Spring Harbo
r Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.

For stable transfection of mammalian cells, depending on the expression vector used and the transfection technique, only a small percentage of cells will have the foreign DN.
It is known not to integrate A into their genome. To identify and select for these integrants, a gene encoding a selectable marker (eg, resistance to antibiotics) is often introduced into the host cell along with the gene of interest.
Suitable selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. The nucleic acid encoding the selectable marker is AB
It may be introduced into the host cell by being placed on the same vector encoding the CB12 transporter protein, or may be placed on another vector and introduced. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive and other cells will die).

Host cells of the invention, eg, cultured prokaryotic or eukaryotic host cells, can be utilized to produce (ie, express) the ABCB12 transporter protein. Accordingly, the invention further provides methods for producing ABCB12 transporter proteins using the host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (in which a recombinant expression vector encoding the ABCB12 transporter protein has been introduced) in a suitable medium so that the ABCB12 transporter protein is produced. including. In another example, the method further comprises the step of isolating the ABCB12 transporter protein from the medium or host cells.

In addition, the host cells of the invention may be used to produce non-human transgenic animals. For example, in one embodiment, the host cell of the present invention is a fertilized egg or embryonic stem cell into which the ABCB12 coding sequence has been introduced. Next, using such a host cell, a non-human transgenic animal in which an exogenous ABCB12 transporter sequence has been introduced into their genome, or a homologous recombination in which the endogenous ABCB12 transporter sequence has been modified is used. An animal can be produced. Such animals are useful for studying ABCB12 transporter function and / or activity and for identifying and / or evaluating modulators of ABCB12 transporter activity. "Transgenic animal" used here
Is a rodent such as a non-human animal, preferably a mammal, more preferably a rat or mouse, in which one or more of the cells of the animal contains the transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is an exogenous DNA that integrates into the genome of the cell from which the transgenic animal originates,
It remains in the mature animal's genome and directs the expression of the encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, "homologous recombination animal" refers to homologous recombination that occurs between an exogenous DNA molecule and an endogenous gene that was introduced into a cell such as the embryonic cell of the animal before the animal developed. Is a non-human animal, preferably a mammal, more preferably a mouse, in which the endogenous ABCB12 transporter gene has been altered.

The transgenic animal of the present invention is obtained by introducing the nucleic acid encoding ABCB12 into the male pronucleus of a fertilized egg by microinjection, retrovirus infection, etc., and developing this egg in a pseudo-pregnant female litter animal. By doing so, it can be manufactured. The ABCB12 transporter cDNA sequence of SEQ ID NO: 1 can be introduced into the genome of a non-human animal as a transgene. Alternatively, a non-human homologue of the human ABCB12 transporter gene, such as the mouse or rat ABCB12 transporter gene, may be utilized as the transgene. Alternatively, another AB
ABCB12 transporter gene homologues, such as CB12 transporter family members (see subsection I above).
It may be isolated and utilized as a transgene based on the hybridization of SEQ ID NO: 1 or 3 to the ABCB12 transporter cDNA sequence (as detailed in). further,
An intron sequence and a polyadenylation signal may also be included in the transgene to improve the transgene expression efficiency. Tissue-specific regulatory sequences operably linked to the ABCB12 transporter transgene can direct specific cells to express the ABCB12 transporter protein. Methods of producing transgenic animals through manipulation of embryos and microinjection, particularly using animals such as mice, are conventional in the art, and also include, for example:
Both are U.S. Pat.Nos. 4,736,866 and 4,870,009 to Radner et al., U.S. Pat.No. 4,873,191 to Wagner et al., And Hogan, B., Manipulating the Mouse Embryo, (Co
ld Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1986). Similar methods are used to generate other transgenic animals. A transgenic founder animal has ABC in its genome.
Presence of the B12 derivative transgene and / or A in the tissue or cells of the animal
It can be identified based on the expression of BCB12 transporter mRNA. Thus, transgenic founder animals can be used to breed additional animals carrying this transgene. In addition, transgenic animals carrying transgenes encoding the ABCB12 transporter protein can be transformed into other transgenic animals carrying other transgenes, such as those carrying transgenes encoding neurotoxic polypeptides such as β-amyloid. You can also mate.

To create homologous recombinant animals, deletions, additions or substitutions are introduced to introduce ABCB.
A vector is created that contains at least a portion of the ABCB12 transporter gene such that the 12 transporter gene has been altered, eg, functionally disrupted. The ABCB12 transporter electron may be a human gene (eg, cDNA of SEQ ID NO: 3), but more preferably
It may be a non-human homologue of the human ABCB12 transporter gene (eg, a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO: 1). For example, the mouse ABCB12 transporter gene can be used to construct homologous recombinant nucleic acid molecules, such as vectors, suitable for altering the endogenous ABCB12 transporter gene in the mouse genome. In one preferred embodiment, the endogenous ABCB12 transporter gene is functionally disrupted upon homologous recombination (ie, no longer encodes a functional protein; also referred to herein as a "knockout" vector). ), Designing this homologous recombination nucleic acid molecule. Alternatively, when homologous recombination occurs, the endogenous ABCB12 transporter gene may be mutated or altered, but still appear to encode a functional protein (e.g., alteration of the upstream regulatory region results in endogenous The expression of the ABCB12 transporter protein can be altered), and homologous recombinant nucleic acid molecules can also be designed. In the homologous recombination nucleic acid molecule, the altered portion of the ABCB12 transporter gene is flanked by additional ABCB12 transporter gene nucleic acid sequences at its 5'and 3'ends, so that homologous recombination occurs. Occurs between the exogenous ABCB12 transporter gene possessed by this homologous recombinant nucleic acid molecule and the endogenous ABCB12 transporter gene in cells such as embryonic stem cells. The additional flanking ABCB12 transporter nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both 5'and 3'ends) are included in the homologous recombination nucleic acid molecule (for a description of homologous recombination vectors see eg Thomas, KR and Capecchi.
, MR (1987) Cell 51: 503). This homologous recombination nucleic acid molecule was introduced into cells such as embryonic stem cell lines (by electroporation etc.), and the cells in which the introduced ABCB12 transporter gene was homologously recombined with the endogenous ABCB12 transporter gene were selected. Sort (see, eg, Li, E. et al. (1992) Cell 69: 915). The sorted cells are then injected into undifferentiated embryo cells of an animal (eg mouse) to form an assembled chimera (eg Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, EJ Robertson, ed. (IRL, Oxford, 1987) pp.113-152
See). Next, transplant the chimeric embryo into a suitable pseudopregnant female litter animal,
Bring the embryo to term. Breeding animals in which all of the animal's cells contain homologously recombined DNA as a result of germline transmission of the transgene, using a progeny that carries the homologously recombined DNA in the germ cells. You can Vectors, etc.
The method for constructing a homologous recombinant nucleic acid molecule or a homologous recombinant animal is further described in Bradle
y, A. (1991) Current Opinion in Biotechnology 2: 823-829 and PCT International Publication No. WO90 / 11354 by Le Moureck et al .; WO91 / 01140 by Smithys et al .; WO92 / 0968 by Gizulstra et al .; and Burns et al. There is an explanation in WO93 / 04169.

In another example, transgenic non-human animals can be produced that contain selected systems so that they can regulate the expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of this cre / loxP recombinase system, see, for example, Lakso et al. (1992) Proc. Natl. Acad. Sci. U.
See SA 89: 6232-6236. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al. (1991) Science 251: 1351-1355. Using the cre / loxP recombinase system). In order to regulate the expression, an animal containing a transgene encoding both Cre recombinase and a given protein is required, such animal containing one of the transgenes encoding a given protein and the other Can be obtained by creating a "dual" transgenic animal, such as by crossing two transgenic animals containing a transgene encoding a recombinase.

In addition, a clone of the non-human transgenic animal described herein was cloned into Wilmut.
, I. et al. (1997) Nature 385: 810-813 and PCT International Publication WO97 / 07668 and WO97.
It may be produced based on the method described in / 07669. Briefly, cells such as somatic cells are isolated from transgenic animals and induced to exit the growth cycle and enter G ₀ phase. The resting cells are then fused, for example by utilizing an electric pulse, to a denuclearized oocyte taken from an animal of the same species from which the resting cell was isolated. The oocytes thus reconstructed are cultured, grown into morula or undifferentiated embryo cells, and then transferred to pseudopregnant female litter animals. The offspring of this female litter will be clones of the animal from which cells such as somatic cells have been isolated.

IV. Pharmaceutical composition ABCB12 transporter nucleic acid molecule, fragment of ABCB12 transporter protein, anti-ABCB12 transporter antibody (also referred to herein as "effective compound"), expressible nucleic acid encoding ABCB12 transporter (or fragment thereof), or , Any compound identified as a modulator of the ABCB12 transporter (as described herein) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein or antibody of interest and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein includes any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent and the like compatible with drug administration. Is intended and intended. The use of such media and agents for pharmaceutically active substances is known in the art. Its use in compositions has been discussed, except where the conventional medium or agent is not compatible with the active compound. Supplementary active compounds can also be included in the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following ingredients: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, Glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid or phosphoric acid Agents and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremopho
r) EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and mold. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants and the like. Microbial activity can be prevented by a variety of antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. To prolong the absorption of the injectable composition, the composition may include agents that delay absorption such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound (eg, a fragment of the ABCB12 transporter protein or anti-ABCB12 transporter antibody) in the required amount in a suitable solvent, with any of the ingredients enumerated above as required. It can be prepared by adding together with one or a combination and filtering. Generally, the dispersant is prepared by adding the active compound into a sterile vehicle which contains the base ABCB12 transporter dispersion medium and the other necessary ingredients listed above. Is prepared. For sterile powders for the preparation of sterile injectable solutions,
Preferred methods of preparation are vacuum drying and freeze drying, which results in a powder of the active ingredient and the desired additional ingredients coming out of their solution, which were previously sterile filtered.

Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be added with excipients and may be utilized in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is orally utilized and after rinsing It may be spit out or swallowed. Pharmaceutically compatible binders and / or adjuvant substances may also be included as part of the composition. Tablets, pills, capsules, troches, etc. may include any of the following ingredients or compounds with similar properties: Binders such as microcrystalline cellulose, tragacanth or gelatin.
Excipients such as starch or nipples; disintegrants such as alginic acid, primogel, or corn starch; lubricants such as magnesium stearate or sterotes; propellants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or Fragrances such as peppermint, methyl salicylate, or orange flavors.

For administration by inhalation, the compounds are delivered in the form of a pressurized container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or an aerosol spray such as from a nebulizer.

Systemic administration may be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, including, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can also be accomplished using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, ointment, gel, cream or the like by a method known in the art.

The compounds may be prepared in the form of suppositories or retention enemas (eg, with conventional suppository bases such as glycerides such as cocoa butter) for rectal delivery.

In one example, the compounds are prepared with carriers that will protect the active compound against rapid elimination from the body, including implants and microencapsulated delivery systems, such as controlled release formulations. For example, vinyl acetate ethylene, polyanhydride, polyglycolic acid,
Biodegradable and biocompatible polymers can be utilized such as collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are known to those of skill in the art. In addition, materials may be obtained from Alza Corporation and Nova Pharmaceuticals. Additionally, liposome suspensions (including liposomes that target infected cells with monoclonal antibodies against viral antigens) may be used as pharmaceutically acceptable carriers. These are, for example, U.S. Pat.
It can be prepared by methods known in the art, as described in 811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units as unit dosages tailored to the subject to be treated, where each unit is the desired unit in association with the required pharmaceutical carrier. It contains a predetermined amount of active compound calculated to produce a therapeutic effect. Details of the dosage unit form of the invention are
Determined by the intrinsic properties of the active compound, the particular therapeutic effect sought to be achieved, and the limitations inherent in the technology of formulating such a compound for personal treatment,
It also depends directly on these.

The toxicity and therapeutic effect of such compounds can be measured in cell culture or in such a way as to determine, for example, the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The determination can be made using standard pharmaceutical techniques in laboratory animals. The ratio of toxic to therapeutic dose is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit greater therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but in order to reduce potential damage to non-infected cells, and thus reduce side effects, design delivery systems that target such compounds to the site of diseased tissue. So be careful.

The data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form used and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Doses may be formulated in animal models to provide a circulating plasma concentration range that includes the cell culture-determined IC50 (ie, the concentration of the test compound that inhibits symptoms by half to maximum). Such information can also be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors include, for example, intravenous injection, local administration (US Pat. No. 5,328,47
Can be delivered to the subject by stereotactic injection (see eg Che).
n et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057).
. The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent and can also comprise a delayed release matrix with the gene delivery vehicle embedded therein. Alternatively, where the complete gene delivery vector can be made intact from recombinant cells such as retroviral vectors, the pharmaceutical formulation can include one or more cells to serve as the gene delivery system.

The pharmaceutical composition may be included in a container, pack or dispenser together with instructions for administration.

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive agents (eg diagnostic assays, prognostic assays, Observational clinical trials and pharmacogenetics); and c) methods of treatment (eg therapeutic and prophylactic agents).

The isolated nucleic acid molecules of the invention may be expressed, for example, by expressing the ABCB12 transporter protein (through a recombinant expression vector in a host cell for gene therapy applications), as described in detail below, ABCB12 transporter mRNA (eg in a biological sample) or AB
It can be used to detect genetic alterations in the CB12 transporter gene and modulate ABCB12 transporter activity. The present ABCB12 transporter proteins can be used to treat diseases characterized by insufficient or excessive production of ABCB12 transporter substrates or production of ABCB12 transporter inhibitors. In addition, the ABCB12 transporter protein is used to
Screening for 12 transporter substrates, screening for drugs or compounds that modulate ABCB12 transporter activity, and further for insufficient or excessive production of ABCB12 transporter protein or ABCB12 transporter wild-type. Diseases characterized by the production of a type of ABCB12 transporter protein with aberrant or unwanted activity relative to the type protein can be treated. Furthermore, the anti-ABCB12 transporter antibodies of the present invention can be used to detect and isolate ABCB12 transporter proteins, regulate the bioavailability of ABCB12 transporter proteins, and modulate ABCB12 transporter activity. it can.

A. Screening Assay: The present invention binds to the ABCB12 transporter protein, expresses the ABCB12 transporter or ABCB.
A modulator having a stimulatory action or an inhibitory action on 12 transporter activity, or a stimulatory action or an inhibitory action on the expression or activity of ABCB12 transporter substrate, that is, a candidate compound or a test compound or Methods are provided for identifying agents (eg, peptides, peptidomimetics, small molecules, or other agents) (also referred to herein as "screening assays").

In one example, the invention provides an assay for screening candidate or test compounds that are substrates for ABCB12 transporter proteins or polypeptides or biologically active portions thereof. In another embodiment, the invention provides an assay for screening candidate or test compounds that bind to or modulate the activity of an ABCB12 transporter protein or polypeptide or a biologically active portion thereof. The test compounds of the invention are: biological libraries; spatially addressable (original: spatially addressa
ble) Parallel solid-phase or liquid-phase libraries: known in the art, including synthetic library methods that require inverse multilayer integration; "one-bead-one compound" library methods; and synthetic library methods using affinity chromatography sorting. It may be obtained using any of the various methods in combinatorial library methods. Biological library methods are limited to peptide libraries, but four other methods are available for compound peptide, non-peptide oligomer or small molecule libraries (Lam, K.
S. (1997) Anticancer Drug Des. 12: 145).

Examples of methods for synthesizing molecular libraries are in the art, eg, Dewitt et al. (1
993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. Med. Chem. 37: 267.
8: Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem
. Int. Ed. Engl. 33: 2059; Carrell et al. (1994) Angew. Chem. Int. Ed. En
gl. 33: 2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233.

Compound libraries are in solution (eg, Houghten (1992) Biotechniques 13: 412).
-421) or on beads (Lam (1991) Nature 354: 82-84), on chips (Fodor (19
93) Nature 364: 555-556), bacteria (Radner et al., US Pat. No. 5,223,409), on spores (Radner et al., US Pat. No. 5,223,409), on plasmids (Cull et al. (19).
92) Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith
(1990) Science 249: 386-390); (Devlin (1990) Science 249: 404-406); Cwir
la et al. (1990) Proc. Natl. Acad. Sci. 87: 6378-6382); (Felici (1991).
J. Mol. Biol. 222: 301-310); (Radner, supra).

In one embodiment, the assay is a cell-based assay, wherein the assay involves contacting cells expressing the ABCB12 transporter protein or a biologically active portion thereof with a test compound, The ability of test compounds to modulate ABCB12 transporter activity is investigated. To test the ability of the test compound to modulate ABCB12 transporter activity,
For example, intracellular transport of organic anions, organic cations, cytotoxic anticancer agents, intracellular calcium, potassium, phosphatidylcholine, sodium concentration, neuronal membrane depolarization, neurotoxic polypeptide (eg β-amyloid), or ABCB12 transport. It is possible by observing the activity of transcription factors that are regulated by the body. The cell may be of mammalian origin, such as a neural cell. The ability of the test compound to modulate the binding of the ABCB12 transporter to the substrate, or the ability to bind to the ABCB12 transporter,
You can look it up. To test the ability of a test compound to modulate the binding of the ABCB12 transporter to the substrate, for example, conjugating a radioisotope or enzyme label to the ABCB12 transporter substrate to bind the ABCB12 transporter substrate to the ABCB12 transporter. Binding can be examined by detecting the labeled ABCB12 transporter substrate as a complex.
To test the ability of a test compound to bind to the ABCB12 transporter, for example, the compound is conjugated to a radioisotope or enzyme label and the binding of the compound to the ABCB12 transporter is tested using a labeled ABCB12 transporter compound. It is possible if it can be examined by detecting it as a complex. For example, for compounds (eg, ABCB12 transporter substrate),
It can be directly or indirectly labeled with ¹²⁵ I, ³⁵ S, ¹⁴ C, ³³ P, ³² P, or ³ H, and this radioisotope can be directly factored in the radiation emission or detected by scintillation counting. it can. Alternatively, the compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by examining the conversion of a suitable substrate to product.

In one example, suitable compounds include, but are not limited to, verapamil, desmethoxyverapamil, chloroquine, quinine, chinchonidine, primaquine, tamoxifen, dihydrocyclosporin, yohimbine, corinanthine, reserpine, physostigmine, acridine, acridine. Orange, quinacrine, trifluoroperazine, chlorpromazine, propanolol, atropine, tryptoamine,
Forskolin, 1,9-dideoxyforskolin, cyclosporine, (U.S. Patent No. 4,117,118 (1978)), PSC-833 (cyclosporin D, 6-[(2S, 4R, 6E) -4-methyl
-2- (Methylamino) -3-oxo-6-octenoic acid]-(9CI), [US Pat. No. 5,525,59
No. 0] [ACS 121584-18-7], Keller et al., "SDZ PSC 833, non-immunosuppress
ive cyclosporine: its potency in overcoming p-glycoprotein-mediated mult
idrug resistance of murine leukemia ", Int J Cancer 50: 593-597 (1992)), RU
-486 (17β-Hydroxy-11β- [4-dimethylaminophenyl] -17α prop-1-
Inyl estra-4,9-dien-3-one), RU-49953 (17β-hydroxy-11β, 17α- [
4-Dimethylaminophenyl] -17αprop-1-ynyl estra-4,9dien-3one), S9778 (6- {4- [2,2-di ()-ethylamino] -1-piperidinyl} -N, N ', di-2-propenyl-1,3,5-triazine-2,4-diamine, bismethanesulfonate, [US Pat. No. 5,225,411; EP 466586] [ACS # 140945-01-3]; Dhainaut et al ., "New tri
azine derivatives as potent modulators of multidrug resistance, "J Medic
inal Chemistry 35: 2481-2496 (1992)), MS-209 (5- [3-4- (2,2-diphenylacetyl) piperazin-1-yl] -2-hydroxypropoxy] quinoline sesquifumarate, [USA Patent No. 5,405,843 (continuation of No. 5,112,817)], [ACS # 158681-49-3], S
ato et al., "Reversal of multidrug resistance by a novel quinoline deriv
ative, MS-209, Cancer Chemother Pharmacol 35: 271-277 (1995)), MS-073 (Fuk
azawa et al., European Patent Application 0363212 (1989), FK-506 (Tanaka et al.,
M. Physicochemical properties of FK-506, a novel immunosupressant isolat
ed from Streptomyces isukubaensis "Transplantation Proceedings. 19 (5 Sup
pl 6): 11-6, (1987); Naito et al., "Reversal of multidrug resistance by a
n immunosuppressive agent FK-506, "Cancer Chemother & Pharmacol. 29: 195-
200 (1992); Pourtier-Manzanedo et al., "FK-506 (fujimycin) reverses the m
ultidrug resistance of tumor cells in vitro, "Anti-Cancer Drugs 2: 279-8
3 (1991); Epand & Epand, "The new potent immunosuppressant FK-506 revers
es multidrug resistance in Chinese hamster ovary cells, "Anti-Cancer Dru
g Design 6: 189-93 (1991)), VX-710 (2-piperidinecarboxylic acid, 1- [oxo (3,4,5
--Trimethoxyphenyl) acetyl] -3- (3-pyridinyl) -1- [3- (3-pyridinyl) propyl] butyl ester [ACS 159997-94-1] [US Pat. No. 5,620,971] German
n et al., "Chemosensitization and drug accumulation effects of VX-710, v
erapamil, cyclosporin A, MS-209 and GF120918 in multifrug resistance-ass
ociated protein MRP "Anti-Cancer Drugs 8, 141-155 (1997); Germann et al.
, "Cellular and biochemical characterization of VX-710 as a chemosensiti
zer: reversal of P- glycoprotein-mediated multidrug resistance in vitro "
Anti-Cancer Drugs 8, 125-140 (1997)), VX-853 ([US Pat. No. 5,543,423])
(ACS # 190454-58-1), AHC-52 (methyl 2- (N-benzyl-N-methylamino) ethyl-2
, 6-Dimethyl-4- (2-isopropylpyrazolo [1,5-a] pyridin-3-yl) -1,4-dihydropyridine-3,5-dicarboxylate; [Japanese Patent 63-135381; Europe Patent No. 0270926] [ACS 119666-09-0] Shinoda et al., "In vivo circumvention
of vincristine resistance in mice with P388 leukemia using a novel compo
und, AHC-52, "Cancer Res 49: 1722-6 (1989)), GF-120918 (9,10-dihydro-5-
Methoxy-9-oxo-N- [4- [2-1,2,3,4-tetrahydro-6,7-dimethoxyisoquinol-2-yl) ethyl] phenyl] -4acridinecarboxamide, [US Pat.
, 604,237] [ACS # 143664-11-3] Hyafil et al., "In vitro and in vivo re.
versal of multidrug resistance by GF120918, an acridonecarboxamide deriv
ative, "Cancer Res 53: 4595-4602 (1993)), and XR-9051 (3-[(3Z, 6Z) -6-benzylidene-1-methyl-2,5-dioxopiperazine-3-ylidenemethyl]- N- [4- [2- (6
, 7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl) ethyl] phenyl] benzamide hydrochloride, [ACS # 57-22-7].

Further within the scope of the present invention, ABCB12 without the labeling of any of the interactants.
Included is examining the ability of the compound (eg, ABCB12 transporter substrate) to interact with the transporter. For example, a microphysiometer can be used to detect the interaction of a compound with the ABCB12 transporter without labeling either the compound or ABCB12 McConnell. HM et al. (1992) Science 257: 1906-1912. . As used herein, a "microphysiometer" (eg, a site sensor) is an analyzer that measures the rate at which cells acidify their environment using a potentiometric sensor (LAPS) that can be optically designated. This change in the acidification rate can be used as an index of the interaction between the compound and ABCB12.

In another example, the assay is a cell-based assay, wherein cells expressing an ABCB12 transporter target molecule (eg, ABCB12 transporter substrate) are contacted with a test compound, The step of examining the ability of this test compound to modulate (eg, stimulate or inhibit) the activity of the ABCB12 transporter target molecule is included. To determine the ability of this test compound to modulate the activity of the ABCB12 transporter target molecule, use the AB
Yes, by examining the ability of the CB12 transporter protein to bind or interact with the ABCB12 transporter target molecule.

To determine the ability of the ABCB12 transporter protein or biologically active fragment thereof to bind or interact with an ABCB12 transporter target molecule, one of the methods described above for direct binding studies can be used, It is possible. In one preferred embodiment, AB
The ability of the CB12 transporter protein to bind or interact with the ABCB12 transporter target molecule can be determined by examining the activity of this target molecule. For example, the target intracellular second messenger (ie intracellular Ca ²⁺ , diacylglycerol, IP ₃ , etc.)
Of the target, functionally linked to a nucleic acid encoding a detectable marker, such as luciferase, to detect the catalytic / enzymatic activity of the target, suitable substrate
-Detecting the induction of reporter genes (including responsive regulators), detecting intracellular transport of reference compounds or neurotoxic polypeptides (eg β-amyloid), or targeting cellular responses that are regulated by the target. The activity of the target molecule can be examined by detection or the like.

In yet another embodiment, the assay of the present invention is a cell-free assay, wherein an ABCB12 transporter protein or biologically active portion thereof is contacted with a test compound, which test compound Examine the ability to bind the ABCB12 transporter protein or its biologically active portion. Suitable biologically active portions of the ABCB12 transporter protein for use in the assays of the invention include fragments that participate in interactions with non-ABCB12 transporter molecules, such as fragments with high surface probability scores (eg, See FIG. 2). Binding of the test compound to the ABCB12 transporter protein may be determined directly as described herein or indirectly. In one preferred embodiment, the assay involves contacting an ABCB12 transporter protein or a biologically active portion thereof with a known compound that binds ABCB12 to form an assay mixture, the assay mixture being tested. And contacting the test compound with the ABCB12 transporter protein to determine the ability of the test compound to interact with the ABCB12 transporter protein. Involves examining the ability of a test compound to preferentially bind to the ABCB12 transporter or a biologically active portion thereof compared to known compounds.

In another example, the assay is a cell-free assay, except that in this assay, AB
The CB12 transporter protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the ABCB12 transporter protein or biologically active portion thereof is examined. To test the ability of a test compound to modulate the activity of the ABCB12 transporter protein, the ABCB12 transporter target molecule is loaded with the ABCB12 transporter protein, for example, by one of the methods described above for direct binding. It is possible by examining the ability to bind. Alternatively, for example,
ATP binding may be measured. In order to examine the ability of the ABCB12 transporter protein to bind to the ABCB12 transporter target molecule, a technique such as real-time biomolecular interaction analysis (BIA) may be used. Sjolander,
S and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345 and Szabo et al. (1
995) Curr. Opin. Struct. Biol. 5: 699-705. As used herein, "BIA" is a technique for investigating a biospecific interaction in real time without labeling any of the interacting substances (for example, BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indicator of real-time reactions between biomolecules.

In an alternative example, the ability of a test compound to modulate the activity of an ABCB12 transporter protein can be determined by further testing this ABCB12 transporter protein for effectors downstream of the ABCB12 transporter target molecule. This is possible by examining their ability to modulate activity. For example, the activity of the effector molecule on a suitable target may be investigated, or the binding of the effector to a suitable target may be investigated, as described above.

In yet another embodiment, the cell-free assay method described above involves contacting an ABCB12 transporter protein or a biologically active portion thereof with a known compound that binds to the ABCB12 transporter protein to form an assay mixture. A step of contacting the assay mixture with a test compound, and examining the ability of the test compound to interact with the ABCB12 transporter protein, where it interacts with the ABCB12 transporter protein. The step of examining the ability of the test compound to inhibit includes the ability of the ABCB12 transporter protein to bind preferentially to the ABCB12 transporter target molecule or modulate its activity.

The cell-free assay method of the present invention is compatible with the use of both soluble and / or membrane-bound isolated proteins (eg, ABCB12 transporter proteins or biologically active portions thereof). In the case of a cell-free assay method using a membrane-bound isolated protein,
It may be preferable to utilize a solubilizer so that the membrane-bound isolated protein of interest is maintained in solution. Examples of such solubilizers include, for example, n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl.
-N-methylglucamide, decanoyl-N-methylglucamide, Triton (R) X-1
00, Triton (R) X-114, Thesit (R), isotridecipoly (ethylene glycol ether) _n , 3-[(3-colamidopropyl) dimethylaminio] -1-propanesulfonate (CHAPS), 3 -[(3-Colamidopropyl) dimethylaminio] -2-hydroxy-1-propanesulfonate (CHAPSO) or N-dodecyl = N, N-dimethyl-3-ammonio-1-propanesulfonate.

In two or more embodiments of the assay methods of the invention described above, immobilization of either the ABCB12 transporter or its target molecule results in complexation from one or both of the uncomplexed forms of these proteins. It would be convenient and preferable to separate the formed shapes and to automate the assay. Binding of the test compound to the ABCB12 transporter protein, or the interaction of the ABCB12 transporter protein with the target molecule in the presence and absence of the candidate compound, is suitable if it accommodates the reactants. It may be performed in any container. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one example, a fusion protein may be made with one additional domain that allows one or both of these proteins to bind to the matrix. For example, a glutathione-S-transferase / ABCB12 transporter fusion protein or a glutathione-S-transferase / target fusion protein is used as a glutathione sepharose beads (Sigma Chemical Co., St. Louis, Mo.) or a glutathione derivatized trace amount. Adsorb to a quantitation plate and then combine the beads or plate with the test compound or with the test compound and either the non-adsorbed target protein or the ABCB12 transporter protein and allow the mixture to lead to complex formation. Incubation may be carried out under conditions (eg physiological conditions of salt and pH). After incubation, the beads or wells of the microtiter plate are washed to wash away unbound components, in the case of beads the matrix is fixed and the complex is examined directly or indirectly, for example as described above. Alternatively, the complex may be cleaved from the matrix and the level of ABCB12 transporter binding or activity examined using standard techniques.

Other techniques for immobilizing proteins on matrices may be used in the screening assays of the invention. For example, either the ABCB12 transporter protein or the ABCB12 transporter target molecule can be immobilized utilizing conjugation of biotin and streptavidin. The biotinylated ABCB12 transporter protein or target molecule was labeled with biotin-NHS (
N-hydroxy-succinimide) prepared using techniques known in the art (eg, Biotinylation Kit from Pierce Chemicals, Rockford, IL) and coated with streptavidin (Pierce Chemical). (Made by the company). Alternatively, an antibody that is reactive to the ABCB12 transporter protein or target molecule but does not interfere with the binding of the ABCB12 transporter protein to its target molecule is derivatized and does not bind to the wells of the plate. The target or ABCB12 transporter protein may be captured in this well by antibody conjugation.
Methods of detecting such complexes, including those described above for GST-immobilized complexes, include immunodetection of the complex using an antibody reactive to the ABCB12 transporter protein or target molecule, or ABCB12. There are enzyme-linked assays that rely on the detection of enzyme activity associated with transporter proteins or target molecules.

In another example, modulators of ABCB12 transporter expression are identified by contacting cells with a candidate compound and examining the expression of ABCB12 transporter mRNA or protein in the cells. The expression level of ABCB12 transporter mRNA or protein in the presence of the candidate compound is compared to the expression level of ABCB12 transporter mRNA or protein in the absence of the candidate compound. Thus, based on this comparison, the candidate compound can be identified as a modulator of ABCB12 transporter expression. For example, if the expression of ABCB12 transporter mRNA or protein is higher (statistically significantly higher) in the presence of the candidate compound than in the absence thereof, the candidate compound is expressed as ABCB12 transporter mRNA or protein. Identified as a stimulant of. Conversely, ABCB12 is more present when the candidate compound is present than when it is absent.
If transporter mRNA or protein expression is low (statistically significantly low), this candidate compound is identified as an inhibitor of ABCB12 transporter mRNA or protein expression. The expression level of ABCB12 transporter mRNA or protein in a cell is
It can be examined by the method described here for the detection of mRNA or protein.

In yet another aspect of the invention, the ABCB12 transporter protein is utilized as a "bait protein" in a two or three hybrid assay (eg, US Pat. No. 5,283,317; Zervos et al. (1993). Cell 72: 223-232; Madura et al.
(1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biotechniqu.
es 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent W.
O94 / 10300), binds or interacts with the ABCB12 transporter (see “ABCB12”).
Other proteins involved in ABCB12 transporter activity can be identified along with the "binding protein" or "ABCB12-bp"). Such ABCB12 binding proteins may also be involved in signal transduction by the ABCB12 transporter protein or ABCB12 transporter target, eg as a downstream factor of the ABCB12 transduced signaling pathway. Otherwise, such an ABCB12 binding protein may be an ABCB12 transporter inhibitor.

The two-hybrid system described above is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, this assay utilizes two different DNA constructs. In one artifact, A
The gene encoding the BCB12 transporter protein is associated with a known transcription factor (eg GAL-4).
It is fused to the gene encoding the DNA binding domain. In the other construct, a single DNA sequence from a library of DNA sequences encoding an unknown protein ("prey" or "sample") is a gene encoding the activation domain of the known transcription factors. Is fused to. If these "bait" and "prey" proteins are able to interact in vitro to form an ABCB12-dependent complex, the DNA binding and activation domains of this transcription factor are closely located. It will be. This proximity can be used to cause transcription of a reporter gene (eg, LacZ) operably linked to a transcriptional regulatory site in response to a transcription factor. Expression of the reporter gene can be detected and cell colonies containing a functional transcription factor isolated, which colony can be used to obtain a cloned gene encoding a protein that interacts with the ABCB12 transporter protein.

In another aspect, the invention features a combination of two or more assay methods described herein. For example, cell-based or cell-free assays may be used to identify modulators and the ability of the modulators to modulate the activity of the ABCB12 transporter protein may be confirmed in vitro, such as in animals.

The present invention further relates to novel agents identified in the screening assays described above. Therefore, it is within the scope of the invention to utilize the agents identified as described herein in suitable animal models. For example, agents identified as described herein (eg, ABCB12 transporter modulators, antisense ABCB12 transporter nucleic acid molecules, ABCB12 specific antibodies, or ABCB12 binding partners, etc.) can be used in animal models to produce such effects. Efficacy, toxicity or side effects when treated with a substance can be investigated. Alternatively, agents identified as described herein can be used in animal models to study the mechanism of action of such agents. The invention also relates to the use of the novel agents identified in the screening assays described above for the treatments described herein.

B. Detection Assays Portions or fragments of the cDNA sequences identified here (and the corresponding complete gene sequences can be used as polynucleotide reagents in a variety of ways. For example, using these sequences: (1) those on the chromosome Mapping each gene and thus finding the genetic regions associated with genetic diseases; (ii) identifying individuals from microscopic biological samples (tissue typing); and (iii) helping forensic identification of biological samples,
be able to. These uses are explained in the following subsections.

1. Chromosome Mapping Once the sequence of a gene (or a portion of that sequence) has been isolated, the sequence can be used to map the location of the gene on the chromosome. This process is called chromosome mapping. Thus, a portion or fragment of the ABCB12 transporter nucleotide sequence described herein can be used to map the location of the ABCB12 transporter gene on the chromosome. Mapping the ABCB12 transporter sequences onto the chromosome is an important first step in correlating these sequences with genes associated with disease.

Briefly, to map the ABCB12 transporter gene to the chromosome, ABCB12
A PCR primer (preferably 15-25 base pairs long) is made from the 12-transporter nucleotide sequence. Computational analysis of the ABCB12 transporter sequence to predict primers that do not span more than one exon in genomic DNA prevents the amplification process from becoming complicated. Next, these primers may be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the ABCB12 transporter sequence will yield an amplified fragment.

Somatic cell hybrids are made by fusing somatic cells taken from different mammals (eg, human and mouse cells). Human and mouse cell hybrids gradually lose human chromosomes in a random order as they grow and divide, but retain mouse chromosomes. Since mouse cells do not have a specific enzyme, using a medium in which this mouse cell cannot grow and human cells can grow, only the human chromosome containing the gene encoding the required enzyme is retained. . A variety of media can be used to establish a panel of hybrid cell lines. Each cell line in a panel contains one or a small number of human chromosomes in parallel and a complete set of mouse chromosomes, so that individual genes can be easily mapped to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220: 919-924). Somatic cell hybrids containing only human chromosome fragments can be prepared by using human chromosomes having translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid technique for assigning specific sequences to specific chromosomes. Just use one thermal cycler,
Three or more sequences can be allocated per day. Using the ABCB12 transporter nucleotide sequence to design an oligonucleotide primer,
Sublocalization is possible with a panel of fragments taken from a particular chromosome. Other mapping strategies that may also be used to map the ABCB12 transporter sequence onto its chromosome include (Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA,
87: 6223-27), in situ hybridization, prescreening using labeled and flow-sorted chromosomes, and preselection method by hybridization to a chromosome-specific cDNA library.

[0167] Furthermore, a DNA sequence in the metaphase chromosome spread was subjected to fluorescence in situ
By using the hybridization method (FISH), the chromosome position can be accurately known in one step. Chromosome spreads can be made using cells whose division is blocked at metaphase by a chemical substance such as colcemid that disrupts the mitotic spindle. This chromosome is quickly trypsinized and stained with Giemsa's solution. Chromosomes can be individually identified because a pattern of light and dark bands occurs on each chromosome. The FISH method can be used for DNA sequences as short as 500 or 600 bases. However, larger clones of more than 1,000 bases are more likely to bind to unique chromosomal locations and simple detection provides sufficient signal strength. Preferably, with 1,000 bases, and more preferably 2,000 bases,
It will be sufficient to get good results at a reasonable time. For a review of this technology, see Verma et al., Human Chromosomes: A Manual of BABCB12 transporter Tec.
See hniques (Pergamon Press, New York 1988).

[0168] Reagents for chromosome mapping may be used to individually mark a single chromosome, or a site on that chromosome, using a panel of reagents to map multiple sites and / or Multiple chromosomes may be marked. In practice, reagents corresponding to the non-codon region of the gene are preferred for mapping purposes. Codon sequences are more likely to be conserved within gene families, thus increasing the likelihood of cross-hybridization during chromosome mapping.

If a sequence can be accurately mapped to a position on the chromosome, the physical position of the sequence on the chromosome can be correlated with the gene map data. (Such data is available, for example, from John Hopkins University Welch, V. McKusick, Mendelian Inh, available online through the Medical Library.
Can be found in eritance in Man. ) Next, the relationship between a gene and a disease that has been mapped to the same chromosomal region is described in, for example, Egeland, J. et a. (1987) Nat.
ure, 325: 783-787 and can be identified by linkage analysis (co-inheritance of physically adjacent genes).

[0170] In addition, differences in DNA sequences between individuals with and without disease associated with the ABCB12 transporter gene can be examined. If a mutation is found in some or all of the affected individuals and not in any unaffected individuals, then the mutation may be the cause of the particular disease. To compare affected and unaffected individuals, one generally first needs to be visible in chromosomal spreads, such as deletions or translocations, or be detectable using PCR based on DNA sequences. There is a step to investigate structural changes in the chromosome. Finally, complete sequencing of genes from several individuals can be performed to confirm the presence of the mutation and distinguish it from polymorphisms.

2. Tissue Typing The ABCB12 transporter sequences of the present invention can also be used to identify individuals from minute biological samples. For example, the US military is considering using Restricted Fragment Length Polymorphism (RFLP) to identify personnel. In this technique, a person's genomic DNA is digested with one or more restriction enzymes, probed with Southern blots, and unique bands are found for identification. This method does not have the limitations of the current "dog license", which can be lost, replaced, or stolen and is difficult to clearly identify. The sequences of the invention are useful as additional DNA markers for RFLP (US Pat. No. 5,272,057).
There is a commentary in the issue).

Furthermore, the sequence of the present invention can be used to represent a given part of the genome of an individual for each base.
It can be used to provide alternative techniques such as DNA sequencing. Thus, the ABCB12 transporter nucleotide sequence described herein can be used to generate two PCR primers from the 5'and 3'ends of this sequence. These primers can thus be used to amplify individual DNA and sequence it.

A panel of corresponding DNA sequences taken from an individual made in this manner is:
It becomes unique personal identification information. This is because each individual has a unique set of such DNA sequences due to allelic differences. Using the sequences of the invention, such identifying sequences can be obtained from individuals and organizations. Of the present invention
The ABCB12 transporter nucleotide sequence uniquely represents a number of parts of the human genome. There is some allelic variation in these codon regions and more frequently in non-codon regions. It is predicted that there will be allelic variation between human genomes approximately once every 500 bases. To some extent, each of the sequences described herein can be used as a target for comparison of DNA taken from an individual for identification purposes. Because there are more polymorphisms in the non-codon regions, fewer numbers of sequences are needed to distinguish individuals. Using the non-codon sequence of SEQ ID NO: 1, individuals can be unambiguously identified with a group of primers, perhaps 10 to 1000, each producing a non-codon amplified sequence of 100 bases.
If a predictive codon sequence is used, such as that of SEQ ID NO: 3, then a primer number of 500 to 2,000 would be more suitable for a clear identification of the individual.

When a panel of reagents based on the ABCB12 transporter nucleotide sequences described herein is used to create a unique identification database for an individual, this same reagent can be used to identify tissues taken from that individual. May be used later. Using this unique identification database, a person can be clearly identified regardless of life or death based on a very small amount of tissue material.

3. Use of Partial ABCB12 Transporter Sequences in Forensic Biology In addition, DNA-based identification techniques can be used in forensic biology. Forensic biology
For example, in the field of science that uses genotyping of biological evidence found at crime scenes as a means of unequivocally identifying criminals. To perform such identification, PCR technology is used to analyze DNA sequences collected from a very small amount of biological samples such as tissues found in crime scenes such as hair or skin and body fluids such as blood, saliva or semen. Amplify. This amplified sequence can then be compared to a standard to identify the origin of the biological sample.

The sequences of the invention can also be used to generate polynucleotide reagents, such as PCR primers, that target specific loci in the human genome, which reagents allow the formation of another "discriminating marker" (ie, a specific individual). (For example, by providing another unique DNA sequence), the reliability of forensic identification based on DNA can be increased. As described above, the actual nucleotide sequence information can be used for identification as accurate information instead of the pattern formed by the fragments obtained by restriction enzyme digestion. Sequences aimed at the non-codon region of SEQ ID NO: 1 are particularly suitable for this use. This is because there is a higher number of polymorphisms in the non-codon regions, making it easier to distinguish individuals using this technique. Examples of polynucleotide reagents include an ABCB12 transporter nucleotide sequence or a portion thereof, such as an SE of at least 20 bases, preferably at least 30 bases in length.
There is a fragment derived from the non-codon region of Q ID NO: 1.

In addition, the ABCB12 transporter nucleotide sequences described herein are polynucleotides, such as labeled or labelable probes, that can be used to identify in situ hybridization techniques and specific tissues such as brain tissue. It can also be used to make reagents. This would be very useful if the forensic pathologist has obtained tissue of unknown origin. Such a panel of ABCB12 transporter probes can be used to identify tissue by species and / or organ type.

In a similar manner, these reagents, such as the ABCB12 transporter primer or probe, are
It can also be used to screen cultures for contamination (ie, screening cultures for mixed cell types).

C. Predictive Drugs: The present invention further relates to the field of predictive drugs in which diagnostic tests, prognostic tests, and observatory clinical trials are used for prognostic (predictive) purposes to prophylactically treat an individual. . Therefore, one embodiment of the present invention is to investigate the expression of ABCB12 transporter protein and / or nucleic acid in a biological sample (blood, serum, cells, tissues, etc.) or the activity of ABCB12 transporter, and It relates to a diagnostic assay for investigating whether a person has a disease or disorder, or is at risk of developing a disorder associated with abnormal or unwanted ABCB12 transporter expression or activity. The invention further provides a prognostic (or prognostic) assay to determine if an individual is at risk of developing a disorder associated with ABCB12 transporter protein, nucleic acid expression or activity. For example, mutations in the ABCB12 transporter gene may be assayed in biological samples. Such assays can be used for prognostic or prognostic purposes, thus preventing prophylactic treatment of an individual prior to the onset of a disorder characterized by or associated with ABCB12 transporter protein, nucleic acid expression or activity. can do.

Another aspect of the invention relates to observing in a clinical trial the effect of an agent (eg, drug, compound) on the expression or activity of the ABCB12 transporter.

[0181]   These and other agents are further detailed in the following sections.

1. Diagnostic Assay One example of a method for detecting the presence or absence of an ABCB12 transporter protein or nucleic acid in a biological sample is the step of obtaining a biological sample from a test subject and detecting the presence of the ABCB12 transporter protein or nucleic acid in this biological sample. Thus, contacting the biological sample with a compound or agent capable of detecting ABCB12 transporter protein or nucleic acid encoding ABCB12 transporter protein (eg, mRNA, genomic DNA). ABCB12 transporter mR
A suitable agent for detecting NA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to ABCB12 transporter mRNA or genomic DNA. The nucleic acid probe can be, for example, a full length ABCB12 transporter nucleic acid, such as the nucleic acid of SEQ ID NO: 1, or sufficient to specifically hybridize to ABCB12 transporter mRNA or genomic DNA under stringent conditions. However, it may be a part thereof, such as an oligonucleotide having a length of at least 15, 30, 50, 100, 250 or 500 nucleotides. Other probes suitable for use in the diagnostic assays of the invention are described herein.

A suitable agent for detecting the ABCB12 transporter protein is an antibody capable of binding to the ABCB12 transporter protein, preferably a detectably labeled antibody. The antibody may be polyclonal, but more preferably it is monoclonal. An intact antibody, or a fragment thereof (eg, Fab or F (ab ′) ₂ ) can also be used. The term "labeled" with respect to a probe or antibody is a direct labeling of a probe or antibody, or a direct label, which attaches (ie physically associates) a detectable substance to the probe or antibody. It is intended to include indirectly labeling the probe or antibody with its reactivity with another reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and method of end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids separated from a subject, as well as tissues, cells and fluids present in the subject. That is, the detection method of the present invention can be used to detect ABCB12 transporter mRNA, protein or genomic DNA in a biological sample not only in vitro but also in vitro. For example, in vitro techniques for detecting ABCB12 transporter mRNA include
There are Northern hybridizations and in situ hybridizations. In vitro techniques for detecting the ABCB12 transporter protein include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation and immunofluorescence. In vitro techniques for detecting ABCB12 transporter genomic DNA include Southern hybridizations. In addition, in vitro techniques for detecting ABCB12 transporter proteins include the introduction of labeled anti-ABCB12 transporter antibodies into a subject. For example,
The antibody may be labeled with a radioactive marker and its presence and location in the subject may be detected by standard imaging techniques.

In one example, the biological sample of interest contains protein molecules taken from a test subject. Alternatively, the biological sample may contain a test subject mRNA molecule or a test subject genomic DNA molecule. A suitable biological sample is a serum sample taken from a subject by conventional means.

In another example, the method comprises obtaining a control biological sample from a control subject,
Contacting the control sample with a compound or agent capable of detecting ABCB12 transporter protein, mRNA or genomic DNA so that the presence of ABCB12 transporter protein, mRNA or genomic DNA is detected in the biological sample; Comparing the presence of ABCB12 transporter protein, mRNA or genomic DNA in the sample with the presence of ABCB12 transporter protein, mRNA or genomic DNA in the test sample.

The invention further includes a kit for detecting the presence of ABCB12 transporter in a biological sample. For example, the kit includes a labeled compound or agent capable of detecting ABCB12 transporter protein or mRNA in a biological sample; ABCB in the sample
Means for determining the amount of 12 transporter; and means for comparing the amount of ABCB12 transporter in the sample to a standard may be included. The compound or agent may be packaged in a suitable container. Further, the kit may include instructions for using the kit to detect ABCB12 transporter protein or nucleic acid.

2. Prognostic Assays The diagnostic methods described herein should be used to further identify subjects with or at risk of developing a disease or disorder associated with abnormal or unwanted ABCB12 transporter expression or activity. You can As used herein, the term “aberrant” includes ABCB12 transporter expression or activity that deviates from wild-type ABCB12 transporter expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, or expression or activity that does not follow the wild-type developmental or subcellular expression pattern. For example, aberrant ABCB12 transporter expression or activity may result in mutations in the ABCB12 transporter gene that result in under- or overexpression of the ABCB12 transporter gene, or in cases where such mutations result in non-abnormality. A functional ABCB12 transporter protein is generated, or a protein that does not interact with, for example, an ABCB12 transporter ligand, or
It is intended to include the case where a protein is produced that functions in a manner that does not occur in the wild type, such as one that interacts with a non-ABCB12 transporter ligand. As used herein, the term “unwanted” includes unwanted phenomena involved in biological reactions. For example, the term unwanted includes undesired ABCB12 transporter expression or activity in a subject.

The assays described herein, such as the diagnostic assays described above or the following assays, identify subjects with or at risk of developing disorders associated with ABCB12 transporter protein activity or dysregulation of nucleic acid expression. Can be used to identify. Alternatively, the prognostic assay can be utilized to identify subjects who have or are at risk of developing disorders associated with the misregulation of ABCB12 transporter protein activity or nucleic acid expression. Thus, the present invention provides a method of identifying a disease or disorder associated with aberrant or unwanted ABCB12 transporter expression or activity, wherein a test sample is obtained from a subject, and ABCB12 Detecting a transporter protein or nucleic acid (eg, mRNA or genomic DNA), where the presence of ABCB12 transporter protein or nucleic acid has a disease or disorder associated with aberrant or unwanted ABCB12 transporter expression or activity Or, it can be used as a criterion for a subject at risk of developing the disease. As used herein, a "test sample" refers to a biological sample collected from a subject of interest. For example, the test sample may be a biological fluid (eg serum), cell sample, or tissue.

In addition, the prognostic assays described herein can be used to treat diseases or disorders associated with abnormal or unwanted ABCB12 transporter expression or activity, such as cancer when cancer cells develop multidrug resistance. To achieve this, one subject may have an agent (e.g., agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule,
Or other drug candidates) can be administered. in this way,
The present invention provides a method for investigating whether a disorder of a subject associated with aberrant or unwanted ABCB12 transporter expression or activity can be effectively treated with an agent. A test sample is obtained and the expression or activity of the ABCB12 transporter protein or nucleic acid is detected (e.g., at this time, the abundance of the ABCB12 transporter protein or nucleic acid expression or activity is abnormal or unwanted ABCB12 transporter expression or activity). This will be a guide for the subject who can administer the agent to treat the related disorder.)

Furthermore, the method of the present invention can be used to detect genetic alterations in the ABCB12 transporter gene,
Thus, it can be determined whether a subject with a genetic alteration is at risk of developing a disorder characterized by misregulation of ABCB12 transporter protein activity or nucleic acid expression. In a preferred embodiment, the method comprises at least one of a change affecting the integrity of the gene encoding the ABCB12-protein, or a misexpression of the ABCB12 transporter gene, in a cell sample taken from the subject. Detecting the presence or absence of a gene change characterized by. For example, one such genetic change is
) Deletion of one or more nucleotides from the ABCB12 transporter gene; 2) addition of one or more nucleotides to the ABCB12 transporter gene; 3) substitution of one or more nucleotides of the ABCB12 transporter gene, 4 ) Chromosomal rearrangement of ABCB12 transporter gene; 5) Abnormal modification of certain ABCB12 transporter gene, such as change in level of messenger RNA transcript of certain ABCB12 transporter gene, 6) Abnormal modification of methylation pattern of genomic DNA , 7) Presence of non-wild type splicing pattern of messenger RNA transcript of ABCB12 transporter gene, 8) Non-wild type level of ABCB12-protein, 9) ABC
It can be detected by confirming the presence of at least one of allelic loss of the B12 transporter gene and 10) inappropriate post-translational modification of ABCB12-protein. As described herein, there are many assays known in the art that can be used to detect alterations in the ABCB12 transporter gene. A suitable biological sample is a tissue or serum sample isolated from a subject by conventional means.

In some embodiments, the detection of changes includes polymerase chain reaction (PCR), eg, anchor PCR or RACE PCR (eg, US Pat. Nos. 4,683,195 and 4,683,
202) or, alternatively, ligation chain reaction (LCR) (
For example, Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al.
(1994) Proc. Natl. Acad. Sci. USA 91: 360-364), the latter being particularly useful for detecting point mutations in the ABCB12-gene. (Abravaya et al. (1995) Nucleic
Acids Res. 23: 675-682). The method comprises the steps of taking a sample of cells from a subject, isolating a nucleic acid (eg genomic nucleic acid, mRNA or both) from cells in the sample, the nucleic acid sample (if present) Under the condition that hybridization and amplification of the ABCB12 gene occur, the step of contacting with one or more primers that specifically hybridize to the ABCB12 transporter gene and detecting the presence or absence of the amplification product, or Detecting the size and comparing its length to a control sample. It is anticipated that PCR and / or LCR would be preferable to utilize as the preliminary amplification step in combination with any of the techniques used to detect the mutations described herein.

An alternative amplification method is the self-sustained sequence replication method (Guatelli, JC et al., (1990) Proc. Natl. Acad. Sci. USA 87: 1874-.
1878), transcription amplification system (Kwoh, DY et al., (1989) Proc. Natl. Acad. Sci. US
A 86: 1173-1177), Q-beta replicase (Lizardi, PM et al. (1988) Bio
-Technology 6: 1197), or some other nucleic acid amplification method, after which the amplified molecule is detected using techniques known to those of skill in the art. These detection schemes are
It is particularly useful for detecting such nucleic acids when the number of nucleic acid molecules present is low.

In one alternative embodiment, mutations in the ABCB12 transporter gene of sample cells can be identified by alterations in the restriction enzyme cleavage pattern. For example, sample DNA and control DNA are isolated (selectively (amplified), digested with one or more restriction endonucleases, and fragment lengths are examined by gel electrophoresis and compared. The difference in fragment length with DNA is indicative of some mutation in the sample DNA. Occurrence or loss can be scored for the presence of a particular mutation.

In another example, a gene mutation in the ABCB12 transporter hybridizes sample and control nucleic acids, such as DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes. Can be specified by (Cronin, M. T.
.et al. (1996) Human Mutation 7: 244-255; Kozal, MJ et al. (1996) Nat
ure Medicine 2: 753-759). For example, the gene mutation in the ABCB12 transporter is
As described by onin MT et al., they can be distinguished by a two-dimensional array containing photogenerated DNA probes. Briefly, the probes of the first hybridization array were used to scan the long range of DNA contained in the samples and controls,
By making a linear array of probes with overlapping sequences, it is possible to identify base changes that occur between sequences. By this step, point mutations can be identified. Following this step, a second hybridization array can be used to characterize a particular mutation by utilizing a smaller, specialized probe array that is complementary to any detected variants or mutations. . Each mutation array is
It consists of a set of parallel probes, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another example, any of a variety of sequencing reactions known in the art can be used to directly sequence the ABCB12 transporter gene, and the sequence of the sample ABCB12 transporter is compared to the corresponding wild type. Mutations can be detected by comparison to the (control) sequence of.
An example of a sequencing reaction is Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. U.
SA 74: 560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74: 5463.) Further, mass spectrometry (for example, PCT International Publication No. WO94 / 1).
6101; Cohen et al. (1996) Adv. Chromatogr. 36: 127-162; and Griffin et al.
(1993) Appl. Biochem. Biotechnol. 38: 147-159), any of a variety of automated sequencing methods may be used to perform diagnostic assays.
Conceivable.

Another method of detecting mutations in the ABCB12 transporter gene utilizes protection from cleaving agents to detect miss-paired bases in RNA / RNA or RNA / DNA heteroduplexes. Method(
Myers et al. (1985) Science 230: 1242). Generally, in the art using "mispair cleavage", RNA or DNA containing the wild type ABCB12 transporter sequence was first obtained from a tissue sample. , To form a heteroduplex formed by hybridizing to RNA or DNA that may be mutated. An agent that cleaves a single-stranded region of this duplex, such as might be present due to a base pair mispairing between the control and sample strands, resulting in this double strand. Processed duplex. For example, RNA / DNA duplexes can be treated with RNase, and DN
When the A / DNA hybrid is treated with S1 nuclease, the mismatched region can be enzymatically digested. In another example, either DNA / DNA or RNA / DNA duplexes are treated with hydroxyamine or osmium tetraacetic acid and piperidine to digest the mismatched regions. The mutation site can be determined by separating the substances produced after digestion of the mismatched region by size on a denaturing polyacrylamide gel. For example, Cotton et a. (1988) Proc. Natl. Acad. Sci USA 85: 4397; Saleeba et
See al. (1992) Methods Enzymol. 217: 286-295. In a preferred embodiment, control DNA or RNA may be labeled for detection.

In yet another example, the mispaired cleavage reaction described above was performed on AB samples obtained from cell samples.
To detect and map point mutations in the CB12 transporter cDNA, one or more proteins that recognize mispaired base pairs in double-stranded DNA in a defined system (so-called "DNA mispairing"). Utilizing repair “enzymes.” For example, the mutY enzyme of E. coli cleaves G / A mispairing at A, and thymidine DNA glycosylase from HeLa cells cleaves G / T mispairing at T ( Hsu et a. (1994) Carcinogenesis 15: 1657-1662) .In one embodiment, a probe based on an ABCB12 transporter sequence, such as the wild-type ABCB12 transporter sequence, is hybridized to cDNA or other DNA products taken from test cells. Soybeans: This duplex is treated with a DNA mispair repair enzyme and if a cleavage product occurs, the product can be detected by electrophoresis protocols, etc. See, eg, US Pat. No. 5,459,039.

In another example, alterations in electrophoretic mobility will be used to identify mutations in the ABCB12 transporter gene. For example, single-stranded conformational polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et a. (198
9) Proc Natl. Acad. Sci USA: 86: 2766, also Cotton (1993) Mutat. Res. 285:
125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9: 73-79). Single stranded DNA fragments of the sample and control ABCB12 transporter nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies by sequence, so that even single base changes can be detected based on changes in electrophoretic mobility. The DNA fragment may be labeled or detected using a labeled probe.
Utilizing RNA (rather than DNA) whose secondary structure is more sensitive to sequence changes may increase the sensitivity of this assay. In one preferred embodiment, the method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al. , (1991) Trends Genet 7: 5).

In yet another example, the movement of mutant or wild-type fragments in polyacrylamide gels containing denaturant gradients is assayed using denaturant gradient gel electrophoresis (DGGE). Myers et al. (1985) Nature 313: 495). When DGGE is used as an analysis method, for example, GC consisting of GC-rich DNA that melts at a high temperature of about 40 base pairs
DNA will be modified so that it will not be completely denatured, such as by adding a clamp by PCR. In a further example, a temperature gradient is used instead of a denaturant gradient to discriminate between mobility differences between control and sample DNA (Rosenbaum and Reissner (1987).
Biophys Chem 265: 12753).

Other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization methods, selective amplification methods, or selective primer extension methods.
For example, create an oligonucleotide primer with a central known mutation and hybridize it to the target DNA under conditions that allow it to hybridize only when there is a perfect match (Saiki et al. (1986). Nature 324: 1
63). Such allele-specific oligonucleotides are labeled with PCR-amplified target D
When hybridized to NA or when this oligonucleotide is attached to the hybridizing membrane and hybridizes to the labeled target DNA, it hybridizes to a number of different mutations.

Alternatively, allele-specific amplification techniques that rely on selective PCR amplification methods may be used in combination with the present invention. Oligonucleotides used as primers for specific amplification have the mutation of interest at the center of the molecule (as amplification depends on differential hybridization) (Gibbs et al. (1992) Mol. Cell Probes 6: 1)
Or, it may be at the most 3'end of one primer under suitable conditions if the polymerase extension reaction can be hindered or reduced due to mispairing (Prossner (1993) Tibtech 11: 238). In addition, it may be preferable to introduce new restriction sites into the mutated region for cleavage-based detection (Gasparini et al. (19
92) Mol. Cell Probes 6: 1). In some examples, it is expected that amplification may be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sc).
i USA 88: 189). In such a case, the ligation reaction occurs only when there is a perfect match at the 3'end of the 5'sequence. Therefore, by checking the presence or absence of amplification, it is possible to determine whether there is a known mutation at a specific site. Can be detected.

The methods described herein may be carried out, for example, utilizing a pre-packaged diagnostic kit comprising at least one probe nucleic acid or antibody reagent described herein, which kit may be, for example, a clinical kit. It would be convenient to use in situ to diagnose patients who show symptoms or a family history of a disease or disorder involving the ABCB12 transporter gene.

In addition, any cell type or tissue that expresses the ABCB12 transporter may be used in the prognostic assays described herein.

3. Observation of effects during clinical trials The effect of an agent (eg, drug) on the expression or activity of one ABCB12 transporter protein is not only observed in the screening for ABCB12 transporter drugs,
It can also be observed in clinical trials. For example, in the screening assay described here, the effect of an agent determined to enhance ABCB12 transporter gene expression or protein level or upregulate ABCB12 transporter activity was evaluated by determining the effect of ABCB12 transporter gene expression or protein level. Can be observed in clinical trials in subjects where a decrease in or down-regulation of ABCB12 transporter activity is seen. Conversely, the effect of the agent determined by the screening assay to reduce ABCB12 transporter gene expression or protein level, or downregulate ABCB12 transporter activity is increased ABCB12 transporter gene expression or protein level, or It can also be observed in clinical trials in subjects where upregulation of ABCB12 transporter activity is seen. In such clinical studies, the expression or activity of the ABCB12 transporter gene, and preferably other genes shown to be involved in such diseases as ABCB12-related, is determined by the "readout" or marker of the phenotype of a particular cell. Can be used as

For example, without limitation, treatment with an agent (eg, compound, drug or small molecule) that modulates ABCB12 transporter activity (identified by the screening assay described herein) modulates intracellularly. Genes containing ABCB12 can be identified. Thus, to study the effects of agents on ABCB12-related diseases, such as in clinical trials, cells are isolated, RNA is made and analyzed to determine whether the ABCB12 transporter gene or ABCB12-related disease is associated with it. The expression levels of other genes that are present can be examined respectively. The level of gene expression (eg, pattern of gene expression) can be quantified by Northern blot analysis or RT-PCR as described herein, or, alternatively, the amount of protein produced can be determined here. It can be obtained by measuring by one of the described methods or by measuring the activity level of ABCB12 transporter or other gene. In this method, the gene expression pattern can serve as a marker that is an indicator of the physiological response of cells to the agent. Therefore, this response state may be examined at various times before and during treatment of the individual with the agent.

In certain preferred embodiments, the invention is directed to certain agents (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecules, or others identified in the screening assays described herein). Of drug candidates) and the effect of observing the effect of treating the subject with the method (i.
) Collecting a pre-dose sample from the subject prior to administration of the agent;
i) detecting the expression level of ABCB12 transporter protein, mRNA or genomic DNA in said pre-dose sample; (iii) collecting one or more post-dose samples from the subject; (iv) ABCB12 Transporter protein, mRNA or genomic DNA
Detecting the expression level or activity level of said protein in said post-administration sample, and ABCB12 transporter protein, mRNA or genomic DN
Comparing the expression or activity level of A, and (vi) correspondingly altering the administration of the agent to the subject. For example, to increase the expression or activity of the ABCB12 transporter to a level higher than that detected, ie to increase the effect of the agent, it may be preferable to increase the administration of the agent. Conversely, in order to reduce the expression or activity of the ABCB12 transporter to a lower level than detected, ie reduce the effect of the agent, it may be preferable to reduce the administration of the agent.
Based on these examples, ABCB12 transporter expression or activity may be available as an indicator of the effect of an agent, even in the absence of an observable phenotypic response.

D. Methods of Treatment The present invention provides a prophylactic method of treating a subject at risk of (ie, susceptible to) a disease associated with aberrant or unwanted ABCB12 transporter expression or activity, or having a disease. It provides both therapeutic methods. Such treatments, both prophylactic and therapeutic, may be specifically adapted or improved based on the findings obtained in the field of pharmacogenomics. As used herein, "pharmacogenomics" refers to the application of genomic technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and the market. More specifically,
The term refers to how a gene in a patient determines his or her response to a drug (eg, to study a patient's "drug response phenotype" or "drug response genotype". In another aspect, the invention also provides for the prophylactic or therapeutic treatment of an individual with either an ABCB12 transporter molecule or an ABCB12 transporter modulator of the invention based on the individual's drug response genotype. Pharmacogenomics allow clinicians or physicians to target prophylactic or therapeutic treatments to the patients who would most benefit from the treatment and also to assess toxicity. Treatment of patients who may experience side effects associated with certain drugs can be avoided.

1. Prophylaxis In one aspect, the invention provides for abnormal or unwanted ABCB12 transporter by administering to the subject an ABCB12 transporter or an agent that modulates ABCB12 transporter expression or at least one ABCB12 transporter activity. Provided are methods of preventing in a subject a disease or condition associated with transporter expression or activity. Subjects at risk of disease due to or contributing to aberrant or unwanted ABCB12 transporter expression or activity may be identified, eg, by any combination of the diagnostic or prognostic assays described herein. Prophylactic agent administration may be performed prior to the onset of symptoms characterized by ABCB12 transporter abnormalities so as to prevent or slow the progression of the disease or disorder. ABCB12 Transporter Depending on the type of abnormality, for example, ABCB12, AB
A CB12 transporter agonist or ABCB12 transporter antagonist agent may be used to treat a subject. Suitable agents can be determined based on the screening assays described herein.

[0209] 2. Therapeutic Methods Another aspect of the invention pertains to methods of modulating ABCB12 transporter expression or activity for therapeutic purposes. Therefore, in one embodiment, the modulation method of the present invention comprises:
Contacting the cell with the ABCB12 transporter or an agent that modulates one or more ABCB12 transporter protein activity associated with the cell. Agents that modulate ABCB12 transporter protein activity include, for example, nucleic acids or proteins, naturally occurring target molecules of ABCB12 transporter proteins (eg, ABCB12 transporter substrates), ABCB12 transporter antibodies,
The agents described herein may be ABCB12 transporter agonists or antagonists, peptidomimetics of ABCB12 transporter agonists or antagonists, or other small molecules. In one embodiment, the agent stimulates one or more ABCB12 transporter activities. Examples of such stimulants include active
There are ABCB12 transporter proteins and nucleic acid molecules that have been introduced into cells that encode the ABCB12 transporter. In another embodiment, the agent inhibits one or more ABCB12 transporter activities. Examples of such inhibitors include Antisense AB
There are CB12 transporter nucleic acid molecules, anti-ABCB12 transporter antibodies, and ABCB12 transporter inhibitors. These modulation methods may be performed in vitro (eg, by culturing cells with the agent of interest) or in vitro (eg, by administering the agent to a subject). Accordingly, the invention provides methods for treating an individual suffering from a disease or disorder characterized by aberrant or unwanted expression or activity of an ABCB12 transporter protein or nucleic acid molecule. In one example, the method involves adding an agent that modulates (eg, up-regulates or down-regulates) ABCB12 transporter expression or activity (eg, an agent identified in the screening assays described herein). One or a combination, including the steps of administering. In another embodiment, the method comprises administering an ABCB12 transporter protein or nucleic acid molecule as a therapeutic method to compensate for reduced, aberrant, or unwanted ABCB12 transporter expression or activity.

If the ABCB12 transporter is abnormally down-regulated and / or if beneficial effects of increasing the activity of ABCB12 transporter are expected to be beneficial, ABCB12 transporter activity may be stimulated. desirable. For example, it may be desirable to stimulate ABCB12 transporter activity when the ABCB12 transporter is down-regulated and / or where beneficial effects of increasing the activity of ABCB12 transporter are expected. Similarly, ABCB12 transporter activity may be inhibited if the ABCB12 transporter is aberrantly upregulated and / or if a beneficial effect in reducing ABCB12 transporter activity is expected. desirable.

[0211] In one embodiment, agents found to inhibit ABCB12 transporter activity are used in combination with another treatment to allow treatment targeting across the blood brain barrier.

3. Pharmacogenomics ABCB12 transporter molecule or agent of the present invention, or having a stimulatory or inhibitory effect on ABCB12 transporter activity (eg, expression of ABCB12 transporter gene) identified by the screening assay described herein. Abnormal or unwanted AB
It can be administered to an individual to treat (prophylactically or therapeutically) an ABCB12-related disorder associated with CB12 transporter activity. From such a treatment relationship, pharmacogenomics (ie, a study of the relationship between an individual's genotype and that individual's response to foreign compounds or agents) may be considered. Differences in metabolism of therapeutic agents can alter the relationship between dose and blood concentration of the pharmacologically effective agent, resulting in severe toxicity or failure of treatment. Therefore, the doctor or clinician
In determining whether to administer an ABCB12 transporter molecule or an ABCB12 transporter modulator, and in adjusting the dosage and / or therapeutic regimen for treatment with the ABCB12 transporter molecule or ABCB12 transporter modulator. Consider application of findings from relevant pharmacogenomic studies.

Pharmacogenomics studies clinically significant genetic variability in the response to drugs due to altered drug properties and abnormal effects in patients. For example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol
.23 (10-11): 983-985 and Linder, MW et al. (1997) Clin. Chem. 43 (2):
See 254-266. Generally, two types of pharmacogenetic states can be distinguished. A genetic condition that is transmitted as a single factor that alters the way a drug acts on the body (alters the action of the drug), or simply alters the way the body acts on the drug (alters the metabolism of the drug). A genetic condition, which is transmitted as a factor. These pharmacogenetic conditions can occur either as rare genetic defects or as naturally occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common hereditary enzymatic disease that can occur after ingestion of oxidants (eg antimalarials, sulfonamides, analgesics, nitrofuran, etc.) , Hemolysis occurs as a major clinical complication after eating broad beans.

One of the pharmacogenomics methods for identifying genes that predict drug response, known as "genome-wide association," is based primarily on known gene-related markers (eg, each having two variants, 60,000 to 10 on the human genome
It relies on a high resolution map of the human genome consisting of "double allele" gene marker maps, etc., consisting of 0,000 polymorphic or variable sites. Such high-resolution gene maps were compared to a map of the genome of each patient in a statistically significant number of Phase II / III drug trials to identify markers associated with a particular observed drug response or side effect. May be identified. Alternatively, such high resolution maps can be generated from combinations of millions of known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, "SNP" is the normal change that occurs at a single nucleotide base in a range of DNA. For example, SNPs are considered to occur once every 1000 bases of DNA. SNPs may be involved in the disease process, but most may not be involved in the disease. Looking at genetic maps based on the frequency of such SNPs, populations can be classified into several gene categories based on the particular pattern of SNPs in their individual genomes. In this way, treatment regimens can be tailored to a population of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals. Put in.

Alternatively, a method termed the “candidate gene approach”, can be used to identify genes that predict drug response. In this way, if the gene encoding a drug target is known (eg, the ABCB12 transporter protein of the invention), all variants normally found in that gene can be very easily identified in the population. Yes, it can be determined whether having one version of another gene for another is associated with a particular drug response.

In an exemplary embodiment, the activity of drug metabolizing enzymes is a major factor in determining both the strength and duration of drug action. Drug-metabolizing enzymes (eg N-acetyltransferase 2 (
The discovery of gene polymorphisms in NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) does not produce the expected drug effect after ingesting standard and safe doses of drug or exaggerated drug response. It was explained that some patients showed severe toxicity and other patients had severe toxicity. These polymorphisms are associated with high metabolic group (EM) and low metabolic group (PM) in the population.
) Appears in two phenotypes. The frequency of PM varies between individual populations. For example, the gene encoding CYP2D6 is highly polymorphic and several mutations have been found in PM, all of which lack functional CYP2D6. In the low metabolism group of CYP2D6 and CYP2C19, exaggerated drug response and side effects occur very frequently even when the standard dose is administered. PM shows no therapeutic response when metabolites are the effective therapeutic component, as demonstrated by the analgesic effect of codeine mediated by the metabolite morphine formed in CYP2D6. Extremely opposite are the so-called ultra-rapid metabolizers, which do not respond at standard doses. Recently, the molecular basis of ultrafast metabolism has been found to be due to amplification of the CYP2D6 gene.

Alternatively, a method termed “gene expression profiling” can be utilized to identify genes that predict drug response. For example, gene expression in an animal to which a drug (for example, ABCB12 transporter molecule or ABCB12 transporter modulator of the present invention) is administered can be used as an index indicating whether or not the gene pathway associated with toxicity has been switched on.

Information obtained from more than one of the above pharmacogenomics methods may be used to determine the appropriate dosage and treatment regimen for prophylactic or therapeutic treatment of an individual. This finding can be used to select dosages or agents to treat a subject with an ABCB12 transporter molecule or ABCB12 transporter modulator, such as a modulator identified in one of the exemplary screening assays described herein. It is possible to avoid an unfavorable reaction or a failure of treatment in the case of carrying out, and to enhance the treatment efficiency or prevention efficiency.

The following examples further exemplify the present invention, but the following examples should not be construed as limiting.

[0220]

【Example】

Example 1 Isolation and Cloning of Human ABCB12 Transporter cDNA This example describes the isolation and cloning of the gene encoding the novel human ABCB12 transporter.

The human ABCB12 transporter cDNA was analyzed by polymerase chain reaction (PCR) and rat nucleotide sequence (GenBank Accession No. AJ003004, Hirsch-Ernst et al., (1998) Biochem.
Biophy Res Chem 249: 151-155) and two partial human nucleotide sequences (GenBank Accession Nos. AF070598 and U66673, Yu et al., (1997) Genome Res 7: 353-3.
58; Andersson et al., (1996) Anal Biochem 5: 107-113; Allikmets et al., (
1996) Hum Mol Genet 5: 1649-1655) was used for isolation.

First, in order to prepare a double-stranded (ds) cDNA library, 15 mg of normal human full-length RNA (commercially available from Invitrogen, order number D6030-01) was synthesized with first and second strands. Phenol-chloroform extraction, alcohol precipitation, Clontech MARA
The THON cDNA amplification kit (Clontech, order number K1802-1) was used according to the manufacturer's instructions and ligated to a double stranded cDNA adapter.

Next, the polymerase chain reaction (PCR) was performed using 2 ml of ds cDNA synthesized as described above.
Primers P1, P2, and P3 (as described below) and Clontech Advantage
Using the reagents provided by the cDNA PCR kit (Clontech, order number K1905-1), the final volume was 20 ml according to the kit. The parameters of the cycle are
Denaturation at 94 ° C for 1 minute, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C (P1 and P2) or 70 ° C (P3 and adapter-specific primer) for 30 seconds, extension reaction at 70 ° C for 2 minutes,
This was done for 30 cycles and the final extension reaction step was 70 ° C. for 5 minutes.

[0224]

[Table 1]

A second PCR reaction was performed using the following method to isolate the 5'untranslated region (UTR) of the human ABCB12 transporter cDNA. Briefly, the PCR reaction was performed using 5 ml (master plate) or 1 ml (subplate) of human brain cDNA library (Origene Technologies, order number LAB-1001) and appropriate reaction conditions (10 mM Tris-HCl pH8.3).
, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.1 mM primers, 2% DMSO, 1.25 units of AmpliTac DNA Polymerase (Perkin Elmer Co.) to a final concentration of 25 ml. Cycling parameters were: denaturation at 94 ° C for 2 minutes, denaturation at 94 ° C for 30 seconds, annealing at 65 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute, 30 cycles of the final extension reaction step. Was 72 ° C for 5 minutes.

Two cDNA fragments were isolated using the technique described above. The first cDNA fragment (hsA14.2) was derived from the human brain double-stranded (ds) cDNA library using primers P1 and P2.
Was obtained by the polymerase chain reaction (PCR) with 1964 base pairs (bp). The second cDNA fragment (hsA14.1) was obtained using PCR, an oligonucleotide specific for the human brain ds cDNA library and an adapter attached to the 5'and 3'ends of the primer P3 and the cDNA library, and the results The resulting fragment was 1725 bp. Each of the above cDNA fragments was subcloned and the nucleotide sequence was revealed using an automated DNA sequencing engine (LICOR). Fragment
The overlap between hsA14.1 and hsA14.2 was 967 bp. The nucleic acid sequence of the isolated fragment was predicted to encode the human ortholog of the mammalian ABC half-transporter UMAT, which is widely expressed in rat. The hsA14.1 and hsA14.2 fragments were found to encode the 3'end and 5'end of this gene, respectively.

The above fragments together, in addition to the 5'and 3'untranslated regions (UTRs), also contain a 2893 bp clone containing the full-length human ABCB12 transporter gene coding sequence (hsA14.
3) could be isolated. The nucleotide sequence encoding the human ABCB12 transporter protein is shown in Figure 1 and is set forth as SEQ ID NO: 1. The longest open reading frame of the human ABCB12 transporter begins at sequence GCCATGG, which corresponds to the eukaryotic consensus translation initiation motif, at nucleotide position 165 (ATG), and ends at the TGA stop signal at position 2694. The coding region of SEQ ID NO: 1 (open reading frame) is described as SEQ ID NO: 3. The putative polyadenylation signal (AATAAA) is in the 3'untranslated region at nucleotide position 2844 (Figure 1).
The open reading frame, beginning with the first ATG in frame, is predicted to encode a polypeptide with a molecular weight of 94 kDa and 843 amino acids. The amino acid sequence of this full-length polypeptide is shown in Figure 2 and is set forth in SEQ ID NO: 2.

Example 2 Characterization of a Novel Human ABCB12 Transporter Molecule In this example, the amino acid sequence of the human ABCB12 transporter polypeptide was compared to the amino acid sequences of known polypeptides to identify various motifs.

In particular, by comparing the nucleic acid sequence of an isolated clone (eg, the deduced amino acid sequence) with a public database (eg, GenBank), the isolated clone was found to be ubiquitously expressed in rat mammalian ABC. Half Transporter (UMAT) Polypeptide (Accession Number AJ003004, Hirsch-Ernst et al., 1998 Biochem. Biophys. Res. Comm. 249
: 151-155) and homology (85% at the amino acid level (BLAST for non-redundant SwissProt sequences
86% at nucleotide level (non-overlapping GenBank + EMBL + DDBJ +)
It was revealed that it encodes a novel human ABC transporter polypeptide (ABCB12) having a BLASTn program for PDB sequence). Therefore, the novel human ABC transporter molecule of the present invention (ABCB12) is also referred to herein as human UMAT polypeptide.

The human ABCB12 transporter polypeptides of the present invention belong to the ATP binding cassette (ABC) superfamily. The members of this family are structurally and functionally related for their ability to catalyze the translocation of biological membrane substrates. The deduced sequence of 843 amino acids consists of an N-terminal membrane anchor domain and a conserved single C-terminal nucleotide binding fold, which identifies the human ABCB12 polypeptide as an ABC half transporter (Figure 2 and See SEQ ID NO: 2). The identity of the novel human ABCB12 transporter protein was confirmed using bioinformatics software known in the art.
No more than 34% of the most recent human homologues were revealed (pairwise global cluster W alignment). The closest human homolog is ABC7
(BLASTp program for non-redundant SwissProt sequences). Applying pairwise global clustered W alignments revealed 34% identity in the amino acid sequences of ABCB12 and ABC7.

The ABCB12 transporter polypeptide was also analyzed for potential transmembrane segments. Bioinformatics tools (Predict Protein Server, Heidelberg, Germany, http://dodo.cpmc.columbia.edu/predictprotein
/ Reference), a region that is presumed to be a transmembrane region of the N-terminal sequence of this polypeptide from the plot of the hydrophobicity index and the two-dimensional fold model obtained using
The sites were identified, and 6 of them had a helical two-dimensional fold structure, suggesting that the human ABCB12 transporter is a membrane-anchored protein. The N-terminal portion of this protein (amino acids 1-550) does not preserve the characteristics of ABCB12,
Amino acids 1-550 show only 13% identity with ABC7, the closest human homologue to ABCB12. However, the C-terminal sequence of ABCB12 (amino acids 601-843) contains a large cluster of conserved residues that define the ATP binding domain (see, eg, Figures 2 and 3).

[0232]

[Table 2]

Another noteworthy feature of the human ABCB12 transporter was the presence of Walker A motifs, Walker B motifs, and ABC signature motifs suggesting ATPase / ATP binding function (see Figure 2; Patel et al. al., 1998, Trends Cell Biol.,
8: 65-71). Thus, the human ABCB12 transporter polypeptide exhibits typical features that are conserved among ABC transporters. The structural organization of the ABC transporter protein shows variations on common themes. One common theme is that this transporter exists with 12 hydrophobic transmembrane segments, either present as a single polypeptide chain or as an assembly of 1/2 or 1/4 molecules. It has a basic structure that it has two hydrophilic ATP binding sites.

The amino acid sequence of human ABCB12 thus defines the human ABCB12 transporter as a novel hemitransporter. Although not necessarily, the human ABCB12 transporter may function as part of the dimer transporter structure. To date, the few uncharacterized hemitransporters in mammalian cells have been found to be localized to the membrane of the intracellular compartment. Bioinformatics modeling tools known in the art suggest that the predicted protein N-terminal portion and the ATP-binding cassette are in the "outer" conformation. Therefore, the human ABCB12 transporter is likely to be associated with intracellular membrane structure.

Example 3: Tissue distribution of human ABCB12 transporter mRNA This example describes the tissue distribution of human ABCB12 transporter mRNA, which can be determined by Northern blot hybridization and in situ hybridization.

Northern blot hybridizations are performed with various RNA samples under standard conditions and washed under stringent conditions, ie at 65 ° C. with 0.2XSSC. The DNA probe is radiolabeled with ³² P-dCTP using Prime-It kit (Stratagene, La Jolla, Calif.) According to the manufacturer's instructions. Human mRNA containing filters (Multi Tissue Northern I and Multi Tissue Northern II, Clontech, Palo Alto, Calif.) Were probed in ExpressHyb Hybridization Solution (Clontech) at high stringency according to the manufacturer's recommendations. To wash.

For in situ analysis, various tissues, eg, brains such as rat or mouse brain, are first frozen on dry ice. A circular section of 10 mm thick tissue was
Postfix with 4% formaldehyde in PC-treated 1X phosphate buffered saline for 10 minutes at room temperature, then wash twice with DEPC-treated 1X phosphate buffered saline and once with 0.1M triethanolamine-HCL (pH 8.0). . 0.25% acetic anhydride-0.1M triethanolamine-H
After incubating in Cl for 10 minutes, the sections were DEPC 2X SSC (1X SSC was 0.15M).
Wash with NaCl and 0.015 M sodium citrate). The tissue is then washed successively with ethanol, dehydrated, incubated in 100% chloroform for 5 minutes,
Wash in 100% ethanol for 1 minute, 95% ethanol for 1 minute and air dry.

Hybridization is performed with ³⁵ S radiolabeled (5 × 10 ⁷ cpm / ml) cRNA probe. Probes are 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% denatured salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1X Denhardt's solution, 50%
55 in the presence of a solution containing formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS) and 0.1% sodium thiosulfate.
Incubate at 18 ° C for 18 hours.

After hybridization, slides are washed with 2X SSC. Then the section 37
At 10 ° C., sequentially in TNE (solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl and 1 mM EDTA) for 10 minutes, in TNE containing 10 mg RNase A per ml TNE for 30 minutes, and finally TNE. Incubate for 10 minutes. The slides are then washed in 2X SSC at room temperature, 2X SSC at 50 ° C for 1 hour, 0.2X SSC at 55 ° C for 1 hour, 0.2XS
Wash with SC at 60 ° C for 1 hour. The sections were then dehydrated by rapid passage through continuous ethanol-0.3M sodium acetate concentrate, then air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours, then dipped in NB-2 photographic emulsion. Exposure at 4 ° C for 7 days, then develop and counterstain. Electrical Northern analyzes suggest that the ABCB12 transporter mRNA is highly neuron-specific.

Example 4: Expression of Recombinant Human ABCB12 Transporter Polypeptide in Bacterial Cells In this example, the human ABCB12 transporter was engineered into recombinant glutathione-S-transferase (G
ST) Expressed in E. coli as a fusion polypeptide and the fusion polypeptide isolated and characterized. In particular, the human ABCB12 transporter is fused to GST and this fusion polypeptide is expressed in E. coli, eg the PEB199 strain. The human ABCB12 transporter polypeptide is predicted to be about 94 kDa in molecular weight and the GST is predicted to be 26 kDa, so the fusion polypeptide is predicted to be about 120 kDa. IPTG induces expression of GST-ABCB12 transporter fusion protein in PEB199. The recombinant fusion polypeptide is purified from the derived crude bacterial lysate of strain PEB199 on affinity chromatography glutathione beads. Analysis of the polypeptide purified from the bacterial lysate by polyacrylamide gel electrophoresis gives the molecular weight of the fusion polypeptide.
If desired, the polypeptides may be prepared with membranes such as micelles or membrane vesicles using techniques known in the art.

Example 5 Expression of Recombinant Human ABCB12 Transporter Polypeptide in Mammalian Cells To express ABCB12 transporter cells in mammalian cells such as COS cells, Invitrogen (San Diego, Calif.) PcDNA / Amp vector was used. To use.
This vector is SV40 origin of replication, ampicillin resistance gene, E. coli origin of replication,
It contains the CMV promoter followed by a polylinker region, and the SV40 intron and polyadenylation site. A DNA fragment encoding the entire ABCB12 transporter protein and an HA tag fused in frame to the 3'end of this fragment (Wilson et al. (19
84) Cell 37: 767) or FLAG tag is cloned into the polylinker region of the vector, thereby placing expression of the recombinant protein under the control of the CMV promoter.

For the construction of plasmids, the ABCB12 transporter DNA sequence (eg SEQ ID NO: 3) is amplified by PCR using two primers. The 5'primer contains a restriction site of interest, which precedes the ABCB12 transporter coding sequence of approximately 20 nucleotides starting from the start codon, and the 3'terminal sequence is a sequence complementary to other restriction sites of interest, the translation termination. Codon,
Contains the HA or FLAG tag and the last 20 nucleotides of the ABCB12 transporter coding sequence. The PCR amplified fragment and the pCDNA / Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). It is preferred that the two restriction sites selected are different so that the ABCB12 transporter gene is inserted in the correct orientation. The ligation mixture was transformed into E. coli cells (HB101, DH5a, SURE from Stratagene Cloning Systems, La Jolla, CA can be used) and the transformed culture was plated onto ampicillin medium plates to obtain resistant colonies. Select. Plasmid DNA is isolated from transformants and the presence of the correct fragment is verified by restriction analysis.

Then, using the calcium phosphate or calcium chloride coprecipitation method, the DEAE dextran mediated transfection method, the lipofection method, or the electroporation method,
Eukaryotic cells such as COS cells are transfected with the ABCB12 transporter pcDNA / Amp plasmid DNA. Other suitable methods for transfecting host cells are Sambro
ok, J., Fritsh, EF, and Maniatis, T. Molecualr Cloning: A Laboratosy M
anual.2nd, ed., Cold Spring Harbor laboratory, Cold Spring Harbor Labor
atory Press, Cold Spring Harbor, NY, 1989. Expression of the ABCB12 transporter polypeptide was carried out using a radiolabeled (
³⁵ S-methionine or ³⁵ S-cysteine from NEN, Boston, Mass., And immunoprecipitation (Harlow, E. and Lane, D. Antibodies)
: A Laboratory Manual, Cold Spring Harbor Laboratoy Press, Cold Spring H
Arbor, NY, 1988). Briefly, cells are labeled with ³⁵ S-methionine (or ³⁵ S-cysteine) for 8 hours. The culture medium is then collected and the cells are washed with detergent (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM).
Dissolve in Tris, pH 7.5). Both cell lysates and culture medium are precipitated with HA-specific monoclonal antibodies. Then, the precipitated polypeptide is analyzed by SDS-PAGE.

In addition, the DNA containing the ABCB12 transporter coding sequence was cloned into pCDNA using appropriate restriction sites.
Clone directly into the polylinker of the / Amp vector. The resulting plasmid is transduced into COS cells by the method described above, and the expression of ABCB12 transporter polypeptide is detected by immunoprecipitation using a radiolabel and ABCB12 transporter-specific monoclonal antibody.

Example 6 In Vitro and In Vitro Screening Assays for Modulators of the Human ABCB12 Transporter This example describes in vitro and in vitro screening assays for modulators of the human ABCB12 transporter molecule.

Briefly, the ABCB12 transporter is expressed in mammalian cells as described above, setting the baseline for cellular transport of reference molecules such as b-amyloid or other small molecules. The cells are then incubated with the test compound and changes in cell traffic of the reference molecule are measured using standard techniques. A test compound that alters the transport of a detectable reference molecule is then identified as a candidate compound that alters ABCB12 transporter activity. The lead compound may then be tested, for example, on eukaryotic cells, such as cells of neural origin, or on brain tissue taken from test animals. Methods for performing such assays are known in the art. Preferably, lead test compounds that inhibit ABCB12-mediated transport of b-amyloid and the like are further tested in vitro. Mouse models of various neurological disorders such as amyloid disease are also known in the art (for example, Hardy et al., (1998) Science 282: 1075-1079; Goate, et al., (1
991) Nature 349: 704; Games, et al., (1995) Nature 373: 523; and Suzuki,
et al., (1994) Science 264: 1336).

Alternatively, transgenic mouse models that overexpress the ABCB12 transporter polypeptide, such as the human ABCB12 transporter polypeptide or the corresponding mouse polypeptide, are generated using the techniques described herein. Then human ABCB12
To test for modulators of transporter polypeptides, the animal may be tested directly or the animal may be used as a cell source or tissue source, eg, a brain tissue source. Preferably, the animal is given a test compound to be tested directly and the resulting physiological phenomenon monitored, eg, the presence of b-amyloid in cerebrospinal fluid (CSF). In applications related to this in vitro assay, an animal may be bred with an animal that overexpresses an undesired polypeptide, eg, an amyloid polypeptide, and changes in polypeptide transport (such as in cerebrospinal fluid) compared to controls. Compare and measure. Ideally, these test animals are further monitored for the presence of modulators of the ABCB12 transporter.

In a modification of this assay, the activity of the ABCB12 transporter is measured with respect to the ability of a detectable reference molecule to cross the brain barrier. In yet another modification of this assay, the animal is tested for the spread of multidrug resistant cells or tissues and / or for its susceptibility to cytotoxic agents suitable for treating neoplasms.

Thus, these animals are an in vivo assay system that tests the ability of ABCB12 transporters and / or ABCB12 transporter modulators to prevent, treat or delay disease onset.

Equivalents One of ordinary skill in the art would be able to recognize or ascertain numerous equivalents to the particular embodiments of the invention described herein using only routine experimentation. Such equivalents are within the scope of the following claims.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the cDNA sequence of the human ABCB12 transporter and the predicted amino acid sequence. The nucleotide sequence corresponds to nucleic acids 1 to 2893 of SEQ ID NO: 1. SEQ ID NO: 3 shows the coding region without the 5'and 3'untranslated regions of the human ABCB12 transporter gene.

FIG. 2 is an amino acid sequence of the ABCB12 transporter molecule corresponding to amino acids 1 to 843 of SEQ ID NO: 2.

FIG. 3 shows the amino acid sequence of human ABCB12 transporter polypeptide and the alignment of the polypeptides found in various species.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12P 21/02 C 4H045 5/10 C12Q 1/02 C12P 21/02 1/68 A C12Q 1 / 02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33/566 C12N 15/00 ZNAA 33/566 5/00 A (31) Priority claim No. 09 / 641,353 (32) Priority date August 17, 2000 (August 17, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY) , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW , ML, MR, NE, SN, TD, TG) AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE , AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Wilson Ka Liona Canada V6R3B9 British Columbia Vancouver 16th Avenue 3461 W F Term (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA80 CA04 CA09 CA20 DA02 DA06 EA04 GA11 HA01 HA12 4B06 Q13 Q18 QAQ AQ QQ20 QQ42 QQ53 QR32 QR56 QR62 QR77 QS25 QS34 4B064 AG01 CA02 CA10 CA19 CC24 DA01 DA13 4B065 AA26X AA90X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4H045 AA10 AA11 AA30 BA10 CA40 DA75 EA20 FA73 FA50 FA72 FA73 FA50

Claims (25)

[Claims] 1. A nucleic acid molecule comprising a) the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence, and b) a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence. An isolated nucleic acid molecule selected from the group. 2. An isolated nucleic acid molecule encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. 3. An isolated nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. 4. A) a nucleic acid molecule comprising a nucleotide sequence which is at least 87% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3 or its complementary sequence, b) the nucleotide sequence of SEQ ID NO: 1 or 3. Or a nucleic acid molecule containing a fragment consisting of at least 150 nucleotides of a nucleic acid containing its complementary sequence, c) a polypeptide containing an amino acid sequence that is at least about 86% homologous to the amino acid sequence of SEQ ID NO: 2 D) a nucleic acid molecule encoding a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, said fragment comprising at least 50 amino acid sequences of SEQ ID NO: 2 An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising contiguous amino acid residues. 5. An isolated nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid molecule of any one of claims 1, 2, 3, or 4. 6. An isolated nucleic acid molecule comprising a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid molecule of any one of claims 1, 2, 3, or 4. 7. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1, 2, 3 or 4 and a nucleotide sequence encoding a heterologous polypeptide. 8. A vector comprising the nucleic acid molecule of any one of claims 1, 2, 3 or 4. 9. The vector according to claim 8, which is an expression vector. 10. A host cell transfected with the expression vector according to claim 9. 11. By culturing the host cell of claim 10 in a suitable culture medium,
A method of producing a polypeptide comprising the step of producing the polypeptide. 12. A fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, comprising at least 15 contiguous amino acids of SEQ ID NO: 2, b) SEQ ID NO. A naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein said polypeptide is encoded by a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule consisting of SEQ ID NO: 1 or 3. An allelic variant, c) a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 50% homologous to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or 3, d) SEQ ID An isolated polypeptide selected from the group consisting of polypeptides comprising an amino acid sequence that is at least 50% homologous to the amino acid sequence of NO: 2. 13. The isolated polypeptide of claim 12, which comprises the amino acid sequence of SEQ ID NO: 2. 14. The polypeptide of claim 12, further comprising a heterologous amino acid sequence. 15. An antibody that selectively binds to the polypeptide of claim 12. 16. A method of detecting the presence of the polypeptide of claim 12 in a sample, comprising: a) contacting the sample with a compound that selectively binds to the polypeptide; b. ) Determining whether the compound binds to the polypeptide in the sample, thereby detecting the presence of the polypeptide of claim 12 in the sample. 17. The method of claim 16, wherein the compound that binds to the polypeptide is an antibody. 18. A kit comprising a compound that selectively binds to the polypeptide of claim 12 and instructions for use. 19. A method for detecting the presence of a nucleic acid molecule according to any one of claims 1, 2, 3 or 4 in a sample, comprising: a) a nucleic acid probe that selectively hybridizes to the nucleic acid molecule. Or a step of contacting said sample with a primer, b) determining whether said nucleic acid probe or primer binds to a nucleic acid molecule in said sample, and thereby any of claims 1, 2, 3, or 4 Detecting the presence of any one nucleic acid molecule in said sample. 20. The method of claim 19, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe. 21. A kit comprising a compound that selectively binds to the nucleic acid molecule of any one of claims 1, 2, 3 or 4 and instructions for use. 22. A method of identifying a compound that binds to the polypeptide of claim 12, comprising: a) contacting the polypeptide or cells expressing the polypeptide with a test compound. And b) determining if the polypeptide binds to the test compound. 23. The binding of the test compound to the polypeptide comprises: a) detecting binding by directly detecting binding of the test compound to the polypeptide; b) detecting binding using a competitive binding assay. And c) is detected by a method selected from the group consisting of detection of binding using an assay for ABCB12 transporter activity. 24. A method of modulating the activity of a polypeptide of claim 12, wherein the compound that binds to the polypeptide in a concentration sufficient to modulate the activity of the polypeptide comprises the polypeptide. Or a method comprising contacting cells expressing said polypeptide. 25. a) contacting the polypeptide according to claim 12 with a test compound, and b) determining the effect of the test compound on the activity of the polypeptide, and thereby the activity of the polypeptide. Identifying compounds that modulate the activity of the polypeptide of claim 12, wherein the method of identifying compounds that modulate the activity of the polypeptide of claim 12.