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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• NUCLEIC ACID SEQUENCE ENCODING THE SAME AND USES THEREOF
• the present invention relates to novel vitamin D receptor related (VDRR) polypeptides.
• VDRR vitamin D receptor related
• Nucleic acid sequences encoding the same, expression vectors containing such sequences and host cells transformed with such expression vectors are also disclosed, as are methods for the expression of the novel VDRR polypeptides of the invention, and uses thereof.
• ONRs orphan nuclear receptors
• the present invention relates to novel vitamin D receptor related (VDRR) polypeptides, and formulations containing the same. Nucleic acid sequences encoding the VDRR polypeptides, expression vectors containing such sequences and host cells transformed with such expression vectors are also disclosed, as are methods for the expression of the novel VDRR polypeptides of the invention.
• the invention further relates to VDRR polypeptides for use as medicaments, and use of substances affecting VDRR signal transduction for the manufacture of medicaments for treating metabolic, proliferative or inflammatory condi- tions.
• FIG. 1 The cDNA sequence encoding the novel nuclear receptor polypeptide vitamin D receptor related gamma (VDRRg) is shown.
• Figure 2 Evolutionary neighbor-joining tree for VDRRg as given by DBD-HMM alignment.
• FIG. 3 Evolutionary neighbor-joining tree for VDRRg as given by LBD-HMM alignment.
• Figure 4 The deduced amino acid sequence of VDRRg is shown.
• FIG. 5 Expression of VDRRg in adult human tissues.
• FIG. 6 Vitamin D3 transactivate a GAL4-DBD VDR-LBD fusion protein but not a GAL4-DBD/VDRR ⁇ -LBD fusion protein in transient transfections of CV-1 cells.
• the number on the left hand side refer to relative luciferase activity of the GAL4-luciferase reporter gene.
• Figure 7 The cDNA sequence encoding VDRRg-2 with an alternatively spliced 5'- end compared to VDRRg is shown.
• FIG. 1 The effect of pregnenolone 16 ⁇ -carbonitrile (PCN), dexamethasone and an antiprogestin (RU486) as activators of VDRRg are shown.
• PCN pregnenolone 16 ⁇ -carbonitrile
• RU486 antiprogestin
• Figure 12 Percent similarity between the new genes VDRRg- 1 and VDRRg-2 and the known genes XOR-6. HVDR, CAR-1 and CAR-2.
• Figure 13 Percent identity between the new genes VDRRg- 1 and VDRRg-2 and the known genes XOR-6. HVDR, CAR-1 and CAR-2.
• the nucleic acid encoding the VDRR polypeptide contains a ligand-binding domain (LBD) characterized by the following amino acid sequence similarity, relative to the LBDs of hVDR and xONRl, respectively: (i) at least about 30% amino acid sequence similarity with the LBD of hVDR, suitably at least 35%) amino acid sequence similarity with the LBD of hVDR; and (ii) at least about 40% amino acid sequence similarity with the LBD of xONRl, suitably at least 45% amino acid sequence similarity with the LBD of xONRl . More particularly, the amino acid sequence similarity relative to the LBDs of hVDR and xONR 1 , respectively is
• amino acid sequence similarity' * refers to: lOOx Consensus Lenght divided by Consencus Length + Mismatsches + Gaps.
• amino acid sequence identity can also be used. Amino acid sequence identity is calculated by comparing the absolute amino acid residue identity. In Figure 13 the amino acid sequence identity between the new genes VDRRg- 1 and VDRRg-2 and the known genes are shown.
• nucleic acid sequences of the present invention are substatially the same as those given in Fig. 1 or Fig. 7, the same or alleles thereof.
• the present invention also relates to a nucleic acid probe for the detection of a nucleic acid sequence encoding a VDRR polypeptide in a sample.
• the probe comprises at least 14 contiguous nucleotides, and preferably at least 28 contiguous nucleo- tides, of the nucleic acid sequences given in Fig. 1 or Fig. 7.
• the nucleic acid probe can be used in a method for identifying clones encoding a VDRR polypeptide, wherein the method comprises screening a genomic or cDNA library with the probe under low stringency hybridization conditions, and identifying those clones which display a substantial degree of hybridization to said probe.
• the present invention further relates to an isolated or recombinant VDRR polypeptide.
• the polypeptide can be full-length, at which the sequence of amino acids is identical to the corresponding sequence found in mammals in general, and in human beings in particular.
• the polypeptide can also be a truncated, extended or mutated form of the full-length polypeptide. Truncated and extended forms relate to VDRR polypeptides where one or more amino acids are missing or have been added, respectively, at the N terminal end of the polypeptide chain. Mutated forms relate to VDRR polypep-tides where one or more amino acid has been substituted by another amino acid.
• the isolated or recombinant VDRR polypeptide exhibits the amino acid sequences given in Fig. 4 or Fig. 8.
• N-terminal sequence of the present nucleic acids encoding VDRR polypeptides may vary.
• various N-terminal isoforms are envisaged, e.g. any of cd, ⁇ 2, ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ l or ⁇ 2 as disclosed in Fig. 7B of Transcription Factors 3: nuclear receptors. Protein Profile, vol. 2, issue 11 (1995), pp. 1173-1235. This review of nuclear receptors generally is hereby inco- ⁇ orated by reference. More specifically, Vitamin D receptors and related o ⁇ hans, e.g. ONR1, are discussed at p. 1 191-1992.
• the present invention further relates to pharmaceutical formulations comprising an isolated or recombinant VDRR polypeptide, and one or more therapeutically acceptable excipients.
• excipients that can be used are carbohydrates, e.g. monosaccharides, disaccharides and sugar alcohols, such as saccharose and sorbitol. Further examples include amino acids, e.g. histidine and arginine.
• surfactants e.g. polyoxyethylene sorbitan fatty acid esters, inorganic salts, e.g. sodium chloride and calcium chloride, and complexing agents, e.g. EDTA and citric acid.
• the present formulation can be in the form of an aqueous solution ready-for-use, or dried, particularly lyophilized. In the latter case, the formulation is reconstituted with a liquid, e.g. sterile water or saline, before use.
• a liquid e.g. sterile water or saline
• the present invention further relates to a process for recombinant production of a VDRR polypeptide, by expressing the claimed isolated or recombinant contiguous nucleic acid sequence encoding a Vitamin D receptor related (VDRR) polypeptide in a suitable host cell, preferably an eukaryotic cell.
• VDRR Vitamin D receptor related
• the present invention further relates to method for identifying a ligand to a VDRR, e.g. by a cell-based reporter assay, transgenic-animal reporter assay or in vz ' tr ⁇ -binding assay. It also relates to a method for identifying a substance for treatment of a condition affected by a VDRR polypeptide, comprising screening for an agonist or an antagonist of VDRR polypeptide signal transduction to be used for treating metabolic, proliferative or inflammatory conditions.
• the present invention further relates to a method for treating metabolic, proliferative or inflammatory conditions by introducing into a mammal a nucleic acid vector encoding for expression of a VDRR polypeptide.
• the nucleic acid vector is capable of transforming a cell in vivo and expressing said polypeptide in said transformed cell.
• the present invention further relates to a method for treatment of a metabolic, proliferative or inflammatory condition by administration of a therapeutically effective amount of a substance affecting VDRR signal transduction, specifically a VDRR polypeptide.
• the term "isolated" in connection with VDRR polypeptides or nucleic acids encoding the same relates to nucleic acids or polypeptides that have been isolated from a natural source, e.g. the liver, small intestine or colon of a human being.
• the isolated VDRR polypeptides or nucleic acids of the present invention are unique in the sense that they are not found in a pure or separated form in nature.
• Use of the term "isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free environment or in a different cellular environment.
• nucleic acid or polypeptide should be essentially free of non-amino acid or non-nucleic acid material naturally associated with the respective product. In this context, essentially free relates to more than 80%, suitably more than 90%, and preferably more than 95% purity.
• the inventors of the present invention have su ⁇ risingly isolated a novel nucleic acid sequence, and a polypeptide encoded by said nucleic acid sequence.
• a novel cDNA encoding a polypeptide designated VDRR ⁇ has been cloned and characterized.
• This polypeptide is, based on amino acid sequence similarity, a novel member of the nuclear (hormone) receptor supergene family.
• the number of amino acid residues in this part of the DBD is six (Cys-X6-Cys-X9-Cys-X2-Cys) as shown in Figs.4 and 8.
• the only other nuclear receptor like sequences found in the TREMBLE data base with the same number of amino acid residues between the two cys residues are two sequences (Q20097 and Q18155) from the worm C. elegans (Q20097 and Q18155).
• the entire DBD of these putative C. elegans nuclear receptors are only distantly related to the DBD of VDRRg.
• VDRR ⁇ relates to the various polypeptides corresponding to the differentially spliced VDRR ⁇ cDNAs including VDRR ⁇ - 1 and VDRR ⁇ -2.
• VDRR ⁇ cDNA and VDRR ⁇ relates specifically to VDRR ⁇ - 1 cDNA and VDRR ⁇ - 1 , respectively.
• VDRR ⁇ cDNA and VDRR ⁇ relates specifically to VDRR ⁇ -2 cDNA and VDRR ⁇ -2, respectively.
• the VDRR ⁇ - 1 cDNA does not contain a classical AUG initiation codon but instead may initiate at an alternative CUG codon.
• This putative non-AUG start site is located in a favorable sequence context for efficient initiation from alternative start sites and is in frame with the entire open reading frame and preceded by a stop codon.
• VDRRs in general, and more specifically the VDRR ⁇ may be important in
• VDRR ⁇ The high amino acid sequence identity of VDRR ⁇ with the VDR both in the DNA- binding domain (DBD) and ligand-binding domain (LBD) indicate that these two receptors may also have overlapping yet distinct functional characteristics.
• retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have similar amino acid sequence identities in the DBD and LBD region as the VDR and VDRR ⁇ .
• RARs and RXRs have been shown to have distinct functional similarities such that both receptors bind 9-cis retinoic acid and have overlapping DNA-binding specificities and accordingly regulate overlapping gene networks.
• the substance affecting VDRR signal transduction can be any small chemical molecule of natural or synthetic origin, e.g. a carbohydrate such as an aromatic compound.
• the small molecule may have a molecular weight in the range of from about 100 up to about 500 Da.
• the small chemical molecule has a molecular weight in the range of from 200 up to 400 Da.
• the small chemical molecule has a molecular weight of about 300 Da.
• VDRR ⁇ polypeptides including VDRR ⁇ - 1 and VDRR ⁇ -2, have been shown to be activated e.g. by pregnenolones and estradiol (weakly), but not by certain other steroid hormones such as cortisol, aldosterone, progesterone and estrogen, and most likely not by progestines and glucocorticoids.
• human VDRR ⁇ is not activated by pregnenolone 16 ⁇ -carbonitrile (PCN), a glucocorticoid antagonist.
• PCN pregnenolone 16 ⁇ -carbonitrile
• human VDRR ⁇ can also be designated human pregnenolone activated (nuclear) receptors (hPAR).
• Information about pregnenolone can be found e.g. in the Merck Index, 11th ed., Merck & Co., Inc. Rahway, N.J., USA, p. 7735, 1989.
• Genes coding for polypeptides may be cloned by inco ⁇ orating a DNA fragment coding for the polypeptide into a recombinant DNA vehicle, e.g. a vector, and transforming suitable prokaryotic or eukaryo-tic host cells.
• a recombinant DNA vehicle e.g. a vector
• suitable prokaryotic or eukaryo-tic host cells e.g.
• the clone was found to encode a putative ligand-binding domain (LBD) with 54% and 44% similarity to xONR-1 and to the vitamin D receptor (VDR), respectively.
• LBD putative ligand-binding domain
• VDR vitamin D receptor
• VDRRg mRNA Expression of VDRRg mRNA in human tissues
• VDRR ⁇ or RXR ⁇ cDNAs were transcribed using T7 polymerase and translated in vitro in TNT reticulocyte lysates (Promega, Madison, WI, USA).
• T7 polymerase T7 polymerase
• RXR ⁇ cDNAs TNT reticulocyte lysates
• a native gel mobility assay was employed essentially as described (Berkenstam et al., Cell, 69, 401-412, 1992) in which in vitro translated VDRR ⁇ was incubated in the presence or absence of in vitro translated RXR ⁇ with different 32P-labelled direct repeats (DR-1 to DR-5) as indicated in Fig. 9.
• the direct repeats were derived from the DR-5 element in the RAR- ⁇ 2 promoter (de The et al., Nature, 343, 177-180, 1990) and modified to be separated by one to five nucleotides (Pettersson et al., Mechanisms of Dev., 54, 1-13, 1995). Protein-DNA complexes were separated on native 5%> polyacryl-amide/0.25xTBE gels followed by autoradiography. As shown in Fig. 9, of the five DRs tested efficient VDRR ⁇ binding could only be detected with DRs separated by three or four nucleotides and only in the presence of RXR. However, weaker RXR-dependent binding could also be observed to DR-2 and DR-1 elements.

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