# Patent

## Title
SOGA polynucleotides and polypeptides and uses thereof

## Abstract

The present invention relates to the identification of polynucleotides and polypeptides involved in insulin and adiponectin signaling and regulation of glucose production. The invention further relates to the use of the identified polynucleotides and polypeptides, and inhibitors of the polynucleotides and polypeptides, in the regulation of glucose production and the monitoring and treatment of metabolic disorders such as diabetes.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. §371 national phase application of PCT Application PCT/US2010/037,472, filed Jun. 4, 2010, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/184,392, filed Jun. 5, 2009. The entire contents of each of these applications is incorporated herein by reference.


STATEMENT OF FEDERAL SUPPORT
This invention was made, in part, with government support under grant numbers DK075573, DK056350, and ES010126 from the National Institutes of Health. The United States government has certain rights to this invention.


FIELD OF THE INVENTION
The present invention relates to the identification of polynucleotides and polypeptides involved in insulin and adiponectin signaling and regulation of glucose production. The invention further relates to the use of the identified polynucleotides and polypeptides, and inhibitors of the polynucleotides and polypeptides, in the regulation of glucose production and the monitoring and treatment of metabolic disorders such as diabetes.
BACKGROUND OF THE INVENTION
Adipose tissue exerts a powerful effect on glucose metabolism by regulating the concentration of circulating adiponectin (Goldline et al., Lancet 362:1431 (2003)). High adiponectin in the lean state is linked to elevated insulin sensitivity whereas low adiponectin in the obese state is linked to insulin resistance and diabetes (Arita et al., Biochem. Biophys. Res. Commun. 257:79 (1999); Hotta et al., Artererioscler. Thromb. Vase. Biol. 20:1595 (2000); Maeda et al., Diabetes 50:2094 (2001); Weyer et al., J. Clin. Endocrinol. Metab. 2001, 86:1930 (2001)). Endogenous glucose production is elevated in diabetes (Wahren et al., Annu. Rev. Nutr. 27:329 (2007)). Studies in mice and liver cells show that adiponectin lowers glucose production by increasing the insulin sensitivity of the liver (Berg et al., Nat. Med. 7:947 (2001); Combs et al., J. Clin. Invest. 108:1875 (2001); Combs et al., Endocrinology 145:367 (2004)).
The signal transduction pathway of adiponectin is currently linked to (a) adiponectin receptors that bind to the full-length or the carboxy-terminal ‘globular’ fragment of adiponectin, (b) binding of the intracellular domains of adiponectin receptors 1 and 2 to the adaptor APPL1 and (c) the activation of AMPK, a signaling intermediate that reduces the gene expression of rate limiting enzymes for glucose production (Combs et al., J. Clin. Invest. 108:1875-(2001); Combs et al., Endocrinology 145:367 (2004); Tomas et al., Proc. Natl. Acad. Sci. USA 99:16309 (2002); Yamauchi et al., Nat. Med. 8:1288 (2002); Shklyaev et al., Proc. Natl. Acad. Sci. USA 100:14217 (2003); Nawrocki et al., J. Biol. Chem. 281:2654 (2006); Andreelli et al., Endocrinology 147:2432 (2006); Mao et al., Nat. Cell Biol. 8:516 (2006); Brooks et al., J. Biol. Chem. 282:35069 (2007); Yoon et al., Exp. Mol. Med. 41:577 (2009); Wang et al., J. Biol. Chem. 282:7991 (2007)). However, the inhibition of glucose production by this pathway is not completely clear.
Glucose production depends on autophagy, a regulated mechanism of intracellular degradation that is inhibited by insulin (Amherdt et al., J. Clin. Invest. 54:188 (1974)). Autophagy provides the biochemical intermediates for glucose production through the hydrolysis of proteins, glycogen and triglycerides (Mortimore et al., Annu. Rev. Nutr. 7:539 (1987); Kotoulas et al., Pathol. Res. Pract. 202:631 (2006); Singh et al., Nature 458:1131 (2009)). Insulin inhibition of autophagy in isolated hepatocytes is linked to the activation of mTOR (Blommaart et al., J. Biol. Chem. 270:2320 (1995); Kanazawa et al., J. Biol. Chem. 279:8452 (2004)). Hence, reports that AMPK, an essential mediator of adiponectin action, inhibits mTOR and stimulates autophagy are perplexing (Shaw et al., Cancer Cell 6:91 (2004); Meley et al., J. Biol. Chem. 281:34870 (2006); Xu et al., Cell Death Differ. 14:1948 (2007); Liang et al., Nat. Cell Biol. 9:218-(2007); Meijer et al., Autophagy 3:238 (2007); Cheng et al., J. Biol. Chem. 279:15719 (2004); Hoyer-Hansen et al., Mol. Cell. 25:193 (2007)).
The present invention addresses previous shortcomings in the art by providing a novel polynucleotide and polypeptide that connects insulin, adiponectin, and glucose production and that can be used for diagnostic and therapeutic methods.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the identification of a novel polypeptide named Suppressor of Glucose by Autophagy (SOGA), also known as Target of Adiponectin (TOA), and the role it plays in insulin and adiponectin signaling and glucose production. The invention is based further on the use of this polypeptide, polynucleotides encoding the polypeptide, and inhibitors thereof, in the regulation of glucose production and the monitoring and treatment of metabolic disorders related to glucose levels, such as diabetes.
Accordingly, as one aspect, the invention provides an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising a nucleotide sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100%) identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1 and 3 and encoding a functional SOGA polypeptide;
(b) a polynucleotide that hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1 and 3 under stringent hybridization conditions and encodes a functional SOGA polypeptide;
(c) a polynucleotide encoding a functional SOGA polypeptide comprising an amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 4; and
(d) a functional fragment of any of (a) to (c).
The invention further relates to vectors and cells comprising the polynucleotides of the invention, and methods of recombinantly expressing the polypeptides of the invention.
Another aspect of the invention relates to isolated SOGA polypeptides or functional fragments thereof encoded by the isolated polynucleotides of the invention. Functional fragments include, without limitation, C-terminal fragments of about 80 kDa and about 25 kDa. In some embodiments, the polypeptide is part of a fusion protein.
A further aspect of the invention relates to agents that inhibit the expression and/or activity of SOGA polypeptides or polynucleotides, including antibodies, antisense oligonucleotides, ribozymes, siRNAs, and small molecules.
An additional aspect of the invention relates to pharmaceutical compositions comprising the polypeptides, polynucleotides, or inhibitory agents of the invention.
A further aspect of the invention relates to non-human animals genetically modified to express the polypeptide of the invention or to inhibit expression of the polypeptide of the invention.
Another aspect of the invention relates to methods of decreasing glucose production in a cell or decreasing autophagy in a cell, comprising contacting the cell with the polypeptides or polynucleotides of the invention.
A further aspect of the invention relates to methods of decreasing blood glucose levels in a subject or of increasing insulin sensitivity in a subject, comprising delivering to the subject the polypeptides or polynucleotides of the invention.
Another aspect of the invention relates to methods of increasing glucose production in a cell or increasing autophagy in a cell, comprising contacting the cell with an agent that decreases the expression and/or activity of the polypeptides or polynucleotides of the invention.
Another aspect of the invention relates to methods of increasing blood glucose levels in a subject or of decreasing insulin sensitivity in a subject, comprising delivering to the subject an agent that decreases the activity of the polypeptides or polynucleotides of the invention.
An additional aspect of the invention relates to a method of measuring the response of a subject to a treatment for diabetes, comprising determining the circulating level of the polypeptides of the invention in the subject after administration of the treatment and comparing it to the circulating level of the polypeptide in the subject before administration of the treatment.
Another aspect of the invention relates to a method of predicting the clinical outcome of a diabetes treatment in a subject, comprising determining the circulating level of the polypeptide of the invention in the subject after administration of the treatment and comparing it to the circulating level of the polypeptide in the subject before administration of the treatment.
Another aspect of the invention relates to a method of identifying an agent that binds to the polypeptides of the invention, comprising contacting the polypeptide or a functional fragment thereof with a test agent under conditions whereby binding between the polypeptide or a functional fragment thereof and the test agent can occur; and detecting binding between the polypeptide or a functional fragment thereof and the test agent.
An additional aspect of the invention relates to a method of identifying an agent that modulates the activity of polypeptides of the invention, comprising contacting the polypeptide or a functional fragment thereof with a test agent under conditions whereby modulation of the activity of the polypeptide or a functional fragment thereof can occur; and detecting modulation of the activity of the polypeptide or a functional fragment thereof upon contact with the test agent as compared to activity of the polypeptide or a functional fragment thereof in the absence of contact with the test agent.
A further aspect of the invention relates to a kit comprising a reagent for determining the expression and/or activity of the polypeptides and/or polynucleotide of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an amino acid sequence analysis of SOGA for conserved functional domains.
FIG. 2 shows the current model of autophagocytosis and the autophagy machinery showing mTOR and ATG 16 in black.
FIG. 3 shows proteolytic cleavage of SOGA yielding a circulating 25 kDa C-terminal fragment.
FIG. 4 shows that antisera from two different rabbits immunized with two different peptide antigens, 476 and 477, detected a 25 kDa band in mouse plasma.
FIG. 5 shows that the concentration of SOGA in plasma corresponded with circulating levels of adiponectin.
FIG. 6 shows western blot and densitometry of adiponectin and SOGA in ob/ob control mice and ob/ob mice treated with pioglitazone.
FIG. 7 shows western blot and densitometry of adiponectin and SOGA in ad libitum and calorie restricted fed C57B1 mice.
FIG. 8 shows western blot and densitometry of adiponectin and SOGA in rapamycin and control fed C57B1 mice.
FIG. 9 shows FPLC fraction analysis of mouse plasma for SOGA.
FIG. 10 show the sequence (SEQ ID NO:2) and predicted functional domains of SOGA.
FIGS. 11A-11D show the function and regulation of SOGA in primary hepatocytes.
FIGS. 12A-12C show detection of circulating SOGA in mice.
FIGS. 13A-13B show detection of recombinant SOGA.
FIGS. 14A-14D show the circulating levels of adiponectin and SOGA in humans and mice.
FIGS. 15A-15B show the circulating levels of SOGA in relation to insulin in humans and mice.



DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.
Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.
Nucleotide sequences are presented herein by single strand only, in the 5′ to 3′ direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage.
Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).
I. Definitions
As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.
The term “consists essentially of” (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5′ and/or 3′ or N-terminal and/or C-terminal ends of the recited sequence such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids on both ends added together. The term “materially altered,” as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in the ability to inhibit glucose production of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.
A “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of diabetes, reduction in glucose levels or increase in insulin sensitivity). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
By the terms “treat,” “treating,” or “treatment of,” it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.
The term “control sample,” as used herein, refers to a tissue or cell sample that is used to compare the level of expression and/or activity of a SOGA polypeptide to the level of expression and/or activity in a sample of interest. The control sample may be, for example, from a normal (i.e., non-diseased) portion of the same tissue or cell type in the subject, from a different tissue or cell type in the subject, from a matched individual, or may be a standard derived from the average of measurements taken from a population of subjects. In another embodiment, the control sample may be from the disease tissue of the subject, e.g., at the time of diagnosis, prior to treatment, or after a stage of treatment.
As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention.
An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5′ non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.
The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose. In certain embodiments, the polypeptide is at least about 50% pure, e.g., at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more pure.
An isolated cell refers to a cell that is separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention. Thus, an isolated cell can be delivered to and/or introduced into a subject. In some embodiments, an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.
The term “fragment,” as applied to a polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, 500, or more consecutive nucleotides of a nucleic acid according to the invention. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of less than about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, or 500 consecutive nucleotides of a nucleic acid according to the invention.
The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 300, 400, 500, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of less than about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, or 500 consecutive nucleotides of a nucleic acid according to the invention.
The term “functional SOGA polypeptide,” as applied herein, refers to a polypeptide that substantially retains at least one biological activity normally associated with the naturally occurring SOGA polypeptide (e.g., the ability to inhibit glucose production, protein binding, ligand or receptor binding). In particular embodiments, the “functional” polypeptide substantially retains all of the activities possessed by the naturally occurring polypeptide. By “substantially retains” biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A “non-functional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as protein binding and suppression of glucose production can be measured using assays that are well known in the art and as described herein. In certain embodiments, the “activity” of a SOGA polypeptide is defined as the ability to inhibit glucose production in a population of isolated hepatocytes (either primary hepatocytes or a hepatocyte cell line).
The term “functional fragment,” as applied to a polypeptide, refers to a fragment that substantially retains at least one biological activity of the full length polypeptide, e.g., the ability to inhibit glucose production. By “substantially retains” biological activity, it is meant that the fragment retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the full length polypeptide (and can even have a higher level of activity than the full length polypeptide). A “non-functional” fragment is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).
The term “functional fragment,” as applied to a polynucleotide, refers to a polynucleotide that encodes a functional fragment of a polypeptide.
A “vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A “replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker. A “recombinant” vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.
Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).
Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).
In some embodiments, a polynucleotide of this invention can be delivered to a cell in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous nucleotide sequences into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
In various embodiments, other molecules can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from nucleic acid binding proteins (e.g., WO96/25508), and/or a cationic polymer (e.g., WO95/21931).
It is also possible to introduce a vector in viva as naked nucleic acid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated nucleic acid delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).
The term “transfection” or “transduction” means the uptake of exogenous or heterologous nucleic acid (RNA and/or DNA) by a cell. A cell has been “transfected” or “transduced” with an exogenous or heterologous nucleic acid when such nucleic acid has been introduced or delivered inside the cell. A cell has been “transformed” by exogenous or heterologous nucleic acid when the transfected or transduced nucleic acid imparts a phenotypic change in the cell and/or a change in an activity or function of the cell. The transforming nucleic acid can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell or it can be present as a stable plasmid.
As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.
A “fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. Illustrative fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to all or a portion of glutathione-S-transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.
By the term “express” or “expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.
II. SOGA Polynucleotides and Polypeptides
In one aspect, the invention relates to an isolated polynucleotide encoding a SOGA polypeptide or a functional fragment thereof. In one embodiment, the SOGA polypeptide is a mammalian SOGA polypeptide, e.g., human or mouse. The cDNA, polypeptide, and genomic sequences of mouse SOGA have been deposited in GenBank under Accession No. H977045 and are disclosed herein as SEQ ID NOS:1, 2, and 10, respectively. The cDNA, polypeptide, and genomic sequences of human SOGA are disclosed herein as SEQ ID NOS:3, 4, and 11, respectively. The polynucleotide can comprise cDNA sequences, genomic sequences, synthetic sequences, or combinations thereof.








Mouse SOGA cDNA Sequence



(SEQ ID NO: 1)










agttgggcctggagctggcgctgagcagcgacgccgagtctgcggcgggcggcccggcgg
  60



 


ggacccgcaccgggcagccgccccagccagcgcagtcggggcagcagcctccgcggcccc
 120


 


ccgcctccccggatgagccgtcggtggccgcatcgtcggtgggcagcagccgcttgccat
 180


 


tcagcgcctcgctagccttctccgacctcaccgaggagatgctggactgtgggcccggag
 240


 


gcttggtgcgggagctggaagagctgcgttccgagaacgactatctcaaggatgagattg
 300


 


aggagctacgggctgagatgctggagatgcgggatgtctacatggaggaagacgtgtatc
 360


 


agctgcagtaccgactgcgtaaggctgagcgccgcagcctccgcgctgcccagacaggcc
 420


 


aggttgatggggaactcatccgaggtctggaacaggacgtcaaggtctctaaggacatct
 480


 


ccatgcggcttcacaaggagctggaggtggtggagaagaagcggatgaggctggaggagg
 540


 


agaacgaggggcttcgacagaggctcattgagacagagctggccaagcaggtgctacaga
 600


 


cggagctggatcgtcccagagagcattccttgaagaaaagaggaacccggtctctgggga
 660


 


agacagataagaagcctactgcacaggaggatagtgcagacctgaagtgccagctgcatt
 720


 


ttgcaaaggaggagtcggccctcatgtgcaagaagctcaccaagttggctaaggagaacg
 780


 


acagcatgaaggaggagctgctcaagtacagatcgctctatggggacctggatgcagccc
 840


 


tgtcggcagaggagctggcggatgctccgcactcccgtgagactgagctgaaggtgcacc
 900


 


tgaagctggtggaggaggaggccaacctgctgagccggcgcatagtggagctggaggtgg
 960


 


agaaccgtggcctgcgagccgagatggacgacatgaaggaccacgggggtggcgggggtc
1020


 


ccgaggccaggctggccttctcttctctgggtggtgagtgcggggagagcctagccgagt
1080


 


tgcggcgccacctgcagttcgtggaagaggaggctgagctgctgaggcgctcctcagctg
1140


 


agctggaggaccagaacaagttgctgctgaacgagctggccaaataccgctcggagcacg
1200


 


agctggacgtgacgctgtcggaggacagctgctccgtgctcagcgagccctcgcaggagg
1260


 


agctggcagccgccaagctgcagatcggcgagctcagcggcaaggtcaagaagctgcagt
1320


 


atgagaaccgcgtgctcctctccaatctgcagcgctgtgacctggcctcctgccagagca
1380


 


cacgccccatgctggagacggacgctgaggctggggactctgcgcagtgcgtgcctgccc
1440


 


ctctgggtgagacgctggagccccacgccgcccggctgtgcagggcccgtgaagccgagg
1500


 


cgctgcccggcctacgggagcaggccgctttggtcagcaaggccatcgacgtcctggtgg
1560


 


ctgatgccaatggcttctcagtcggcctccgcctgtgcctggacaatgagtgtgctgact
T620


 


tgcgactgcacgaggcgcctgacaacagcgagggccccagggatgccaagctcatccacg
1680


 


ccatcctggtgcggctgagtgtgttgcaacaggagctgaacgccttcacccgcaaggcag
1740


 


atgtggccttggggagctctggcaaggagcagcctgagcccttccctgctctgcctgcct
1800


 


tgggctcccagggccctgctaaggagatcacgctgtccaaagaccttggctctgacttcc
1860


 


agccacctgacttcagagacctgcttgagtgggagcccaggatccgagaggccttccgta
1920


 


ccggggacttggagtccaagcctgaccctagtcggaacttcaggccctaccgagctgaag
1980


 


ataacgattcttatgcctctgagatcaaggatcttcagctggtcctggccgaggcccacg
2040


 


acagcctccggggcttgcaagagcagctgtcccaggagcggcagctccggaaggaggagg
2100


 


ctgacagcttcaaccagaaaatyguccagctgaaggaagaccagcagagggcgctgctga
2160


 


gacgggagtttgagctgcagagtctgagcctccagcggcgactggagcagaagttctgga
2220


 


gccaagagaagaacatcctggtgcaggagtcccagcagttcaagcacaactttctgctgc
2280


 


tcttcatgaagctccggtggttcctgaagcgctggcggcagggcaaggttctgcccagcg
2340


 


aagaggatgacttcctggaggtgaacagcatgaaggaactgtacctgctgatggaggaag
2400


 


aggagatgaacgcccagcactcggataacaaggcctgcacaggggagagctggacccaga
2460


 


acacgcctaatgagtgcatcaagaccctggccgacatgaaggtcaccctgaaggagctgt
2520


 


gctggctgctccaggacgagcgtcggggtctgactgaacttcagcagcagatcgcaaagg
2580


 


ccaaggccacctgggagacagagcgtgcagagctcaagggccacgcctcgcagatggagc
2640


 


tgaaggctgggaagggtgccagtgagaggcccgggcctgactggaaggctgcactgcaga
2700


 


gagagcgggaggagcagcaacacctcctggcagagtcctacagcgccgtcatggagctga
2760


 


cgaggcagctgcagctgagcgagcgccactggagacaggagaagctgcagctggtggagc
2820


 


ggctgcagggagaaaagcagcaggtggagcagcaggtgaaggagctgcagaaccgcctca
2880


 


gtcagttgcagaaggctgccgagccctgggtcctgaagcactcagacatggagaagcaag
2940


 


acaacagctggaaagaggcacgaagtgagaagacccatgacaaggagggtgtctctgaag
3000


 


ctgagctcgggggaactggcutaaagaggaccaaatcagtctcctccatgtctgagtttg
3060


 


aaagtttgctcgactgctccccgtaccttgctggcggggatgcccggaacaagaagctgc
3120


 


ccaacggccctgcttttgcctttgtgagtactgagccagtggagcctgagaaagacgcca
3180


 


aggagaaggcggggctttccacccgggactgtagccacattggtagcttggcctgtcagg
3240


 


aacctgcagggagacagatgcagcgcagctacacggctccagacaagacgggaatccgag
3300


 


tctactatagtccgccagtggctcggcgcctgggtgtccctgtggtccatgacaaggagg
3360


 


gcaagatcctcattgagccaggcttcctcttcactaccgccaagcccaaggagccagccg
3420


 


aggctgacgggctggccgagagctcctacagccggtggctttgcaatttctcccggcagc
3480


 


ggctggatggaggatccggggccagcacctcgggttccggacctgctttccccgccttgc
3540


 


atgactttgagatgtcgggcaacatgagtgacgacatgaaggagatcaccaactgcgtgc
3600


 


ggcaggccatgcgctccggctctctggagaggaaggtaaagaacacatccagccagacgg
3660


 


taggcgtggccaccgtgggcacccagaccattcggacggtcagtgtaggtcttcagaccg
3720


 


acccaccccgcagcagcctccacagcaagagctggtcaccccgcagctcctcgcttgtgc
3780


 


ctgtgcgcagcaagcagatctcttcctccctggacaaggtccattctcgcattgagcggc
3840


 


catgttgctcgcccaagtacggctcacccaagctccagagacgatcggtgtccaagctgg
3900


 


atagcaccaaggaccgcagcctgtggaacctgcaccagggcaagcaaaatggctccgcct
3960


 


gggctcgctccaccaccacacgggatagccctgtactgaggaacatcaatgatgggcttt
4020


 


ctagcctctctagtgtggtggagcactctgggagcaccgagtctgtgtggaaactgggca
4080


 


tgtctgaggcccgaaccaaacctgagcctcccaagtatggcattgttcaggagttcttcc
4140


 


ggaacgtgtgtggccgggcaccgagccccactactgcagcaggcgaggaaagctgcaaga
4200


 


aaccagagcccccttcgccagccagctaccatcaacccgagggtgtatccaggatcctga
4260


 


acaagaaggcggccaaggcaggtggtagcgaagaggtcagacccaccatgctgtcccagg
4320


 


tggggaaggatggcatccttcgggatggagatggatccttgatccttcccagtgaggatg
4380


 


ccgtatgtgactgtagcgcccagtcacttgcctcctgcttcatccggccatcccgcaaca
4440


 


ccatccggcactctccttccaagtgcaggctgcacccttcagagtcaggctggggcgggg
4500


 


aggagagggcagctceccagtgagtccctgagcaaccaagcacccacctcaagcagccca
4560


 


gacccCggagatgaggcaagggctcgtgtcctcagcctcaghccatccaggaggaatggc
4620


 


agctgtgccactgccacagaagagctttcacattaaggtaaagcaaggtgtcttgctgac
4680


 


tgctgggcagtgacctctgatttccaggggaagaca
4716


 








Mouse SOGA Polypeptide Sequence



(SEQ ID NO: 2)










MLDCGPGGLVHKLEELRSENDYLKDEIEELRAEMLEHKDVYMEBDVYQLQYRLRKAERRS
  60



 


LRAAQTGQVDGELIRGLEQDVKVSKDISMRLKKELEVVEKKRMRLEEENKGLRQRLIETE
 120


 


LAKQVLQTELDRPREHSLKKRGTRSLGKTDKKPTAQEDSADLKCQLHKAKEESALMCKKL
 180


 


TKLAKENDSHKEELLKYRSLYGDLDAALSAEELADAPHSRETEIKVHLKLVEEEANLLSR
 240


 


RIVELEVKNRGLRAEMDDMKDHGGGGGPEARLAFSSLGGECGESLAELRRHLQFVEREAE
 300


 


LLRRSSAELEDQNKLLLNELAKYRSEHELDVTLSEDSCSVLSEPSQEELAAAKLQIGELS
 360


 


GKVKKLQYENRVLLSNLQRCDLASCQSTRPMLETDAEAGDSAQCVPAPLGETLEPHAARL
 420


 


CRAREAEALPGLHKQAALVSKAIDVLVADANGFSVGLRLCLDNKCADLRLHEAPDNSEGP
 480


 


RDAKLIHAILVRLSVLQQELNAFTRKADVALGSSGKEQPEPKPALPALGSQGPAKEIMLS
 540


 


KDLGSDFQPPDFRDLLEWEPRIREAFRTGDLESKPDPSRNFRPYRAEDNDSYASEIKDLQ
 600


 


LVLAEAHDSLRGLQEQLSQERQLRKKEADSPNQKMVQLKEDQQRALLRREFELQSLSLQR
 660


 


RLEQKTOSOEKNILVQKSQQPKHNFLLLFMKLRWFLKRWRQGKVTPSKKDDFLEVNSMKE
 720


 


LYLLMEEEEMNAQHSDNXACTGESWTQNTPNECIKTLADMKVTLKSLCWLLQDERRGLTE
 780


 


LQQQFAKAKATWETERAELKGHASQMELKAGKGASERPGPDWKAALQREREEQQHLLAES
 840


 


YSAVMELTRQLQLSERHWSQEKLQLVERLQGEKQQVEQQVKELQNRLSQLQKAAEPWVLK
 900


 


HSDMEKQDNSWKEAHSEKTHDKEGVSEAELGGTGLKRTKSVSSMSEFESLLDCSPYLAGG
 960


 


DARNKKLPNGPAFAFVSTEPVEPEKDAKEKAGLSTRDCSHIGSLACQBPAGRQMQRSYTA
1020


 


PDKTGIRVYYSPPVARRLGVPVVHDKEGKILIEPGFLFTTAKPKKSAEADGLAESSYSRW
1080


 


LCNFSRQRLDGGSGASTSGSGPAFPALHDFEMSGNMSDDMKEITNCVRQAMRSGSLERKV
1140


 


KNTSSQTVGVATVGTQTIRTVSVGLQTDPPRSSLHSKSWSPRSSSLVSVRSKQISSSLDK
1200


 


VHSRIERPCCSPKYGSPKLQRRSVSKLDSTKDRSLWNLHQGKQNGSAWARSTTTRDSPVL
1260


 


RNINDGLSSLFSVVEHSGSTKSVWKLGHSEARTKPEPPKYGIVQKKFRNVCGRAPSPTTA
1320


 


AGEESCKKPEPLSPASYHOPEGVGRILNKKAAKAGGSEEVRPTMLSQVGKDGILRDGDGS
1380


 


LILPSEDAVCDCSAQSLASCFIRPSRKTIRHSPSKCRLHPSESGWGGEERAAPQ
1434


 








Human SOGA cDNA Sequence



(SEQ ID NO: 3)










cgctgagcagcgacgccgagtccgcggccgggggcccggcgggggtccgtacggggcagc
  60



 


cggcccagcccgcgccctccgcgcagcagcccccgcggccgcccgcctccccggacgagc
 120


 


cgtcggtggccgcgtcgtcggtgggcagcagccgcttgccgctcagcgcctcgcttgcct
 180


 


tctccgacctcaccgaggagatgctggactgcgggcccagcggcttggtgcgggagctgg
 240


 


aggagctgcgctcggagaacgactatctcaaggacgagattgaggagctgcgggccgaga
 300


 


tgccggagatgcgggacgtctatatggaggaggacgtgtatcagctgcagtaccggctgc
 360


 


gcaaagccgagcgccgcagtctccgtgccgcccagaccggccaggtggacggcgagctta
 420


 


tccgtggtctggagcaggatgtcaaggtctctaaggacatctccatgcggctgcataagg
 480


 


agctcgaggtggtggagaagaaacgggcgcggccggaggaggagaacgaagagcttcgtc
 540


 


agcggctcatcgagactgagetggctaagcaggtgctgcagacggagctggagcgaccga
 600


 


gagagcattccttgaagaaaagaggaacccgctccctggggaaggccgataagaagactt
 660


 


tggtgcaggaggacagtgcagacctgaagtgccagttgcactttgcaaaggaggagtcag
 720


 


ccctcatgtgcaagaagctcactaagcttgccaaggagaatgacagcatgaaggaggagc
 780


 


tgcCgaagtaccgctcgctctatggggacctggacagcgcgctgtcagccgaggagctgg
 840


 


ccgatgccccccactcgcgggagaccgagctgaaggtgcacctgaagctggtggaggagg
 900


 


aagccaacctgccgagccgccgcatcgtggagctggaggtggagaaccgaggcctgcggg
 960


 


ctgagatggacgacatgaaggatcatggaggtggctgtgggggtcctgaggcacgcctgg
1020


 


ccCtctccgcgctgggtggcggagagtgcggggagagcttggcagagctgcggcgacacc
1080


 


tgcagtttgtcgaagaggaggccgagctgcCgcggcgctcctctgccgagctcgaggacc
1140


 


agaacaagctgctgctgaacgagctggccaagttccgctcggagcacgagctggacgtgg
1200


 


cgctgtcggaggacagctgttctgtgctcagcgaaccttcacaggaggagctggcggccg
1260


 


ccaagctgcagatcggcgagctcagcggcaaggtcaagaagcLgcagtacgagaaccgcg
1320


 


tgctcctctccaacctccagcgctgtgacctcgcctcctgccagagtacgcggcccatgc
1380


 


tggagacggacgccgaggccggggactctgcccagtgtgtgcctgctcccctgggcgaga
1440


 


cacacgagCcccacgcggtccgactctgcagagccagggaggccgaggtgctgcctgggc
1500


 


tgagagagcaggccgccctggtcagtaaggccatcgatgtcctggtggctgatgccaatg
1560


 


gctccacggctggcctccggctgtgtctggacaacgagtgtgctgacttccggctgcatg
1620


 


aggcccccgacaacagcgagggccccagggacaccaagctcatccatgccatcctggtgc
1680


 


gcctgagcgtgctgcagcaggagctgaatgccttcacgcggaaggcagatgcagtcctcg
1740


 


ggtgctctgtcaaggaacagcaggagtccttctcatcactgccccccttgggctcccagg
1800


 


ggctctctaaggagattcttctggcaaaagaccttggctcagactttcagccacctgact
1860


 


tcagggacctgccggaatgggagcccaggatccgagaggctttccgcactggtgacttgg
1920


 


actctaagcccgaccccagccggagcttcaggccttaccgagctgaagacaatgattcct
1980


 


atgcctctgagatcaaggagctgcagctggtgctggctgaggcccacgacagcctccggg
2040


 


gcttgcaagagcagctctcccaggagcggcagctacgaaaggaggaggccgacaatttca
2100


 


accagaaaatggtccsgotgaaggaggaccagcagagggcgctcctgaggcgggagtttg
2160


 


agctgcagagtctgagcctccagcggaggctggagcagaaattctggagccaggagaaga
2220


 


acatgccggtgcaggagtcccagcaattcaagcacaacttcctgctgctcttcatgaagc
2280


 


tcaggtggttcctcaagcgctggcggcagggcaaggttttgcccagcgaaggggatgact
2340


 


tcctcgaggtgaacagcatgaaggagctgtacttgctgatggaggaagaggagataaacg
2400


 


ctcagcattctgataacaaggcctgcacgggggacagctggacccagaacacgcccaatg
2460


 


agtacatcaagacactggccgacatgaaggtgacgctgaaggagctgtgctggctgctcc
2520


 


gggatgaacgccgtggtctgacggagcttcagcaacagtttgccaaggccaaggctacct
2580


 


gggagacagagcgggcagagctcaagggccatacctcccagatggagctgaagacaggga
2640


 


agggggccggggagcgggcagggcccgactggaaggcagccctacagcgggagcgtgagg
2700


 


agcagcagcacctcctagctgagtcctacagcgctgtcatggagctgactcggcagctgc
2760


 


agaccagtgagcgcaactggagccaggaaaagctgcagctggtggagcggctgcagggtg
2820


 


agaageagcaggtggagcagcaggtgaaggagctgcagaaccgcctaagccagctgcaga
2880


 


aggctgccgacccctgggtcctgaagcactcggagctggagaagcaggacaacagctgga
2940


 


aggagacacgcagtgagaagatccacgacaaggaggctgtttccgaagttgagcttggag
3000


 


gaaatggtttaaagagaaccaaatctgtttcttccatgtctgagtttgaaagtttgctcg
3060


 


actgttccccttacctcgctggcggagatgcccggggcaagaagctgcctaacaaccctg
3120


 


cctttggctttgtgagctccgagccaggggatccagagaaagacaccaaggagaagcctg
3180


 


ggctctcgtcgagggactgcaaccacctgggtgccctggcctgccaggaccccccaggga
3240


 


ggcagatgcagcgcagctacacggctcctgacaagaegggcatccgagtctactatagtc
3300


 


ccccggtggcccggcgcctcggagtccctgtggttcatgacaaagagggcaagatcatta
3360


 


tcgagcccggcttcctcttcaccacagccaagcccaaagagtcggccgaggctgatgggc
3420


 


tggctgagagctcctatggtcggtggctctgcaacttctcacggcagcgcctggacggag
3480


 


gctcagcgggcagcccctcggcggccgggcctggcttcccagcggccctgcatgactttg
3540


 


agatgtcaggcaacatgagtgatgacatgaaggagatcaccaactgtgtgcgccaggcca
3600


 


tgcgctccggctcactggagaggaaagtgaagagcacatccagccagacggtgggcctgg
3660


 


ccagtgtgggcacacagaccatccgcacggtcagcgtgggcctgcagaccgacccacccc
3720


 


gcagcagcctccatggcaaggcctggtcaccccgcagctcttcgctcgtgtctgtgcgca
3780


 


gcaagcagatctcctcctccctggacaaggtccattcgcgcatcgagcggccctgctgct
3840


 


cccccaagtatggctcaccaaagctccagaggcggtctgtgtccaagctggacagcagca
3900


 


aggaccgcagcctgtggaacctgcaccagggcaagcagaacggctcggcctgggcccgct
3960


 


ccaccaccacgcgggacagccctgtattgagaaacatcaacgatggactctccagcctct
4020


 


tcagtgtggtggagcactcagggagcacggagtctgtctggaaactaggcatgtctgaga
4080


 


cgcgggccaagcccgagcctcccaagtacggcattgtgcaggaattcttccgtaatgtgt
4140


 


gtggccgggcaccgagccccacctcatcagcaggagaggagggcaccaagaagccagagc
4200


 


ccctctccccagccagctaccatcagccagagggtgtggccaggatcctgaacaagaagg
4260


 


cagccaagttgggcagcagtgaggaggtcagactcaccatgctcccccaggtggggaagg
4320


 


atggtgCcctccgggacggagatggagccgtggtccttcccaatgaggacgctgtttgtg
4380


 


actgtagtacccagtctctcacctcctgcttcgcccgatcgCcccgctctgccatccgcc
4440


 


artctccttccaagtgcaggctgcacccttcagagtccagctggggtggggaggagaggg
4500


 


cactcccccccagcgagtgacagagcagccaagctccccgcctcaaccagcccagcccct
4560


 


ggatagcagaagggaaccagcagagacgagacgaggtgaggcgaggggctgtgtcctcag
4620


 


cattgcctggccctggagggacagcagtgatgccactgccagaatgcagctttcacatca
4680


 


aggtaaagccgggtctcctgctggcccctgggtggtgagcttcgacttcccaggggaagg
4740


 


cagtgagtgggagagagaccaaacctgggottcccaagcatccactgagagatctgtcaa
4800


 


gagccgatccctgggtcctaagagagagccttgcctggttctgcccatgccaccctcttg
4860


 


ga
4862


 








Human SOGA Polypeptide Sequence



(SKQ ID NO: 4)










MLDCGPSGLVRELEELRSENDYLKDEIEBLRAEMLEMRDVYMKEDVYQLQYRLHKAERRS
  60



 


LRAAQTGQVDGELIRGLEQDVKVSKDISMRLHKELEVVEKKRARLEEENEELRQRLIETE
 120


 


IAKQVLOTELERPRKHSLKKRGTRSLGKADKKTLVQEDSADLKCQLHKAKEESALMCKKL
 180


 


TKLAKENDSMKEELLKYRSLYGDLDSALSAEELADAPHSRETELKVHLKLVEEEANLLSR
 240


 


RIVELEVENRGLRAEMDDMKDHGGGCGGPEARLAFSALGGGECGESLAELRRHLQFVEEE
 300


 


AELLRRSSAKLEDQNKLLLNELAKPRSKHRLDVALSEDSCSVLSEPSQKKIAAAKLQIGE
 360


 


LSGKVKKLQYENRVLLSNLQRCDIASCQSTRPMLETDAEAGDSAQCVPAPLGETHESHAV
 420


 


RLCRAREAEVLPGLREQAALVSKAIDVLVADANGFTAGLRLCLDNECADFRLHEAPDNSE
 480


 


GPRDTKLIHArLVRLSVLQQELNAFTRKADAVLGCSVKEQOESFSSLPPLGSQGLSKEIL
 540


 


LAKDLGSDFQPPDFRDLPEWEPRIREAFRTGDLDSKPDPSRSPRPYRAEDNOSYASEIKE
 600


 


LQLVLAKAHDSLRGLQEQLSQERQLRKKEADNFNQKHVQLKEDQQRALLRREFELQSLGL
 660


 


QRRLEQKFWSQEKNMLVQESQQFKHNFLLLFMKLRWFLKRWRQGKVLPSEGDDFLEVNSM
 720


 


KELYLLMEEEEINAQHSDNKACTGDSWTQNTPNEYIKTLADMKVTLKELCWLLRDERRGL
 780


 


TELQQQFAKAKATWETERAELKGHTSQMELKTGKGAGERAGPDWKAALQREREEQQHLLA
 840


 


KSYSAVMELTROLQISERNWSQKKLQLVERLQGEKQQVEQQVKELQNRLSQLQKAADPWV
 900


 


LKHSELEKQDNSWKETRSEKIHDKEAVSEVELGGNGLKRTKSVSSMSEFESLLDCSPYIA
 960


 


GGDARGKKLPNNPAFGFVSSEPGDPEKDTKEKPGLSSHUCNHLGALACODPPGRQMQRSY
1020


 


TAPDKTGIRVYYSPPVARRLGVPVVHDKEGKIIIEPGFLFTTAKPKESAKADGLAESSYG
1080


 


RWLCNFSRQRLDGGSAGSPSAAGPGFPAALHDFEMSGNMSDDMKEITNCVRQAMRSGSLE
1140


 


RKVKSTSSQTVGLASVGTQTIRTVSVGLQTDPPRSSLHGKAWSPRSSSLVSVRSKQTSSS
1200


 


LDKVHSRIERPCCSPKyGSPKLQRRSVSKLDSSKDRSLWNLHQGKQNGSAWARSTTTRDS
1250


 


PVLRNINDGLSSLFSVVEHSGSTESVWKLGMSKTRAKPEPPKYGIVQEFFRNVCGRAPSP
1320


 


TSSAGEEGTKKPEPLSPASYHQPEGVARILNKKAAKLGSSEKVRLTMLPQVGKDGVIRDG
1380


 


DGAVVLPNEDAVCDCSTQSLTSCFARSSRSAIRHSPSKCRLHPSESSWGGEERALPPSE
1439






One embodiment of the invention is an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising a nucleotide sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100%) identical to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1 and 3 and encoding a functional SOGA polypeptide;
(b) a polynucleotide that hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1 and 3 under stringent hybridization conditions and encodes a functional SOGA polypeptide;
(c) a polynucleotide encoding a functional SOGA polypeptide comprising an amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100%) identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 4; and
(d) a functional fragment of any of (a) to (c).
In another embodiment, the isolated polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:1 and 3 or a fragment thereof that encodes a functional SOGA polypeptide;
(b) a polynucleotide encoding a functional SOGA polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 4 or a functional fragment thereof; and
(c) a polynucleotide comprising a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) above due to the degeneracy of the genetic code.
In one aspect, the invention relates to SOGA polypeptides and functional fragments or homologs thereof. The SOGA polypeptide can be from any species expressing SOGA, such as mammalian SOGA, e.g., human or mouse SOGA. As used herein, the term “homolog” is used to refer to a polypeptide which differs from a naturally occurring polypeptide by minor modifications to the naturally occurring polypeptide, but which significantly retains a biological activity of the naturally occurring polypeptide. Minor modifications include, without limitation, changes in one or a few amino acid side chains, changes to one or a few amino acids (including deletions, insertions, and substitutions), changes in stereochemistry of one or a few atoms, and minor derivatizations, including, without limitation, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation, and addition of glycosylphosphatidyl inositol. The term “substantially retains,” as used herein, refers to a fragment, homolog, or other variant of a polypeptide that retains at least about 20% of the activity of the naturally occurring polypeptide (e.g., inhibition of glucose production), e.g., about 30%, 40%, 50% or more. SOGA activity can be measured as disclosed herein. Other biological activities may include enzyme activity, receptor binding, ligand binding, a cell signal transduction event, etc.
Functional fragments of SOGA polypeptide include any fragment that substantially retains at least one biological activity of full length SOGA polypeptide. In one embodiment, the functional fragment is a C-terminal fragment of SOGA. In certain embodiments, the C-terminal fragment begins immediately after the internal signal sequence of SOGA. In other embodiments, the functional fragment is a C-terminal fragment of about 80 kDa or 25 kDa.
In exemplary embodiments, the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of the polypeptide disclosed herein and in the GenBank accession numbers listed above or a functional fragment thereof. In another embodiment, the isolated polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 70% identical, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the disclosed amino acid sequence or a functional fragment thereof (and polynucleotide sequences encoding the same).
The polypeptide of the invention also include functional portions or fragments (and polynucleotide sequences encoding the same). The length of the fragment is not critical as long as it substantially retains at least one biological activity of the polypeptide. Illustrative fragments comprise at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, 500, or more contiguous amino acids of a SOGA polypeptide.
Likewise, those skilled in the art will appreciate that the present invention also encompasses fusion polypeptides (and polynucleotide sequences encoding the same) comprising a SOGA polypeptide or a functional fragment thereof. For example, it may be useful to express the polypeptide (or functional fragment) as a fusion protein that can be recognized by a commercially available antibody (e.g., FLAG motifs) or as a fusion protein that can otherwise be more easily purified (e.g., by addition of a poly-His tail). Additionally, fusion proteins that enhance the stability of the polypeptide may be produced, e.g., fusion proteins comprising maltose binding protein or glutathione-S-transferase. As another alternative, the fusion protein can comprise a reporter molecule. In other embodiments, the fusion protein can comprise a polypeptide that provides a function or activity that is the same as or different from the activity of the polypeptide, e.g., a targeting, binding, or enzymatic activity or function.
Likewise, it will be understood that the SOGA polypeptides specifically disclosed herein will typically tolerate substitutions in the amino acid sequence and substantially retain biological activity. To identify polypeptides of the invention other than those specifically disclosed herein, amino acid substitutions may be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
Amino acid substitutions other than those disclosed herein may be achieved by changing the codons of the DNA sequence (or RNA sequence), according to the following codon table.










TABLE 1





Amino Acid


Codons







Alanine
Ala
A
GCA GCC GCG GCT


 


Cysteine
Cys
C
TGC TGT


 


Aspartic acid
Asp
D
GAC GAT


 


Glutamic acid
Glu
E
GAA GAG


 


Phenylalanine
Phe
F
TTC TTT


 


Glycine
Gly
G
GGA GGC GGG GGT


 


Histidine
His
H
CAC CAT


 


Isoleucine
Ile
I
ATA ATC ATT


 


Lysine
Lys
K
AAA AAG


 


Leucine
Leu
L
TTA TTG CTA CTC CTG CTT


 


Methionine
Met
M
ATG


 


Asparagine
Asn
N
AAC AAT


 


Proline
Pro
P
CCA CCC CCG CCT


 


Glutamine
Gln
Q
CAA CAG


 


Arginine
Arg
R
AGA AGG CGA CGC CGG CGT


 


Serine
Ser
S
AGC ACT TCA TCC TCG TCT


 


Threonine
Thr
T
ACA ACC ACG ACT


 


Valine
Val
V
GTA GTC GTG GTT


 


Tryptophan
Trp
W
TGG


 


Tyrosine
Tyr
Y
TAC TAT









In identifying amino acid sequences encoding polypeptides other than those specifically disclosed herein, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (see, Kyte and Doolittle, J. Mol. Biol. 157:105 (1982); incorporated herein by reference in its entirety). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, id.), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); praline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). Accordingly, the hydropathic index of the amino acid (or amino acid sequence) may be considered when modifying the polypeptides specifically disclosed herein.
It is also understood in the art that the substitution of amino acids can be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (incorporated herein by reference in its entirety) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Thus, the hydrophilicity of the amino acid (or amino acid sequence) may be considered when identifying additional polypeptides beyond those specifically disclosed herein.
In embodiments of the invention, the polynucleotide encoding the SOGA polypeptide (or functional fragment) will hybridize to the nucleic acid sequences specifically disclosed herein or fragments thereof under standard conditions as known by those skilled in the art and encode a functional polypeptide or functional fragment thereof.
For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45% formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditions represented by a wash stringency of 50% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) to the polynucleotide sequences encoding the SOGA polypeptides or functional fragments thereof specifically disclosed herein. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989).
In other embodiments, polynucleotide sequences encoding the SOGA polypeptides have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleic acid sequences disclosed herein and in the GenBank accession numbers listed above or functional fragments thereof and encode a functional polypeptide or functional fragment thereof.
Further, it will be appreciated by those skilled in the art that there can be variability in the polynucleotides that encode the polypeptides (and fragments thereof) of the present invention due to the degeneracy of the genetic code. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same polypeptide, is well known in the literature (See, e.g., Table 1).
Likewise, the polypeptides (and fragments thereof) of the invention include polypeptides that have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amino acid sequence identity with the disclosed polypeptide sequences.
As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 (1984), preferably using the default settings, or by inspection.
An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5:151 (1989).
Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Meth. Enzymol., 266:460 (1996); blastmustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
An additional useful algorithm is gapped BLAST as reported by Altschul et al., Nucleic Acids Res. 25:3389 (1997).
A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
In a similar manner, percent nucleic acid sequence identity with respect to the coding sequence of the polypeptides disclosed herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.
The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the polypeptides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.
In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of “0,” which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the “shorter” sequence in the aligned region and multiplying by 100. The “longer” sequence is the one having the most actual residues in the aligned region.
Those skilled in the art will appreciate that the isolated polynucleotides encoding the polypeptides of the invention will typically be associated with appropriate expression control sequences, e.g., transcription/translation control signals and polyadenylation signals.
It will further be appreciated that a variety of promoter/enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter can be constitutive or inducible, depending on the pattern of expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. The promoter is chosen so that it will function in the target cell(s) of interest.
To illustrate, the polypeptide coding sequence can be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, an albumin promoter, an Elongation Factor 1-α (EF1-α) promoter, a PγK promoter, a MFG promoter, or a Rous sarcoma virus promoter.
Inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements, and other promoters regulated by exogenously supplied compounds, including without limitation, the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (see WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA 93:3346 (1996)); the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547 (1992)); the tetracycline-inducible system (Gossen et al., Science 268:1766 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15:239 (1997); Wang et al., Gene Ther., 4:432 (1997)); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865 (1997)).
Other tissue-specific promoters or regulatory promoters include, but are not limited to, promoters that typically confer tissue-specificity in hepatocytes. These include, but are not limited to, promoters for albumin, hepatocyte nuclear factors, transthyretin, α1-antitrypsin, and the hepatitis B virus core promoter. In other embodiments, the promoters typically confer tissue specific in renal cells. These include, but are not limited to, promoters for ksp-cadherin, erythropoietin, γ-glutamyl transpeptidase, kidney androgen-regulated protein, vacuolar H+-ATPase B1 subunit, and AQP2. In other embodiments, the promoters typically confer tissue specific in muscle cells, e.g., skeletal muscle and/or cardiac muscle. Skeletal muscle cell promoters include, but are not limited to, promoters for β-actin, Pitx3, creatine kinase, and myosin light chain. Cardiac muscle cell promoters include, but are not limited to, promoters for cardiac actin, cardiac troponin T, troponin C, myosin light chain-2, and α-myosin heavy chain.
Moreover, specific initiation signals are generally required for efficient translation of inserted polypeptide coding sequences. These translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.
The present invention further provides cells comprising the isolated polynucleotides and polypeptides of the invention. The cell may be a cultured cell or a cell in vivo, e.g., for use in therapeutic methods, diagnostic methods, screening methods, methods for studying the biological action of SOGA polypeptides, in methods of producing the polypeptides, or in methods of maintaining or amplifying the polynucleotides of the invention, etc. In another embodiment, the cell is an ex vivo cell that has been isolated from a subject. The ex vivo cell may be modified and then reintroduced into the subject for diagnostic or therapeutic purposes.
In particular embodiments, the cell is an untransformed cell or a cell from a cell line of a gluconeogenic tissue, such as liver, kidney, skeletal muscle, or cardiac muscle.
The isolated polynucleotide can be incorporated into an expression vector. Expression vectors compatible with various host cells are well known in the art and contain suitable elements for transcription and translation of nucleic acids. Typically, an expression vector contains an “expression cassette,” which includes, in the 5′ to 3′ direction, a promoter, a coding sequence encoding a SOGA polypeptide or functional fragment thereof operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.
Non-limiting examples of promoters of this invention include CYC1, E1TS3, GAL1, GAL4, GAL10, ADH1, PGK, P1105, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet, trp, IPL, IPR, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-, seed specific-, pollen specific-, ovary specific-, pathogenesis or disease related-promoters, cauliflower mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific promoters, root specific promoters, chitinase, stress inducible promoters, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells).
Further examples of animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis and/or disease-related promoters, and promoters that exhibit tissue specificity, such as the elastase I gene control region, which is active in pancreatic acinar cells; the insulin gene control region active in pancreatic beta cells, the immunoglobulin gene control region active in lymphoid cells, the mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; the albumin gene promoter, the Apo AI and Apo All control regions active in liver, the alpha-fetoprotein gene control region active in liver, the alpha 1-antitrypsin gene control region active in the liver, the beta-globin gene control region active in myeloid cells, the myelin basic protein gene control region active in oligodendrocyte cells in the brain, the myosin light chain-2 gene control region active in skeletal muscle, and the gonadotropic releasing hormone gene control region active in the hypothalamus, the pyruvate kinase promoter, the villin promoter, the promoter of the fatty acid binding intestinal protein, the promoter of smooth muscle cell α-actin, and the like. In addition, any of these expression sequences of this invention can be modified by addition of enhancer and/or regulatory sequences and the like.
Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor I (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.
Termination control regions, i.e., terminator or polyadenylation sequences, may be derived from various genes native to the preferred hosts. In some embodiments of the invention, the termination control region may comprise or be derived from a synthetic sequence, a synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.
It will be apparent to those skilled in the art that any suitable vector can be used to deliver the polynucleotide to a cell or subject. The vector can be delivered to cells in vivo. In other embodiments, the vector can be delivered to cells ex vivo, and then cells containing the vector are delivered to the subject. The choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro versus in vivo delivery, level and persistence of expression desired, intended purpose (e.g., for therapy or screening), the target cell or organ, route of delivery, size of the isolated polynucleotide, safety concerns, and the like.
Suitable vectors include plasmid vectors, viral vectors (e.g., retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus and other parvoviruses, lentivirus, poxvirus, or herpes simplex virus), lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors, and the like.
Any viral vector that is known in the art can be used in the present invention. Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found in Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York) and other standard laboratory manuals (e.g., Vectors for Gene Therapy. In: Current Protocols in Human Genetics. John Wiley and Sons, Inc.: 1997).
Non-viral transfer methods can also be employed. Many non-viral methods of nucleic acid transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In particular embodiments, non-viral nucleic acid delivery systems rely on endocytic pathways for the uptake of the nucleic acid molecule by the targeted cell. Exemplary nucleic acid delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
In particular embodiments, plasmid vectors are used in the practice of the present invention. For example, naked plasmids can be introduced into muscle cells by injection into the tissue. Expression can extend over many months, although the number of positive cells is typically low (Wolff et al., Science 247:247 (1989)). Cationic lipids have been demonstrated to aid in introduction of nucleic acids into some cells in culture (Feigner and Ringold, Nature 337:387 (1989)). Injection of cationic lipid plasmid DNA complexes into the circulation of mice has been shown to result in expression of the DNA in lung (Brigham et al., Am. J. Med. Sci. 298:278 (1989)). One advantage of plasmid DNA is that it can be introduced into non-replicating cells.
In a representative embodiment, a nucleic acid molecule (e.g., a plasmid) can be entrapped in a lipid particle bearing positive charges on its surface and, optionally, tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., No Shinkei Geka 20:547 (1992); PCT publication WO 91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
Liposomes that consist of amphiphilic cationic molecules are useful as non-viral vectors for nucleic acid delivery in vitro and in vivo (reviewed in Crystal, Science 270:404 (1995); Blaese et al., Cancer Gene Ther. 2:291 (1995); Behr et al., Bioconjugate Chem. 5:382 (1994); Remy et al., Bioconjugate Chem. 5:647 (1994); and Gao et al., Gene Therapy 2:710 (1995)). The positively charged liposomes are believed to complex with negatively charged nucleic acids via electrostatic interactions to form lipid:nucleic acid complexes. The lipid:nucleic acid complexes have several advantages as nucleic acid transfer vectors. Unlike viral vectors, the lipid:nucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they can evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency. A number of publications have demonstrated that amphiphilic cationic lipids can mediate nucleic acid delivery in vivo and in vitro (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987); Loeffler et al., Meth. Enzymol. 217:599 (1993); Feigner et al., J. Biol. Chem. 269:2550 (1.994)).
Several groups have reported the use of amphiphilic cationic lipid:nucleic acid complexes for in vivo transfection both in animals and in humans (reviewed in Gao et al., Gene Therapy 2:710 (1995); Zhu et al., Science 261:209 (1993); and Thierry et al., Proc. Natl. Acad. Sci. USA 92:9742 (1995)). U.S. Pat. No. 6,410,049 describes a method of preparing cationic lipid:nucleic acid complexes that have a prolonged shelf life.
Expression vectors can be designed for expression of polypeptides in prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in bacterial cells such as E. coli, insect cells (e.g., the baculovirus expression system), yeast cells, plant cells or mammalian cells. Some suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Examples of bacterial vectors include pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., EMBO J. 6:229 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933 (1982)), pJRY88 (Schultz et al., Gene 54:113 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors available for expression of nucleic acids to produce proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156 (1983)) and the pVL series (Lucklow and Summers Virology 170:31 (1989)).
Examples of mammalian expression vectors include pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187 (1987)). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.
Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).
In addition to the regulatory control sequences discussed above, the recombinant expression vector can contain additional nucleotide sequences. For example, the recombinant expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acids (e.g., DNA and RNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
If stable integration is desired, often only a small fraction of cells (in particular, mammalian cells) integrate the foreign DNA into their genome. In order to identify and select integrants, a nucleic acid that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the nucleic acid of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that comprising the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
Polypeptides and fragments of the invention can be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety. Likewise, the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable “carrier” proteins prior to administration.
Another embodiment of the invention relates to homologs of the polypeptides of the invention that are peptidomimetic compounds that are designed based upon the amino acid sequences of the functional polypeptide fragments. Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a “peptide motif”) that is substantially the same as the three-dimensional conformation of a selected peptide. The peptide motif provides the peptidomimetic compound with biological activities qualitatively identical to that of the functional fragment from which the peptidomimetic was derived. Peptidomimetic compounds can have additional characteristics that enhance their therapeutic utility, such as increased cell permeability and prolonged biological half-life.
The peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based. Several types of chemical bonds, e.g., ester, thioester, thioamide, retroamide, reduced carbon A, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics.
III. Inhibitors of SOGA Polypeptides and Polynucleotides
As one aspect, the invention provides agents that inhibit the expression and/or activity of SOGA polypeptides or polynucleotides. These agents can be used to inhibit or down-regulate the SOGA signaling pathway, e.g., in a cell or a subject.
In one embodiment of the invention, decreasing the expression and/or activity of a SOGA polypeptide comprises decreasing the level of a nucleic acid (DNA or RNA) encoding the polypeptide or the level of expression of the polypeptide from the nucleic acid. Numerous methods for reducing the level and/or expression of polynucleotides in vitro or in vivo are known. For example, the nucleotide sequences for the human and mouse SOGA polypeptides are disclosed herein. An antisense nucleotide sequence or nucleic acid encoding an antisense nucleotide sequence can be generated to any portion thereof in accordance with known techniques.
The term “antisense nucleotide sequence” or “antisense oligonucleotide” as used herein, refers to a nucleotide sequence that is complementary to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids that express the same can be made in accordance with conventional techniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al. The antisense nucleotide sequence can be complementary to the entire nucleotide sequence encoding the polypeptide or a portion thereof of at least 10, 20, 40, 50, 75, 100, 150, 200, 300, or 500 contiguous bases or more and will reduce the level of polypeptide production.
Those skilled in the art will appreciate that it is not necessary that the antisense nucleotide sequence be fully complementary to the target sequence as long as the degree of sequence similarity is sufficient for the antisense nucleotide sequence to hybridize to its target and reduce production of the polypeptide. As is known in the art, a higher degree of sequence similarity is generally required for short antisense nucleotide sequences, whereas a greater degree of mismatched bases will be tolerated by longer antisense nucleotide sequences.
For example, hybridization of such nucleotide sequences can be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45% formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and/or conditions represented by a wash stringency of 50% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) to the nucleotide sequences specifically disclosed herein. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989).
In other embodiments, antisense nucleotide sequences of the invention have at least about 70%, 80%, 90%, 95%, 97%, 98% or higher sequence similarity with the complement of the coding sequences specifically disclosed herein and will reduce the level of polypeptide production.
In other embodiments, the antisense nucleotide sequence can be directed against any coding sequence, the silencing of which results in a modulation of a SOGA polypeptide.
The length of the antisense nucleotide sequence (i.e., the number of nucleotides therein) is not critical as long as it binds selectively to the intended location and reduces transcription and/or translation of the target sequence, and can be determined in accordance with routine procedures. In general, the antisense nucleotide sequence will be from about eight, ten or twelve nucleotides in length up to about 20, 30, 50, 75 or 100 nucleotides, or longer, in length.
An antisense nucleotide sequence can be constructed using chemical synthesis and enzymatic ligation reactions by procedures known in the art. For example, an antisense nucleotide sequence can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleotide sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleotide sequence include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethyl guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleotide sequence can be produced using an expression vector into which a nucleic acid has been cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
The antisense nucleotide sequences of the invention further include nucleotide sequences wherein at least one, or all, of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues can be modified as described. In another non-limiting example, the antisense nucleotide sequence is a nucleotide sequence in which one, or all, of the nucleotides contain a 2′ lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides can be modified as described. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989); Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al., Nucleic Acids Res. 18:3537 (1990); Sproat et al., Nucleic Acids Res. 17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011 (1988); incorporated by reference herein in their entireties for their teaching of methods of making antisense molecules, including those containing modified nucleotide bases).
Triple helix base-pairing methods can also be employed to inhibit production of SOGA polypeptides. Triple helix pairing is believed to work by inhibiting the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., (1994) In: Huber et al., Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
Small Interference (si) RNA, also known as RNA interference (RNAi) molecules, provides another approach for modulating the expression of SOGA polypeptides. The siRNA can be directed against polynucleotide sequences encoding the SOGA polypeptides or any other sequence that results in modulation of the expression of SOGA polypeptides.
siRNA is a mechanism of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) corresponding to a coding sequence of interest is introduced into a cell or an organism, resulting in degradation of the corresponding mRNA. The mechanism by which siRNA achieves gene silencing has been reviewed in Sharp et al., Genes Dev. 15:485 (2001); and Hammond et al., Nature Rev. Gen. 2:110 (2001)). The siRNA effect persists for multiple cell divisions before gene expression is regained. siRNA is therefore a powerful method for making targeted knockouts or “knockdowns” at the RNA level. siRNA has proven successful in human cells, including human embryonic kidney and HeLa cells (see, e.g., Elbashir et al., Nature 411:494 (2001)). In one embodiment, silencing can be induced in mammalian cells by enforcing endogenous expression of RNA hairpins (see Paddison et al., Proc. Natl. Acad. Sci. USA 99:1443 (2002)). In another embodiment, transfection of small (21-23 nt) dsRNA specifically inhibits nucleic acid expression (reviewed in Caplen, Trends Biotechnol. 20:49 (2002)).
siRNA technology utilizes standard molecular biology methods. dsRNA corresponding to all or a part of a target coding sequence to be inactivated can be produced by standard methods, e.g., by simultaneous transcription of both strands of a template DNA (corresponding to the target sequence) with T7 RNA polymerase. Kits for production of dsRNA for use in siRNA are available commercially, e.g., from New England Biolabs, Inc. Methods of transfection of dsRNA or plasmids engineered to make dsRNA are routine in the art.
MicroRNA (miRNA), single stranded RNA molecules of about 21-23 nucleotides in length, can be used in a similar fashion to siRNA to modulate gene expression (see U.S. Pat. No. 7,217,807).
Silencing effects similar to those produced by siRNA have been reported in mammalian cells with transfection of a mRNA-cDNA hybrid construct (Lin et al., Biochem. Biophys. Res. Commun. 281:639 (2001)), providing yet another strategy for silencing a coding sequence of interest.
The expression of SOGA polypeptides can also be inhibited using ribozymes. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim et al., Proc. Natl. Acad. Sci. USA 84:8788 (1987); Gerlach et al., Nature 328:802 (1987); Forster and Symons, Cell 49:211 (1987)). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, J. Mol. Biol. 216:585 (1990); Reinhold-Ilurek and Shub, Nature 357:173 (1992)). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, Nature 338:217 (1989)). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., Proc. Natl. Acad. Sci. USA 88:10591 (1991); Sarver et al., Science 247:1222 (1990); Sioud et al., J. Mol. Biol. 223:831 (1992)).
In another embodiment of the invention, decreasing the expression and/or activity of SOGA polypeptides comprises decreasing the activity of the polypeptide. Polypeptide activity can be modulated by interaction with an antibody or antibody fragment. The antibody or antibody fragment can bind to the polypeptide or to any other polypeptide of interest, as long as the binding between the antibody or the antibody fragment and the target polypeptide results in modulation of the activity of the SOGA polypeptide.
The term “antibody” or “antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody can be monoclonal or polyclonal and can be of any species of origin, including (for example) mouse, rat, rabbit, horse, goat, sheep, camel, or human, or can be a chimeric antibody. See, e.g., Walker et al., Molec. Immunol. 26:403 (1989). The antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also be chemically constructed according to the method disclosed in U.S. Pat. No. 4,676,980.
Antibody fragments included within the scope of the present invention include, for example, Fab, Fab′, F(ab′)2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Such fragments can be produced by known techniques. For example, F(ab′)2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 254:1275 (1989)).
Antibodies of the invention may be altered or mutated for compatibility with species other than the species in which the antibody was produced. For example, antibodies may be humanized or camelized. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593 (1992)).
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues (e.g., all of the CDRs or a portion thereof) and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al, and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10:779 (1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812 (1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65 (1995).
Polyclonal antibodies used to carry out the present invention can be produced by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen to which a monoclonal antibody to the target binds, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures.
Monoclonal antibodies used to carry out the present invention can be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265:495 (1975). For example, a solution containing the appropriate antigen can be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. The hybridoma cells are then grown in a suitable medium and the supernatant screened for monoclonal antibodies having the desired specificity. Monoclonal Fab fragments can be produced in E. coli by recombinant techniques known to those skilled in the art. See, e.g., Huse, Science 246:1275 (1989).
Antibodies specific to the target polypeptide can also be obtained by phage display techniques known in the art.
Various immunoassays can be used for screening to identify antibodies having the desired specificity for the polypeptides of this invention. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known in the art. Such immunoassays typically involve the measurement of complex formation between an antigen and its specific antibody (e.g., antigen/antibody complex formation). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the polypeptides or peptides of this invention can be used as well as a competitive binding assay.
Antibodies can be conjugated to a solid support (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques. Antibodies can likewise be conjugated to detectable groups such as radiolabels (e.g., 35S, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescence labels (e.g., fluorescein) in accordance with known techniques. Determination of the formation of an antibody/antigen complex in the methods of this invention can be by detection of, for example, precipitation, agglutination, flocculation, radioactivity, color development or change, fluorescence, luminescence, etc., as is well known in the art.
In one embodiment, the activity of SAGA polypeptides is inhibited using aptamers. Recently, small structured single-stranded RNAs, also known as RNA aptamers, have emerged as viable alternatives to small-molecule and antibody-based therapy (Que-Gewirth et al., Gene Ther. 14:283 (2007); Ireson et al., Mol. Cancer. Ther. 5:2957 (2006)). RNA aptamers specifically bind target proteins with high affinity, are quite stable, lack immunogenicity, and elicit biological responses. Aptamers are evolved by means of an iterative selection method called SELEX (systematic evolution of ligands by exponential enrichment) to specifically recognize and tightly bind their targets by means of well-defined complementary three-dimensional structures.
RNA aptamers represent a unique emerging class of therapeutic agents (Que-Gewirth et al., Gene Ther. 14:283 (2007); Ireson et al., Mol. Cancer. Ther. 5:2957 (2006)). They are relatively short (12-30 nucleotide) single-stranded RNA oligonucleotides that assume a stable three-dimensional shape to tightly and specifically bind selected protein targets to elicit a biological response. In contrast to antisense oligonucleotides, RNA aptamers can effectively target extracellular targets. Like antibodies, aptamers possess binding affinities in the low nanomolar to picomolar range. In addition, aptamers are heat stable, lack immunogenicity, and possess minimal interbatch variability. Chemical modifications, such as amino or fluoro substitutions at the 2′ position of pyrimidines, may reduce degradation by nucleases. The biodistribution and clearance of aptamers can also be altered by chemical addition of moieties such as polyethylene glycol and cholesterol. Further, SELEX allows selection from libraries consisting of up to 1015 ligands to generate high-affinity oligonucleotide ligands to purified biochemical targets.
In another embodiment, the method of decreasing the activity of a SOGA polypeptide comprises delivering to a cell or to a subject an agent that decreases the activity of a SOGA polypeptide, the agent administered in an amount effective to modulate the activity of the polypeptide. The agent can interact directly with the SOGA polypeptide to decrease the activity of the polypeptide. Alternatively, the agent can interact with any other polypeptide, nucleic acid or other molecule if such interaction results in a decrease of the activity of the SOGA.
The term “agent” as used herein is intended to be interpreted broadly and encompasses organic and inorganic molecules. Organic compounds include, but are not limited to, small molecules, polypeptides, lipids, carbohydrates, coenzymes, aptamers, and nucleic acid molecules (e.g., gene delivery vectors, antisense oligonucleotides, siRNA, all as described above).
Polypeptides include, but are not limited to, antibodies (described in more detail above) and enzymes. Nucleic acids include, but are not limited to, DNA, RNA and DNA-RNA chimeric molecules. Suitable RNA molecules include siRNA, antisense RNA molecules and ribozymes (all of which are described in more detail above). The nucleic acid can further encode any polypeptide such that administration of the nucleic acid and production of the polypeptide results in a decrease of the activity of a SOGA polypeptide.
The agent can further be an agent that is identified by any of the screening methods described below.
In one embodiment of the invention, the agent is a modulator of the insulin and/or adiponectin signaling pathways that directly or indirectly inhibits SOGA expression and/or activity. For example, the agent can be an activator of AMPK such as AICAR (N1-(β-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide). In another embodiment, the agent can be a PI3 kinase inhibitor such as LY294002. In a further embodiment, the agent can be an inhibitor of adiponectin such as rapamycin.
IV. Inhibition of Glucose Production
Increases in SOGA polypeptide levels and/or activity result in the inhibition of glucose production in cells. Thus, the SOGA polypeptides and polynucleotides of the invention can be used in methods in which a decrease in glucose production is desired for research, diagnostic, and/or therapeutic proposes. These methods can be carried using techniques to increase the expression and/or activity of SOGA polypeptides in a cell, in a tissue, and/or in a subject.
One aspect of the invention relates to a method of decreasing glucose production in a cell, comprising contacting said cell with a polynucleotide, polypeptide, or fusion protein of the invention in an amount effective to decrease glucose production in the cell.
Another aspect of the invention relates to a method of decreasing autophagy in a cell, comprising contacting said cell with a polynucleotide, polypeptide, or fusion protein of the invention in an amount effective to decrease autophagy in said cell.
The cells to be contacted can be in vitro, ex vivo, or in vivo (e.g., in an animal model of disease or a patient). Cells can be contacted with a polynucleotide or polypeptide of the invention by any means known in the art and as described herein.
A further aspect of the invention relates to a method of decreasing blood glucose levels in a subject, comprising delivering to said subject a polynucleotide, polypeptide, or fusion protein of the invention in an amount effective to decrease the blood glucose levels in said subject.
Another aspect of the invention relates to a method of increasing insulin sensitivity in a subject, comprising delivering to said subject a polynucleotide, polypeptide, or fusion protein of the invention in an amount effective to increase insulin sensitivity in said subject.
In one embodiment, the subject is one that is in need of decreased glucose levels and/or increased insulin sensitivity. The subject can currently have or be at risk for a carbohydrate-related metabolic disorder such as diabetes mellitus (Type I or Type II), alcoholic ketoacidosis, diabetic ketoacidosis, nonketotic hyperosmolar syndrome, and new onset diabetes (NOD), such as in cancer patients undergoing chemotherapy, immunosuppressed patients, post-operative patients, and trauma patients. In certain embodiments, the methods of the invention encompass methods of treating a subject having a carbohydrate-related metabolic disorder such as diabetes, comprising delivering to said subject a polynucleotide, polypeptide, or fusion protein of the invention in an amount effective to treat the disorder.
In one embodiment, increasing the expression and/or activity of a SOGA polypeptide comprises delivering a nucleic acid encoding the polypeptide or a fragment or homolog thereof to the cell or tissue or subject. In another embodiment, increasing the expression and/or activity of a SOGA polypeptide comprises delivering the polypeptide itself or a fragment or homolog thereof to the cell or tissue or subject.
In one embodiment, the methods comprise delivering to the subject an isolated SOGA polypeptide. In exemplary embodiments, the polypeptide comprises, consists essentially of or consists of the amino acid sequence of the polypeptide disclosed herein or a functional fragment thereof. In another embodiment, the isolated polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 70% identical, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the disclosed amino acid sequence or a functional fragment thereof (and polynucleotide sequences encoding the same).
In one embodiment, the polynucleotides, polypeptides, or homologs thereof of the invention are administered directly to the subject. Generally, the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. They can be delivered directly to a site involved in gluconeogenesis, such as the liver, kidney, and/or muscle. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01-100.0 μg/kg. Wide variations in the needed dosage are to be expected in view of the variety of polypeptides and fragments available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of the polypeptide in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
According to certain embodiments, the polynucleotides or vectors can be targeted to specific cells or tissues in vivo. Targeting delivery vehicles, including liposomes and viral vector systems are known in the art. For example, a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind. Targeting liposomes are described, for example, in Ho et al., Biochemistry 25:5500 (1986); Ho et al., J. Biol. Chem. 262:13979 (1987); Ho et al., J. Biol. Chem. 262:13973 (1987); and U.S. Pat. No. 4,957,735 to Huang et al., each of which is incorporated herein by reference in its entirety). Enveloped viral vectors can be modified to deliver a nucleic acid molecule to a target cell by modifying or substituting an envelope protein such that the virus infects a specific cell type. In adenoviral vectors, the gene encoding the attachment fibers can be modified to encode a protein domain, that binds to a cell-specific receptor. Herpesvirus vectors naturally target the cells of the central and peripheral nervous system. Alternatively, the route of administration can be used to target a specific cell or tissue. For example, intracoronary administration of an adenoviral vector has been shown to be effective for the delivery of a gene to cardiac myocytes (Maurice et al., J. Clin. Invest. 104:21 (1999)). Intravenous delivery of cholesterol-containing cationic liposomes has been shown to preferentially target pulmonary tissues (Liu et al., Nature Biotechnol. 15:167 (1997)), and effectively mediate transfer and expression of genes in vivo. Other examples of successful targeted in vivo delivery of nucleic acid molecules are known in the art. Finally, a recombinant nucleic acid molecule can be selectively (i.e., preferentially, substantially exclusively) expressed in a target cell by selecting a transcription control sequence, and preferably, a promoter, which is selectively induced in the target cell and remains substantially inactive in non-target cells.
The polypeptides and polynucleotides of the present invention can optionally be delivered in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered concurrently with the polypeptides and polynucleotides of the invention. As used herein, the word “concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other). In one embodiment, the polypeptides and polynucleotides of the invention are administered in conjunction with anti-diabetic agents, including without limitation, (1) PPARγ agonists such as glitazones (e.g., ciglitazone, darglitazone, englitazone, isaglitazone (MCC-555), pioglitazone, rosiglitazone, troglitazone, BRL49653, CLX-0921, 5-BTZD, GW-0207, LG-100641, and LY-300512; (2) biguanides such as buformin, metformin, and phenformin; (3) protein tyrosine phosphatase-1B (PTP-1B) inhibitors such as ISIS 113715; (4) sulfonylureas such as acetohexamide, chlorpropamide, diabinese, glibenclamide, glypizide, glyburide, glimepiride, gliclazide, glipentide, gliquidone, glisolamide, tolazamide, and tolbutamide; (5) meglitinides such as repaglinide and nateglinide; (6) alpha glucoside hydrolase inhibitors such as acarbose, adiposine, camiglibose, emiglitate, miglitol, voglibose, pradimicin-Q, salbostatin, CKD-711, MDL-25,637, MDL-73,945, and MOR 14; (7) alpha-amylase inhibitors such as tendamistat, trestatin, and Al-3688; (8) insulin secretagogues such as linogliride and A4166; (9) fatty acid oxidation inhibitors such as clomoxir and etomoxir; (10) adenosine A2 antagonists such as midaglizole, isaglidole, deriglidole, idazoxan, earoxan, and fluparoxan; (11) insulin or insulin mimetics such as biota, LP-100, novarapid, insulin detemir, insulin lispro, insulin glargine, insulin zinc suspension (lente and ultralente), Lys-Pro insulin, GLP-1 (73-7) (insulintropin), and GLP-1 (7-36)-NH2); (12) non-thiazolidinediones such as JT-501 and farglitazar (GW-2570/G1-262579); (13) PPARα/γ dual agonists such as BVT-142, CLX-0940, GW-1536, GW1929, GW-2433, KRP-297, L-796449, LR-90, MK-0767, SB 219994, muraglitazar and reglitazar (ITT-501); (14) other insulin sensitizing drugs; (15) VPAC2 receptor agonists; (16) GLK modulators such as those disclosed in WO 03/015774; (17) retinoid modulators such as those disclosed in WO 03/000249; (18) GSK 3beta/GSK 3 inhibitors such as 4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine; (19) glycogen phosphorylase (HGLPa) inhibitors such as those disclosed in WO 03/037864; (20) ATP consumption promoters such as those disclosed in WO 03/007990; (21) TRB3 inhibitors, (22) vanilloid receptor ligands such as those disclosed in WO 03/049702, (23) hypoglycemic agents such as those disclosed in WO 03/015781 and WO 03/040114; and (24) Insulin-responsive DNA binding protein-1 (IRDBP-1) as disclosed in WO 03/057827.
V. Stimulation of Glucose Production
Decreases in SOGA polypeptide levels and/or activity result in the stimulation of glucose production in cells. Thus, inhibitors of the SOGA polypeptides and polynucleotides of the invention can be used in methods in which an increase in glucose production is desired for research, diagnostic, and/or therapeutic proposes. These methods can be carried using techniques to decrease the expression and/or activity of SOGA polypeptides in a cell, in a tissue, and/or in a subject,
One aspect of the invention relates to a method of increasing glucose production in a cell, comprising contacting said cell with an agent that decreases the activity of a polynucleotide or polypeptide of the invention in an amount effective to increase glucose production in the cell.
Another aspect of the invention relates to a method of increasing autophagy in a cell, comprising contacting said cell with an agent that decreases the activity of a polynucleotide or polypeptide of the invention in an amount effective to increase autophagy in said cell.
The cells to be contacted can be in vitro, ex vivo, or in vivo (e.g., in an animal model of disease or a patient). Cells can be contacted with an agent by any means known in the art and as described herein.
A further aspect of the invention relates to a method of increasing blood glucose levels in a subject, comprising delivering to said subject an agent that decreases the activity of a polynucleotide or polypeptide of the invention in an amount effective to increase the blood glucose levels in said subject.
Another aspect of the invention relates to a method of decreasing insulin sensitivity in a subject, comprising delivering to said subject an agent that decreases the activity of a polynucleotide or polypeptide of the invention in an amount effective to decrease insulin sensitivity in said subject.
In one embodiment, the subject is one that is in need of increased glucose levels and/or decreased insulin sensitivity. The subject can currently have or be at risk for a carbohydrate-related metabolic disorder such as hypoglycemia, e.g., as a result of sepsis, malaria, or injection of insulin.
Agents that can be used in the methods of the invention include, without limitation, an antisense oligonucleotide, ribozyme, or siRNA that targets a SOGA polynucleotide, an antibody or antibody fragment that binds to a SOGA polypeptide, agents that modulate the insulin and/or adiponectin signaling pathways, and agents identified by the screening methods described below.
The agents of the present invention can optionally be delivered in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered concurrently with the agents of the invention.
VI. Monitoring of Responsiveness to Treatment
The increased levels of SOGA polypeptide in response to administration of insulin and adiponectin provides the basis for monitoring responsiveness of a subject to anti-diabetic treatments. It is known that insulin treatment of diabetics is not effective 100% of the time and that certain drugs may induce adiponectin but do not necessarily lower glucose. Measuring the induction of SOGA in response to an anti-diabetic treatment may provide insight into the ability of a subject to respond to the treatment and can help identify subjects that are likely to respond or not respond to a particular treatment.
One aspect of the invention relates to a method of measuring the response of a subject to a treatment for diabetes, comprising determining the circulating level of a SOGA polypeptide or a functional fragment thereof in said subject after administration of the treatment and comparing it to the circulating level of the polypeptide or a functional fragment thereof in said subject before administration of the treatment.
Another aspect of the invention relates to a method of predicting the clinical outcome of a diabetes treatment in a subject, comprising determining the circulating level of a SOGA polypeptide or a functional fragment thereof in said subject after administration of the treatment and comparing it to the circulating level of the polypeptide or a functional fragment thereof in said subject before administration of the treatment.
In these methods, an increase in circulating levels of SOGA polypeptide or a functional fragment thereof subsequent to administration of an anti-diabetic treatment is indicative that the subject will respond to the treatment (e.g., the treatment will lower glucose levels). Conversely, if the circulating level of SOGA does not increase or increases less than a “normal” amount, the subject may not respond favorably to the treatment. The magnitude of the increase in SOGA polypeptide (e.g., a “normal” increase as compared to a “less than normal” increase in SOGA) can be classified based on average numbers in a population of similar subjects.
In one embodiment, determining the level of a SOGA polypeptide comprises determining the level the polypeptide. Determining the level of a polypeptide can be carried out by any means known in the art and as described herein, such as Western blots, immunoblots, immunoprecipitation, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbant assays, and radioimmunoassays. Assays for expression and/or activity can be carried out automatically or partially automatically in a machine or apparatus designed to perform such assays, e.g., using computer-assisted methods. The results of the assays can be stored in a computer database and analyzed to produce predictive results. In some embodiments, the data can be analyzed, e.g., by comparing intra-patient results over time or before and after treatment or comparing inter-patient results to determine baseline and/or abnormal values in a population.
In a further embodiment, determining the level of a SOGA polypeptide comprises determining the activity of the polypeptides. The activity may be any activity associated with the polypeptide, including, without limitation, inhibition of glucose production, enzyme activity, protein interaction, receptor binding, ligand binding, a cell signal transduction event, etc.
In one embodiment, determining the level of a SOGA polypeptide comprises determining the level of a nucleic acid encoding the polypeptide. Determining the level of a nucleic acid can be carried out by any means known in the art and as described herein, such as Northern blots, dot blots, PCR, RT-PCR, quantitative PCR, sequence analysis, gene microarray analysis, in situ hybridization, and detection of a reporter gene.
One aspect of the invention relates to kits useful for carrying out the methods of the invention. One embodiment relates to kits for determining the level of expression and/or activity of SOGA, e.g., to assess responsiveness to anti-diabetic treatment, comprising a reagent for determining the expression and/or activity of a SOGA polypeptide or a functional fragment thereof. The reagents may be nucleic acids (e.g., an oligonucleotide that specifically hybridizes to a nucleic acid encoding a SOGA polypeptide and can be used as a hybridization probe or an amplification primer), antibodies (e.g., one the specifically binds to a SOGA polypeptide), or other agents that specifically recognize the polynucleotides or polypeptides of the invention.
The reagents can be conjugated to a detectable tag or detectable label. Such a tag can be any suitable tag which allows for detection of the reagents and includes, but is not limited to, any composition or label detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
In addition, the reagents can be immobilized on a substrate. Such a substrate can include any suitable substrate for immobilization of a detection reagent such as would be used in any of the previously described methods of detection. Briefly, a substrate suitable for immobilization of a detection reagent includes any solid support, such as any solid organic, biopolymer or inorganic support that can form a bond with the detection reagent without significantly effecting the activity and/or ability of the detection reagent to detect the desired target molecule. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized intact whole cells, and stabilized crude whole cell/membrane homogenates. Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex®), agarose, collagen and chitin. Exemplary inorganic supports include glass beads (porous and nonporous), stainless steel, metal oxides (e.g., porous ceramics such as ZrO2, TiO2, Al2O3, and NiO) and sand.
The kits may further comprise other components useful for detecting expression or activity, e.g., buffers, cells, culture medium, enzymes, labeling reagents, containers, etc.
In one embodiment, the kit comprises an array of reagents for determining expression and/or activity. The array can comprise a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a polynucleotide or polypeptide of the invention. The array can have a density of at least, or less than, 10, 20 50, 100, 200, 500, 700, 1,000, 2,000, 5,000 or 10,000 or more addresses/cm2, and ranges between. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to addresses of the plurality can be disposed on the array.
In one embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a polynucleotide of the invention, e.g., the sense or anti-sense strand. Each address of the subset can include a capture probe that hybridizes to a different region of a polynucleotide. An array can be generated by any of a variety of methods. Appropriate methods include, e.g., photolithographic methods (e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).
In another embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a polypeptide of the invention or fragment thereof. The polypeptide capture probe can be a naturally-occurring interaction partner of a SOGA polypeptide. In one embodiment, the polypeptide is an antibody, e.g., an antibody specific for a SOGA polypeptide, such as a polyclonal antibody, a monoclonal antibody, or a single-chain antibody.
VII. Screening Assays and Animal Models
The identification of polynucleotides and polypeptides that are involved in insulin and adiponectin signaling and glucose regulation provides targets that can be used to screen for agents that regulate glucose production as well as models for studying these pathways in vitro or in animals.
One aspect of the invention relates to a method of identifying an agent that binds to a SOGA polypeptide or a functional fragment thereof of the invention, comprising:
contacting the polypeptide or a functional fragment thereof with a test agent under conditions whereby binding between the polypeptide or a functional fragment thereof and the test agent can occur; and
detecting binding between the polypeptide or a functional fragment thereof and the test agent.
Another aspect of the invention relates to a method of identifying an agent that modulates the activity of a SOGA polypeptide or a functional fragment thereof of the invention, comprising:
contacting the polypeptide or a functional fragment thereof with a test agent under conditions whereby modulation of the activity of the polypeptide or a functional fragment thereof can occur; and
detecting modulation of the activity of the polypeptide or a functional fragment thereof upon contact with the test agent as compared to activity of the polypeptide or a functional fragment thereof in the absence of contact with the test agent.
In each aspect above, the assay may be a cell-based or cell-free assay. In one embodiment, the cell may be a primary cell, e.g., an endothelial cell or a tumor cell, such as a breast tumor cell. In another embodiment, the cell is from a cell line, e.g., a hepatocyte, kidney, or muscle cell line or a tumor cell line. The cell may be contacted with the agent in vitro (e.g., in a culture dish) or in an animal (e.g., a transgenic animal or an animal model). In one embodiment, the detected increase or decrease in expression and/or activity is statistically significant, e.g., at least p<0.05, e.g., p<0.01, 0.005, or 0.001. In another embodiment, the detected increase or decrease is at least about 10%, 20%, 30%, 40%, 50%, 60&, 70%, 80%, 90%, 100% or more.
Any desired end-point can be detected in a screening assay, e.g., binding to the polypeptide, gene or RNA, modulation of the activity of the polypeptide, modulation of glucose-related pathways, and/or interference with binding by a known regulator of a polynucleotide or polypeptide. Methods of detecting the foregoing activities are known in the art and include the methods disclosed herein.
Any agent of interest can be screened according to the present invention. Suitable test agents include organic and inorganic molecules. Suitable organic molecules can include but are not limited to small molecules (compounds less than about 1000 Daltons), polypeptides (including enzymes, antibodies, and Fab′ fragments), carbohydrates, lipids, coenzymes, and nucleic acid molecules (including DNA, RNA, and chimerics and analogs thereof) and nucleotides and nucleotide analogs. In particular embodiments, the agent is an antisense nucleic acid, an siRNA, or a ribozyme that inhibits production of a SOGA polypeptide.
Further, the methods of the invention can be practiced to screen an agent library, e.g., a small molecule library, a combinatorial chemical compound library, a polypeptide library, a cDNA library, a library of antisense nucleic acids, and the like, or an arrayed collection of agents such as polypeptide and nucleic acid arrays.
In one representative embodiment, the invention provides methods of screening test agents to identify a test agent that binds to a SOGA polypeptide or functional fragment thereof. Agents that are identified as binding to the polypeptide or functional fragment can be subject to further screening (e.g., for modulation of glucose production) using the methods described herein or other suitable techniques.
Also provided are methods of screening agents to identify those that modulate the activity of a SOGA polypeptide or functional fragment thereof. The term “modulate” is intended to refer to agents that enhance (e.g., increase) or inhibit (e.g., reduce) the activity of the polypeptide (or functional fragment). For example, the interaction of the polypeptide or functional fragment with a binding partner can be evaluated. As another alternative, physical methods, such as NMR, can be used to assess biological function. Activity of the SOGA polypeptides or functional fragment can be evaluated by any method known in the art, including the methods disclosed herein.
Agents that are identified as modulators of activity can optionally be further screened using the methods described herein (e.g., for binding to the SOGA polypeptide or functional fragment thereof, polynucleotide or RNA, modulation of glucose, and the like). The agent can directly interact with the polypeptide or functional fragment, polynucleotide or mRNA and thereby modulate its activity. Alternatively, the agent can interact with any other polypeptide, nucleic acid or other molecule as long as the interaction results in a modulation of the activity of the SOGA polypeptide or functional fragment.
With respect to cell-free binding assays, test agents can be synthesized or otherwise affixed to a solid substrate, such as plastic pins, glass slides, plastic wells, and the like. For example, the test agents can be immobilized utilizing conjugation of biotin and streptavidin by techniques well known in the art. The test agents are contacted with the polypeptide or functional fragment thereof and washed. Bound polypeptide can be detected using standard techniques in the art (e.g., by radioactive or fluorescence labeling of the polypeptide or functional fragment, by ELISA methods, and the like).
Alternatively, the target can be immobilized to a solid substrate and the test agents contacted with the bound polypeptide or functional fragment thereof. Identifying those test agents that bind to and/or modulate the SOGA polypeptide or functional fragment can be carried out with routine techniques. For example, the test agents can be immobilized utilizing conjugation of biotin and streptavidin by techniques well known in the art. As another illustrative example, antibodies reactive with the polypeptide or functional fragment can be bound to the wells of the plate, and the polypeptide trapped in the wells by antibody conjugation. Preparations of test agents can be incubated in the polypeptide (or functional fragment)-presenting wells and the amount of complex trapped in the well can be quantitated.
In another representative embodiment, a fusion protein can be provided which comprises a domain that facilitates binding of the polypeptide to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with cell lysates (e.g., 35S-labeled) and the test agent, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel detected directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of SOGA polypeptide or functional fragment thereof found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
Another technique for agent screening provides for high throughput screening of agents having suitable binding affinity to the polypeptide of interest, as described in published PCT application WO84/03564. In this method, a large number of different small test agents are synthesized on a solid substrate, such as plastic pins or some other surface. The test agents are reacted with the SOGA polypeptide or functional fragment thereof and washed. Bound polypeptide is then detected by methods well known in the art. Purified polypeptide or a functional fragment can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
With respect to cell-based assays, any suitable cell can be used, including bacteria, yeast, insect cells (e.g., with a baculovirus expression system), avian cells, mammalian cells, or plant cells. In exemplary embodiments, the assay is carried out in a cell line that naturally expresses the polynucleotide or produces the polypeptide, e.g., hepatocytes or renal cells. Further, in other embodiments, it is desirable to use nontransformed cells (e.g., primary cells) as transformation may alter the function of the polypeptide.
The screening assay can be used to detect agents that bind to or modulate the activity of the native SOGA polypeptide (e.g., polypeptide that is normally produced by the cell). Alternatively, the cell can be modified to express (e.g., overexpress) a recombinant SOGA polypeptide or functional fragment thereof. According to this embodiment, the cell can be transiently or stably transformed with a polynucleotide encoding the SOGA polypeptide or functional fragment, but is preferably stably transformed, for example, by stable integration into the genome of the organism or by expression from a stably maintained episome (e.g., Epstein Barr Virus derived episomes). In another embodiment, a polynucleotide encoding a reporter molecule can be linked to a regulatory element of the polynucleotide encoding a SOGA polypeptide and used to identify compounds that modulate expression of the polypeptide.
In a cell-based assay, the agent to be screened can interact directly with the SOGA polypeptide or functional fragment thereof (i.e., bind to it) and modulate the activity thereof. Alternatively, the agent can be one that modulates polypeptide activity (or the activity of a functional fragment) at the nucleic acid level. To illustrate, the agent can modulate transcription of the gene (or transgene), modulate the accumulation of mRNA (e.g., by affecting the rate of transcription and/or turnover of the mRNA), and/or modulate the rate and/or amount of translation of the mRNA transcript.
As a further type of cell-based binding assay, the SOGA polypeptide or functional fragment thereof can be used as a “bait protein” in a two-hybrid or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223 (1993); Madura et al., J. Biol. Chem. 268:12046 (1993); Bartel et al., Biotechniques 14:920 (1993); Iwabuchi et al., Oncogene 8:1693 (1993); and PCT publication WO94/10300), to identify other polypeptides that bind to or interact with the polypeptide of the invention or functional fragment thereof.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the polynucleotide that encodes the SOGA polypeptide or functional fragment thereof is fused to a nucleic acid encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, optionally from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a nucleic acid that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact in vivo, forming a complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter sequence (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the nucleic acid encoding the polypeptide that exhibited binding to the SOGA polypeptide or functional fragment.
As another cell-based assay, the invention provides a method of screening an agent for modulation of glucose production. In particular embodiments, the cell comprises an isolated polynucleotide encoding the SOGA polypeptide or functional fragment thereof. According to this embodiment, it is preferred that the isolated polynucleotide encoding the polypeptide or functional fragment is stably incorporated into the cell (i.e., by stable integration into the genome of the organism or by expression from a stably maintained episome such as Epstein Barr Virus derived episomes).
Screening assays can also be carried out in vivo in animals. Thus, as still a further aspect, the invention provides a transgenic non-human animal comprising an isolated polynucleotide encoding a SOGA polypeptide or functional fragment thereof, which can be produced according to methods well-known in the art. The transgenic non-human animal can be from any species, including avians and non-human mammals. According to this aspect of the invention, suitable non-human mammals include mice, rats, rabbits, guinea pigs, goats, sheep, pigs, and cattle. Suitable avians include chickens, ducks, geese, quail, turkeys, and pheasants.
The polynucleotide encoding the polypeptide or functional fragment can be stably incorporated into cells within the transgenic animal (typically, by stable integration into the genome or by stably maintained episomal constructs). It is not necessary that every cell contain the transgene, and the animal can be a chimera of modified and unmodified cells, as long as a sufficient number of cells comprise and express the polynucleotide encoding the polypeptide or functional fragment so that the animal is a useful screening tool.
Exemplary methods of using the transgenic non-human animals of the invention for in vivo screening of agents that modulate glucose production and/or the activity of a SOGA polypeptide comprise administering a test agent to a transgenic non-human animal (e.g., a mammal such as a mouse) comprising an isolated polynucleotide encoding a SOGA polypeptide or functional fragment thereof stably incorporated into the genome and detecting whether the test agent modulates glucose levels and/or polypeptide activity (or the activity of a functional fragment). It is known in the art how to measure these responses in vivo.
Methods of making transgenic animals are known in the art. DNA or RNA constructs can be introduced into the germ line of an avian or mammal to make a transgenic animal. For example, one or several copies of the construct can be incorporated into the genome of an embryo by standard transgenic techniques.
In an exemplary embodiment, a transgenic non-human animal is produced by introducing a transgene into the germ line of the non-human animal, Transgenes can be introduced into embryonal target cells at various developmental stages. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal used should, if possible, be selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
Introduction of the transgene into the embryo can be accomplished by any of a variety of means known in the art such as microinjection, electroporation, lipofection, or a viral vector. For example, the transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s). Following introduction of the transgene construct into the fertilized egg, the egg can be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. One common method is to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.
The progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of a segment of tissue. An embryo having one or more copies of the exogenous cloned construct stably integrated into the genome can be used to establish a permanent transgenic animal line.
Transgenically altered animals can be assayed after birth for the incorporation of the construct into the genome of the offspring. This can be done by hybridizing a probe corresponding to the polynucleotide sequence coding for the polypeptide or a segment thereof onto chromosomal material from the progeny. Those progeny found to contain at least one copy of the construct in their genome are grown to maturity.
Methods of producing transgenic avians are also known in the art, see, e.g., U.S. Pat. No. 5,162,215.
In particular embodiments, to create an animal model in which the activity or expression of a SOGA polypeptide is decreased, it is desirable to inactivate, replace or knock-out the endogenous gene encoding the polypeptide by homologous recombination with a transgene using embryonic stem cells. In this context, a transgene is meant to refer to heterologous nucleic acid that upon insertion within or adjacent to the gene results in a decrease or inactivation of gene expression or polypeptide amount or activity.
A knock-out of a gene means an alteration in the sequence of a gene that results in a decrease of function of the gene, preferably such that the gene expression or polypeptide amount or activity is undetectable or insignificant. Knock-outs as used herein also include conditional knock-outs, where alteration of the gene can occur upon, for example, exposure of the animal to a substance that promotes gene alteration (e.g., tetracycline or ecdysone), introduction of an enzyme that promotes recombination at a gene site (e.g., Cre in the Cre-lox system), or other method for directing the gene alteration postnatally. Knock-out animals may be prepared using methods known to those of skill in the art. See, for example, Hogan, et al. (1986) Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
A knock-out construct is a nucleic acid sequence, such as a DNA or RNA construct, which, when introduced into a cell, results in suppression (partial or complete) of expression of a polypeptide encoded by endogenous DNA in the cell. A knock-out construct as used herein may include a construct containing a first fragment from the 5′ end of the gene encoding a SOGA polypeptide, a second fragment from the 3′ end of the gene and a DNA fragment encoding a selectable marker positioned between the first and second fragments. It should be understood by the skilled artisan that any suitable 5′ and 3′ fragments of a gene may be used as long as the expression of the corresponding gene is partially or completely suppressed by insertion of the transgene. Suitable selectable markers include, but are not limited to, neomycin, puromycin and hygromycin. In addition, the construct may contain a marker, such as diphtheria toxin A or thymidine kinase, for increasing the frequency of obtaining correctly targeted cells. Suitable vectors include, but are not limited to, pBLUESCRIPT, pBR322, and pGEM7.
Alternatively, a knock-out construct may contain RNA molecules such as antisense RNA, siRNA, and the like to decrease the expression of a gene encoding a SOGA polypeptide. Typically, for stable expression the RNA molecule is placed under the control of a promoter. The promoter may be regulated, if deficiencies in the protein of interest may lead to a lethal phenotype, or the promoter may drive constitutive expression of the RNA molecule such that the gene of interest is silenced under all conditions of growth. While homologous recombination between the knock-out construct and the gene of interest may not be necessary when using an RNA molecule to decrease gene expression, it may be advantageous to target the knock-out construct to a particular location in the genome of the host organism so that unintended phenotypes are not generated by random insertion of the knock-out construct.
The knock-out construct may subsequently be incorporated into a viral or nonviral vector for delivery to the host animal or may be introduced into embryonic stem (ES) cells. ES cells are typically selected for their ability to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knock-out construct. Thus, any ES cell line that can do so is suitable for use herein. Suitable cell lines which may be used include, but are not limited to, the 129J ES cell line or the J1 ES cell line. The cells are cultured and prepared for DNA insertion using methods well-known to the skilled artisan (e.g., see Robertson (1987) In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. IRL Press, Washington, D.C.; Bradley et al., Curr. Topics Develop. Biol. 20:357 (1986); Hogan et al., (1986) Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Insertion of the knock-out construct into the ES cells may be accomplished using a variety of methods well-known in the art, including, for example, electroporation, microinjection, and calcium phosphate treatment. For insertion of the DNA or RNA sequence, the knock-out construct nucleic acids are added to the ES cells under appropriate conditions for the insertion method chosen. If the cells are to be electroporated, the ES cells and construct nucleic acids are exposed to an electric pulse using an electroporation machine (electroporator) and following the manufacturer's guidelines for use. After electroporation, the cells are allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct.
Each knock-out construct to be introduced into the cell is first typically linearized if the knock-out construct has been inserted into a vector. Linearization is accomplished by digesting the knock-out construct with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knock-out construct sequence.
Screening for cells which contain the knock-out construct (homologous recombinants) may be done using a variety of methods. For example, as described herein, cells can be processed as needed to render DNA in them available for hybridization with a nucleic acid probe designed to hybridize only to cells containing the construct. For example, cellular DNA can be probed with 32P-labeled DNA which locates outside the targeting fragment. This technique can be used to identify those cells with proper integration of the knock-out construct. The DNA can be extracted from the cells using standard methods (e.g., see, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989)). The DNA may then be analyzed by Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with one or more particular restriction enzymes.
Once appropriate ES cells are identified, they are introduced into an embryo using standard methods. They can be introduced using microinjection, for example. Embryos at the proper stage of development for integration of the ES cell to occur are obtained, such as by perfusion of the uterus of pregnant females. For example, mouse embryos at 3-4 days development can be obtained and injected with ES cells using a micropipet. After introduction of the ES cell into the embryo, the embryo is introduced into the uterus of a pseudopregnant female mouse. The stage of the pseudopregnancy is selected to enhance the chance of successful implantation. In mice, 2-3 days pseudopregnant females are appropriate.
Germline transmission of the knockout construct may be determined using standard methods. Offspring resulting from implantation of embryos containing the ES cells described above are screened for the presence of the desired alteration (e.g., knock-out of the SOGA polypeptide). This may be done, for example, by obtaining DNA from offspring (e.g., tail DNA) to assess for the knock-out construct, using known methods (e.g., Southern analysis, dot blot analysis, PCR analysis). See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989). Offspring identified as chimeras may be crossed with one another to produce homozygous knock-out animals.
Mice are often used as animal models because they are easy to house, relatively inexpensive, and easy to breed. However, other knock-out animals may also be made in accordance with the present invention such as, but not limited to, monkeys, cattle, sheep, pigs, goats, horses, dogs, cats, guinea pigs, rabbits and rats. Accordingly, appropriate vectors and promoters well-known in the art may be selected and used to generate a transgenic animal deficient in expression of a SOGA polypeptide.
In another embodiment, animal models may be created using animals that are not transgenic. For example, animal models of diabetes or obesity are well known in the art and can be used to study the effects of regulators of glucose production.
VIII. Pharmaceutical Compositions
As a further aspect, the invention provides pharmaceutical formulations and methods of administering the same to achieve any of the diagnostic or therapeutic effects (e.g., inhibition or stimulation of glucose production) discussed above. The pharmaceutical formulation may comprise any of the reagents discussed above in a pharmaceutically acceptable carrier, e.g., a polynucleotide encoding a SOGA polypeptide or a fragment thereof or a vector or cell comprising the polynucleotide, a SOGA polypeptide or fragment thereof, an antibody against a SOGA polypeptide, an antisense oligonucleotide, an siRNA molecule, a ribozyme, an aptamer, a peptidomimetic, a small molecule, or any other agent that modulates the activity of a SOGA polypeptide, including agents identified by the screening methods described herein.
By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.
The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
The agents of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the agent (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is preferably formulated with the agent as a unit-dose formulation, for example, a tablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the agent. One or more agents can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.
A further aspect of the invention is a method of treating subjects in vivo, comprising administering to a subject a pharmaceutical composition comprising an agent of the invention in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. Administration of the compounds of the present invention to a human subject or an animal in need thereof can be by any means known in the art for administering agents.
The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, kidney or muscle). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular agent which is being used.
For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). For other methods of administration, the carrier can be either solid or liquid.
For oral administration, the agent can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Agents can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the agent in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the agent in an inert base such as gelatin and glycerin or sucrose and acacia.
Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the agent, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit/dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising an agent of the invention, in a unit dosage form in a sealed container. The agent or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 1 mg to about 10 grams of the agent or salt. When the agent or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the agent or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.
Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the agent with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the agent. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M of the agent.
The agent can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the agent, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles comprising the agent can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles comprising the agent can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
Alternatively, one can administer the agent in a local rather than systemic manner, for example, in a depot or sustained-release formulation.
Further, the present invention provides liposomal formulations of the agents disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the agent or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the agent or salt, the agent or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the agent or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.
The liposomal formulations containing the agents disclosed herein or salts thereof, can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
In the case of water-insoluble agent s, a pharmaceutical composition can be prepared containing the water-insoluble agent, such as for example, in an aqueous base emulsion. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the agent. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin,
In particular embodiments, the agent is administered to the subject in a therapeutically effective amount, as that term is defined above. Dosages of pharmaceutically active agents can be determined by methods known in the art, see, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.). The therapeutically effective dosage of any specific agent will vary somewhat from agent to agent, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the agent, including the cases where a salt is employed. Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg/kg, with all weights being calculated based upon the weight of the agent, including the cases where a salt is employed. A dosage from about 10 mg/kg to about 50 mg/kg can be employed for oral administration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection. Particular dosages are about 1 mal/kg to 50 μmol/kg, and more particularly to about 22 μmol/kg and to 33 μmol/kg of the agent for intravenous or oral administration, respectively.
In particular embodiments of the invention, more than one administration (e.g., two, three, four, or more administrations) can be employed over a variety of time intervals (e.g., hourly, daily, weekly, monthly, etc.) to achieve therapeutic effects.
The present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. The term “avian” as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. The term “mammal” as used herein includes, but is not limited to, humans, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. In other embodiments, the subject is an animal model of diabetes or other metabolic disorder.
The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
Example 1
Identification of SOGA
Type II diabetes is associated with high glucose production. Obesity increases glucose production by lowering circulating levels of the hormone adiponectin. Therefore, type II diabetes can be treated by stimulating the adiponectin signaling pathway. Adiponectin lowers circulating glucose by inhibiting glucose production from the liver. Adiponectin inhibits glucose production by activating AMP-activated kinase (AMPK). AMPK stimulates fatty acid (FA) oxidation. The inhibition of glucose production by a signaling intermediate that increases FA oxidation is counter-intuitive because ATP generated from FA oxidation fuels glucose production. Furthermore, AMPK stimulates autophagy, a regulated mechanism of intracellular degradation that provides the biochemical intermediates for glucose production through the hydrolysis of proteins, glycogen and triglycerides. This deadlock led to the hypothesis that adiponectin inhibits glucose production through a novel mediator. Insulin inhibition of glucose production in the liver is mediated by the suppression of lysosome activity. We treated rat hepatoma cells with full-length recombinant adiponectin and identified the proteins that were bound to APPL1 in a co-immunoprecipitation assay using proteomics analysis. APPL1 was previously identified in a yeast 2-hybrid screen using the intracellular region of the adiponectin receptor. Proteomics analysis revealed a gene we are calling SOGA (also called TOA (Target Of Adiponectin)) that encodes a 161 kDa protein containing (1) a leucine zipper motif that enables binding to the leucine zipper motif of APPL1 and (2) Atg16 and Rab5-binding motifs that enable participation in membrane assembly for autophagy. The hydrolysis of proteins and glycogen by autophagy increases glucose production by producing biochemical intermediates for gluconeogenesis and glycogenolysis. Northern blot analysis revealed that SOGA is ubiquitously expressed as a 3.0 and a 4.5 kb mRNA. Our current hypothesis is that adiponectin stimulation of SOGA (NCBI Accession: FJ977045) can suppress glucose production.
We verified the expression of SOGA in the liver and other tissues by RT-PCR and Northern blot analysis. There are no publications describing SOGA, its gene, mRNA or amino acid sequence. The open reading frame of murine SOGA is derived from 16 exons. SOGA cDNA encodes a 1434 amino acid protein that lacks transmembrane domains. SOGA contains a leucine zipper motif that we predict allows SOGA to bind to the leucine zipper motif of APPL1 in our co-immunoprecipitation experiment (FIG. 1). The predicted regions of interest in SOGA include (1) a leucine zipper motif, (2) ATG16 motifs, (3) a Rab5 motif, (4) a casein kinase domain, (5) multiple myristoylation and glycosylation sites and (6) multiple kinase specific phosphorylation sites (FIG. 1). Amino acid sequence alignment shows that murine SOGA is 91% identical to human SOGA. When substitutions for similar amino acids are taken into account, murine SOGA is 95% identical to human SOGA. SOGA is a highly conserved gene in mammals but absent in lower eukaryotes like yeast. Our current model is that adiponectin signaling triggers SOGA binding to APPL1, a proximal target of the adiponectin receptor. Based on conserved domain predictions, SOGA binding to APPL1 contributes to adiponectin inhibition of protein degradation and glucose production. This may be accomplished through the binding of SOGA to APPL1, the proteolytic cleavage of SOGA and the secretion of its 25 kDa fragment.
The formation of the phagophore, a primary step in autophagy, can lead to the digestion of proteins and glycogen providing the biochemical intermediates for glucose production (FIG. 2). Atg16-Atg5-Atg12 forms a protein complex that is essential for the formation of an autophagosome. Atg12 is covalently conjugated to Atg5 by ubiquitination-like reactions that involve Atg7 and Atg10. Overexpression of Atg5 and Atg12 in yeast causes an increase in autophagy that is absent in mammalian cells, suggesting the existence of a novel protein in higher eukaryotes. Although 31 autophagy-related (Atg) proteins have been identified in yeast, SOGA is highly conserved in mammals but bears little homology to any gene product in yeast. Thus, the study of SOGA can lead to the elucidation of the mechanisms governing autophagy in mammals.
We predicted that SOGA plays a role in adiponectin's inhibition of glucose production based on its binding to APPL1 under adiponectin exposure and the conserved functional domains of SOGA which include (1) a leucine zipper motif that enables SOGA to bind to APPL1, (2) an ATG16 (autophagy 16) motif that enables SOGA to initiate autophagy through the formation of the phagophore, (3) Rab5 motif (a small GTPase) that enables the fusion of the autophagosome and lysosome, (4) casein kinase domain that enables a downstream signaling cascade, (5) myristoylation and glycosylation sites that enable anchoring and (6) multiple kinase-specific phosphorylation sites that enable the modulation of SOGA by kinases and phosphatases (FIG. 1). Further insight into SOGA can increase our understanding of nutrient metabolism and lead to new ways of preventing and treating diabetes.
Species specific (mouse) SOGA peptide antigen (476) was detected with immune but not pre-immune sera from New Zealand White rabbits (FIG. 3, left panel). The signal intensity is proportional to the peptide antigen concentration. Using our rabbit polyclonal antisera (476) that is specific for mouse SOGA, SOGA was detected in mouse plasma at 25 kDa but not in human plasma (FIG. 3, right panel). Antisera from two different rabbits immunized with two different peptide antigens, 476 and 477 specific for mouse SOGA, detected a 25 kDa band in mouse plasma (FIG. 4). Antigen peptides 476 and 477 correspond to overlapping amino acid sequences in mouse SOGA.
The concentration of SOGA in plasma corresponded with circulating levels of adiponectin (FIG. 5). Plasma was sampled from young female C57B1 adiponectin null and wild-type mice. Western blot and densitometry of adiponectin and SOGA in ob/ob control mice and ob/ob mice treated with pioglitazone showed that adiponectin and SOGA were increased in ob/ob mice on pioglitazone compared to controls (FIG. 6). Western blot and densitometry of adiponectin and SOGA in ad libitum and calorie restricted fed C57 mice showed that adiponectin and SOGA were increased in calorie restricted mice compared to those fed ad libitum (P<0.05 for statistical significance) (FIG. 7). Western blot and densitometry of adiponectin and SOGA in rapamycin and control fed C57B1 mice revealed that SOGA was decreased in rapamycin fed mice compared to controls (P<0.05 for statistical significance) (FIG. 8). FPLC fraction analysis of mouse plasma for SOGA was performed (FIG. 9). Graphs show SOGA, triglyceride, and cholesterol levels in FPLC fractions 11-33.
In summary, SOGA (TOA) is a novel protein that we have identified through proteomics and a co-immunoprecipitation assay; it binds to APPL1 under adiponectin exposure. The SOGA gene contains Atg16 and Rab5-binding motifs that are indicative of autophagic activities; it is hypothesized that adiponectin stimulation of SOGA can suppress glucose production. SOGA peptide antigen was detected by immune sera from NZW rabbits; SOGA was detected at 25 kDa in mouse plasma but not human plasma. Two distinct antigens corresponding to overlapping segments of SOGA produced antisera that detected a 25 kDa SOGA. Circulating levels of SOGA were greatly suppressed in adiponectin null (−/−) mice. Adiponectin and SOGA were increased by pioglitazone, and calorie restriction, but were suppressed by rapamycin. FPLC analysis indicates that SOGA circulates below 100 kDa.
Example 2
Experimental Methods
Mass Spectrometry.
McArdle rat hepatoma cells were exposed to adipocyte conditioned media with or without adiponectin (Brooks et al., J. Biol. Chem. 282:35069 (2007)). Cell lysates were digested with proteomics grade trypsin (Sigma) and filtered through YM-10 molecular weight cutoff filters (Millipore, Bedford, Mass.). Tryptic digests were injected into an LCQ-Deca Ion Trap mass spectrometer coupled to a Surveyor HPLC system (Thermo Fisher Scientific, Waltham, Mass.). The solvent, 50% methanol and 0.1% formic acid, was delivered to the spectrometer at 200 μL/min. Peptide masses were acquired in positive mode using electrospray ionization under the following source conditions: spray voltage was 5 kV, sheath gas was 40 (arbitrary units), auxiliary gas was 20 (arbitrary units), and heated capillary temperature was 350° C.
Cloning of Murine SOGA.
Total RNA was obtained from primary mouse hepatocytes using Triazol reagent (Invitrogen). mRNA was isolated using Oligotex mRNA Kit (Qiagen). Primers used to clone SOGA were designed using publically available genomic and mRNA sequence data based on the open reading frame of SOGA peptides detected by mass spectrometry. The 4.7 kb SOGA cDNA was isolated by annealing two PCR products using overlap extension. RNA ligase mediated RACE (Ambion) was used to clone the sequence from the 5′-end of SOGA mRNA. The cDNA for human SOGA was cloned by a similar method.
Antibody Production.
Human- and murine-specific polyclonal antisera were produced in three New Zealand White rabbits (Franklin Rabbitry, N.C.) using a human-specific peptide antigen STQSLTSFARSSRSAIRHSPSKC (SEQ ID NO:5) and two partially overlapping murine-specific peptide antigens CSAQSLASCFIRPSRN (SEQ ID NO:6) and SAQSLASC*FIRPSRNPIRHSPSKC (SEQ ID NO:7), where C* represents acemidomethyl cysteine. Synthetic peptides were purified by HPLC and analyzed on the LCQ-Deca Ion Trap mass spectrometer to confirm their molecular weight. Antigenic peptides (10 mg) were dissolved in 0.1 M NaH2PO4 (pH 7.2)/0.05 M NaCl and conjugated to keyhole limpet hemocyanin (KLH; 4 mg) before injection. KLH conjugated peptides were dissolved in 3 ml of 0.03% trifluoroacetic acid and added to 3 ml complete Freund's adjuvant (Sigma). New Zealand White rabbits (Franklin Rabbitry, Wake Forest, N.C.) were injected intradermally using multiple injection sites. After 5 weeks, each animal was reinjected subcutaneously with KLH conjugated antigen in 1 ml of 50% incomplete Freund's adjuvant (Sigma). Four weeks later, 20 ml of blood were collected and rabbits were reimmunized. Injections and bleedings were performed at monthly intervals thereafter. The antibody production protocol was approved by UNC's Institutional Animal Care and Use Committee (IACUC).
Hepatocyte Studies.
Mouse livers were perfused with a Krebs-Ringer-HEPES buffer containing collagenase (Sigma-Aldrich). Livers were isolated and cells were dispersed by gentle shaking and filtered through sterile nylon gauze. Cells were washed twice with sterile phosphate-buffered saline and purified by centrifugation in 50% isotonic Percoll (Sigma-Aldrich). Cells were resuspended with Krebs-Ringer-HEPES Ca2+ buffer to a total volume of 10 ml. Viability was validated via trypan blue exclusion and routinely exceeded 90%. Freshly isolated mouse hepatocytes were plated at 105 cells per well in 12-well culture plates coated with rat tail collagen I (BD Biosciences). Cells were maintained in Dulbecco's modified Eagle medium (DMEM; Caisson Laboratories), 25 mM glucose and 10% horse serum (HS). Adiponectin was provided from adipocyte conditioned media with or without adiponectin (Brooks et al., J. Biol. Chem. 282:35069 (2007)). SOGA siRNA, AICAR (500 μM) or LY293004 (10 nM) were introduced to the media 48 hours before the measurement of glucose production. siRNA sequences corresponding to base pairs 333-351 and 1988-2007 on the open reading frame of murine SOGA were selected using a rational design algorithm (Invitrogen). Transfection with a pool of 2 siRNAs targeting SOGA had a greater knockdown efficiency than transfecting with the individual siRNAs. Transfection was achieved by electroporation using the Mouse Hepatocyte Nucleofector Kit (LONZA) according to the manufacturer's protocol. In brief, freshly isolated mouse hepatocytes were diluted to 3×106 cells/tube in media without antibiotics and centrifuged at 2,000 rpm for 2 minutes. The supernatant was removed and the cells were resuspended in 100 μl of Nucleofector solution containing 100 nM of siRNA. The cell suspension was transferred to an electroporation cuvette which was placed in a Nucleofector I electroporation device and pulse charge was applied for 2 minutes using program T-28. Hepatocytes received 1.0 ml of media and were transferred to 12 well plates. SOGA expression, valine and glucose production were assayed 72 hours after siRNA transfection. Media was replaced with glucose-free DMEM containing MG-132 (10 μM), an inhibitor of the ubiquitin-proteasome pathway of protein degradation, for 6-8 hours to measure hepatocytes glucose production. Glucose was measured by colorimetric assay (Autokit Glucose CII) (Brooks et al., J. Biol. Chem. 282:35069 (2007)). Valine in the medium was measured by a HPLC (Waters) coupled TSQ-Quantum ultra triple quad mass analyzer (ThermoFinigan) in the Biomarkers Facility Core at UNC. Valine was measured in selected reaction monitoring mode (SRM) using the MS/MS transition of 118→72.
Lysosomal Activity.
Autophagic activity was estimated by lysosome and late autophagosome vacuole staining using LysoTracker Red DND 99 (Invitrogen), a membrane permeable fluorescent labeled basic amine with high affinity for the acidic interior of the lysosome and late autophagosome vacuole (Klionsky et al., Autophagy 4:151 (2008)). Cell medium was removed and replaced with GF/DMEM containing 50 nM LysoTracker Red. Cells were incubated for 30 min at 37° C. and the medium was replaced with GF/DMEM. Digital images were obtained at the Light Microscopy Facility at UNC with an Olympus 1×81 Motorized Inverted Microscope, a 40×11.30 Oil DIC lens, Camera pixel count: Hamamatsu C10600-1013 1344×1024 using the acquisition software Volocity 5.3.2 (Perkin Elmer). Fluorescence Filter Cubes Specifications (Semrock, Inc.) were TXRED-4040B for rhodamine and Texas Red: Exciter 562 nm 20, Dichroic R 530-585/T 601-800, Emitter 642±20. Lysosome and late autophagosome vacuole number was determined from digital images as isolated punctuate staining, greater than background staining intensity threshold, distinct from lipid droplets in clearly demarcated cells containing two nuclei. Spot recognition and enumeration according to the foregoing definition was determined by two individuals.
Mouse Studies.
Mice were housed in ventilated isolator cage systems in a pathogen-free barrier facility maintained at 23° C., 55% humidity on a 12-h light/12-h dark cycle. Mice received a standard chow diet consisting of 73% carbohydrate, 18% protein, 4% fat and 5% ash (Purina). Young (3-6 month old) female C57B1/6J calorie restricted (CR) and ad libitum fed (AL) mice were maintained as previously described (Combs et al., Diabetes 52:268 (2003)). Adjustments were made to ensure that CR mice received 70% of the ad libitum food intake. Blood samples were collected at 1300 from the tail tip using heparinized capillary tubes (Fisher) and stored at −20° C. Male ob/ob mice (FVB background strain) received a daily dose of pioglitazone at 0.6 mg/kg BW in 0.025% (w/w) carboxymethylcellulose by oral gavage for 4 days. Control mice received carboxymethylcellulose by oral gavage for 4 days. Blood was collected from the tail tip on day 5 and analyzed for glucose, adiponectin and 25 kDa SOGA. Immediately after the collection of blood samples, ob/ob mice were sacrificed by cervical dislocation for tissue collection. Northern blot analysis for SOGA mRNA and 18S RNA was performed using 20 μg of liver RNA. NOD mice were bred and housed as previously described (Wong et al., J. Immunol. 176:1637 (2006)). Where indicated, diabetic NOD mice were injected with 5 units of insulin (NPH Human Insulin, Isophane Suspension; 100 U/ml Novolin; Novo Nordisk) 24 hours prior to blood collection. Adiponectin transgenic mice were produced as previously described (Combs et al., Endocrinology 145:367 (2004)). Glucose was measured by colorimetric assay. Adiponectin and SOGA were measured by SDS-PAGE analysis using 1 μl of plasma. The total concentration of protein in plasma, measured by BCA assay (Pierce), did not differ between groups. Experimental procedures were approved by IACUC.
Human Studies.
Thirteen healthy women between the ages of 20-63, body mass indexes (in kg/m2) between 20.2 and 31.9, were included for this study. Inclusion was contingent on a good, age-typical health status, as ascertained by physical examination and standard clinical laboratory tests such as complete blood count, blood chemistries, fasting glucose, insulin, lipid and liver function tests, liver lipid content and the presence of no known chronic disease including diabetes. Subjects were admitted to the Clinical and Translational Research Center of UNC and placed on a balanced weight maintenance diet for 10 days (Fischer et al., Am. J. Clin. Nutr. 85:1275 (2007)). Circulating SOGA and adiponectin were measured from plasma samples collected from an intravenous catheter following an overnight fast. The race-ethnicity distribution of the participants was white (63%), African American (27%), Asian (6%), and Native American (4%), which reflected the local population characteristics of the Raleigh-Durham-Chapel Hill area. Plasma adiponectin and SOGA were determined by SDS-PAGE using polyclonal antisera against human adiponectin and human SOGA, horseradish peroxidase linked secondary anti-rabbit IgG. Circulating adiponectin and SOGA levels were measured by enhanced chemiluminescence (ECL) signal intensity. Human studies were performed under an IRB approved protocol (CTRC-2645; Study: 07-1158).
Statistical Analysis.
Student's t test was used to identify significant differences when data within groups showed a normal distribution and Wilcoxon-Rank Sum test was used when data did not show a normal distribution. P values less than 0.05 were considered significant.
Example 3
Identification of SOGA by Mass Spectrometry
Protein extracts from hepatoma cells exposed to adiponectin were digested with trypsin and analyzed by mass spectrometry. Mass spectrometry revealed a peptide, KVLPSEEDDFLEVNSM (SEQ ID NO:8), encoded by a gene located on chromosome 2 in mice (2411) and chromosome 20 in humans (20q 11). Mouse liver RNA was used to clone the full length 4.7 kb SOGA cDNA (GENBANK ID: H977045). Northern blot analysis, using a probe recognizing the C-terminal end of SOGA, revealed a single dominant 4-5 kb band in the liver. The ORF of the cDNA clone predicts a 161 kDa protein that contains an internal secretory peptide sequence, FKHNFLLLFMKLRWFLKRWRQG (SEQ ID NO:9) (FIG. 10). On the basis of computational methods that incorporate signal peptide and cleavage site predictions, SOGA is cleaved between G at the end of the signal peptide and K at the beginning of at the peptide identified by mass spectrometry (Emanuelsson et al., Nat. Protoc. 2:953 (2007)).
FIG. 10 is a map showing the location of the conserved ATG16 and Rab5-binding motifs, the secretory signal peptide and the species-specific epitope in the predicted 161 kDa SOGA. The map also shows the predicted domains of the 80 kDa peptide detected in vitro and the 25 kDa peptide detected in plasma. FIG. 10 shows the amino acid sequence for murine SOGA (SEQ ID NO:2) showing the location of the Atg16 (232-375) and Rab5-binding (757-886) motifs underlined, the signal peptide (681-702) in bold, the tryptic peptide identified by mass spectrometry (703-718) shaded and the species specific domain (1392-1416) in a box. The position of the internal signal peptide explains why our antibodies, recognizing the species-specific epitope near the C-terminus of SOGA, detect an 80 kDa SOGA peptide rather than the 161 kDa SOGA protein.
Example 4
Function of SOGA in Primary Hepatocytes
Consistent with the predicted position of the cleavage site, rabbit antisera recognizing the species-specific domain on the C-terminal region of murine SOGA recognized a single 80 kDa protein in isolated hepatocytes (FIG. 11A). FIG. 11A shows a representative SDS-PAGE of primary murine hepatocyte samples showing the knockdown of 80 kDa SOGA as a function of time after exposure to siRNA. siRNA suppression of SOGA caused a dramatic increase in lysosome and late autophagic vacuole number (2.0±0.2 per cell compared to 17.5±2.0 per cell where n=25-30 cells per group, p<0.0001) as indicated by isolated punctate acidotropic dye staining which provides correlative data on autophagy (FIG. 11B) (Klionsky et al., Autophagy 4:151 (2008)). FIG. 11B shows representative purified binucleate hepatocyte cultures transfected with control (left) or SOGA siRNA (right) stained with the lysosome-specific fluorescent dye LysoTracker Red. The hypothesis that SOGA inhibits autophagy is further supported by the reduction of total cell protein content 48 hours after siRNA suppression of SOGA (11.2 E 0.6 μg/well compared to 16.3±0.4 μg/well; n=4 per group; p<0.05). FIG. 2C depicts bar graphs showing the effects of adiponectin and SOGA siRNA on glucose and valine secretion in hepatocyte conditioned media (top and middle) and 80 kDa SOGA measured by densitometry of ECL (enhanced chemiluminescent signal) after SDS-PAGE (bottom). Adiponectin exposure caused a 40% increase of SOGA in primary hepatocytes and a 50% reduction in glucose production (FIG. 11C). siRNA suppression of SOGA blocked the inhibition of glucose production and stimulated valine secretion (FIG. 11C), The secretion of valine, an essential amino acid that cannot be metabolized, due to the absence of branched chain aminotransferase in hepatocytes, also suggests an increase in autophagy. These results support the hypothesis that the elevation of SOGA in response to adiponectin exposure is linked to the inhibition of autophagy.
Example 5
Regulation of SOGA in Primary Hepatocytes and the Correlation of Intracellular and Extracellular Levels of SOGA
FIG. 11D depicts bar graphs showing the roles of AMPK and PI3K on adiponectin regulation of intracellular and extracellular SOGA levels. Primary hepatocytes were incubated in the presence or absence of 500 μM AICAR, a stimulator of AMPK, or 10 nM LY294002, a PI3K inhibitor. Bars represent mean values±SEM for n=4 per group where “*” indicates a significant difference compared to control (left bar) at p<0.05 by nonparametric Student's t-test. Grey and black bars indicate whether measurements were made in hepatocyte conditioned media or hepatocytes, respectively. The activation of AMPK by AICAR caused a decrease in SOGA that was blocked by adiponectin exposure (FIG. 11D). On the other hand, the inhibition of PI3K by LY294002 caused a decrease in SOGA that was not blocked by adiponectin (FIG. 11D). These observations suggest that adiponectin increases SOGA through the insulin signaling pathway through a mechanism that can be inhibited by AMPK. Consistent with the identification of an internal secretory signal peptide in SOGA, SDS-PAGE analysis revealed that the 80 kDa SOGA fragment is secreted in hepatocyte conditioned media. The reduction of intracellular SOGA by adiponectin and LY294002 was reflected in the levels of SOGA in hepatocyte conditioned media. These results suggested that extracellular levels of SOGA could be used as a biomarker of its intracellular activity.
Example 6
Circulating SOGA in Mice and Humans
Antisera from 2 different rabbits immunized with two different peptide antigens, 476 and 477, detected a 25 kDa peptide in mouse plasma (FIG. 12A). SDS-PAGE shows the SOGA peptide antigen 476 was detected with immune but not pre-immune sera. The blot exposed to immune sera shows that the signal intensity is proportional to the peptide antigen concentration. FIG. 12B, left panel, shows that mouse-specific polyclonal antisera 476 detected a 25 kDa protein in mouse plasma but not human plasma. FIG. 12B, right panel, shows that antisera from two different rabbits immunized with two different peptide antigens, 476 and 477, detected a 25 kDa peptide in mouse plasma. Peptide antigens 476 and 477 correspond to overlapping amino acid sequences in the species specific epitope of SOGA. Peptide antigens used to produce rabbit antisera, SAQSLASCFIRPSRNPIRFISPSKC (SEQ ID NO:7) (antigen 476) and CSAQSLASCFIRPSRN (SEQ ID NO:6) (antigen 477), were analyzed by mass spectrometry to confirm their amino acid sequence. Rabbit antisera recognizing murine SOGA did not cross-react with any proteins in human plasma. FIG. 12C, top panel, shows a UV absorption plot for plasma proteins generated by HPLC. SDS-PAGE shows that 25 kDa SOGA eluted in fraction 9. For reference, the triglyceride peak (VLDL particle, −400 kDa) and the cholesterol peak (HDL particle, −200 kDa) were observed in fractions 1-2 and 5-6, respectively. HPLC analysis confirms that 25 kDa SOGA circulates as a monomer. FIG. 12C, bottom panel, presents SDS-PAGE showing SOGA precipitated out of HPLC fraction 9 in a 40% ammonium sulfate solution. Due to the presence of cysteine residues within the antigenic motif of SOGA, antibody detection of 25 kDa SOGA required the reduction of the sample with dithiothreitol. Based on the predicted sequence of 25 kDa fragment, the intramolecular disulfide bonds between cysteine residues on the carboxy-terminal end of SOGA should generate a fish hook conformation. Two observations indicate that 25 kDa SOGA circulates as a monomer. First, SOGA was detected at 25 kDa when plasma samples were reduced after SDS-PAGE. Second, by size exclusion chromatography of plasma proteins under native conditions, SOGA eluted at 25 kDa (FIG. 12C).
Recombinant 25 kDa SOGA was produced in E. coli and was detectable with the antibodies raised against full length SOGA. FIG. 13A shows a BsrG1 digest of murine 25 kDa SOGA clone in pET-DEST42 GATEWAY vector in 2% agarose. FIG. 13B shows a SDS-PAGE blot of recombinant 25 kDa murine SOGA, either without or with 6×His tag, produced in IPTG stimulated E. coli transformed with the pET-DEST42 GATEWAY vector. The left panel shows cross reactivity of our murine SOGA antisera with recombinant 25 kDa murine SOGA. The right panel shows a Ponceau red stained blot of total bacterial lysates after SDS-PAGE.
Example 7
Correlation between Circulating Adiponectin and SOGA
To further validate the link between adiponectin and SOGA in vivo, circulating levels of adiponectin and SOGA were measured in (a) healthy human volunteers, (b) wild-type mice after weight reduction by calorie restriction, and (c) pioglitazone treatment in ob/ob mice, a model of type II diabetes. FIG. 14A shows adiponectin and 25 kDa SOGA levels in human plasma from healthy female volunteers (ages 20-63; n=13). Plasma was collected after an overnight fast. Values represent averages from 2 plasma samples taken 10 minutes apart. A correlation coefficient (R2) of 0.82 was found between SOGA and adiponectin. The analysis of human plasma from healthy fasting female volunteers (plasma insulin: 7.1±1.0 μU/ml) showed a positive correlation between circulating levels of adiponectin and SOGA (R2=0.82) (FIG. 14A). FIG. 14B shows the effect of ad libitum (AL) versus 30% calorie restricted (CR) feeding on adiponectin, SOGA and glucose in wild-type mice. Bar graphs show levels of plasma adiponectin (top), 25 kDa SOGA (middle) and glucose (bottom). Calorie restriction, a nutritional intervention that doubled plasma adiponectin, resulted in a 2-fold elevation of circulating SOGA (FIG. 14B). The concentration of plasma glucose in calorie restricted mice compared to ad libitum fed mice was 80±7 mg/dl and 131±10 mg/dl, respectively (FIG. 14B). The complex oligomeric structure, high turnover rate and abundance of circulating adiponectin prevented us from using recombinant adiponectin to study the regulation of SOGA in vivo (Shetty et al., Trends Pharmacol. Sci. 30:234 (2009)). Therefore, oral pioglitazone treatment was used to elevate adiponectin in ob/ob mice, an obese model of type II diabetes. FIG. 14C shows the effect of pioglitazone treatment on liver SOGA mRNA and circulating adiponectin, SOGA and glucose in diabetic ob/ob mice. Mice received a daily dose of pioglitazone (TZD) or placebo (CTL) by oral gavage. Bar graphs show the levels of plasma adiponectin (top), liver SOGA mRNA/18S RNA (second), plasma 25 kDa SOGA (third) and plasma glucose (bottom) after 4 days of treatment. Pioglitazone treatment caused a 40% increase of SOGA mRNA in the liver and a 3-fold elevation of circulating adiponectin and SOGA (FIG. 14C). The concentration of plasma glucose was 155±8 mg/dl in pioglitazone treated ob/ob mice compared to 450±18 mg/dl in untreated ob/ob mice (p<0.05) (FIG. 14C). These results support the hypothesis that adiponectin elevation of SOGA increases insulin sensitivity. Both calorie restriction and pioglitazone treatment have pleiotropic effects beyond the elevation of circulating adiponectin making it difficult to draw any conclusions about the linkage between adiponectin and SOGA. Hence, circulating levels of SOGA between wild-type and adiponectin transgenic mice were compared. FIG. 14D shows circulating levels of adiponectin and SOGA in male adiponectin transgenic mice and their wild type litter mates on a high fat diet. Bars in panels B, C and D represent mean±SEM for n=4-5 per group where “*” indicates a significant difference (p<0.05) by nonparametric Student's t-test. Previous studies have shown that the 3-fold elevation of adiponectin in transgenic mice exerts a protective effect against diabetogenic high fat diet (Combs et a, Endocrinology 14.5:367 (2004); Brooks et al., J. Biol. Chem. 282:35069 (2007)). Consistent with a stimulatory effect of adiponectin, circulating levels of SOGA were higher in adiponectin transgenic mice than their wild type litter mates on a high fat diet (FIG. 14D). These results support the hypothesis that the increase of SOGA in response to adiponectin contributes to the reduction of glucose production in vivo.
Example 8
Correlation between Circulating Insulin and SOGA
Because adiponectin is an insulin sensitizer and the inhibition of the insulin signaling intermediate PI3K blocked the induction of SOGA in isolated hepatocytes (FIG. 11D), we sought to determine whether there is a correlation between circulating insulin and SOGA during (a) feeding and fasting in humans and (b) insulin withdrawal in NOD mice, a model of type I diabetes. FIG. 15A shows the percent change in circulating levels of SOGA in healthy human volunteers (20-43 years old) measured at 8-11 AM, within 2 hours of feeding or following an overnight (10-12 hour) fast. Bars represent mean values±SEM for n=5 and “*” indicates a significant difference at p<0.05 by nonparametric Student's t-test. Consistent with the theory that insulin stimulates SOGA, a 12-hour fast in healthy human volunteers was associated with a 25% decrease in circulating SOGA (FIG. 15A). The reduction of SOGA in the fasted state is consistent with the induction of SOGA by insulin and the role of SOGA in the inhibition of autophagy and glucose production. FIG. 15B shows the effect of insulin withdrawal and insulin injection on SOGA and glucose in NOD mice. Circulating levels of 25 kDa SOGA and glucose in NOD mice without diabetes (Group I), NOD mice with diabetes (Group 2) and NOD mice with diabetes treated by a single injection of insulin 24 hours earlier (Group 3) were measured. Bar graphs show the levels of plasma SOGA (top) and glucose (bottom). Bars show mean±SEM for n=5 per group where “*” indicates significantly lower than Groups 1 and 3, “**” indicates significantly greater than Group 2 and “***” indicates significantly greater than Groups 1 and 3. Statistical significance was determined by Student's t-test where p<0.05. A 3-fold reduction of circulating SOGA in hyperglycemic NOD mice, in comparison to euglycemic NOD mice, also suggests that insulin induces SOGA in vivo (FIG. 15B). In support of the theory that the increase of SOGA in response to insulin contributes to the reduction of plasma glucose, the treatment of type I diabetes by insulin injection was associated with a 2-fold induction of SOGA (FIG. 15B).
The results of this study suggest that the elevation of SOGA in response to adiponectin and insulin can lower liver glucose production through the inhibition of autophagy resulting in a decrease of plasma glucose. The observation that knockdown of SOGA elevated glucose production in primary hepatocytes suggested that SOGA is an inhibitor of glucose production. The elevation of glucose production during the reduction of SOGA was linked to changes in primary hepatocytes that suggested an increase in autophagy such as the reduction in protein content and the elevation of lysosome staining and the secretion of valine, a branched chain amino acid that cannot be synthesized or metabolized in hepatocytes.
The hypothesis that SOGA may interfere with autophagy is supported by the identification of conserved domains found in Atg16 and Rab5-binding proteins (Longatti et al., Cell Death Differ. 16:956 (2009)). Both Atg16 and the Rab5-binding proteins contribute to the early stages of autophagy. Although Atg16 is an essential component of the autophagic machinery, adenoviral overexpression of Atg16 inhibits autophagy in mammalian cells (Matsushita et al., J. Biol. Chem. 282:6763 (2007)); Fujita et al., Mol. Biol. Cell 19:2092 (2008)). The disruption of autophagy by overexpression of Atg16 provides a paradigm that may explain how elevated SOGA inhibits glucose production. Although the current study focuses on the role of SOGA in the liver, it is important to point out that SOGA is also expressed in the other gluconeogenic organs like the kidney and tissues that are rich sources of gluconeogenic substrates like skeletal and cardiac muscle. The elevation of SOGA in extrahepatic tissues may play a critical role in the reduction of glucose production and the amelioration of glucose homeostasis.
Intracellular levels of SOGA in isolated hepatocytes were proportional to the levels of SOGA in hepatocyte conditioned media leading us to propose that circulating levels of SOGA can be used as a biomarker of intracellular SOGA levels. This hypothesis was supported by the elevation of liver SOGA mRNA and circulating SOGA in pioglitazone treated ob/ob mice. Our in vitro experiments suggest that the elevation of circulating SOGA indicates a decrease in glucose production. This interpretation is consistent with the elevation of circulating SOGA after calorie restriction, oral pioglitazone, transgenic elevation of adiponectin, feeding and insulin injection. Although glucose production was not measured in the present study, previous reports in mice, rats and humans show that glucose production is reduced by the elevation of adiponectin in transgenic mice, the implementation of calorie restriction, the treatment of type II diabetes by oral insulin sensitizers and the treatment of type 1 diabetes by insulin (Wahren et al., Annu. Rev. Nutr. 27:329 (2007); Combs et al., J. Clin. Invest. 108:1875 (2001); Combs et al., Endocrinology 145:367 (2004); Barzilai et al., J. Clin. Invest. 101:1353 (2998); Miyazaki et al., J. Clin. Endocrinol. Metab. 89:4312 (2004)).
The elevation of SOGA in calorie restricted, pioglitazone and adiponectin transgenic mice supports the hypothesis that adiponectin induces SOGA. The elevation of SOGA in response to adiponectin was not impaired by pharmacologic inhibition of AMPK in isolated hepatocytes suggesting that the induction of SOGA is an insulin sensitizing effect of adiponectin that is mediated independent of AMPK. Adiponectin mediated increases in SOGA were impaired by pharmacologic inhibition of the insulin signaling intermediate PI3K suggesting that the expression of SOGA is regulated by the insulin signaling pathway. The reduction of circulating SOGA by a 12-hour fast in humans or hyperglycemic NOD mice and the elevation of circulating SOGA by insulin injection support the hypothesis that SOGA is induced by the insulin signaling pathway. Adiponectin could increase SOGA through the insulin signaling pathway via APPL1, an adaptor protein that binds to the intracellular domain of the adiponectin receptors and the catalytic subunit of PI3K (Mao et al., Nat. Cell Biol. 8:516 (2006); Mitsuuchi et al., Oncogene 18:4891 (1999); Yang et al., J. Biol. Chem. 278:16820 (2003)).
Antibodies recognizing the C-terminal region of murine SOGA show that cultured hepatocytes as well as liver samples incubated ex vivo secrete an 80 kDa SOGA fragment rather than a 161 kDa protein predicted by the 4.7 kb cDNA. The size discrepancy is explained by the location of an internal secretory signal peptide, also seen in chicken ovalbumin (Lingappa et al., Nature 281:117 (1979)). The presence of repeated LXXXXXL sequences in the amino terminal portion of the SOGA (amino acids 222-250 and 288-314) suggests a potential feedback mechanism through protein-protein interactions of leucine zipper motifs in SOGA and APPL1. The absence of 25 kDa SOGA in hepatocytes and liver conditioned media suggests that proteolytic cleavage of 80 kDa SOGA depends on an extracellular factor that is inactive or absent in vitro. The incubation of mouse hepatocyte conditioned media containing 80 kDa SOGA with endothelial cells (HUVECs) or human plasma did not yield a 25 kDa fragment. Circulating SOGA may play a physiologic role in glucose homeostasis.
The discovery that circulating levels of adiponectin and SOGA were highly correlated in humans suggests that the measurement of SOGA may be clinically relevant. For example, while TZD drug treatment is almost always effective in the induction of adiponectin, it is only effective in lowering glucose in 70% of type II diabetics (Snitlter et al., Diabetes Care 27:1365 (2004)). Insulin treatment in type I diabetics is also not completely effective 100% of the time. Based on the results presented here, it would not be surprising if specific cases of poor clinical outcomes were associated with poor induction of SOGA.
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

## Claims

1. A method of identifying an agent that binds to or modulates the activity of a mammalian Suppressor of Glucose by Autophagy (SOGA) polypeptide or a functional fragment thereof, comprising:
contacting the polypeptide or a functional fragment thereof with a test agent under conditions whereby binding between the polypeptide or a functional fragment thereof and the test agent can occur or modulation of the activity of the polypeptide or a functional fragment thereof can occur; and
detecting binding between the polypeptide or a functional fragment thereof and the test agent or detecting modulation of the activity of the polypeptide or a functional fragment thereof upon contact with the test agent as compared to activity of the polypeptide or a functional fragment thereof in the absence of contact with the test agent.

2. The method of claim 1, wherein the method is carried out in a cell comprising the polypeptide or a functional fragment thereof.

3. The method of claim 2, wherein the cell comprises an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide or a functional fragment thereof.

4. The method of claim 3, wherein the cell is stably transformed with the isolated polynucleotide.

5. The method of claim 1, wherein the method is carried out as a cell-free assay.

6. The method of claim 1, wherein the method is carried out in a transgenic non-human mammal comprising an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide or a functional fragment thereof.

7. The method of claim 1, wherein the SOGA polypeptide or a functional fragment thereof is a human SOGA polypeptide or a functional fragment thereof.

8. The method of claim 1, wherein the SOGA polypeptide or a functional fragment thereof is a functional fragment of a SOGA polypeptide beginning immediately after an internal signal sequence.

9. The method of claim 1, wherein the SOGA polypeptide or a functional fragment thereof is a C-terminal functional fragment of a SOGA polypeptide of about 80 kDa.

10. The method of claim 1, wherein the SOGA polypeptide or a functional fragment thereof is a C-terminal functional fragment of a SOGA polypeptide of about 25 kDa.