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  Pharmaceutical Patents  

 

Title:  Insulin-responsive DNA binding protein-1 and methods to regulate insulin-responsive genes
United States Patent; 
7,563,775
Issued: 
July 21, 2009

Inventors: 
Villafuerte; Betty C. (Louisville, KY)
Assignee: 
Villafuerte; Betty C. (Lousiville, KY)
Appl. No.: 
10/310,002
Filed: 
December 4, 2002


 

Training Courses --Pharm/Biotech/etc.


Abstract

The present invention relates to the novel protein Insulin-Responsive DNA Binding Protein-1 (IRDBP-1) and nucleotide sequences that encode it. IRDBP-1 binds to nucleic acid regions of genes that respond when cells are exposed to insulin. IRDBP-1 regulates genes important in mediating the insulin response in mammals and in regulating conditions such as diabetes, obesity, insulin-resistant syndrome and cell proliferative disorders. The present invention provides nucleic acids useful as probes for detecting nucleic acids encoding regions of the IRDBP-1 protein. Within the scope of the present invention are recombinant cells, tissues and animals containing non-naturally occurring recombinant nucleic acid molecules encoding IRDBP-1, including expression vectors, antibodies specific for IRDBP-1, assays for IRDBP-1 polypeptide, and methods relating to all of the foregoing, the development of therapeutic and diagnostic agents that mimic, facilitate or inhibit the action of IRDBP-1, and/or are based on relationships to the structure and action of IRDBP-1.

Description of the Invention

Rat and Human IRDBP-1 Nucleic Acids

One aspect of the present invention provides isolated nucleic acids, derivatives and variants thereof that encode human or rat IRDBP-1 proteins, derivatives or variants thereof. IRDBP-1 protein or functionally active derivatives or fragments thereof are particularly useful as direct or indirect modulators of gene expression wherein the genes so modulated comprise an IRE and are capable of responding to fluctuations in insulin levels. The present invention further provides an isolated nucleic acid encoding a fragment of a rat IRDBP-1 protein isolated based on the ability of the expressed protein product thereof to bind to the nucleic acid Insulin Responsive Element (IRE) associated with the rat IGFBP-3 and which has the nucleotide sequence 5'-AATTCAAGGGTATCCAGGAAAGTCTCC-3' (SEQ ID NO: 1). As used herein, IREs are regulatory nucleic acid sequences of insulin-regulated genes that are necessary to enable an insulin-dependent response. The nucleotide sequence of SEQ ID NO: 1 is localized between the -1150 and the -1124 bp positions of the promoter region of the IGFBP-3 encoding gene of the rat.

A rat liver cDNA library using the yeast one-hybrid system was screened using concatemerized IREs of rat IGFBP-3, using methods described by Wang & Reed (1993) Nature 364: 121-126, incorporated herein by reference in its entirety, and discussed in Example 1 below. The cDNA library screening provided a novel 952-bp cDNA (clone 52) encoding a portion of the Insulin-Responsive DNA Binding Protein-1 (IRDBP-1) that was identified and sequenced (SEQ ID NO: 2) (GenBank Accession No. AF439714), as illustrated in FIG. 1 (see Original Patent). The nucleic acid sequence of clone 52 (SEQ ID NO: 2) encodes a polypeptide having the amino acid sequence of SEQ ID NO: 3, as shown in FIG. 2 (see Original Patent), capable of binding to the IRE region of the rat IGFBP-3 (SEQ ID NO: 1), as described in Example 1 and 4.

A clone 52-thioredoxin (Trx) fusion protein also binds to the rat IGFBP-3 IRE SEQ ID NO: 1. The amino acid sequence SEQ ID NO: 3 deduced from the nucleotide sequence (SEQ ID NO: 2) of clone 52 comprises a homeodomain motif typical of transcription factors. Binding by the polypeptide SEQ ID NO: 3 to the IRE of IGFBP-3 (SEQ ID NO: 1) could be competed away by IGFBP-3 IRE nucleic acids but not by nucleic acids of sequences unrelated to the IRE, as shown in Example 1. The interaction between the IRDBP-1-related polypeptide (SEQ ID NO: 3) and the IGFBP-3 IRE nucleic acid (SEQ ID NO: 1) was specific.

The IRDBP-1 polypeptide fragment (SEQ ID NO: 3) encoded by clone 52 also interacts with IREs associated with other insulin-responsive genes besides IGFBP-3, as shown in Examples 4 and 7 below. The polypeptide interacts with the IREs from insulin-responsive genes encoding IGF-1, IGFBP-1, phosphoenol pyruvate carboxykinase (PEPCK), amylase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

IGFBP-1 is a hepatic acute phase reactant protein that coordinates the level of IGF-1 in response to changes in insulin levels (Lee et al. (1993)). Amylase is important for intestinal hydrolysis of carbohydrates. GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, a rate-limiting step in adipose tissue glycolysis. While not being bound by any theory, the naturally occurring IRDBP-1 protein is likely a transcription factor that coordinates the responses of several genes to insulin. The IRDBP-1 polypeptide (SEQ ID NO: 3) of clone 52 can regulate critical genes in target tissues implicated in insulin resistance and insulin secretion. While not wishing to be held to any one theory, it is believed that th enaturally occurring IRDBP-1 polypeptide modulates the pleiotropic actions of insulin in the normal metabolism and storage of ingested carbohydrate and other fuels, in the modulation of intermediary metabolism, and in normal cellular growth and differentiation.

Ribonuclease protection assays (discussed in Example 3) using an antisense RNA probe obtained by transcribing a Kpn1-Xhol fragment of clone 52 and having the nucleic acid sequence SEQ ID NO: 4, as shown in FIG. 3 (see Original Patent), showed that at least one gene, encoding at least one nucleic acid with sequence similarity to a region of the clone 52 cDNA sequence SEQ ID NO: 2 is expressed in at least liver, kidney, brain, small intestine, muscle, and fat pads.

The abundance of a rat RNA transcript capable of hybridizing to the probe having a nucleic acid sequence of the clone 52 (SEQ ID NO: 4) was increased with the addition of physiological concentrations of insulin (10.sup.-9 M) in cell culture. It was also decreased in the livers of diabetic rats, as described in Example 8.

Another aspect of the present invention provides for the use of the isolated cDNA clone 52 (SEQ ID NO: 2) as a probe to screen rat and human cDNA libraries to obtain isolated nucleic acids capable of hybridizing with clone 52, as discussed in Example 5. Nucleic acid regions extending the cDNA sequences in the 5' direction from the isolated human and rat partial cDNA clones were obtained by primer extension reactions such as 5' RACE, and then sequenced.

The present invention further provides rat cDNA clones that hybridize to the clone 52 probe, and were identified and sequenced as SEQ ID NOS: 5, 6, 14 (GenBank Accession Nos: AF 439715, AF439716, and AF439719, respectively) and SEQ ID NO: 44 and shown in FIGS. 4A-4B, 5A-5B, 12A-12B, and 13, respectively (see Original Patent). A first rat IRDBP-1 cDNA clone (SEQ ID NO 5; shown in FIGS. 4A and 4B (see Original Patent)) comprises about 4998 bp, and includes at least one open reading frame (ORF) (SEQ ID NO: 8) as in FIG. 4A (see Original Patent) and which encodes a rat ISRBP-1 protein (SEQ ID NO: 11; FIG. 4). The nucleotides at positions 68-349 of clone 52 (SEQ ID NO: 2) correspond to the nucleotide positions 2123-2404 of SEQ ID NO: 5 as shown in FIGS. 4A-4B. A second rat cDNA clone (SEQ ID NO: 6, shown in FIGS. 5A and 5B (see Original Patent)) is a partial cDNA comprising a partial open-reading frame (ORF) (FIG. 5A) having sequence similarity to a region of SEQ ID NO: 5 (FIG. 4A), and a 3' untranslated region (FIG. 5B) longer than that of SEQ ID NO: 5 (shown in FIG. 5B). Nucleic acid SEQ ID NO: 44 and the protein sequence encoded therein (SEQ ID NO: 47) are shown in FIGS. 6 and 15, respectively (see Original Patent).

The present invention also provides for the human cDNA clones having the nucleic acid sequences SEQ ID NO: 7 as shown in FIGS. 6A-6C, SEQ ID NO: 10 as shown in FIGS. 8A and 8B (see Original Patent)) that were also identified by hybridization with a probe comprising the clone 52 nucleic acid sequence (SEQ ID NO: 2) during the screening of a human cDNA library, and SEQ ID NO: 45 (shown in FIG. 45 (see Original Patent)) generated by 5' RACE extension of the isolated clone SEQ ID NO: 7.

It is contemplated that any nucleic acid of the present invention can comprise one or more regulatory regions, full-length or partial coding regions such as, but not limited to, the fragments SEQ ID NOS: 16-41 (FIG. 7 (see Original Patent)) derived from the IRDBP-1 gene, or any combinations thereof. It is contemplated to be within the scope of the present invention for a probe to be derived from any of SEQ ID NOS: 2, 5-10, 14, 16-41 and 44-45 or a variant or truncated variant thereof. The minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybridization product with the complementary sequence of another nucleic acid molecule under selected stringency conditions.

Embodiments of the present invention may, therefore, include, but are not limited to, nucleic acid molecules such as: a) an IRDBP-1 cDNA molecule derived from the rat and comprising the protein coding region (SEQ ID NO: 8, shown in FIG. 4A) of SEQ ID NO: 5 or the coding region of SEQ ID NO: 44, and a 3' non-coding, or untranslated, region of SEQ ID NOS: 5 or 44; b) an IRDBP-1 cDNA molecule derived from the rat nucleic acid SEQ ID NOS: 5 or 44 and comprising the isolated coding region (SEQ ID NO: 8), or a substantial region thereof; or nucleic acid molecules representing degenerate variants, derivatives, modified sequences and truncated variants such as, but not limited to, SEQ ID NO: 6 shown in FIGS. 5A and 5B, thereof; c) an IRDBP- 1-encoding cDNA molecule derived from the human comprising the protein coding region and a 5' and/or 3' non-coding regions of the sequence SEQ ID NO: 7 (GenBank Accession No. AF439717) as shown in FIG. 6A, or SEQ ID NO: 45 shown in FIG. 14 (see Original Patent); d) a nucleic acid molecule derived from the human IRDBP-1 cDNA sequence SEQ ID NO: 7 and comprising the human IRDBP-1 coding region alone (SEQ ID NO: 9), as depicted in FIG. 6B, or the coding region of SEQ ID NO: 45; and/or nucleic acid molecules representing degenerate variants, derivatives, alternatively spliced variants and modified variants thereof. A variant may be, but is not limited to, the sequence SEQ ID NO: 10 (GenBank Accession No. AF439718) as shown in FIGS. 8A and 8B. Such nucleic acid molecules can include nucleotides in addition to those included in SEQ ID NOS: 2, 5-10, 14, and 44-45 such as, but not limited to, a full-length gene, a full-length coding region, or a nucleic acid molecule encoding a fusion protein. BLASTN algorithm searching of the Genbank database using the human IRDBP-1 nucleic acid sequence SEQ ID NO: 7 or 45 as the search target found that there was almost 100% identity with regions of the human genomic DNA sequence GenBank Accession No. AC005237 from the human chromosome 1p31.31.3-32.2 and at least one human gene encoding the IRDBP-1 transcribed nucleic acid and protein derived therefrom is comprised of at least 26 exons as shown in Table 1, Example 5 (see Original Patent). The present invention, therefore, is intended also to provide isolated nucleic acids comprising at least one exon, or a fragment, variant or derivative thereof, capable of hybridizing with at least one region of the sequences SEQ ID NOS: 2, 5-10, 14, and 44-45 under low, medium or high stringency conditions, wherein the hybridization is specific for an IRDBP-1-encoding nucleic acid, or a fragment, variant or derivative thereof.

One aspect of the invention therefore also provides nucleic acids that hybridize under selected high, medium or low stringency conditions to a nucleic acid that encodes a peptide having all of, a derivative of, or a portion of an amino acid sequence derived from the nucleic acid sequences SEQ ID NOS: 2, 5-10, 14, and 44-45. Appropriate stringency conditions which promote DNA hybridization, for example, 6.times.SSC at about 45.degree. C., followed by a wash of 2.times.SSC at 50.degree. C., are well known to those skilled in the art or can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.times.SSC at 50.degree. C. to a high stringency of about 0.2.times.SSC at 50.degree. C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22.degree. C., to high stringency conditions at about 65.degree. C.

Isolated nucleic acids that differ in sequence from the nucleotide sequences represented in SEQ ID NOS: 2, 5-10, 13, and 44-45 due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids can encode functionally equivalent peptides (i.e., a polypeptide having a biological activity of a IRDBP-1 protein). Isolated nucleic acid sequence variants may also encode non-functional polypeptides, the sequences of which are substantially similar, but not identical, to those of functional variants of IRDBP-1. These isolated nucleic acids may be used to generate variant animals with inactive or functionally modified IRDBP-1 polypeptides or fragments, variants or derivatives thereof.

For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the subject protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the present IRDBP-1 protein of the present invention will exist from one human or animal subject to the next of the same species. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding peptides having an activity of, for example, an IRDBP-1 protein may exist among individuals due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. Nucleic acid variants having sequence differences of about 3-4% may be readily detectable under high or medium stringency hybridization conditions using, for example, any of SEQ ID NOS: 2, 5-10, 13 or 44-45 or fragments thereof, such as SEQ ID NO: 4, as the probe.

Fragments of a nucleic acid encoding an active portion of one of the subject IRDBP-1 proteins are also within the scope of the invention. As used herein, a fragment of the nucleic acid encoding an active portion of a IRDBP-1 protein refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of the protein but which encodes a peptide that possesses agonistic or antagonistic activity relative to a naturally occurring form of the protein.

Nucleic acid fragments within the scope of the invention also include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species for use in screening protocols to detect IRDBP-1 homologs. Comparison of the nucleic acid sequences of rat and human IRDBP-1 show that oligonucleotide primers can be generated that are suitable for detecting and isolating IRDBP-1 clones in other eukaryotes. For example, the cDNA clone 52 (SEQ ID NO: 2) could be used to detect IRDBP-1 homologs in other vertebrate species, such as, but not only, human, mice, rats, chickens. Thus SEQ ID NO: 2 was used to identify a hybridizing human IRDBP-1-encoding cDNA SEQ ID NO: 7 under medium stringency hybridization conditions.

One embodiment of the present invention, therefore, provides a nucleic acid comprising a nucleic acid sequence substantially similar to the clone 52 cDNA sequence (SEQ ID NO: 2) encoding at least a region of a rat IRDBP-1 protein (SEQ ID NO: 3) as shown in FIGS. 1 and 2 respectively, or any variants thereof. The nucleic acid molecules of the present invention can include an isolated deletion mutation corresponding to the IRDBP-1 phenotype, a natural IRDBP-1 gene, an IRDBP-1 cDNA molecule, a degenerate variant, a truncated form thereof, a homolog thereof or any other modified versions.

In another embodiment of the present invention, a nucleic acid is provided comprising a nucleic acid sequence substantially similar to the cDNA sequence for a rat IRDBP-1 (SEQ ID NO: 5) as shown in FIGS. 4A and 4B, or any variant thereof. The nucleic acid molecules of the present invention can include an isolated deletion mutation corresponding to the IRDBP-1 phenotype, a natural IRDBP-1 gene, an IRDBP-1 cDNA molecule, a degenerate variant thereof, a truncated variant thereof or a homolog thereof or any other variant thereof, including a human IRDBP-1-encoding nucleic acid having at least 75% sequence similarity to SEQ ID NOS: 2 or 5.

In yet another embodiment of the present invention, a nucleic acid is provided comprising a nucleic acid sequence substantially similar to the cDNA sequence for a rat IRDBP-1 (SEQ ID NO: 6) shown in FIGS. 5A and 5B comprising a variant of SEQ ID NO: 5.

In yet another embodiment of the present invention, an isolated nucleic acid is provided that comprises the nucleic acid sequence corresponding to a human IRDBP-1 sequence SEQ ID NO: 7 as shown in FIGS. 6A-6C.

In another embodiment of the present invention, an isolated nucleic acid is provided that comprises the nucleic acid sequence corresponding to a variant human IRDBP-1 (SEQ ID NO: 10) as shown in FIGS. 8A and 8B.

In still another embodiment of the present invention, a mammalian IRDBP-1 gene or nucleic acid molecule can be allelic variants of SEQ ID NOS: 2, 5-10 and 44-45. An allelic variant is a gene that occurs essentially at the same locus or loci in the mammalian genome as the genes comprising SEQ ID NOS: 5-10, 14 and 44-45, but which has similar, but not identical, sequences to that of SEQ ID NO: 5-10 and 44-45.

In one embodiment of the present invention, an isolated nucleic acid molecule of the present invention includes a nucleic acid that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical to a rat-derived IRDBP-1-encoding nucleic acid molecule as depicted in SEQ ID NO: 5 or 44, and/or a variant thereof, such as, but not limited to, SEQ ID NOS: 6 and 14 or the human IRDBP-1 nucleic acids SEQ ID NOS: 7, 10 and 45.

In another embodiment of the present invention, an isolated nucleic acid molecule of the present invention includes a nucleic acid that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably. at least about 90%, and even more preferably at least about 95% identical to a human-derived nucleic acid molecule as depicted in SEQ ID NOS: 7 and 45, and/or a variant thereof, such as, but not limited to, SEQ ID NO: 10.

The nucleic acid sequences of a IRDBP-1 nucleic acid molecules (SEQ ID NOS: 2, 5-10, 14 and 44-45) of the present invention allow one skilled in the art to, for example, (a) make copies of those nucleic acid molecules by procedures such as, but not limited to, insertion into a cell for replication by the cell, by chemical synthesis or by procedures such as PCR or LCR, (b) obtain nucleic acid molecules which include at least a portion of such nucleic acid molecules, including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions and the like, (c) obtain IRDBP-1 nucleic acid homologs in other mammalian species such as the dog, cat, cow, pig or primates other than human and, (d) to obtain isolated nucleic acids capable of hybridizing to a mammalian IRDBP-1 nucleic acid and be used to detect the presence of IRDBP-1 nucleic acid sequences by complementation between the probe and the target nucleic acid.

Such nucleic acid homologs can be obtained in a variety of ways including by screening appropriate expression libraries with antibodies of the present invention; using traditional cloning techniques employing oligonucleotide probes made according to the present invention to screen appropriate libraries; amplifying appropriate libraries or DNA using oligonucleotide primers of the present invention in a polymerase chain reaction or other amplification method; and screening public and/or private databases containing genetic sequences using nucleic acid molecules of the present invention to identify targets. Examples of preferred libraries to screen, or from which to amplify nucleic acid molecules, include but are not limited to mammalian BAC libraries, genomic DNA libraries, and cDNA libraries. Similarly, preferred sequence databases useful for screening to identify sequences in other species homologous to IRDBP-1 include, but are not limited to, GenBank and the mammalian Gene Index database of The Institute of Genomics Research (TIGR).

IRDBP-1 Polypeptides

Another aspect of the present invention is to provide protein sequences that comprise a mammalian IRDBP-1 protein, and derivatives and fragments thereof. One embodiment of the present invention, therefore, comprises a protein sequence (SEQ ID NO: 3, as shown in FIG. 2) encoded by the rat cDNA clone 52 nucleic acid sequence SEQ ID NO: 2.

In another embodiment of the present invention, a rat IRDBP-1 protein is provided having an amino acid sequence (SEQ ID NO: 11, illustrated in FIG. 9) derived from the coding region SEQ ID NO: 8, as in FIG. 4A, of the rat cDNA clone IRDBP-1 SEQ ID NO: 5.

In still yet another embodiment of the present invention, a rat IRDBP-1 protein sequence SEQ ID NO: 47, illustrated in FIG. 15 is provided that is encoded by the coding region of human nucleic acid sequence SEQ ID NO: 44 (FIG. 13).

In yet another embodiment of the present invention, a human IRDBP-1 protein sequence (SEQ ID NO: 12, illustrated in FIG. 10) is provided that is encoded by a coding region SEQ ID NO: 9 of the human nucleic acid sequence SEQ ID NO: 7, as shown in FIG. 6B.

In still yet another embodiment of the present invention, a human IRDBP-1 protein sequence SEQ ID NO: 48, illustrated in FIG. 16 is provided that is encoded by the coding region of human nucleic acid sequence SEQ ID NO: 45 (FIG. 14).

In still other embodiments of the present invention, peptide fragments of a human or animal IRDBP-1 protein are provided, wherein the fragments may be immunogenic peptides, capable of inducing an immune response when administered to an animal, and which will be recognized and bound by an antibody or not immunogenic when administered to an animal.

In one embodiment of the present invention, the peptide fragment is an epitope essentially within the carboxy-region of the rat IRDBP-1 protein SEQ ID NO: 3 (as in FIG. 2) and has the amino acid sequence: AcetylatedCys-Thr-Ser-Gln-Asn-Thr-Lys-Ser-Arg-Ty-Iso-Pro-Asn-Gly-Lys-Leu (SEQ ID NO: 15) at amino acid positions 62-76 of the rat IRDBP-1 amino acid sequence SEQ ID NO: 3 shown in FIG. 2.

In another embodiment, the epitope is substantially within the N-region of the IRDBP-1 protein between amino acid positions 233-247 of SEQ ID NO: 44 and having the sequence AcetylatedCys-Arg-Asn-Gly-Gly-Thr-Tyr-Lys-Glu-Thr-Gly-Asp-Glu-Tyr-Arg (SEQ ID NO: 46).

It is further contemplated to be within the scope of the present invention for proteins having substantial similarity to the rat or human protein amino acid sequences SEQ ID NOS: 11, 12, 47 and 48 wherein the proteins retain the capacity to bind to the IGFBP-3 IRE SEQ ID NO: 1. Isolated peptides and polypeptides of the present invention may also include any protein fragments thereof, a protein analogue, or any immunologic fragments thereof.

In another embodiment of the present invention, an IRDBP-1 nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and more preferably still at least about 95% identical to a rat IRDBP-1 protein whose amino acid sequence is disclosed in SEQ ID NO: 11 or 47, as well as allelic variants of an IRDBP-1 nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.

In an embodiment of the present invention, an IRDBP-1 nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and more preferably still at least about 95% identical to a human IRDBP-1 protein whose amino acid sequence is disclosed in SEQ ID NO: 12 and 48, as well as allelic variants of an IRDBP-1 nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.

Isolated peptidyl portions of the subject IRDBP-1 proteins within the scope of the present invention can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, one of the subject IRDBP-1 proteins may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced recombinantly or by chemical synthesis and tested to identify those peptidyl fragments which can function as either agonists or antagonists of, for example, IRDBP-1 binding to nucleic acids. Other fragments such as, for example, SEQ ID NOS: 15 and 46 are especially useful for the generation of antibodies specific for the IRDBP-1 protein or selected regions thereof. In an illustrative embodiment, peptidyl portions of IRDBP-1 can tested for nucleic acid-binding activity, as well as preventing inhibitory ability, by expression as, for example, thioredoxin fusion proteins each of which contains a discrete fragment of the IRDBP-1 protein (see, for example, U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT publication WO94/02502) incorporated herein by reference in their entireties.

Furthermore, it is also possible to modify the structure of an IRDBP-1 polypeptide for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). Such modified peptides are considered functional equivalents of peptides having an activity of, or which antagonize, a IRDBP-1 protein as defined herein. A modified polypeptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine. (see, for example, Biochemistry, 2nd ed, Ed. by L. Stryer, WH Freeman and Co.:1981). Whether a change in the amino acid sequence of a peptide results in a functional IRDBP-1 homolog can be readily determined by assessing the ability of the variant peptide to, for instance, mediate ubiquitination in a fashion similar to the wild-type IRDBP-1. Peptides in which more than one replacement has taken place can readily be tested in the same manner.

In one embodiment of the present invention, therefore, a host cell is transformed with a nucleic acid comprising the sequences SEQ ID NOS: 5-10, 14 or 44-45, or variants thereof. The transformed cell may, but not necessarily, express the transformed nucleic acid to yield rat (SEQ ID NOS: 3, 11, and 47) or human (SEQ ID NOS: 12-13 and 48) IRDBP-1 polypeptides respectively, or any fragment or derivative thereof. A recombinant expression vector suitable for transformation of a host cell means that the recombinant expression vector contains a nucleic acid molecule, or an oligonucleotide fragment thereof, of the present invention coupled to a regulatory sequence selected on the basis of the host cell used for expression. For example, the nucleic acid sequence coding for the IRDBP-1 protein of the present invention may be operatively linked to a regulatory sequence selected to direct expression of the desired protein in an appropriate host cell.

The protein of the present invention may be produced in purified form by any known conventional techniques. For example, rat or human cells may be homogenized and centrifuged. The supernatant is then subjected to sequential ammonium sulfate precipitation and heat treatment. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.

The present invention provides novel compositions comprising nucleotide sequences encoding IRDBP-1 fragments. Also provided are recombinant proteins produced using the novel coding sequences, and methods of using the recombinant proteins.

Recombinant Nucleic Acids Including IRDBP-1-related Sequences and Insertion into Vectors and Mammalian Cells

The DNA nucleic acid molecules of the present invention can be incorporated into cells using conventional recombinant DNA technology. Such techniques are especially useful, for example, for producing IRDBP-1 polypeptides in cells, or to regulate the expression of the naturally occurring IRDBP-1 gene in the recipient cells. The DNA molecule may be inserted into an expression system to which the DNA molecule is heterologous (i.e. not normally present). Alternatively, as described more fully below, the DNA molecule may be introduced into cells which normally contain the DNA molecule, as, for example, to correct a deficiency or defect in IRDBP-1 expression, or where over-expression of the IRDBP-1 protein is desired.

For expression in heterologous systems, the heterologous DNA molecule can be inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen & Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinia virus or adenovirus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus. Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, PGEX, pET series (see Studier, F. W. et. al. (1990) "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes" Gene Expression Technology, vol. 185, which is hereby incorporated by reference) and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.

A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation). Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals that differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgamo (SD) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts & Lauer (1979) Methods in Enzymology 68: 473, which is hereby incorporated by reference in its entirety.

Promoters vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P.sub.R and P.sub.L promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgamo (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

Once the isolated DNA molecule of the present invention has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, and the like.

Recombinant expression vectors can be designed for the expression of the encoded proteins in prokaryotic or eukaryotic cells. The prokaryotic expression system may comprise the host bacterial species E. coli, B. subtilis or any other host cell known to one of skill in the art. Useful vectors may comprise constitutive or inducible promoters to direct expression of either fusion or non-fusion proteins. With fusion vectors, a number of amino acids are usually added to the expressed target gene sequence such as, but not limited to, a protein sequence for thioredoxin. A proteolytic cleavage site may further be introduced at a site between the target recombinant protein and the fusion sequence. Additionally, a region of amino acids such as a polymeric histidine region may be introduced to allow binding to the fusion protein by metallic ions such as nickel bonded to a solid support, and thereby allow purification of the fusion protein. Once the fusion protein has been purified, the cleavage site allows the target recombinant protein to be separated from the fusion sequence. Enzymes suitable for use in cleaving the proteolytic cleavage site includes, but are not limited to, Factor Xa and thrombin. Fusion expression vectors that may be useful in the present invention include pGex (Amrad Corp., Melbourne, Australia), pRIT5 (Pharmacia, Piscataway, N.J.) and pMAL (New England Biolabs, Beverly, Mass.), that fuse glutathione S-transferase, protein A, or maltose E binding protein, respectively, to the target recombinant protein.

Expression of unfused foreign genes in E. coli may be accomplished with recombinant vectors including, but not limited to, the E. coli expression vector pUR278 as described in Ruther et aL (1983) E.M.B.O.J. 2: 1791, incorporated herein by reference in its entirety. Using the pUR278 vector, the nucleotide sequence coding for the IRDBP-1 gene product may be ligated in frame with the lacV coding region to produce a fusion protein.

Expression of a foreign gene can also be obtained using eukaryotic vectors such as mammalian, yeast or insect cells. The use of eukaryotic vectors permits partial or complete post-translational modification such as, but not only, glycosylation and/or the formation of the relevant inter- or intra-chain disulfide bonds. Examples of vectors useful for expression in the yeast Saccharomyces cerevisiae include pYepSecl as in Baldari et al., (1987), E.M.B.O.J, 6: 229-234 and pYES2 (Invitrogen Corp., San Diego, Calif.), incorporated herein by reference in their entirety.

Baculovirus vectors are also available for the expression of proteins in cultured insect cells (F9 cells). The use of recombinant Baculovirus vectors can be, or is, analogous to the methods disclosed in Richardson C. D. ed., (1995), "Baculovirus Expression Protocol" Humana Press Inc.; Smith et al. (1983) Mol. Cell. Biol. 3: 2156-2165; Pennock et al. (1984) Mol. Cell. Biol. 4: 399-406 and incorporated herein by reference in their entirety.

Other vectors useful for expressing the IRDBP-1 protein, or an epitope of a IRDBP-1 protein, include viral vectors. Methods for making a viral recombinant vector useful for expressing the IRDBP-1 protein are analogous to the methods disclosed in U.S. Pat. Nos. 4,603,112; 4,769,330; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 4,722,848; Paoletti, E. (1996) Proc. Natl. Acad. Sci. 93: 11349-11353; Moss (1996) Proc. Natl. Acad. Sci. 93: 11341-11348; Roizman (1996) Proc. Natl. Acad. Sci. 93: 11307-11302; Frolov et al. (1996) Proc. Natl. Acad. Sci. 93: 11371-11377; Grunhaus et al. (1993) Seminars in Virology 3: 237-252 and U.S. Pat. Nos. 5,591,639; 5,589,466; and 5,580,859 relating to DNA expression vectors, inter alia; the contents of which are incorporated herein by reference in their entireties.

One embodiment of the present invention, therefore, is a recombinant viral vector comprising an adenovirus vector capable of expressing in a suitable host cell a polypeptide encoded by at least a region of the nucleic acids SEQ ID NO: 44. The expressed polypeptide is capable of binding to an IRE, wherein the binding can be modulated by insulin, as described in Example 20.

In one embodiment of this aspect of the present invention, the recombinant adenoviral vector (or other suitable vector) may express the IRDBP-1 nucleic acid as an antisense nucleic acid that is not translated but, by hybridizing to a region of the IRDB-1 gene or a transcript thereof, can modulate the level of IRDBP-1 activity in a cell.

Probes, Primers and Sense/Antisense Oligonucleotides Specific for IRDBP-1

Another aspect of the present invention pertains to the use of an isolated nucleic acid molecule for constructing nucleotide probes and primers useful for a variety of functions. For example, synthetic oligonucleotide probes are useful for detecting complementary nucleotide sequences in biological materials such as cells, cell extracts or tissues (as well as in an in situ hybridization technique). Isolated nucleic acids synthesized according to the present invention can determine whether a cell expresses an mRNA transcript encoding the IRDBP-1 protein. The present invention also contemplates the use of antisense nucleic acid molecules, which are designed to be complementary to a coding strand of a nucleic acid (i.e., complementary to an mRNA sequence) or, alternatively, complimentary to a 5' or 3' untranslated region of the mRNA. Another use of synthetic nucleotides is as primers (DNA or RNA) for a polymerase chain reaction (PCR), ligase chain reaction (LCR), or the like.

Synthesized nucleotides can be produced in variable lengths--the number of bases synthesized will depend upon a variety of factors, including the desired use for the probes or primers. Additionally, sense or anti-sense nucleic acids or oligonucleotides can be chemically synthesized using modified nucleotides to increase the biological stability of the molecule or of the binding complex formed between the anti-sense and sense nucleic acids. For example, acridine substituted nucleotides can be synthesized. Protocols for designing isolated nucleotides, nucleotide probes, and/or nucleotide primers are well-known to those of ordinary skill, and can be purchased commercially from a variety of sources (e.g., Sigma Genosys, The Woodlands, Tex. or The Great American Gene Co., Ramona, Calif.).

Nucleotides constructed in accordance with the present invention can be labeled to provide a signal as a means of detection. For example, radioactive elements such as .sup.32P, .sup.3H, and .sup.35S or the like provide sufficient half-life to be useful as radioactive labels. Other materials useful for labeling synthetic nucleotides include fluorescent compounds, enzymes and chemiluminescent moieties. Methods useful in selecting appropriate labels and binding protocols for binding the labels to the synthetic nucleotides are well known to those of skill in the art. Standard immunology manuals such as Promega: Protocol and Applications Guide, 2nd Edition, 1991 (Promega Corp., Madison, Wis.; the content of which is incorporated herein in its entirety) may be consulted to select an appropriate labeling protocol without undue experimentation.

IRDBP-1 Specific Antibodies

It is further contemplated to be within the scope of the present invention to produce and use antibodies specifically reactive with an IRDBP-1 protein or a region thereof. The antibody may be monoclonal or polyclonal and may be produced by conventional methodology using the IRDBP-1 protein, or an immunologic fragment thereof, as an immunogen. For example, a mammal (i.e., a mouse, rabbit, horse, sheep, or goat) may be immunized with a IRDBP-1 protein of the present invention, an immunogenic fragment thereof, or an IRDBP-1 fusion protein or fragment thereof, using an immunization protocol conducive to producing antibodies reactive with the IRDBP-1 protein or a fragment thereof. Following completion of the immunization steps, antiserum reactive with the jointed protein may be collected and, if desired, polyclonal anti-IRDBP-1 antibodies isolated.

One embodiment of the present invention, therefore, is a fragment of an amino acid sequence of the rat IRDBP-1 protein of SEQ ID NOS: 3, 11 or 47, or human IRDBP-1 protein (SEQ ID NOS: 12, 13 or 48) that may be synthesized and used as an immunogen to produce an anti-IRDBP-1 polyclonal antibody. Exemplary sequences of the immunogenic peptide synthesized are: AcetylatedCys-Thr-Ser-Gln-Asn-Thr-Lys-Ser-Arg-Tyr-Ile-Pro-Asn-Gly-Lys-Leu (SEQ ID NO: 15) at amino acid positions 786-800 of the rat IRDBP-1 amino acid sequence SEQ ID NO: 47 and AcetylatedCys-Arg-Asn-Gly-Gly-Thr-Tyr-Lys-Glu-Thr-Gly-Asp-Glu-Tyr-Arg (SEQ ID NO: 46). The polyclonal antibody raised against the peptide SEQ ID NO: 15 was specific for the carboxy region rat IRDBP-1 protein and cross-reacted with the human IRDBP-1 protein. The polyclonal antibody raised against the peptide SEQ ID NO: 46 is specific for the N-region of the rat or human IRDBP-1.

Antibodies that specifically bind, for example, IRDBP-1 epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of IRDBP-1. Anti-IRDBP-1 antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate IRDBP-1 levels in tissue or bodily fluid as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of diabetes or cell proliferation disorders. Likewise, the ability to monitor IRDBP-1 levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of IRDBP-1 can be measured in cells isolated from bodily fluid, such as in samples of cerebral spinal fluid or blood, or can be measured in tissue, such as produced by biopsy. Diagnostic assays using anti-IRDBP-1 antibodies can include, for example, immunoassays designed to aid in early diagnosis of a diabetic, neoplastic or hyperplastic disorder, e.g. the presence of insulin-responsive negative cells in the sample, e.g. to detect cells in which a lesion of the IRDBP-1 gene has occurred.

Another application of anti-IRDBP-1 antibodies is in the immunological screening of cDNA libraries constructed in expression vectors, such as .lamda.gt11, .lamda.gt18-23, .lamda.ZAP, and .lamda.ORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, .lamda.gt11 will produce fusion proteins whose amino termini consist of .beta.-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of IRDBP-1 can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-IRDBP-1 antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of IRDBP-1 homologs can be detected and cloned from other human sources, i.e. to identified other closely homologous human isoforms, as well as to identify IRDBP-1 homologs in other mammals.

It is further contemplated to be within the scope of the present invention for an assay to detect natural serum antibodies specific for the IRDBP-1 protein. These antibodies may be induced as a result of the release of IRDBP-1 or fragments thereof, during the onset of deterioration and destruction of the cells of the islets of Langerhan. The detection of the antibodies will provide a diagnostic indication of the onset of diabetes, cancer and the progressive loss of pancreatic activity.

IRDBP-1-specific Oligonucleotide Probes

Moreover, the nucleotide sequence determined from the cloning of subject IRDBP-1 from a human or animal cell line will further allow for the generation of probes designed for use in identifying IRDBP-1 homologs in other animal cell-types, particularly cells associated with the onset and maintenance of diabetes and obesity, cancer or other transformed or immortalized cells, as well as IRDBP-1 homologs from other non-human mammals.

In addition the present invention contemplates nucleotide probes can be generated from a cloned nucleic acid sequence of the IRDBP-1 protein, which allow for histological screening of intact tissue and tissue samples for the presence of IRDBP-1 mRNA. Similar to the diagnostic uses of anti-IRDBP-1 antibodies, the use of probes directed to IDBP-1 mRNA, or to genomic IRDBP-1 sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, diabetes or other metabolic disorders directly or indirectly attributed to a failure of the cells to respond or over-respond to insulin as well as neoplastic or hyperplastic disorders such as, but not limited to, unwanted cell growth. Used in conjunction with anti-IRDBP-1 antibody immunoassays, the nucleotide probes can help facilitate the determination of the molecular basis for a disorder or ailment that may involve some abnormality associated with expression (or lack thereof) of an IRDBP-1 protein and perturbation of insulin regulation of a gene expression or activity. For instance, nucleic acid molecules complementary to an IRDBP-1 coding sequence can be used to determine if cells contain IRDBP-1 coding sequences using Southern hybridization analysis. Nucleic acid molecules can also be used to determine the level of expression of IRDBP-1 mRNA in cells using Northern analysis as discussed in Example 8.

In a diagnostic embodiment of the present invention, therefore the nucleotide sequence of the isolated DNA molecule of the present invention may be used as a probe in nucleic acid hybridization assays for the detection of the IRDBP-1 gene in various patient, body fluids. The nucleotide sequence of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E. M. (1975) J. Mol. Biol. 98: 508; Northern blots (Thomas et al. (1980) Proc. Natl. Acad. Sci. 77: 5201-05); Colony blots (Grunstein et al, (1975) Proc. Natl. Acad. Sci. 72: 3961-65, which are hereby incorporated by reference). Alternatively, the isolated DNA molecule of the present invention can be used in a gene amplification detection procedure such as a polymerase chain reaction (Erlich et al. (1991) "Recent Advances in the Polymerase Chain Reaction" Science 252: 1643-51, which is hereby incorporated by reference) or in restriction fragment length polymorphism (RFLP) diagnostic techniques, as described in Watson et al., (2d ed. 1992), Recombinant DNA, Scientific American Books, 519-522, 545-547, which is hereby incorporated by reference.

Specifically, for example, the DNA molecules of the invention can be used in prenatal or postnatal diagnosis of the human diseases associated with defects in response to variation in the level of insulin. A probe for the DNA encoding IRDBP-1 can be designed using the DNA molecule of the invention, and used to probe the DNA obtained from amniotic fluid or chorionic tissue and amplified by PCR, LCR or any other known amplification technique for the presence of the IRDBP-1 gene or a variant thereof, as noted above. Similar procedures can be used in postnatal diagnostic work, as, for example, to diagnose the source of an IRDBP-1 deficiency in a person who is diabetic.

For example, the present method provides a method for determining if a subject is at risk for a disorder characterized by unwanted insulin non-responsiveness or cell proliferation. In preferred embodiments, the subject method can be generally characterized as comprising: detecting in a tissue of a subject (e.g. a human patient), the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding IRDBP-1 or (ii) the mis-expression of the IRDBP-1 gene. To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from the IRDBP-1 gene, (ii) an addition of one or more nucleotides to the IRDBP-1 gene, (iii) a substitution of one or more nucleotides of the IRDBP-1 gene, (iv) a gross chromosomal rearrangement of the IRDBP-1 gene, (v) a gross alteration in the level of a messenger RNA transcript of the IRDBP-1 gene, (vi) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the IRDBP-1 gene, and (vii) a non-wild type level of the IRDBP-1 protein. In one aspect of the invention there is provided a probe/primer comprising an oligonucleotide containing a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence derived from nay of the rat or human IRDBP-1 sequences SEQ ID NOS: 2, 5-10, 14 or 44-45, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the IRDBP-1 gene. The probe is exposed to nucleic acid of a tissue sample; and the hybridization of the probe to the sample nucleic acid is detected. In certain embodiments, detection of the lesion comprises utilizing the probe/primer in, for example, a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. 91: 360-364), the later of which can be particularly useful for detecting even point mutations in the IRDBP-1 gene and which are incorporated herein in their entirety. Alternatively, or additionally, the level of IRDBP-1 protein can be detected in an immunoassay.

IRDBP-1 and its Role in the Onset and Maintenance of Obesity

IRDBP-1 can also be used for the treatment of obesity and complications associated with obesity. The organ systems and the specific diseases associated with obesity include the following: (1) cardiovascular system: hypertension, congestive heart failure, cor pulmonale, varicose veins, pulmonary embolism, coronary heart disease; (2) Endocrine: insulin resistance, glucose intolerance, type II diabetes mellitus, dyslipidemia, polycystic ovary syndrome, infertility, amenorrhea; (3) Musculoskeletal: immobility, degenerative arthritis, low back pain; (4) Integument: venous stasis of legs, cellulitis, intertrigo, carbuncles; (5) Respiratory system: dyspnea and fatigue, obstructive sleep apnea, hypoventilation (pickwickian) syndrome; (6) Gastrointestinal: gastroesophageal reflux disease, hepatic steatosis, nonalcoholic steatohepatitis, cholelithiasis, hernias, colon cancer; (7) Psychosocial: work disability, depression; (8) Genitourinary: urinary stress incontinence, hypogonadism, breast and uterine cancer; (9) Neurologic: stroke, meralgia paresthetica, idiopathic interacranial hypertension. Any of the above conditions, when associated with obesity, could be used as indications for the effective use of IRDBP-1 agonists or antagonists.

Using in-situ hybridization to localize IRDBP-1 mRNA in the brain, IRDBP-1 expression was detected in the areas of the brain known to be involved in ingestive, autonomic and neuroendocrine functions of feeding and satiety, as described in the Examples 10-12 below. Regulation of body weight requires a balance among energy intake, expenditure, and storage. The brain appears to define the set point around which body weight is regulated. The levels of IRDBP-1 mRNA in the lateral hypothalamus and the nucleus of the solitary tract are differentially regulated in obese as compared to lean Zucker rats, showing a significant interactive role of IRDBP-1 in modulating body weight.

Gene Therapy Modulation of IRDBP-1 Activity

The IRDBP-1 polypeptides of the invention can be used in therapeutic applications. Since IRDBP-1 increases the transcription of IGFBP-3, IRDBP-1 can be used to treat diseases (e.g., diabetes) associated with low levels of IGFBP-3. Further, many diseases are associated with an excess of circulating IGF-1 or IGF-II, for example, some cancers and type II diabetes. IRDBP-1 can be used in patients with low levels of IGFBP-3 or high levels of IGF. Introduction of the gene encoding IRDBP-1 (or a functional derivative) into cells using either retroviral vectors or liposomes results in increased production of IGFBP-3. Many methods of delivering expressible coding sequences to cells are known in the art.

A useful application of the DNA molecules of the present invention is the possibility of increasing the amount of IRDBP-1 protein present in a mammal by gene transfer (so-called "gene therapy"). Of course, in most instances, this gene would be transferred into the animal host along with promoters, inducers, and the like (which are well known and recognized techniques in the field of genetic engineering, as noted supra) to allow the cell to initiate and continue production of the genetic product protein. The DNA molecule of the present invention can be transferred into the extra-chromosomal or genomic DNA of the host.

Methods for gene therapy are described in U.S. Pat. No. 5,399,346, issued to Anderson et al. and U.S. Pat. No. 5,766,899, issued to Kuo et al. describes methods for gene delivery into liver cells. The use of amphipathic compounds to deliver DNA is described in U.S. Pat. No. 5,744,335 issued to Wolf et al. and which are incorporated herein in their entirety.

It is further contemplated to be within the scope of the present invention for IRDBP-1-expression vectors to be used as a part, of a gene therapy protocol to reconstitute IRDBP-1 function in a cell in which IRDBP-1 is mis-expressed, or alternatively, to provide an antagonist of the naturally-occurring IRDBP-1 or an antisense construct. For instance, expression constructs of the subject IRDBP-1-proteins may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively transfecting cells in vivo with a recombinant IRDBP-1-gene. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO.sub.4 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically.

A preferred approach for in vivo introduction of nucleic acid encoding one of the subject proteins into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") that produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76: 271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding an IRDBP-1 proteins, thereby rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions that can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel et al. (1989) (eds.) Greene Publishing Associates, Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include psiCrip, psiCre, psi2 and psiAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos & Mulligan (1988) Proc. Natl. Acad. Sci. 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. 85: 3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. 87: 6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. 88: 8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150: 4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573), and which are incorporated herein in their entireties.

Furthermore, it has also been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) Proc. Natl. Acad. Sci. 86: 9079-9083; Julan et al. (1992) J. Gen. Virol. 73: 3251-3255; and Goud et al. (1983) Virology 163: 251-254); or coupling cell surface ligands to the viral env proteins (Neda et al. (1991) J. Biol. Chem. 266: 14143-14146), and which are incorporated herein in their entireties. Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector into an amphotropic vector. Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences that control expression of the IRDBP-1-gene of the retroviral vector.

Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 43 1434; and Rosenfeld et al. (1992) Cell 68: 143-155), and which are incorporated herein in their entirety. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (LeMarchand et al. (1992) Proc. Natl. Acad. Sci. 89: 6482-6486), hepatocytes (Herz & Gerard (1993) Proc. Natl. Acad. Sci. 90: 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. 89: 2581-2584), and which are incorporated herein in their entireties. Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. supra; Haj-Ahmand & Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E. J. Murray, (1991) Ed. (Humana, Clifton, N.J.) vol. 7. pp. 109-127), and which are incorporated herein in their entirety. Expression of the inserted IRDBP-1-gene can be under control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery of, for example, the subject IRDBP-1-gene, is the adeno-associated virus (AAV): Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. (1992) Curr. Topics in Micro. and Immunol. 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790), and which are incorporated herein in their entirety.

Other viral vector systems that may have application in gene therapy have been derived from such as, but not limited to, herpes virus, vaccinia virus, and several RNA viruses. In particular, herpes virus vectors may provide a unique strategy for persistence of the recombinant IRDBP-1 gene in cells of the central nervous system.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of an IRDBP-1-protein, or an IRDBP-1 antisense molecule, in the tissue of an animal. Most non-viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject IRDBP-1 gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding one of the subject IRDBP-1 proteins can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) NO Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075), and which are incorporated herein in their entireties. For example, lipofection of papilloma-virus infected epithelial cells can be carried out using liposomes tagged with monoclonal antibodies against, for example, squamous cells.

In similar fashion, the gene delivery system comprises an antibody or cell surface ligand that is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180), and which are incorporated herein in their entireties. For example, an IRDBP-1 gene construct encoding an antagonistic form of the protein, e.g. a dominant negative mutant, can be used to transfect HPV-infected squamous cells in vivo using a soluble polynucleotide carrier comprising an HPV viral coat protein conjugated to a polycation, e.g. poly-lysine (see U.S. Pat. No. 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via receptor-mediated endocytosis can be improved using agents which enhance escape of gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) Proc. Natl. Acad. Sci. 89:7934; and Christiano et al. (1993) Proc. Natl. Acad. Sci. 90:2122), and which are incorporated herein in their entirety.

In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the gene into the target cells relies predominantly on the specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) Proc. Natl. Acad. Sci. 91: 3054-3057), both of which references are incorporated herein in their entireties.

Moreover, the phamnaceutical preparation can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g. retroviral packages, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system. In the case of the latter, methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices. The generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, Mass., 1990); Sabel et al. U.S. Pat. No. 4,883,666; Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Lim U.S. Pat. No. 4,391,909; Sefton U.S. Pat. No. 4,353,888; and Aebischer et al. (1991) Biomaterials 12:50-55), and which are incorporated herein in their entireties.

Further, IRDBP-1 encoding sequences of the invention are useful in increasing production of recombinant IGFBP-3 for treatment of the aforementioned diseases, including GH deficiencies and complications caused by increased unbound IGF, can be accomplished by administration of recombinant IGFBP-3 (for example, produced in cell culture) via pharmaceutical compositions. Production of IGFBP-3 from recombinant cells can be increased by transfecting such cells with an IRDBP-1 encoding sequence either under the control of its own or a heterologous promoter.

IRDBP-1 polypeptides of the present invention are also useful in the treatment of growth hormone disorders, especially those where IGFBP-3 levels are below normal. IRDBP-1 is formulated into a pharmaceutical composition for parenteral administration, and a therapeutical dose is administered, with the result of raising IGFBP-3 and IRDBP-1 levels in the treated patient.

The presence of micro-satellite DNA downstream of the IRDBP-1 coding sequence is also further noted. Expression of the IRDBP-1 coding sequence is greater in the presence than absence of this micro-satellite DNA. Probes and/or primers for analysis of this region may allow the identification of genetic diseases associated with aberrant IRDBP-1 expression.

Antisense/Sense Nucleic Acid Modulation of IRDBP-1 Gene Expression

Another aspect of the invention relates to the use of the isolated nucleic acid in "antisense" therapy. An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a IRDBP-1-protein, e.g. the rat or human IRI)BP-1 nucleic acid sequences represented in SEQ ID NOS: 2, 5-10, 14, and 44-45, as described in Example 21. Alternatively, the antisense construct can be an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding one of the subject IRDBP-1 proteins. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by van der Krol et al. (1988) Biotechniques 6: 958-976; and Stein et al. (1988) Cancer Res. 48: 2659-2668 and which are incorporated herein in their entirety.

Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts. Inhibition of cell proliferation may result, but this condition may be desirable where, for example, proliferation may lead to a pathological condition such as, but not limited to a blockage of a blood vessel after angioplasty, or proliferation of endothelial cells for angiogenesis in tumor formation. An increase in cell regulation may result, but this condition may be desirable where, for example, a deterioration or deficiency in the number of cells results in a pathological condition such as, but not limited to, a progressive decrease in neural cells, or muscular atrophy. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, the oligomers of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous for injection, the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.

Transgenic Animals

Another aspect of the present invention concerns transgenic animals, such as, but not limited to animal models for diabetes, obesity, mood disorders, developmental and, proliferative diseases, that are comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express the subject IRDBP-1 in one or more cells in the animal. In embodiments of the present invention, therefore, the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern. In the present invention, such mosaic expression of the subject IRDBP-1 proteins can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of IRDBP-1 mutations or overexpression that might grossly alter development in small patches of tissue within an otherwise normal embryo. Toward this end, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.

Genetic techniques that allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo are well known to those skilled in the art. For instance, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination a target sequence. As used herein, the phrase "target sequence" refers to a nucleotide sequence that is genetically recombined by a recombinase. The target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity. Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of the subject receptor. For example, excision of a target sequence that interferes with the expression of the receptor can be designed to activate expression of that protein. This interference with expression of the subject protein can result from a variety of mechanisms, such as spatial separation of the IRDBP-1 gene from the promoter element or an internal stop codon. Moreover, the transgene can be made wherein the coding sequence of the IRDBP-1 gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject IRDBP-1 gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element that allow for promoter driven transcriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system of bacteriophage P1 (Lakso et al. (1992) Proc. Natl. Acad. Sci. 89:6232-6236; Orban et al. (1992) Proc. Natl. Acad. Sci. 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCT publication WO 92/15694), and which are incorporated herein in their entireties, can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination. The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase. Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus, the activation of expression of the recombinant UBC9 gene can be regulated via regulation of recombinase expression.

Use of the these recombinase system to regulate expression of, for example, a dominant negative IRDBP-1 gene, or an antisense gene, requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject gene. Animals containing both the Cre recombinase and the IRDBP-1 genes can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., one harboring the IRDBP-1 gene, and the other harboring the recombinase gene.

One advantage derived from initially constructing transgenic animals containing a IRDBP-1 transgene in a recombinase-mediated expressible format derives from the likelihood that the subject IRDBP-1 protein, whether antagonistic or agonistic, will be deleterious upon expression in the transgenic animal. In such an instance, a founder population, in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues, or in a developmentally restricted pattern. Thus, the creation of a founder population in which, for example, an antagonistic IRDBP-1 transgene is silent will allow the study of progeny from that founder in which disruption of IRDBP-1-mediated insulin responsiveness in a particular tissue or at certain developmental stages could result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Operators present in prokaryotic cells have been extensively characterized in vivo and in vitro and can be readily manipulated to place them in any position upstream from or within a gene by standard techniques. Such operators comprise promoter regions and regions which specifically bind proteins such as activators and repressors. One example is the operator region of the lexA gene of E. coli to which the LexA polypeptide binds. Other exemplary prokaryotic regulatory sequences and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat. No. 4,833,080. Thus, as described above for the recombinase-mediated activation, silent transgenic animals can be created which harbor the subject transgene under transcriptional control of a prokaryotic sequence that is not appreciably activated by eukaryotic proteins. Breeding of this transgenic animal with another animal that is transgenic for the corresponding prokaryotic trans-activator, can permit activation of the IRDBP-1 transgene. Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods (such as described above) wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, the IRDBP-1 transgene could remain silent into adulthood until "turned on" by the introduction of the trans-activator.

Additionally, inducible promoters can be employed, such as the tet operator and the metallothionein promoter which can be induced by treatment with tetracycline and zinc ions, respectively (Gossen et al. (1992) Proc. Natl. Acad. Sci. 89:5547-5551; and Walden et al. (1987) Gene 61:317-327), and which are incorporated herein in their entirety.

Methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert recombinase target sequences flanking portions of an endogenous IRDBP-1 gene, such that tissue specific and/or temporal control of inactivation of an IRDBP-1 allele can be controlled as above. Furthermore, the present invention, by making available purified and recombinant forms of the subject IRDBP-1 proteins, will allow the development of assays which can be used to screen for drugs which either agonize or antagonize the function of IRDBP-1 in vivo.

Screening for IRDBP-1 Agonists/Antagonists

Assays for the measurement of IRDBP-1 can be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Such agents can be used, for example, in the treatment of diabetic or feeding disorders, proliferative and/or differentiative disorders, and to modulate cellular metabolism.

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, potential inhibitors of IRDBP-1 function can be detected in a cell-free assay generated by constitution of a functional IRDBP-1/target nucleic acid sequence in a cell lysate.

Another aspect of the present invention concerns three-dimensional molecular models of the subject IRDBP-1 proteins, and their use as templates for the design of agents able to inhibit at least one biological activity of the IRDBP-1 protein. An integral step to designing inhibitors of the subject IRDBP-1 involves construction of computer graphics models of the IRDBP-1 that can be used to design pharmacophores by rational drug design. For instance, for an inhibitor to interact optimally with the subject protein, it will generally be desirable that it have a shape which is at least partly complimentary to that of a particular binding site of the protein, as for example those portions of the human IRDBP-1 that are involved in recognition of a particular region of a nucleic acid sequence. Additionally, other factors, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, and cooperative motions of ligand and enzyme, all influence the binding effect and should be taken into account in attempts to design bioactive inhibitors.

A computer-generated molecular model of the subject protein can be created by homology modeling, and then calculate the structure of the protein and velocities of each atom at a simulation temperature. Computer programs for performing energy minimization routines are commonly used to generate molecular models. For example, both the CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106: 765) algorithms handle all of the molecular system setup, force field calculation, and analysis (see also, Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ Health Perspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475488), and which are incorporated herein in their entirety.

Moreover, a number of programs are presently available for virtual design of IRDBP-1 protein inhibitors. For instance, the increasing availability of biomacromolecule structures of potential pharmacophoric molecules that have been solved crystallographically has prompted the development of a variety of direct computational methods for molecular design, in which the steric and electronic properties of substrate binding sites are used to guide the design of potential inhibitors (Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol. 161: 269-288; Desjarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) Spec. Publ., Roy. Soc. Chem. 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849-857; Desjarlais et al. J. Med. Cam. 29: 2149-2153), and which are incorporated herein in their entireties. Most algorithms of this type provide a method for finding a wide assortment of chemical structures that are complementary to the shape of a binding site of the subject protein. Each of a set of small molecules from a particular data-base, such as the Cambridge Crystallographic Data Bank (CCDB) (Allen et al. (1973) J. Chem. Doc. 13: 119), is individually docked to a nucleic acid or other ligand binding site of the IRDBP-1 protein in a number of geometrically permissible orientations with use of a docking algorithm. In an illustrative embodiment, a set of computer algorithms called DOCK, can be used to characterize the shape of invaginations and grooves that form the active sites and recognition surfaces of the subject protein (Kuntz et al. (1982) J. Mol. Biol. 161: 269-288). The program can also search a database of small molecules for templates whose shapes are complementary to particular binding sites of the protein (Desjarlais et al. (1988) J. Med. Chem. 31: 722-729). These templates normally require modification to achieve good chemical and electrostatic interactions (Desjarlais et al. (1989) ACS Symp. Ser. 413: 60-69). However, the program has been shown to position accurately known cofactors for inhibitors based on shape constraints alone.

Other exemplary virtual drug design programs include GRID (Goodford (1985, J. Med. Chem. 28:849-857); Boobbyer et al. (1989) J. Med. Chem. 32:1083-1094), CLIX Lawrence et al. (1992) Proteins 12:31-41), GROW (Moon et al. (1991) Proteins 11:314-328), the multiple copy simultaneous search method (MCSS) (described by Miranker et al. (1991) Proteins 11: 29-34), and NEWLEAD (Tschinke et al. (1993) J. Med. Chem. 36: 3863,3870), which are incorporated herein in their entireties.
 

Claim 1 of 17 Claims

1. A method of regulating a blood glucose level in a mammal with diabetes mellitus and/or insulin resistance, comprising the step of: increasing an intracellular IRDBP-1 protein level in cells of the mammal by introducing a DNA construct encoding the IRDBP-1 protein into the cells of the mammal, thereby increasing glucose transport into the cells and resulting in regulation of the blood glucose level in the mammal, wherein the IRDBP-1 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 47, and 48, and wherein the step of increasing the intracellular IRDBP-1 level is insulin-independent.

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