JP2006504407A - New therapeutic targets for the treatment of vascular disorders, dyslipidemia and related diseases - Google Patents

Abstract

The present invention deals with compositions and methods for the modulation of HDL levels and / or the treatment of vascular disorders, dyslipidemias or related diseases in patients in need thereof. The invention also includes a method for the identification of therapeutic agents for the treatment of said diseases based on the identification of novel therapeutic targets here. The invention also includes diagnostic and pharmacogenomic compositions and methods using the therapeutic targets set forth herein.

Description

  This application claims priority to US Provisional Application No. 60/391878, filed Jun. 27, 2002, the disclosure of which is incorporated herein by reference in its entirety.

  The present invention relates to the discovery of gene 95, which is a novel therapeutic target for the diagnosis and treatment of diseases, in particular for the diagnosis and treatment of heart diseases such as dyslipidemia and hypoHDL diseases including hypoalphalipoproteinemia. The present invention also relates to the discovery of therapeutic factors useful for the prevention, treatment or otherwise improvement of the above-mentioned diseases and methods for screening and identifying the therapeutic factors.

  Epidemiological studies have shown that plasma high density lipoprotein cholesterol (HDL-C) levels are inversely related to the occurrence of vascular disorders, particularly heart disease (CVD) and coronary artery disease (CAD). HDL-C levels are largely graded and are independent cardiovascular risk factors. The increased protective effect of HDL-C persists until age 80. Low HDL-C is associated with an increased CAD risk, even at normal total plasma cholesterol levels (<5.2 mmol / l). In the face of other dyslipidemias or secondary factors, HDL-C levels are an important predictor of CAD. Low HDL cholesterol (called hypoalphaproteinemia in severe cases) is also implicated in cerebrovascular disease, coronary restenosis and peripheral vascular disorders.

  Pharmacological intervention at low HDL-C levels has not been well documented so far. Currently, there are no FDA approved therapeutics that modulate HDL levels in a direct and effective manner. Current research methods for the regulation of HDL include increased production of ApoA1, increased rate of reverse cholesterol transport (RCT) and decreased catabolism of HDL. For example, increased ApoA1 levels may be possible through small molecule up-regulation of transcription or by injection of ApoA1 protein. Alternatively, up-regulation of RCT may be possible by making ABCA1 agonists and increasing cholesterol efflux from peripheral tissues (see PCT Publication WO 00/55318, incorporated herein by reference). HDL catabolism is regulated by a number of enzymes including cholesteryl ester transfer protein (CETP), which may also be a suitable therapeutic target for HDL regulation.

  The identification of additional genes and corresponding proteins that serve to regulate HDL levels in humans will allow rational selection of therapeutic methods for the pursuit of drug development. The present invention is for the discovery of new targets for therapeutic intervention in the treatment of vascular disorders and dyslipidemia, and for screening and identifying therapeutic agents useful for reducing low HDL levels and associated symptom symptoms. Of the target.

(Brief summary of the invention)
The present invention provides the complete gene 95 cDNA nucleotide sequence (SEQ ID NO: 3) and gene 95 amino acid sequence (SEQ ID NO: 4) from human. The present invention further identifies the role and function of gene 95 as a major regulator of HDL cholesterol levels by identifying mutant forms of genes associated with human hereditary low HDL disease.

  In one aspect, the present invention provides a purified nucleic acid sequence of gene 95 and fragments thereof including purified and semi-purified forms, and also provides a recombinant cell line and virus or cell extract comprising a gene 95 nucleotide component. To do.

  In other embodiments, the present invention provides purified gene 95 protein and fragments thereof including purified and semi-purified forms, and cells or cell extracts having gene 95 biological activity and anti-gene 95 antibodies, such as Methods for producing polypeptides are provided.

  In one aspect, the present invention provides a method for determining whether a candidate compound modulates HDL levels and / or treats vascular disorders or dyslipidemia. This method comprises (a) measuring the biological activity of a gene 95 gene or protein, (b) contacting the source of this biological activity with a candidate compound under conditions that promote this biological activity, And (c) measuring the change in biological activity of the gene or protein as a result of the contact. Here, a change in biological activity is a candidate compound or candidate compound as a compound that regulates the biological activity and is useful for the regulation of HDL levels and / or for the treatment of vascular disorders, dyslipidemia or related diseases. Are identified. Other methods of analyzing gene 95 activity are also specifically contemplated by the present invention.

  In another aspect, the present invention provides a method for computer identification of compounds for modulating HDL levels and / or treating vascular disorders or dyslipidemia. This method (a) determines the active site of the selected gene 95 protein (eg, via X-ray crystallography or other techniques), and (b) interacts with the active site via computer modeling. Identification of the compound, thereby modulating HDL levels and / or identifying the compound or analog thereof as a compound useful for the treatment of vascular disorders, dyslipidemias or related diseases.

  In another aspect, the invention provides for the regulation of HDL levels and / or vascular disorders, dyslipidemias or related diseases comprising administering to a human in need a compound that modulates the activity of the gene 95 gene or protein. Provide a method for the treatment of.

  In a further aspect, the present invention provides for the diagnosis of vascular disorders and dyslipidemia, including the identification of gene 95 gene or protein mutations such as those identified in SEQ ID NOs: 6-9 or Table 1. Means and / or pharmacogenomics of therapeutic agents for treating vascular disorders and dyslipidemia are provided.

(Definition)
As used herein, the following terms have the meanings specified, unless explicitly stated otherwise.

  As used herein, the term “equivalent” in the sense of “gene corresponding to gene 95” is the same RNA that the gene has the specified nucleotide sequence or is encoded by the specified sequence. The term “substantially” means that it is at least about 50% identical to the entire sequence encoding the protein with gene 95 biological activity, and more preferably Is at least about 60%, 70%, 80%, 90% or more identical and includes its conjugated variants as defined elsewhere.

  “Corresponding to gene 95” also refers to homologs or orthologs of genes 95 from other organisms and at least about 50% identity at the nucleotide or amino acid level, more preferably about 60%, Including sequence variants thereof with 70%, 80%, 90% or more homology. Preferably, the gene is derived from a eukaryote, more preferably a mammal, more preferably a rodent or a human. The human genes and proteins described herein are representative of a wide variety of homologs, orthologs and analogs obtained from other species. These homologues, orthologs and / or analogs, and suitable variants thereof can be used in the compositions and methods of the present invention. For example, screening analysis using mouse homologues of the described human genes can also be used in screening analysis to identify potential therapeutic agents for treating humans.

  As such, the present invention is directed to isoform biology when translated, as confirmed on regions by isoforms, homologues, and region bases from these other species. Relates to the use of similar sequences which retain their activity. Said similar sequence preferably shares at least 50% identity with SEQ ID NO: 3 or SEQ ID NO: 4, more preferably 60%, 70%, 80% and most preferably at least about the isoform described herein. Share 90% identity. All of these sequences can be useful in the practice of the methods of the invention provided they retain sufficient therapeutic target biological activity to measure the effect of the test compound in modulating biological activity. The understanding is available to those with ordinary knowledge in the field based on the suggestions disclosed herein.

Further in accordance with the present invention, the term “percent identity” or “percent identity”, when referring to a sequence, refers to the disclosed or claimed sequence of the sequence to which the sequence is compared (“comparison sequence”). (Alignment) with ("reference sequence") means to be compared with the claimed or disclosed sequence. Percent identity is then measured according to the following formula:
Percent identity = 100 [1- (C / R)]
Where C is the number of differences between the reference sequence and the comparison sequence over the entire length of the alignment between the reference sequence and the comparison sequence, where (i) the corresponding aligned base or amino acid in the comparison sequence Each base or amino acid in the reference sequence without any difference between (ii) each gap in the reference sequence and (iii) each aligned base or amino acid in the reference sequence different from the aligned base or amino acid in the comparison sequence And R is the number of bases or amino acids in the reference sequence for the full length alignment with the comparison sequence, including gaps constructed in the reference sequence as bases or amino acids.

  If an alignment exists between the comparison sequence and the reference sequence and the percent identity calculated as above is equal to or greater than the specified minimum percent identity, then the alignment is A comparison sequence has a defined minimum percent identity to a reference sequence, even if it exists if the calculated percent identity is less than or equal to the defined percent identity.

  As used herein, the terms “site”, “segment” and “fragment” when used in reference to a polynucleotide refer to a contiguous sequence of nucleotide residues, which represents a portion of a larger sequence. Form. The term includes products produced by treatment of the polynucleotide with any common endonuclease, or an extension of a polynucleotide that can be synthesized synthetically. These may include exon and intron sequences of the corresponding gene.

  As used herein, “therapeutic target biological activity” (or “biological activity only”) includes, but is not limited to, the embodiments shown in (a) to (d) below: It is a very broad term relating to all directly and indirectly measurable and identifiable biological activities of genes and proteins.

  (A) For a purified therapeutic target protein, the therapeutic target biological activity is the interaction, binding or binding of all biological processes, ligands, proteins, membrane components or other compounds (such as small organic compounds) Including, but not limited to, mode, binding activity relationship, pKa, pD, enzymatic reaction rate, stability and protein function evaluation.

  (B) With respect to therapeutic target biological activities in the cell fraction, reconstituted cell fraction or whole cells, these activities include the binding mode of the ligand or antibody and the signal transduction system, efflux, influx or accumulation of cellular components, This includes, but is not limited to, all measurable results of this effect, such as measurement of stability or degradation, membrane organization and mode, cell proliferation, progression or mode and other direct or indirect effects of therapeutic target activity.

  (C) With respect to therapeutic target genes and transcription, therapeutic target biological activity includes the rate, scale or extent of transcription of DNA that produces therapeutic target mRNA or alternative transcripts, the effect of regulatory proteins of the transcription, the transcription In addition to the influence of regulatory factors on the regulatory proteins, all measurements of mRNA transcripts, post-transcriptional processing, mRNA amount and folding stability and mode, translation of mRNA into polypeptide sequence are included.

  With respect to therapeutic target biological activities in organisms, this includes biological activities identified by deficiencies or deficiencies in disease processes or disorders caused by abnormal therapeutic target biological activities in these organisms. It is not limited. Broadly speaking, therapeutic target biological activity can be measured by any method known in the art for analyzing the biological properties of proteins and genes.

(Detailed description of the invention)
In accordance with the present invention, genes and proteins associated with maintaining human HDL levels are determined by identifying the mutant form of the gene in a family with low HDL levels using the positional cloning method shown in Example 1. Is done.

  The BC-11M family of lines is shown in FIG. The family phenotype used to define linkage segregates the low HDL (low alpha proteinemia) trait in a highly penetrating autosomal dominant species. Other lipid conditions (triglycerides (TG) and low density lipoprotein (LDL)) are normal in this family and there are no other confounding factors (such as obesity and diabetes). There are particularly strong signs of heart disease and coronary artery disease in this family, with many experiencing cardiac accidents (myocardial infarction (MI), stroke and angina) and treatment for symptoms of this disease (coronary artery bypass graft (CABG)) Etc.). Part of this family is Behcet's syndrome (related to lupus and autoimmune diseases), others are celiac disease (gluten intolerance), and the rest have abnormalities in the thyroid.

  Since this family has a wild-type sequence in the coding region of ABCA1, it is distinguished by mutations that cause low HDL in ABCA1 identified so far. In addition, the ABCA1 gene is excluded based on genetic characteristics of this family and based on normal cholesterol efflux measurements in cultured fibroblasts from diseased individuals of this family.

  The lines of the NL-003 family are shown in FIG. Probands (II: 35) have HDL levels below the first percentile (age and gender corrected). Although there is little overweight / obesity (BMI> 25> 30), this condition is not well co-separated from HDL conditions and therefore may not be related.

  The lineage of the NL-120 family is shown in FIG. Again, the proband (II: 01) has an HDL level below the fifth percentile (age and gender corrected). Many low HDL individuals are observed in both NL-003 and NL-120. In the NL-003 family, individuals with low HDL, heart disease and cardiac accident (MI) are observed.

  Therefore, the gene identified according to the present invention is located on the 9th chromosome, on the 9q22 band or in the vicinity thereof. Markers for this locus have been identified and are as follows: 9q22ca7 (SEQ ID NO: 1) and 9q22ca17 (SEQ ID NO: 2). Other markers are available as shared property.

Description of gene 95 and its mutations Gene 95 is known and is described herein as a P1G95, gene 95, G95 or HDL regulatory protein. The nature of gene 95 is disclosed in various sources in public databases.

  Includes Goldenpath Gene ID: ENSG0000013925 and Transcript ID: ENS000000259452 (Goldenpath Assembly of Human Genome Project Working Draft, www.genome.ucsc.edu or ensemble website www.ensembl.org, conducted August 2001) Seen in the edition).

  GenBank Accession Number: AK056453, XM — 088573 (available at www.ncbi.nlm.nih.gov/entrez/query.fcqi). Those of ordinary skill in the art will recognize that it does not provide the exact full-length nucleotide coding sequence of gene 95, nor does it provide the exact amino acid sequence of gene 95 when translated.

  The full length cDNA sequence of gene 95 (P1G95) is set forth in SEQ ID NO: 3 and includes a 4333 bp transcript containing a 5 ′ UTR and a 3 ′ end. The exon / intron structure of gene 95 is shown in FIG. The genes include 10 exons with possible alternative transcripts for exon 6 (“exon 6alt” or E6a) and exon 9 (“exon 9alt” or E9a). FIG. 4 shows the expected 10 exon configuration and other possible transcripts using E6a or E9a.

  The gene 95 protein includes a 516 amino acid protein (SEQ ID NO: 4). The use of an alternative methionine start site can generate a sequence of 489 amino acids. Analysis of the protein sequence of gene 95 shows that it has water-soluble protein characteristics associated with maintaining human serum lipid homeostasis. The protein appears to contain one or more of the following features: N-glycosylation site (PS00001), glycosaminoglycan binding site (PS00002), 4cAMP- and cGMP-dependent protein kinase phosphorylation sites. (PS00004), 12 protein kinase C phosphorylation site (PS00005), 8 casein kinase II phosphorylation site (PS00006), 8N-myristoylation site (PS00008), zinc finger C2H2 type region (domain) (PS50157), Cysteine-rich region (PS50311).

  For SEQ ID NO: 3, residues 1 to 400 correspond to 5'-UTR (untranslated region), while 3'UTR includes from residue 1952 to the end. For the mutant sequence of SEQ ID NO: 6, the T1802G mutation is shown, the 5'-UTR region is indicated by residues 1 to 400, and the 3'-UTR region is indicated by residues 1952 to the end. For the mutant sequence of SEQ ID NO: 8, showing the 1706C1707 mutation, the 5'-UTR region is indicated by residues 1 to 400 and the 3'-UTR region is indicated by residues 1952 to the end.

  Gene 95 has a Rhodanese-like domain that is most closely related to a hypothetical, yet uncharacterized group of bacterial proteins. An exemplary rhodanese-like domain is found at amino acids 304-406. There are no other human genes similar to this gene other than hypothetical or “rhodanese-like” or “sulfurtransferase-related” proteins. The rhodanese domain is found in thiosulfate sulfuryltransferase (eg, rhodanese; IPR001307). Public database searches identified 21 in the human genome as rhodanese domains. One is platelet adhesion molecule and the rest is phosphatase. Other domains are also shown, such as the DnaJ central domain (eg [CXXCXGXG] 4; amino acids 422 to 480), the XS zinc finger domain (eg amino acids 458-465) and the integrase site (eg amino acids 452-465).

FIG. 5 shows the nucleotide and amino acid sequences of individual exons, several sequences from the promoter (genomic) and intron regions (especially splice site junctions). Explanation: Lower case letters are intron regions that do not form part of mature mRNA or cDNA. “...” is an intron region available from public databases and is not included here. Capital letters are nucleotide bases or amino acids (standard notation symbols are used as indicated in the text). “*” Is a stop codon. +1, +2, and +3 are frame shifts of nucleic acid translation. A short reading frame before and after the correct amino acid sequence (short open reading frame) is used for completeness, but does not appear to form part of the gene 95 protein. Here, exon E1 is a part of SEQ ID NO: 18, exon E2 is a part of SEQ ID NO: 19, exon E3 is a part of SEQ ID NO: 20, and exon E4 is a part of SEQ ID NO: 21 , Exon E5 is part of SEQ ID NO: 22, exon E6 is part of SEQ ID NO: 23, exon E7 is part of SEQ ID NO: 24, exon E8 is part of SEQ ID NO: 25, exon E9 is part of SEQ ID NO: 26, exon E10 is part of SEQ ID NO: 27, exon E6a is part of SEQ ID NO: 28, and exon E9a is part of SEQ ID NO: 29. Here, exon 6a is alternative exon 6 (“exon 6alt”) (incomplete protein), while exon 9a is alternative exon 9 (“exon 9alt”) (longer but incomplete) ). In addition, ctttga (SEQ ID NO: 30) indicates the starting point of the new intron sequence. The gene 95 sequence spans approximately 34 kb of genomic DNA. Introns not fully shown can be obtained from the public human genome sequence database, Golden Pass. As indicated in the text, lower case letters indicate introns and promoter regions, and upper case letters indicate nucleotides or amino acids in cDNA or mRNA. Exon 1 and exon 2 have one or more asterisks (" ^* "). There is a putative stop codon. The true starting methionine appears to be the first methionine in exon 2 after the asterisk. An asterisk can also be found in exon 10. The first asterisk is the expected termination site that produces a 516 amino acid polypeptide. The remaining amino acid translations have been shown for completeness and do not appear to be functional proteins. Exon 6a and exon 9a contain a stop codon. If these alternative exons are used, they are likely to be incomplete proteins as shown.

  Homology: In the full-length protein, the closest homologue to human gene 95 has 29-34% identity with rhodanese-related sulfur transferases (Salmonella thphimurium and Brucella melitensis) and dihydrofolate reductase (Schizosaccharomyces pombe). The hypothetical bacterial proteins with the highest homology are Q9RVC9_DEIRA / 119-216; Q55613_SYNY3 / 113-210; Q9Z7H1_CHLPN; 084632_CHLPN / 113-212; 084632_CHLTR / 113-2112; YBFQ protein).

  The present invention also discloses and claims the mouse gene 95 protein (SEQ ID NO: 5), the first non-human mammalian homologue of gene 95. This sequence has approximately 76% sequence identity with human gene 95 over the entire length of the protein. The human and mouse amino acid sequences are aligned in FIG. Common amino acids are shown, with conservative and non-conservative conversions.

Further investigation of short nucleic acid sequence segments from other mammals shows that mammalian species share generally conserved gene 95 sequences at the nucleotide level. In particular,
Human: Bovine is 514/565 (90%) identical based on overlapping sequences. Human: Rats are 338/401 (84%) identical based on overlapping sequences. Human: pigs are 228/265 (86%) identical based on overlapping sequences. Human: Mice are 266/307 (86%) identical based on overlapping sequences.

  Expression pattern: Based on EST validation, the gene has been shown to be expressed in the following tissues and cell lines: teratocarcinoma, brain, placenta, ovarian tumor, lymphoma, melanoma, neuroblastoma. The data of Examples 2 and 3 confirms expression in a wide range of tissues.

  Mutation 1 found in the BC-11M family was identified as 1802T> G (SEQ ID NO: 6) causing the Ser468Ala mutation, SEQ ID NO: 7. Mutations were present in 13 affected family members and not in 10 unaffected family members. The mutation was not present in 198 control chromosomes, nor was it present in 164 chromosomes from high HDL individuals. Both control groups were of ethnicity similar to the BC-11M family. Said mutation can result in the loss of a putative protein kinase A phosphorylation site (PKA-basic-basic-neutral-Ser). This mutation converts a serine residue conserved in the mouse protein.

  Mutation 2 from an individual in the NL-003 family with HDL levels less than one-fifth percent contains the 1706C1707 insertion SEQ ID NO: 8. This insertion is in the second base of the codon for Q436, resulting in 39 additional specific amino acids producing Q436P and the entire premature stop codon 475aa (counted from SEQ ID NO: 9 and methionine at residue 14). Mutation 2 is also observed in individuals from the NL-120 family with less than a fifth percent of HDL. Mutation 2 was not observed in 228 control chromosomes of similar ethnicity.

  FIG. 7 shows a sequence of the amino acid sequences of mutation 1 and mutation 2 and the wild type amino acid sequence of human gene 95.

  Single nucleotide polymorphisms (cSNPs) and other polymorphisms of the coding region of gene 95 have been identified and are shown in Table 1. These cSNPs result in the identified amino acid conversion, if any. Other cSNPs and polymorphisms in the exon or intron region of gene 95 can also be identified by those with ordinary knowledge in the field.

  Due to the processing performed in the conversion of the starting RNA transcript to the final mRNA, the nucleotide sequences disclosed herein may correspond to less than the complete genomic sequence. The gene and cDNA sequences are considered to be corresponding genes because they both encode similar RNA sequences. Thus, for purposes of a non-limiting example only, a gene encoding an RNA transcript that is converted to a short mRNA encodes both of the RNAs and thus into cDNA (using the usual Watson-Crick complementarity law). Complementary RNA, ie RNA encoded by cDNA (eg, the sequences disclosed herein) is considered to be encoded. Thus, the sequences disclosed herein correspond to genes contained in the cells, and these sequences correspond to the same sequences or are complementary to RNAs encoded by these genes, so Used to measure the target level. The genes also include different alleles and splice variants that can be expressed in the cells used in the methods of the invention.

  Therefore, a polynucleotide such as the gene sequence disclosed herein for use in the screening analysis of the present invention is encoded by a polynucleotide having SEQ ID NO: 3 or is complementary by hybridization or the like. RNA having a sequence of at least 50%, preferably at least 70%, 80%, 90%, and more preferably at least 95% or 98% identical to the sequence of such RNA (particularly naturally occurring splice variants) And “corresponding” to gene 95 in the case of encoding conversion (including conversion or non-conversion). In addition, at least 50% identical to SEQ ID NO: 3, preferably at least about 70%, 80%, 90% identical to the sequence, more preferably at least about 98% identical to the sequence, and most preferably a sequence comprising the sequence In particular, all the methods of the present invention are intended as genes corresponding to these sequences. In addition, sequences encoding the same polypeptides and proteins as any of these sequences may also include any of the sequences, regardless of how they are disclosed or limited, regardless of the percent identity of the sequences. It is specifically contemplated by any method of the present invention that depends on or all. Thus, the sequences can be used in the practice of any method disclosed in accordance with the present invention. The sequence also includes an open reading frame as defined herein that lies between genes 95. Said sequence comprises the mutated gene 95 sequence of SEQ ID NO: 6 and SEQ ID NO: 8 and also includes the sequence comprising the alternative exons shown in FIG.

  In accordance with the foregoing, the present invention includes an isolated polynucleotide comprising a polynucleotide sequence or a full complement of this polynucleotide sequence. Wherein said polynucleotide sequence is at least 65%, preferably at least 75%, more preferably at least 85%, especially at least 90%, most preferably 95% or even 98% identical to SEQ ID NO: 3, The sequence of SEQ ID NO: 3 is preferable. The present invention also includes an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4.

  With respect to the gene 95 protein (SEQ ID NO: 4), the present invention includes all of the protein, polypeptide and amino acid sequences corresponding to SEQ ID NO: 4. Said sequence corresponding to SEQ ID NO: 4 comprises a full-length protein or a fragment thereof that retains the biological activity of the gene 95 protein. Said sequence is preferably at least about 50% identical to SEQ ID NO: 4, more preferably about 60%, 70%, 80% and most preferably at least about 90% or 95% identical to SEQ ID NO: 4. The sequence comprises an ortholog or homologue of the gene 95 protein from any organism, preferably a eukaryote, more preferably a mammal, most preferably a rodent or human, and at least about 50%, 60% of the sequence. %, 70%, 80%, and most preferably comprises an amino acid sequence having a sequence identity of at least about 90%. Said sequences include the mutant sequences SEQ ID NO: 7 and SEQ ID NO: 9, the sequence corresponding to the alternative reading frame of the gene 95 nucleotide sequence and the sequence found in the alternative splice exon in FIG.

  In accordance with the foregoing, the present invention provides at least 65%, preferably at least 75%, more preferably at least 85%, particularly at least 90%, most preferably 95% or even 98% of the amino acid sequence shown in SEQ ID NO: 4. It includes isolated polypeptides that contain amino acid sequences with identity. Here, the polypeptide comprising the sequence of SEQ ID NO: 4 is particularly preferred.

  The invention also relates to a nucleic acid vector comprising an isolated polynucleotide comprising the nucleic acid vector and a recombinant host cell. The invention also includes a method for producing the polypeptide of SEQ ID NO: 4, comprising culturing the host cell of claim 49 under conditions that support production of the polypeptide. All of these methods are well known to those having ordinary knowledge in the art, taking into account the disclosure herein.

Screening analysis using gene 95 The present invention provides screening analysis using gene 95 genes and / or proteins for use in the identification of therapeutic agents, particularly agents that antagonize low HDL levels. One protocol involves the analysis of chemicals for the ability to up or down regulate the activity of gene 95 to increase HDL activity, such as by increasing HDL levels, particularly plasma levels, in animals including humans. Gene 95 gene, an active fragment thereof, a substance that converts the activity of a protein, a substance that can regulate the activity of a polypeptide encoded by gene 95, or a polypeptide that functions as a transcription factor for regulating the activity of the gene Alternatively, other related gene segments such as enhancers or other regulatory genetic elements that regulate the activity of gene 95 working in a cis- or trans-type manner are identified thereby, reverse cholesterol transport or other lipid-related processes Useful in the prevention, treatment or improvement of diseases associated with low HDL levels, or the effects of low HDL levels, and / or heart disease, atherosclerosis and other diseases Prove that it is useful for diseases such as That.

  In accordance with the invention disclosed herein, suffering from low HDL-C, vascular disorders, dyslipidemia, atherosclerosis or related diseases by administration of a compound that modulates biological activity or gene 95 expression Compositions and methods are provided for patient treatment or for improving reverse cholesterol transport in the body.

  The present invention is particularly intended for screening analysis, for example, where a wide range of compounds can be screened for activity in modulating therapeutic target biological activity. For all of the analyzes disclosed herein, the modulation can include an increase or decrease in therapeutic target biological activity.

  Those with ordinary knowledge in the field can identify measurable biological activity of therapeutic targets that can be effectively incorporated into low or high throughput screening analyses. Non-limiting examples of the analysis are disclosed herein for purposes of illustration. Based on these suggestions, other examples of therapeutic target screening analyzes could be performed as is.

  The present invention also provides an analysis for the identification of said therapeutic compounds and analogs thereof. Compounds that modulate the biological activity of the gene 95 gene or protein are considered useful in the present invention. Typical screening analyzes for the identification of said compounds (in vitro or in vivo) and computer-assisted analysis (in silico) are detailed below. The screening analysis of the present invention simplifies the evaluation, identification and development of therapeutic agents for the treatment and prevention of low HDL and / or vascular disorders, dyslipidemia or related diseases. In general, screening methods are easy for selection of natural product extracts or synthetic compounds of interest from large populations (eg, chemical libraries) that are further evaluated and condensed into several active center structures. Provide a simple way. Numerous analogs of the central structure can be developed and tested to identify preferred analogs with improved therapeutic properties.

The present invention also relates to a method for identifying a substance that modulates the activity of a polypeptide whose activity converts high density lipoprotein (HDL) activity, said method comprising:
a) contacting the candidate compound with a polypeptide encoded by a polynucleotide corresponding to gene 95 under conditions that promote said activity;
b) measuring a change in biological activity of the polypeptide as a result of the contact;
Here, said change in activity identifies the test compound as a substance that modulates said polypeptide biological activity.

  In one embodiment of this process, the change in biological activity in step b) is a decrease or increase in the activity of the polypeptide. In a preferred embodiment, the activity is measured by measuring the activity of the enzyme, for example, where the polypeptide itself has an enzymatic activity that can be analyzed directly or indirectly. The analysis can be performed in vitro or in vivo.

  In a preferred embodiment, the polypeptide has the amino acid sequence of SEQ ID NO: 4, and in another embodiment, the polynucleotide has SEQ ID NO: 3.

  In another embodiment, the polypeptide is present in cell extracts, liposomes or intact cells. The invention specifically contemplates examples where the cell is a recombinant cell line comprising a heterologous gene 95 configuration. In another embodiment, the cell or extract is treated by means other than genetic recombination techniques, eg, where the activity of the polypeptide is enhanced, and the cell contains or is on the surface of the polypeptide. However, the polypeptide is present by physical insertion of the polypeptide into the membrane or cytoplasm of the cell, not through the expression of genes contained in the cell.

The present invention also relates to a method for identifying a substance that modulates gene 95 activity, wherein said method comprises:
a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to gene 95 under conditions that support the activity of said polypeptide;
b) measuring a change in the activity of the polypeptide as a result of the contact;
The change in activity here identifies the test compound as a substance that modulates gene 95 activity.

  The change in activity measured in step b) above can be a decrease or increase in activity. In one embodiment, the activity is measured by measuring the activity of the enzyme. In another embodiment of the method, the polypeptide is present in a qualitative bilayer, wherein the lipid bilayer comprises a portion of a liposome. In addition, the polypeptide can be part of an intact cell. For example, here intact cells are cells that have been treated to contain the polypeptide, for example, intact cells here are recombinant cells that have been genetically modified to express the polypeptide, Preferably here the cell does not express the polypeptide without the recombination. In all cases, the intact cell is preferably a mammalian cell.

  The invention also relates to a method for identifying an HDL-enhancing agent, said method comprising administering to an animal an effective amount of a substance found to have modulatory activity using any of the assays disclosed herein, Detecting an increase in plasma HDL activity in said animal as a result of administration, thereby identifying substances useful for enhancing HDL activity.

  In a preferred embodiment, the animal exhibits low HDL activity prior to administration of the substance. In another preferred embodiment, the HDL enhancing activity is an increase in HDL levels in the animal, most preferably an increase in plasma HDL levels, and preferably wherein the animal is a human patient.

  The invention further relates to a method for treating said disease in an animal afflicted with a low HDL-related disease, wherein said method has been found to have HDL enhancing activity in said animal using any assay of the invention Administration of an effective amount of the substance. In a preferred embodiment, the animal to be treated is a human patient. In a further preferred embodiment, the disease to be treated is selected from the group consisting of low HDL disease, vascular disorders and dyslipidemia.

  Substances identified according to the process of the present invention are also useful for the treatment or prevention of coronary artery disease, regardless of the patient's HDL status. For example, patients with normal HDL levels who have a family history of coronary artery disease may be advised to take therapeutic agents according to the present invention to increase HDL levels and further reduce the risk of coronary artery disease. Let's go. Thus, the patient need not have dyslipidemia in order to be suitable for treatment according to the present invention. In general, the screening methods of the present invention include screening of any compound for therapeutically active substance by application of any in vitro or in vivo test system. Examples of methods useful for the identification of the compounds are detailed herein.

  The method of the present invention simplifies the evaluation, identification and development of active substances for the treatment and prevention of low HDL levels and heart disease (CVD) and dyslipidemia. In general, screening methods provide a rapid method for selection of natural product extracts or compounds of interest from large populations that are further evaluated and condensed into several active and selective materials. Positive candidates from this population are subsequently purified and evaluated in the methods of the invention to measure their HDL-elevation, anti-CVD or anti-dyslipidemic activity, or both. Positive candidates can be used directly as therapeutic agents. Alternatively, they can provide structures that provide information for structure-activity relationship (SAR) analysis and the development of further analogs that are preferred therapeutics.

Evaluation of gene 95 as a sulfur transferase involved in HDL regulation While human genetic information has demonstrated that the function of gene 95 maintains human HDL levels, the specific mechanism of function of gene 95 is still Not proved. Without being bound by any theory about the mechanism of action, the inventors suggest that gene 95 can function as a sulfur transferase or a similar type of enzyme. This is based on a comparison of rhodanese domains found in proteins. Rhodanese domains can be classified as active for sulfurtransferase activity, as active for phosphatase activity, or as catalytically inactive. Human and mouse G95 contain consensus for sulfur transferase within the rhodanese domain. A positive match for phosphatase within the rhodanese domain is found in humans but not conserved in mouse G95. In addition, another human gene with this match is noted as sulfur transferase activity.

  Several types of evidence link these activities with HDL metabolism. For example, the thioredoxin binding protein Txnip is known to be associated with lipid metabolism based on the hyperlipidemic mouse phenotype.

  It is known that screening assays using gene 95 sulfur transferase biological activity can be designed to use known substrates of sulfur transferases such as cyanide, thioredoxin and dihydrolipoate. Alternatively, the screening analysis can be designed based on the aforementioned sulfur transferase analysis. In one colorimetric assay for the thiocyanate product, the assay measures the conversion of thiosulfate and cyanide to sulfite and thiocyanate. In the analysis of NADPH oxidation by thioredoxin reductase, the assay measures the conversion of thiosulfate and thioredoxin to sulfite and thioredoxin persulfide. Compounds that act or antagonize these transformations through gene 95 interactions are modulators of the present invention.

  Functional analysis may be based on the activity of a polypeptide encoded by one or more polynucleotides disclosed herein as corresponding to gene 95. The polynucleotide includes a candidate having high homology, such as a polynucleotide at least 90% or 95% identical to the gene or a polypeptide having high homology with a polypeptide encoded by the gene. The analysis can use drug screening techniques such as (but not limited to) the ability of various dyes to change color in response to changes in analytical conditions due to polypeptide activity.

  Drug screening analysis can also be based on the ability of gene 95 polypeptides to interact with other proteins. The interacting protein can be identified by various methods known in the art, including, for example, radioimmunoprecipitation, co-immunoprecipitation, and yeast two-hybrid screening. The ability of test compounds to act or antagonize protein-protein interactions is a standard form of screening analysis that can be used. The interaction can be analyzed by methods including but not limited to fluorescence polarization or scintillation proximity. Drug selection can be performed based on functions or characteristics found in the production of transformed or knockout mice, or based on overexpression of proteins or protein fragments in mammalian cells in vitro. Furthermore, yeast or C.I. Expression of mammalian (eg human) gene 95 polypeptide in elegans allows screening of candidate compounds in wild type and mutation systems, and selection of mutations that enhance or suppress the low HDL phenotype. Modified gene selection can also be performed in transformed or knockout mice.

  For those of ordinary skill in the art, the disclosure herein facilitates incorporation of gene 95 into standard screening assays to identify compounds that interact with and / or modulate gene 95. It will be clear. Such analyzes include protein / compound binding analysis, gene expression analysis and many other types. In addition to the standard analysis, the known features of gene 95 suggest a more specific analysis that can be used. For example, a putative phosphorylation site is a useful modulator of the present invention, and an analysis that measures the tendency of a test compound to affect the rate, amount, or timing of gene 95 phosphorylation is also shown here. It can be a potential therapeutic agent for a disease or an analogue thereof. The features of gene 95 that can be used for screening are: N-glycosylation site, glycosaminoglycan binding site, 4cAMP- and cGMP-dependent protein kinase phosphorylation site, 12 protein kinase C phosphorylation site, 8 casein kinase It includes an II phosphorylation site, an 8N-myristoylation site, a zinc finger C2H2 type domain, and a cysteine-rich region. Other features that may be useful for measurement include the rhodanese-like domain, the DnaJ central domain, the XS zinc finger domain and the integrase site.

In accordance with the foregoing, the present invention relates to a method for identifying a substance that modulates the activity of a polynucleotide whose expression modifies high density lipoprotein (HDL) activity in vivo, said method comprising:
a) contacting a test compound with a polynucleotide corresponding to gene 95 under conditions that promote said polynucleotide expression;
b) measuring a change in expression of the polynucleotide as a result of the contact;
Here, said change in expression identifies the test compound as a substance whose expression modulates the activity of a polynucleotide whose expression modifies high density lipoprotein (HDL) activity.

  In a specific embodiment of the process, the change in expression in step b) can be a decrease or increase in the expression of the polynucleotide or gene.

In another aspect, the invention relates to a method for identifying a substance that modulates gene 95 activity, said method comprising:
a) contacting a test compound with a genetic construct comprising a reporter gene operably linked to the gene 95 promoter under conditions that support transcription of said reporter gene;
b) measuring a change in transcription of the reporter gene as a result of the contact;
Here, the change in transcription indicates that the test compound is a substance that regulates gene 95 activity.

  The measured change can be an increase or decrease in the transcription. Here, transcription is determined by measuring the amount of expression product encoded by the reporter gene, and the expression product includes RNA or polypeptide. In a preferred embodiment, the reporter gene can be present in intact cells such as liposomes or mammalian cells. A preferred embodiment of said method involves the use of a mammalian gene 95 promoter, such as a human promoter, preferably the promoter shown in SEQ ID NO: 15, or a mouse promoter, preferably a promoter with the promoter sequence of SEQ ID NO: 14. In all cases, the reporter gene is usually a gene whose expression is readily measured, and is other than gene 95 itself. However, the latter is not excluded for use as a reporter gene.

  The process is particularly used to identify substances that are effective against low HDL levels and diseases and symptoms thereof. Said substances or chemical analogues thereof are also useful for regulating the gene 95 gene or protein and / or for the treatment or prevention of heart disease. One example of a structure comprising the human gene 95 promoter sequence is shown in SEQ ID NO: 15, where the promoter sequence (first about 1500 bases) is at the beginning of the sequence and exon 1 (starting at base 1502) is Followed by intron 1, followed by exon 2 at the end of the sequence. On the other hand, the sequence incorporating the mouse promoter (approximately 1500 nucleotide residues) is shown in SEQ ID NO: 14, with the promoter at the start of the sequence, followed by exon 1 (starting from residue 1501) followed by intron 1 There is exon 2 at the end of the sequence. Exon sequences were found in the mRNA. The present invention includes the exon and promoter sequences.

  In another aspect, the invention provides a method for computer identification of compounds for modulation of HDL levels and / or treatment of vascular disorders or dyslipidemia. The method includes (a) determining the active site of gene 95 protein (eg, via X-ray crystallography or other techniques), and (b) identifying compounds that interact with the active site via computer modeling. The identification of compounds or analogs thereof as compounds useful for the modulation of HDL levels and / or for the treatment of vascular disorders, dyslipidemias or related diseases.

The invention further relates to a method for identifying a therapeutic agent, said method comprising:
a) contacting a chemical with a polynucleotide corresponding to gene 95 under conditions that support expression of said polynucleotide;
b) measuring a change in expression of the polynucleotide as a result of the contact;
Here, the change in expression identifies a therapeutic substance.

In a further embodiment, the invention includes a method for identifying a therapeutic agent that modulates the activity of a polypeptide that affects high density lipoprotein (HDL) activity in vivo, said method comprising:
a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to gene 95 under conditions that support the activity of said polypeptide;
b) measuring a change in the activity of the polypeptide as a result of the contact;
Here, the change in activity identifies the test compound as a therapeutic agent that modulates the activity of the polypeptide that affects HDL activity.

  In these preferred embodiments, the polypeptide corresponds to SEQ ID NO: 4 and / or the polynucleotide corresponds to SEQ ID NO: 3.

  Preferably, mammalian, most preferably rodent or human gene 95 polypeptides can be used as antigens to generate antibodies, including monoclonal antibodies. The antibodies may be useful for a wide range of purposes including, but not limited to, therapeutic use to modulate gene 95 biological activity, functional studies, and drug screening analysis and diagnostics development. Observation of the effect of substances (eg, small organic compounds) on the expression or biological activity of gene 95 polypeptides identified according to the present invention can be applied not only in basic drug screening but also in clinical trials. For example, the effectiveness of a substance found to increase or decrease gene expression, protein level or biological activity by the screening analysis disclosed herein may be due to inappropriate gene expression, protein level or biological activity. Can be observed in clinical trials of subjects exhibiting low HDL levels. Alternatively, the effectiveness of a gene found by screening analysis to regulate gene 95 and structurally and functionally related genes, genes with high homology with them, protein levels or biological activity is: It can be observed in clinical trials of subjects who show reduced corrected gene expression, protein levels, or biological activity. In the clinical trial, the expression or activity of the gene or polypeptide disclosed herein, preferably other genes associated with, for example, heart disease can be used to elucidate the effectiveness of a particular drug.

  For example, and without limitation, a substance that modulates the activity of gene 95, or any expression product thereof, or the activity of a polypeptide that modulates the activity of said gene (eg, identified in the screening analysis disclosed herein) Genes that are regulated in cells by treatment with (eg, compounds, drugs or small molecules) can be identified. Thus, for example, to study cholesterol levels or the effects of substances on heart disease in clinical trials, cells are isolated, RNA is adjusted, and the level of expression of gene 95 and other genes associated with similar or related diseases Can be analyzed. The level of gene expression can be determined by Northern blot analysis or RT-PCR, or by measuring the amount of protein produced by one of many methods known in the art, or the gene or other Can be quantified by measuring the level of biological activity of the polypeptide encoded by the gene. Thus, the gene expression can serve as a label that is an indicator of the physiological response of the cell to the substance. Thus, this responsive state can be measured at various times before and during treatment of an individual with the substance.

  In preferred embodiments, substances (eg, agonists, antagonists, peptidomimetics, proteins, peptides, anti-gene 95 antibodies, antisense molecules, nucleic acids, small molecules, or others identified by the screening analysis disclosed herein) A method for observing the effectiveness of treatment of a subject with a drug candidate) comprising: (i) obtaining a pre-administration sample from the subject prior to administration of the substance; and (ii) administration Detecting the HDL activity level in the pre-sample or the expression level of gene 95 protein, mRNA or genomic DNA; (iii) obtaining one or more post-administration samples from the subject; (iv) in the post-administration sample Detecting the level of expression or activity of gene 95 protein, mRNA, or genomic DNA or HDL, (v Comparing the protein, mRNA or the level of expression or activity of genomic DNA, in the post administration sample and the corresponding pre-dose sample, comprising the step of modifying the administration of the substance to the subject accordingly (vi). For example, increasing administration of the substance may increase the expression or activity of gene 95 or its encoded polypeptide, or increase HDL activity, such as plasma HDL levels, to a level higher than the detected level; That is, it would be desirable to increase the effectiveness of the material. Alternatively, a decrease in administration of the substance is desirable to reduce the expression or activity of the polypeptide or gene, where overexpression contributes to a decrease in HDL activity or level.

  The gene 95 gene disclosed herein as being associated with HDL regulation, such as induction of low HDL levels in animals, can be used, or a fragment thereof can be used as a tool for protein expression, wherein Genes can be used to encode proteins in appropriate cells in vitro or in vivo (gene therapy) or to produce sufficient amounts of protein for use in in vitro analysis for drug screening. Can be replicated in a possible expression vector. Expression systems that can be used include baculoviruses, herpesviruses, adenoviruses, adeno-associated viruses, bacterial systems, and eukaryotic systems such as CHO cells. Naked DNA and DNA-liposome complexes can also be used.

  Analysis of the activity promotes binding to intracellular interacting proteins, interaction with proteins that up-regulate gene or polypeptide activity, interaction with HDL particles or components, interaction with HDL or components thereof Includes measurement of interactions with other proteins and cholesterol efflux. Furthermore, the analysis can be based on the molecular mechanics of macromolecules, metabolites and ions using fluorescence-protein biosensors. Alternatively, the effect of candidate modulators on expression or activity can be achieved using the same general techniques combined with standard immunological detection techniques such as Western blotting or immunoprecipitation using specific antibodies. It can be measured at the level of production. In addition, useful cholesterol modulating or anti-CVD therapeutic modulators are identified as inducing activity changes associated with increased HDL levels, particularly increased HDL levels in the plasma of test subjects.

  Candidate modulators (ie, test compounds) are purified (or substantially purified) molecules, or mixtures of compounds (eg, combinatorial libraries, or extracts or supernatants obtained from cells) ). In mixed compound analysis, gene expression continues until it is shown that a single compound or minimal set of compounds modulates the activity or expression of a gene or protein in a manner that has a beneficial effect on HDL levels (eg, standard purification techniques, For example, HPLC or FPLC (produced by Ausubel et al.) Are tested against a declining population of candidate compound pools.

  Specific compounds that will regulate gene expression or gene transcription levels in the cells of gene 95 include antisense nucleic acids, ribozymes and other nucleic acid compositions, and also include the genes (exons or introns of said genes). A sequence-specific binding compound or an RNA transcript of gene 95 that specifically hybridizes. These specific compounds are compounds of the present invention and are useful for the treatment of the aforementioned diseases. The design and manufacture of the compounds can be easily performed by those having ordinary knowledge in the field based on the disclosure of the present specification.

  Specific compounds that modulate the activity of the gene 95 polypeptide include antibodies (polyclonal or monoclonal) that specifically bind to an epitope of the polypeptide. These specific compounds are compounds of the present invention and are useful in the treatment of the above diseases. The design and manufacture of such compounds is well known to those having ordinary knowledge in the field.

  One or more epitopes of gene 95, or epitopes of conserved variants of gene 95, or peptide fragments of gene 95 are also encompassed by the present invention. The antibodies include polyclonal antibodies, monoclonal antibodies (MAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ′) 2 fragments, fragments produced by Fab expression libraries, anti-idiotypes (anti-anti-types). Id) including but not limited to antibodies, and epitope binding fragments of any of the above. These can be used to treat any of the diseases disclosed herein.

  As is well known to those with ordinary knowledge in the field, the use of fully humanized antibodies is generally less immunogenic and has a longer half-life when administered to humans. Generally preferred. The main available technologies are the UltraMab human antibody development system (sm) handled by Medarex, Inc (Princeton, NJ) using transformed mice with the entire set of human genes for antibodies, and mouse antibody gene expression Abgenix, Inc., which uses a mouse genetically engineered strain that is repressed and functionally replaced with human antibody gene expression, while leaving the rest of the mouse immune system intact. (Fremont, CA) 's Xenomous technology.

  These mice can be induced to generate fully humanized polyclonal antibodies that are converted to large scale products using hybridoma technology. In one embodiment, the method comprises a transformed non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene, eg, transformation, with purification or enrichment production of gene 95 protein or fragment thereof. Immunizing mice. The animal's B cells (eg, splenic B cells) are subsequently acquired and fused with myeloma cells to form immortal hybridoma cells that secrete human monoclonal antibodies to the gene 95 protein.

  Humanized antibodies can also be produced, for example, by replacing an immunogenic site of an antibody with a corresponding but non-immunogenic site (ie, a chimeric antibody) (Robinson, RR et al., International Patent Publication). PCT / US 86/02269; Akira, K. et al., European patent application 184,187; Taniguchi, M., European patent application 171, 496; Morrison, SL et al., European patent application 173,494; Neuberger, MS et al., PCT application WO86 / 01533; Cabilly, S. et al., European patent application 125,023; Better, M. et al., Science 240; 1988); Liu, A. Y. et al., J Immunol.139: 3521-3526 (1987); Sun, LK et al., Proc.Natl.Acad.Sci.USA84: 214-218 (1987); Nishimura, Y. et al., Canc. 47: 999-1005 (1987); Wood, CR, et al., Nature 314: 446-449 (1985); Shaw et al., J. Natl. Cancer Inst., 80: 1553-1559 (1988). ). A general view of “humanized” chimeric antibodies is described by Morrison, S .; L. (Science, 229: 1202-1207 (1985)) and Oi, V .; T.A. et al. , BioTechniques 4: 214 (1986). Alternatively, suitable “humanized” antibodies can be produced by CDR or CEA substitution (Jones, PT et al., Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534 (1988); Beidler, CB et al., J. Immunol. 141: 4053-4060 (1988)).

  For the production of antibodies, various host cells have been deleted of gene 95, gene 95 peptide (eg one corresponding to the functional domain of the protein), incomplete gene 95 polypeptide (one or more domains). Gene 95), a functional equivalent of gene 95 or a mutant of gene 95 can be immunized. Such host animals include, but are not limited to, rabbits, mice, goats and rats, to name a few. Various adjuvants can be used to increase the immunological response depending on the host species, such as Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surfaces such as lysolecithin Contains active substances, pluronic polyols, polyvalent anions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human auxiliary substances such as BCG (Bacille Calmette Guerin) and Corynebacterium valvum However, it is not limited to these. Polyclonal antibodies are a heterogeneous population of antibody molecules obtained from the sera of immunized animals.

  There are a variety of expression systems that can be used for the production of complete antibodies and antibody fragments. These include bacterial or mammalian cell culture and transformed animals or plants. The generally preferred expression system is determined by the intended application and the desired production, as known to those with ordinary knowledge in the field. For example, animal cell cultures and recombinant expression systems are desirable where antibody glycosylation is desired, while bacterial expression systems are more useful for the production of non-glycosylated antibodies, Fab fragments and the like. Chad, HE and Chamow, SM. 2001. Curr. Opin. Biotech. 12: 188-193.

  In order to generate fully human monoclonal antibodies against gene 95, HuMab antibodies can be immunized using generation of purified gene 95 or fragments thereof according to any standard method. The HuMab mouse is a human immunoglobulin gene minilocus that encodes unrearranged human heavy chain (mu and gamma) and kappa light chain immunoglobulin sequences with targeted mutations that render the endogenous mu and kappa chain loci inactive. (Miniloci) (Lonberg, N. et al. (1994) Nature. 368 (6474): 856-859). These mice show reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes produce high affinity human IgG kappa monoclonals, Subject to class switching and somatic mutation.

  Preferably, the mice are 6 to 16 weeks old at the time of the first immunization. Initial immunization is performed intraperitoneally (IP) with the antigen in complete Freund's adjuvant, followed by IP immunization with the antigen in incomplete Freund's adjuvant (up to a total of 6) every other week. Is done. The immune response can be observed throughout the course of the immunization protocol with plasma samples obtained by retroorbital bleeding. Plasma can be selected, for example, by ELISA or flow cytometry, and mice with sufficient titers of anti-gene 95 human immunoglobulin can be used for fusion. Mice are challenged intravenously 3 days prior to dissection and spleen removal. A few fusions for each antigen are required for success.

For the generation of hybridomas that produce human monoclonal antibodies against gene 95, mouse splenocytes are first isolated and fused with a mouse myeloma cell line based on standard protocols using PEG. The resulting hybridomas are subsequently screened for production of antigen specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused with one-sixth P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. . Cells are seeded at approximately 2 × 10 ⁵ in a flat bottom microtiter plate, followed by 20% fetal calf serum, 18% “653” conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM. L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin and 1 × HAT (Sigma; HAT is post-fusion Cultured for 2 weeks in a selective medium containing (added at 24 hours). Two weeks later, the cells are cultured in a medium in which HAT is replaced with HT. Each well is then sorted by ELISA for human antigene 95 monoclonal IgM and IgG antibodies. When extensive hybridoma growth occurs, medium is usually observed after 10 to 14 days. If the antibody-secreting hybridoma is removed, screened again, and positive for human IgG, the anti-gene 95 antibody can at least double sub-clone (subclone) by limiting dilution. Stable subclones are then cultured in vitro to produce small amounts of antibody in tissue culture media for characterization.

To purify human anti-gene 95 antibodies, selected hybridomas can be grown in 2 liter spinner flasks for monoclonal antibody purification. The supernatant is filtered and concentrated prior to affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ.). The eluted IgG can be examined by gel electrophoresis and high performance liquid chromatography to ensure purity. Buffer may be converted into PBS, and the concentration can be determined by _{OD 280} using 1.43 extinction coefficient. Monoclonal antibodies are aliquoted and stored at −80 ° C. until use according to the methods of the present invention.

  Other compositions that can increase HDL levels and / or serve to treat the diseases disclosed herein are nucleic acid constructs that include gene 95 genes and are combined in a gene therapy delivery vehicle. Said transport means may be a simple aqueous buffer (for naked DNA transport), a virus-related transport means or a non-viral (particularly lipid-related) transport means. These specific compounds are the compounds of the present invention and are useful in the treatment of the aforementioned diseases. The design and manufacture of the compounds can be easily performed by those having ordinary knowledge in the field based on the present specification.

  Agents, antagonists or mimetics that appear to be effective in modulating the level of cellular expression or activity are useful in animal models (eg, primates, mice, pigs, dogs, rabbits or chickens) It can be confirmed. For example, the compound can improve low HDL levels in mouse or chicken hypoalphaproteinemia.

  Compounds that promote increased expression or activity of genes or polypeptides that have the function of promoting HDL production would be particularly useful in the present invention. The molecule can be used as a therapeutic agent, for example, to increase the level or activity of HDL, thereby treating an animal (eg, human) low HDL condition.

  The present invention allows the identification of other HDL-related genes that play a role in the regulation of HDL activity and therefore whose mutation may be important in the study of other genes associated with HDL levels. Thus, for example, mutations that act to stabilize the gene 95 protein or otherwise contribute to increased HDL levels are valuable. The mutation can be incorporated into any protein therapy or gene therapy performed for the treatment of low HDL-C levels. Similarly, compounds that increase the stability of the wild type gene 95 polypeptide or reduce its catabolism may also be useful in the treatment of any other condition as a result of low HDL-C or low HDL levels. . The mutant form of gene 95 identified here is also useful for this purpose. Additional mutations and compounds can be identified using the methods disclosed herein.

  In one example, cells expressing a gene 95 polypeptide with a mutation are transiently metabolically labeled during translation, and the half-life of the polypeptide is measured using standard techniques. Mutations that increase the half-life of the polypeptide are those that increase protein stability. These mutations can subsequently be evaluated for HDL-related biological activity. They can also be used to identify proteins that affect the stability of the corresponding mRNA or other proteins. Subsequently, these factors or compounds that affect the stability of these factors can be analyzed to bind the polypeptide. In other embodiments, cells expressing the wild type gene 95 polypeptide are transiently metabolically labeled during translation and contacted with a candidate compound, and the half-life of the polypeptide is measured using standard techniques. Is done. Compounds that increase the half-life of the polypeptide are useful compounds in the present invention.

  In other examples, treatment with an agent of the invention can be combined with other HDL increases or other conditions requiring anti-CVD or anti-dyslipidemia treatment, or HDL management.

  Analysis of the present invention includes protein-based analysis. Polypeptides encoded by the gene 95 gene (purified or unpurified) include other proteins (including but not limited to proteins that appear to interact specifically with proteins known to be associated with HDL activity) It can be used for analysis to measure the ability to bind proteins. Some examples include ABCA1 protein and apolipoprotein. The effect of the compound on this binding is subsequently measured.

  In other types of binding assays for polypeptides encoded by the genes disclosed herein, gene 95 polypeptides (or polypeptide fragments thereof, or epitope-tagged forms or fragments thereof) are obtained from appropriate sources (eg, , From prokaryotic expression systems, eukaryotic cells, cell-free systems, or by immunoprecipitation from expressing cells). The polypeptide is then bound to a suitable support, such as nitrocellulose or an antibody, or a metal agarose column in the case of a his-tagged version of the polypeptide, for example. Binding to the support is preferably performed under conditions that allow the protein associated with the polypeptide to maintain an association with the polypeptide. The conditions can include the use of a buffer that minimizes interference with protein-protein interactions. The binding step can be performed in the presence or absence of a compound that has been tested for the ability to interfere with the interaction between the polypeptide and other molecules. If necessary, other proteins (eg, cell lysates) are added and fully associated with the polypeptide. The immobilized polypeptide is then washed to remove proteins or other cellular constituents that can bind non-specifically to the polypeptide or support. Subsequently, the immobilized polypeptide is dissociated from the support and the bound protein is dissociated (eg, by heating), or alternatively, the bound protein can be removed from the support without dissociation of the polypeptide from the support. Dissociated from the peptide. Dissociated proteins and other cellular components are analyzed by, for example, SDS-PAGE gel electrophoresis, Western blotting and detection using specific antibodies, phosphoamino acid analysis, protease digestion, protein sequencing or isoelectric focusing can do. The normal and mutated forms of the polypeptide can be used in these analyzes to obtain further information about where in the polypeptide a particular factor binds. In addition, when incompletely purified polypeptides are used, comparison of the normal and mutant forms of the protein can be used to help identify the true binding protein.

  In a particular embodiment of the analysis, the analysis is performed using a purified or semi-purified protein or other molecule known to interact with a polypeptide encoded by a polynucleotide corresponding to gene 95. . This analysis may include the following steps.

1. Obtaining a gene 95 polypeptide and binding an appropriate fluorescent label to said polypeptide;
2. Labeling the interacting protein (or other molecule) with a second different fluorescent label. Dyes that form different disappearance patterns depending on whether they are close to each other or physically separated (that is, they disappear when they are close to each other, and emit fluorescence when they are not close to each other) )Use of;
3. 3. exposure of the interacting molecule to the immobilized polypeptide in the presence or absence of a compound to be tested for the ability to interfere with the two interactions; Collection of fluorescence readout information.

  Another analysis includes fluorescence resonance energy transfer (FRET) analysis. This analysis can be performed as follows.

1. Provision of the gene 95 protein or appropriate polypeptide fragment thereof and binding of an appropriate FRET donor (eg, nitro-benzoxadiazole (NBD));
2. Labeling interacting proteins (or other molecules) with FRET acceptors (eg rhodamine);
3. 3. exposure to the donor-label polypeptide in the presence or absence of a compound to be tested for the ability of the acceptor-label interaction molecule to interfere with the two interactions; Measurement of fluorescence resonance energy transfer.

  Disappearance and FRET analysis are related. Either one can be used in any case, depending on which fluorescent set is used in the analysis.

  Gene 95 can work by converting membrane permeability, such as membrane permeability to ions. Said activity can be analyzed for the use of vesicles such as liposomes or intact cells. Here, the structure contains the gene 95 polypeptide of the present invention, and the polypeptide is expressed in the vesicle, preferably an intact cell, a mammalian recombinant cell, etc., and the permeability of the membrane of the cell is Measured in the presence or absence of expression. Similarly, the permeability can subsequently be analyzed in the presence and absence of chemicals known to modulate the activity of gene 95. Therefore, the usefulness of these substances in enhancing the activity of proteins known to act on the membrane transport can be easily measured. Similarly, the ability of these substances to affect the transport of other molecules such as lipids, especially HDL across the membrane, is also easily measured.

  In performing the analysis, a test cell such as the mammalian recombinant cell that expresses the gene 95 or a polynucleotide corresponding to the gene has a reporter molecule that can detect ions (when its specific ion is bound, its fluorescence characteristics are A variable fluorescent ion indicator, etc.) is attached, or alternatively an external medium is attached to the molecule. Molecules that exhibit different properties when inside the vesicles than when they are outside the vesicles can be used. For example, molecules that have disappearance properties at high concentrations and do not have disappearance properties at low concentrations may be suitable. Alternatively, the effect of transporting ions (Ca, K, Na, metals, etc.) across the membrane can be measured if the effect eliminates or does not eliminate the dye contained in the medium or cells. can do. In the presence or absence of a compound to be tested for its ability to affect this process, the movement of the charged molecule (or its ability to move or its rate of movement) can be measured.

  In another analysis, the absorption of radioisotopes into or from vesicles can be measured. The vesicles are separated from the extravesicular medium and the radioactivity in and in the vesicles is quantified and compared. As already indicated, the present invention also relates to assays that can use transcription factors of one or more genes disclosed herein. The relationship between the polynucleotide to be tested and the binding agent can be determined using any system that discriminates between protein-bound and non-protein-bound DNA (eg, gel retardation assay). The effect of the compound on the binding of the factor to the DNA is determined using the analysis. In addition to binding analysis, in vitro analysis in which the regulatory region of the gene binds to the reporter gene can also be performed. In a preferred embodiment, the polynucleotide corresponds to gene 95, most preferably wherein the polynucleotide has SEQ ID NO: 3.

  The present invention includes a recombinant cell line comprising the gene 95 gene construct. Recombinant cell lines expressing the corresponding protein are tested to identify the relevant biological activity of the protein that can be modulated by exposure to the test compound. The compounds are systematically screened to assess whether they modulate the identified biological activity, and those that work effectively are therapeutic agents according to the present invention, or analogs thereof.

  Recombinant cell lines are also preferred for preparation of purified protein when purified protein analysis is required. Those with ordinary knowledge in this field are capable of producing recombinant cell lines and extracting protein fractions containing highly purified proteins. These samples can be used for various binding assays to identify compounds that interact with the protein. The interacting compound is a therapeutic substance of the invention or an analogue thereof.

  The present invention also includes an upstream untranslated region of gene 95 and a promoter region. The segments of the polo motor region are included in FIG. A wider portion of this promoter region can be identified by those having ordinary knowledge in the art using this disclosure. The 5'UTR (untranslated region) is disclosed in SEQ ID NOs: 3, 6, 8 and FIG. The genomic region or untranslated region can be included in a plasmid used in the analysis to identify compounds that modulate the expression of the identified gene. In one of the above analyses, the upstream genomic region binds to the reporter gene and is integrated into the expression plasmid. The plasmid is placed in the cell (transfected) and the recombinant cell is exposed to the test compound. These compounds that increase or decrease the expression of the reporter gene are thus gene / protein regulators and are considered therapeutic agents of the invention.

  The genes disclosed herein can also be associated with the regulation of cholesterol efflux. The transport-based analysis for the activity can be performed in vivo or in vitro. For example, the analysis can be based on any part of a reverse cholesterol transport process that is easily reconstituted in culture, such as cholesterol or phospholipid efflux. Alternatively, the analysis can be based on pure cholesterol transport throughout the organism, as determined using a labeled substrate (such as cholesterol).

  For high throughput screening, fluorescent lipids can be used for measurement of lipid efflux. For phospholipids, the fluorescent precursor C6-NBD-phosphatidic acid can be used. This lipid is taken up by cells and dephosphorylated by phosphatidic acid phosphohydrolase. The product NBD-diglyceride is consequently a precursor for the synthesis of glycerophospholipid-like phosphatidylcholine. NBD-phosphatidylcholine efflux can be observed by detecting the fluorescence resonance energy transfer (FRET) of NBD to the appropriate acceptor in the cell culture. Suitable acceptors include rhodamine labeled phosphatidylethanolamine, a phospholipid that is not readily taken up by cells. The use of single chain precursors eliminates the need for phospholipid transport proteins in the culture medium. With respect to cholesterol, NBD-cholesterol esters can be reconstituted into LDL. The LDL can efficiently transport this lipid to cells via the LDL receptor pathway. NBD-cholesterol ester is hydrolyzed in the lysosome, resulting in NBD-cholesterol that can be transported to the original plasma membrane and shed from the cell. The efflux can be observed by the FRET analysis, in which the NBD transports its fluorescence resonance energy to the rhodamine-phosphatidylethanolamine acceptor.

  Animal models are also useful for the analysis of the present invention. Compounds determined to be active in any of the above analyzes subsequently include the animals to determine whether HDL levels are increased by compounds administered in approximately effective amounts in pigs, rabbits or chickens. Screen in a non-limiting available animal model system. Test compounds are administered to these animals according to standard methods. Test compounds can also be tested in mice with mutations that interfere with cholesterol transport. In addition, compounds can be screened for the ability to enhance the interaction between the polypeptide encoded by the polynucleotide corresponding to gene 95 and HDL particle components such as ApoAI, ApoAII or ApoE.

  The cholesterol efflux assay measures the ability of cells to transport cholesterol to extracellular acceptor molecules and is usually dependent on the presence of transport molecules such as ABCA1. Thus, the genes disclosed herein may be involved in regulating ABCA1 activity. For example, herein, a polypeptide encoded by a polynucleotide disclosed herein binds to and modulates the activity of ABCA1 or other HDL-related proteins. In this method, cells have radiolabeled cholesterol by any of several biochemical processes (Marcil et al., Arterioscler. Thromb. Vasc. Biol. 19: 159-169, 1999). Cholesterol efflux is then measured after incubation for various times (usually 0 to 24 hours) in the presence of HDL3 or purified ApoAI. Cholesterol efflux is measured as the percentage of total cholesterol in the culture after various times of culture. Compounds that modulate cholesterol efflux in this assay may act on gene 95 protein targets. Preferably, the cell is a recombinant cell line comprising the gene 95 construct.

  The present invention also provides a transgenic animal, such as a mouse, which has one or both alleles of inactivated gene 95 (eg, by homologous recombination), or conversely, one or more additional copies of gene 95. Or mice with additional mutated copies of gene 95. Such mice, including transgenic mice, represent a useful animal model for selecting compounds that increase HDL-C levels. The mice are also useful for experimental purposes to determine the role of gene 95 in biological systems. The animal can be made using standard techniques. In addition to the initial screening of test compounds, animals with different types of said genes may be used to verify the efficacy and safety of the drug or substance originally identified using one of the other screening methods disclosed herein. Useful for further testing. Cells taken from the animal and placed in the culture medium can also be exposed to the test compound. HDL-C levels can be measured using standard techniques such as those disclosed herein.

  Compounds initially identified as useful for increasing HDL activity using one or more assays of the invention can be administered with a pharmaceutically acceptable diluent, carrier or excipient in a single dosage form. . Conventional pharmaceutical practice can be used to provide a suitable dosage form or formulation for administering the composition to a patient. Oral administration is preferred, but any suitable route of administration such as parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intracerebroventricular, intracapsular, intrathecal, intracisternal, intraperitoneal, nasal cavity Of these, aerosol or oral administration can be used. The therapeutic dosage form can be in the form of a solution or suspension. For oral administration, the dosage form can be in the form of a tablet or capsule. Also, for intranasal administration, the dosage form can be in the form of a powder, nasal spray or aerosol.

Successful gene 95 modulating therapeutics typically have some or all of the following criteria: Oral availability should be 20% or higher (thus a minimum of 20% is absorbed into the bloodstream). Animal model efficacy should be less than about 2 mg / kg and target human dosage should be between 50 and 250 mg / 70 kg, although dosages outside this range are acceptable. The therapeutic index (or the ratio of addictive to drug dose) is greater than 100. Efficacy (as indicated by the IC ₅₀ value) is less than about 10 μM, preferably 1 μM or less, most preferably 100 nM or less. The substance exhibits reversible binding (ie competitive inhibition) and is reasonably selective for unrelated targets or related targets that cause unwanted side effects. The required dosage is only once or twice a day, or at mealtime.

  Methods well known in the art for making dosage forms are described, for example, in Remington: The Science and Practice of Pharmacy, (19th ed.) Ed. A. R. Gennaro AR. , 1995, Mack Publishing Company, Easton, PA. Dosage forms for parenteral administration include, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the agents of the present invention include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Inhalation dosage forms may contain excipients such as lactose. Alternatively, it may be an aqueous solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Or it may be an oily solution for administration in the form of nasal drops or as a gel.

  New drugs for the treatment of abnormal cholesterol levels and / or CVD or dyslipidemia usually come from large libraries of both natural or synthetic (or semi-synthetic) extracts or according to methods known in the art. Identified from chemical libraries, including combinatorial chemical libraries. Those with ordinary knowledge in this field or in the field of drug discovery and development will understand that the exact source of the test extract or compound is not essential to the screening methods of the invention. Thus, virtually any chemical extract or compound can be screened using the exemplary methods disclosed herein. Examples of the extract or compound include, but are not limited to, plant-derived, fungal-derived, prokaryotic-derived or animal-derived extracts, fermentation broth and synthetic compounds, as well as preparation of existing compounds. Many methods can also be used to generate random or direct synthesis (eg, semi-synthetic or complete synthesis) of any compound including, but not limited to, saccharide-derived, lipid-derived, peptide-derived, and nucleic acid-derived compounds. . Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be found in Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographics Institute (Ft. Pierce, FL) and PharmaMar, U. . S. A. Commercially available from a number of sources, including (Cambridge, MA). In addition, natural and synthetic production libraries are made, if necessary, according to methods known in the art, for example by standard extraction and fractionation methods. Additionally, any library or compound can be readily prepared using standard chemistry, physics or biochemistry methods as needed.

  In addition, those with ordinary knowledge in the field of drug discovery and development may have dereplication (eg, taxonomic dereplication, biological dereplication, and chemical dereplication or any combination thereof) Alternatively, it is readily understood that methods for replication or repeated removal of known substances for their HDL elevation, anti-CVD and / or anti-dyslipidemic activity can be used whenever possible.

  If the crude extract appears to have cholesterol modulating activity and / or anti-CVD activity, then further fractionation of the positive dominant extract can be used to isolate the chemical components responsible for the observed effect. is necessary. Thus, the purpose of the extraction, fractionation and purification process is the careful characterization and identification of chemical components in the crude extract with cholesterol modulating or anti-CVD activity. The same in vivo and in vitro analysis disclosed herein for detection of activity in confusion of compounds can be used to purify the active ingredient and to test its derivatives. Methods for fractionating and purifying the heterogeneous extract are known in the art. If necessary, compounds that have been shown to be useful substances for the treatment of pathogenicity are chemically prepared according to methods known in the art. Compounds that are determined to be therapeutically valuable are subsequently analyzed using standard animal models known in the art.

  The present invention further includes the use of the gene 95 protein or protein fragment as a therapeutic substance and also includes a regulated form of gene 95 that retains the biological activity of gene 95. These therapeutics can be administered to patients in need thereof, who will benefit from increased local, local or whole body gene 95 biological activity when administered. It is. Such patients include, but are not limited to, patients suffering from low HDL, dyslipidemia, heart disease and other diseases.

  Functional gene 95 protein and soluble derivatives thereof, such as peptides corresponding to the functional domain of gene 95 or a modified form of gene 95 protein can be produced by standard methods known in the art. Administration to the patient can be performed intravenously, orally, intraperitoneally or by other methods known in the art.

Diagnosis and pharmacogenomics using gene 95 Relative and absolute gene expression, and biological activity, and mutational analysis can each be used as a diagnostic tool for low HDL. Thus, the determination of genetic subtyping of a gene sequence can be achieved by determining whether a low HDL phenotype is associated with a particular protein function, similar to the family used according to the present invention, It can be used when a family is subtyped. This diagnostic process can result in tailoring of drug treatment according to the patient's genotype, referred to herein as pharmacogenomics, including the prediction of side effects from administration of HDL-enhancing drugs. Pharmacogenomics allows for the selection of substances (eg, drugs) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (eg, an individual's genotype determines an individual's ability to respond to a particular substance). Inspected to determine).

  The invention further relates to a process for diagnosing the presence of a disease, in particular in its early stages, or diagnosing the risk of developing the disease. Thus, in another aspect, the invention relates to a process for diagnosing the presence of a disease in a patient suspected of suffering from the disease, said process detecting a mutation in gene 95 gene in said patient's genome Including that. In a preferred embodiment, the invention relates to said diagnostic process, wherein said mutation is a mutation in the nucleic acid sequence shown in SEQ ID NO: 6 and / or SEQ ID NO: 8 and / or Table 1.

  In a particular embodiment of the process, the mutation is detected in a sample of DNA obtained from the patient. The sample can be obtained by methods commonly used to obtain the sample, such as a blood sample, biopsy or other method. Further in accordance with the process of the present invention, the detection can be performed in vivo, for example, where the in situ method is used to detect the presence of a patient's mutant gene 95 gene or nucleic acid sequence. Diseases that can be detected are not limited to low HDL, dyslipidemia, heart disease and other diseases already disclosed and included.

  In accordance with the foregoing, the present invention relates to a process for diagnosing the presence of a gene 95-related disease in a patient suspected of suffering from a gene 95-related disease, said process comprising the gene 95 gene in the patient's genome or mRNA. Includes detection of mutations.

  In a preferred embodiment the invention relates to said process, wherein said mutation is detected in a sample of DNA taken from a patient, most preferably wherein said mutation is SEQ ID NO: 6 or SEQ ID NO: 8 or Table 1. Mutation.

  Many different techniques are known for detecting the presence or absence of the mutation. In a preferred embodiment, the detection is performed by measuring the ability of the nucleic acid probe to contain at least 15 contiguous nucleotides complementary to the mutation site of the sequence of SEQ ID NO: 1. Most preferably, the probe comprises a minimum of 30 contiguous nucleotides, particularly where the probe comprises a minimum of 50 contiguous nucleotides.

  The process disclosed herein is also useful for determining the risk of developing one or more of the above-mentioned diseases in a patient. Accordingly, the present invention also relates to a process for determining a patient's risk of developing a disease, wherein the patient is suspected of being at risk, and the gene 95 in the patient's genome or mRNA is Includes detection of mutations in the gene. The detection can be performed by the in vitro or in vivo method. In a preferred embodiment, the disease is low HDL, dyslipidemia and / or heart disease. In a preferred embodiment, the mutation is a mutation in the gene corresponding to SEQ ID NO: 1.

  Diagnosing a disease using detection of gene 95 protein in a tissue or body sample, in particular mutant gene 95 protein, or mutation or normal gene 95 protein level is also an example of the present invention. is there. Said detection can be obtained by various methods but usually involves the use of anti-gene 95 antibodies in a standard ELISA assay. Based on the present disclosure, one with ordinary knowledge in the field can develop diagnostic means for detecting mutations or normal genes 95 in patient samples for the diagnosis of various diseases.

  Substances or regulators that have a stimulatory or inhibitory effect on the gene 95 gene or gene product can be administered to an individual for the treatment of diseases associated with low HDL activity or levels. In conjunction with the treatment, the pharmacogenomics of the individual (ie, a study of the relationship between the individual's genotype and the individual's response to a foreign compound or drug) can be considered. Differences in therapeutic efficacy can be made useful for toxicity or therapeutic deficiencies by changing the relationship between medication and blood levels of pharmacologically active agents. Thus, the pharmacogenomics of an individual allows the selection of substances (eg, drugs) that are effective for prophylactic or therapeutic treatment taking into account the genotype of the individual. The pharmacogenomics can further be used to determine appropriate dosages and treatment regimens. Accordingly, an individual's gene complement for the genes disclosed herein can be determined to screen for suitable substances for the therapeutic or prophylactic treatment of said individual.

  Pharmacogenomics addresses clinically important genetic variations in response to drugs that cause altered drug properties and abnormal effects in diseased humans (Eichelbaum, M., Clin. Exp. Pharmacol. Physiol., 23). : 983-985, 1996; Linder, MW, Clin. Chem., 43: 254-266, 1997). In general, a distinction can be made between two types of pharmacogenomic states. Genetic conditions are transmitted as a single factor that changes the way the drug works in the body (altered drug action), or genetic conditions change the way the body works in the drug (altered drug metabolism) It was transmitted as a factor. Altered drug action can occur in patients with polymorphisms (eg, single nucleotide polymorphisms or SNPs) in the promoter, intron or exon sequences of gene 95. Thus, measurement of the presence and prevalence of polymorphisms allows prediction of patient response to a particular therapeutic substance. In particular, polymorphisms in the promoter region can be important in determining HDL deficiency and CVD risk. In addition to mutations, polymorphisms in gene 95 can also be associated with the identification and use of substances identified using the analysis disclosed herein.

  In addition, different substances may have different abilities to act on the genes of the characteristic gene set. For example, if a potential therapeutic substance, such as substance A, causes a decrease in the expression of a gene or group of genes associated with low HDL levels, in such cases, a lower amount of mRNA is expressed from the gene, Alternatively, if less protein is produced from the mRNA, but a second potential substance, such as substance B, reduces the expression by half while regulating the activity of the same or related gene as the gene, In such a case, only half of the mRNA is transcribed compared to the substance A, or only half of the protein is translated from the mRNA. Therefore, substance B is considered to have twice the therapeutic potential of substance A.

  The present invention also relates to a process comprising a method for the production of a product comprising the generation of test data, said process for identifying said substance (ie a therapeutic substance identified according to the analytical method disclosed herein) Identifying a substance according to one of the disclosed processes, wherein the product is data collected for the substance as a result of the identification process or analysis, wherein the data is It is sufficient to convey the chemical characteristics and / or structure and / or properties of the substance. For example, the present invention specifically uses the assay to select a compound having a desired polypeptide, eg, an enzyme, a regulatory activity or a gene expression modulating activity, and identifies the compound, followed by a user of the assay of the present invention. The information (ie, information about structure, dosage, etc.) is communicated to other users utilizing the information to reproduce and administer the substance for therapeutic or research purposes according to the present invention. The situation is intended. For example, the user of the analysis (User 1) selects a number of test compounds without knowing the structure or identity of the compound (eg, a number of code numbers are used here, the first user is the code (Only numbered samples are provided), after performing the screening process using one or more analytical processes of the present invention, followed by a specific or specific method Information sufficient to identify a compound with modulatory activity (eg, a code number with the corresponding result) is communicated to the next user (user 2). This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.

In accordance with the foregoing, the present invention relates to a method for generating test data relating to the modulating activity of a test compound, said method comprising:
(A) a polynucleotide construct comprising a reporter gene operably linked to a gene 95 polynucleotide or gene 95 polypeptide or gene 95 promoter; and the polynucleotide or reporter gene is transcribed, or the polypeptide Contact under conditions that activate
(B) measuring the activity of the polypeptide as a result of the contact, or a change in transcription of the polynucleotide or reporter gene;
(C) generating test data relating to the modulatory activity of the test compound based on a change in transcription of the measured polynucleotide or reporter gene or a change in activity of the measured polypeptide, wherein the change comprises a regulatory activity Indicates.

Example 1
Mutation in gene 95 that causes low HDL in humans Methods Patient identification and sample collection The BC origin of the family of Dutch origin was identified as part of a collaboration between the Department of Genetic Medicine at the University of British Columbia and St. Paul Hospital in Vancouver, British Columbia. It was. This relative was identified based on a hypoalphaproteinemia phenotype (<5% percentage) in the absence of other lipids and clinical abnormalities (eg obesity and diabetes). The history of coronary artery disease was evident at the confirmation stage in this relative. Additional Dutch relatives NL-003, NL-120 were identified at the Academic Clinical Center in Amsterdam as part of a patient referral program to lipid medicine based on abnormal cholesterol levels. A control population of normolidemic individuals (HDL is higher than 1/20 percent and lower than 1/80 percent) was used to confirm the nature responsible for the mutation.

Labeling and genotyping Information on gene labeling was obtained from Genome Database and GenBank. All labels used for genome scanning and fine mapping were previously localized with a Marshfield map. “X” was added to many of the internal label names. Genomic DNA was genotyped using an Applied Biosystems Prism 3100 Genomic Analyzer (Applied Biosystems) running GeneMapper software. Allele Mendelian inheritance was verified using the PedCheck program.

Linkage analysis Two-point linkage analysis was performed using the MLINK program v4.1p of the FASTLINK software package, and multipoint linkage analysis was performed using VITESSE v1.0. An autosomal dominant inheritance model was used and it was assumed that 10% of the total low HDL (<1/5 percent) in the population was due to genetic mutations. Disease allele frequency appears to be 0.25% in non-carriers with 98% penetration and 4.5% phenotypic copy rate. The marker allele frequency was estimated from a huge sample in the US and Europe. Haplotypes were manually reconstructed and cross-validated using SIMWALK v2.8.

Results Linkage analysis of the BC11M family Twenty-four individuals of the BC11M family are incorporated into a 400 marker genome scan of 12 multiplexed lines that segregated isolated hypoalphaproteinemia of the dominant genotype. Capability analysis indicated that BC11M relatives were suitable for identification of HDL loci using proximity-based linkage (MaxE (Zmax 5cM) = 4.762) in an autosomal dominant model. Two-point linkage analysis of the genome scan data gave a suggestive LOD score for chromosome 9 (D9S1677 LOD = 2.285). For this LOD score, multipoint linkage analysis, penetrating genotyping and haplotype identification using additional markers were subsequently performed on a wide range of lines. The region on chromosome 9 was supported by multipoint linkage analysis, and multipoint LOD reached 8.0 (interval 1; D9S1780 to 9q22xca17) showing only the effect of an interval of 20.91 cM (Table 2). This interval will be further divided into subregions based on the chromosomal breakpoints of unaffected individuals (Interval 2; 9q22xca7 to 9q22x-ca17) (Interval 3; D9S1812 to D9S1803x) (Table 2). Interval 2 was the focus of most extensive mutation detection.

(Example 2)
Tissue segmentation of gene 95 using the Origin gene panel 24 serial dilutions (4 log range to ensure linear amplification) placed in multiwell PCR plates, cDNA replication was performed in Rockville, MD Obtained from. PCR was performed using gene specific primers for gene 95 (sometimes "G95" here). Reactions were in 10 μl, 1.0 μl of 10 × buffer (Roche Molecular Biochemicals, Indianapolis, Ind., USA), 0.25 mM of each dNTP (Boehringer Mannheim), 5 pmoles of forward primer and reverse. ) Primers, and 0.25 units of Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA).

  The amplified fragment is between exons 4 and 6 and has a size of 230 bp.

  DNA Engineer Tetrad cyclers (MJ Research, Inc., Waltham, MA, USA) were used for PCR amplification. PCR conditions were 94 ° C for 3 minutes denaturation step followed by 35 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute. A final extension step of 7 minutes at 72 ° C. was performed and the samples were stored at 10 ° C. PCR products were analyzed by electrophoresis on 2% agarose gels in 1 × TBE. A 100 bp ladder (Gibco-BRL, Rockville, MD, USA) was used as the size standard.

  The relative amount of G95 cDNA is shown in Table XX. H = high, M = medium, L = low.

(Example 3)
Tissue Expression by Northern Blot Quantification and sizing of the gene 95 transcript was analyzed by use of human 12-lane multi-tissue Northern blot (BD Biosciences Clontech, Palo Alto, CA, USA). The human blot was hybridized with a 401 bp probe for exons 6 to 9 of gene 95. As expected, a 4.4 kb band was identified. Actin was used as a control.

The results (FIG. 9) show that gene 95 is ubiquitously expressed at the highest level in muscle and heart. These results were confirmed by Northern blot analysis of mouse tissues (data not shown). In one of the above implementations, fibroblasts were used in a 6-well format to obtain 50-70% confluency on the next day, 2 × 10 ⁵ cells approximately 24 hours prior to cholesterol treatment. Each well was seeded. Cells were treated with a final concentration of 30 μg / ml cholesterol for 24 hours, followed by total RNA preparation. The control was gene 95 expression obtained from an unaffected cell line.

Example 4
Gene 95 expression in patient fibroblasts A Ser to Ala mutation in exon 10 of gene 95 was present in four patient fibroblasts (400, 558, 549 and 575). We decided to compare the expression of gene 95 in these fibroblasts with normal controls (226, 475 and 485). This analysis was performed in the absence or presence of exogenous cholesterol to investigate the relationship between lipid metabolism and gene 95. Quantitative real-time PCR (or “TaqMan”) analysis was performed as follows. The human gene 95 primer and its probe were designed using Primer Express software (Applied Biosystems, Foster City, Calif.).

RT-PCR is 25 ul containing 120 ng human gene 95 total RNA or 50 ng human apoA1 RNA, 200 μM primer, and 600 μM probe in 1 × Taqman one-step RT-PCR master mix (Applied Biosystem, Calif.). Of ABI 7900 sequence detection system. GAPDH or 18s (human) (Applied Biosystems) was used as an internal control. Data quantification and analysis was performed according to the manufacturer's protocol. Briefly, the calibrator and sample ΔC _T were obtained by subtracting the VIC C _T value of 18sRNA or GAPDH from the FAM C _T value of the relevant gene. ΔΔC _T was calculated by subtracting the average ΔC _T (calibrator) value from ΔC _T (sample). The amount of mRNA for the calibrator is expressed as a 1 × sample, and all other amounts are expressed as a number of percentage changes associated with the calibrator. Each sample was analyzed in triplicate and standard error (SE) was calculated. RT-PCR reactions without RNA and reactions with RNA but without reverse transcriptase were used as negative controls. The size of the PCR product was confirmed by agarose gel.

  Overall, an extremely low level of expression of gene 95 was found in all fibroblasts including controls. TaqMan results indicated that expression of gene 95 in two patient cell lines (400 and 575) was down-regulated by approximately 50% compared to controls with or without cholesterol treatment. Expression of gene 95 in patient 558 was not significantly different from control. However, the expression of gene 95 at 549 was increased over the control (data not shown).

  A trend towards a decrease in gene 95 expression in mutation carriers was noted. Messenger RNA expression levels will be sensitive to cell culture conditions, cell density and passage number. Without wishing to be tied to any theory about the mechanism of action, the decreasing trend of gene 95 mRNA levels in this mutation carrier would be the underlying mechanism of low HDL status in these patients.

System diagram of BC-11M family System diagram of the NL-003 family System diagram of NL-120 family Diagram showing the incomplete organization assembled from known fragments of gene 95, including the expected 10 exon configuration of gene 95 cDNA and other possible transcripts with E6a or E9a. Diagram showing exon, intron and amino acid translation of gene 95 Diagram showing exon, intron and amino acid translation of gene 95 Diagram showing exon, intron and amino acid translation of gene 95 Diagram showing exon, intron and amino acid translation of gene 95 Diagram showing exon, intron and amino acid translation of gene 95 Diagram showing exon, intron and amino acid translation of gene 95 Diagram showing exon, intron and amino acid translation of gene 95 The figure which shows the alignment (alignment) of the amino acid sequence of the gene 95 of a human (sequence number 4) and a mouse | mouth (sequence number 5). The figure which shows alignment (alignment) with the wild type amino acid sequence (sequence number 4) of the amino acid sequence of the mutation 1 (sequence number 7) and the mutation 2 (sequence number 16) of the human gene 95 Diagram showing the predicted exon binding of gene 95 with the positions of mutations and polymorphisms identified according to the present invention The figure which shows the Northern blot using the gene 95 probe which shows the result of Example 3

Claims (50)

A method for identifying a substance that modulates gene 95 activity comprising:
a) contacting a test compound with a genetic construct comprising a reporter gene operably linked to the gene 95 promoter under conditions that support transcription of said reporter gene;
b) measuring a change in transcription of the reporter gene as a result of the contact;
Wherein said transcriptional change indicates that the test compound is a substance that modulates gene 95 activity.   The method of claim 1, wherein the measured change in transcription of step b) is a reduction in transcription.   The method of claim 1, wherein the measured change in transcription of step b) is an increase in transcription.   2. The method of claim 1, wherein transcription is measured by measuring the amount of expression product encoded by the reporter gene.   5. The method according to claim 4, wherein the expression product is RNA.   5. The method according to claim 4, wherein the expression product is a polypeptide.   2. The method of claim 1, wherein the reporter gene is in a liposome.   2. The method of claim 1, wherein the reporter gene is in an intact cell.   9. The method of claim 8, wherein the intact cell is a mammalian cell.   2. The method of claim 1, wherein the promoter is a mammalian gene 95 promoter.   11. The method of claim 10, wherein the gene 95 promoter is a human promoter.   12. The method of claim 11, wherein the human promoter comprises the promoter sequence of SEQ ID NO: 15.   11. The method according to claim 10, wherein the gene 95 promoter is a mouse promoter.   14. The method of claim 13, wherein the mouse promoter comprises the promoter sequence of SEQ ID NO: 14.   The method of claim 1, wherein the reporter gene is not gene 95. A method for identifying a substance that modulates gene 95 activity comprising:
a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to gene 95 under conditions that support the activity of said polypeptide;
b) measuring a change in the activity of the polypeptide as a result of the contact;
Wherein the change in activity identifies the test compound as a substance that modulates gene 95 activity.   17. A method according to claim 16, wherein the measured change in activity of step b) is a decrease in activity.   17. A method according to claim 16, characterized in that the change in activity measured in step b) is an increase in activity.   The method of claim 16, wherein the activity is measured by measuring the activity of an enzyme.   17. The method of claim 16, wherein the polypeptide is present in a lipid bilayer.   21. The method of claim 20, wherein the lipid bilayer is part of a liposome.   17. The method of claim 16, wherein the polypeptide is part of an intact cell.   24. The method of claim 22, wherein the intact cell is a cell that has been treated to contain the polypeptide.   23. The method according to claim 22, wherein the intact cell is a recombinant cell that has been genetically modified to express the polypeptide.   25. The method of claim 24, wherein the cell does not express the polypeptide when the recombination treatment is absent.   24. The method of claim 22, wherein the cell is a mammalian cell.   50. A method for identifying an HDL-enhancing agent, said method comprising the step of: identifying a substance found to have regulatory activity in an animal using the analysis according to any one of claims 1, 13, 24 or 49. Administering an effective amount and detecting an increase in plasma HDL activity in said animal as a result of said administration, thereby identifying a substance useful for enhancing HDL activity.   28. The method of claim 27, wherein the animal exhibits low HDL activity prior to administration of the substance.   28. The method of claim 27, wherein the HDL enhancing activity is an increase in HDL levels in the animal.   30. The method of claim 29, wherein the increase in HDL level is an increase in plasma HDL level.   30. The method of claim 29, wherein the animal is a human patient.   28. A method for treating said disease in an animal afflicted with a low HDL-related disease, said method comprising: an effective amount of a substance found to have HDL enhancing activity in said animal using the analytical method of claim 27 Administering.   33. The method of claim 32, wherein the animal is a human patient.   35. The method of claim 32, wherein the disease is selected from the group consisting of low HDL disease, vascular disorder and dyslipidemia. A method for generating test data relating to the modulating activity of a test compound, said method comprising:
(A) a polynucleotide construct comprising a reporter gene operably linked to a gene 95 polynucleotide or gene 95 polypeptide or gene 95 promoter and a test compound, the polynucleotide or reporter gene is transcribed, or the polynucleotide Contacting under conditions that activate the peptide,
(B) measuring the activity of the polypeptide as a result of the contact, or a change in transcription of the polynucleotide or reporter gene;
(C) generating test data relating to the modulating activity of the test compound based on a measured change in transcription of the polynucleotide or reporter gene or a measured change in activity of the polypeptide,
Wherein the change is indicative of regulatory activity.   A method for the treatment or prevention of heart disease, said method comprising the step of treating or preventing a dose of a compound that modulates gene 95 protein biological activity or expression in a patient in need of treatment or prevention of heart disease. Administering. Administering the method. A method for identifying a therapeutic agent, the method comprising:
a) contacting a chemical with a polynucleotide corresponding to gene 95 under conditions that support expression of said polynucleotide;
b) measuring a change in expression of the polynucleotide as a result of the contact;
Wherein said change in expression identifies a therapeutic agent. A method for identifying a therapeutic agent that modulates the activity of a polypeptide that affects high density lipoprotein (HDL) activity in vivo, said method comprising:
a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to gene 95 under conditions that support the activity of said polypeptide;
b) measuring a change in the activity of the polypeptide as a result of the contact;
Wherein said change in activity identifies said test compound as a therapeutic agent that modulates the activity of a polypeptide that acts on HDL activity.   40. The method of claim 38, wherein the polypeptide corresponds to SEQ ID NO: 4.   40. The method of claim 38, wherein the polynucleotide corresponds to SEQ ID NO: 3.   An isolated polynucleotide comprising a polynucleotide sequence or a complete complement of the polynucleotide sequence, wherein said polynucleotide sequence is at least 95% identical to SEQ ID NO: 3.   An isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4.   An isolated polynucleotide comprising a polynucleotide having the sequence shown in SEQ ID NO: 3.   An isolated polypeptide comprising an amino acid sequence having at least 95% identity with the amino acid sequence set forth in SEQ ID NO: 4.   45. The isolated polypeptide of claim 44, wherein the isolated polypeptide comprises an amino acid sequence having at least 95% identity with the amino acid sequence set forth in SEQ ID NO: 4. Isolated polypeptide.   An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4.   An isolated polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.   42. A nucleic acid vector comprising the isolated polynucleotide of claim 41.   49. A recombinant host cell comprising the vector of claim 48.   A method for producing the polypeptide of SEQ ID NO: 4, characterized in that it comprises culturing the host cell of claim 49 under conditions that support the production of said polypeptide. Method.