Patent Grant
10036016
This invention is directed to; inter alia, a method for inducing glucose uptake in a muscle cell by inhibiting Trim32 protein and/or increasing the abundance of plakoglobin in the muscle cell.
Growth of skeletal and cardiac muscles, like that of dividing cells, is largely dependent on signaling through the Insulin/PI3K/Akt/FoxO pathway. Conversely, the atrophy of specific muscles upon disuse, wasting, or denervation and the systemic muscle wasting in fasting and disease states (e.g. cancer cachexia, sepsis, and untreated diabetes) results from reduced activity of this pathway. This rapid loss of muscle mass results primarily through accelerated degradation of myofibrillar and soluble proteins, but in most catabolic states (e.g. fasting), protein synthesis also decreases.
FoxO (forkhead box O) transcription factors, one of the main downstream mediators of PI3K (phosphatidylinositol-3 kinase)/Akt [also known as PKB (protein kinase B)] signal transduction pathway, play an important role in modulating cellular homoeostasis. Recent studies have revealed the significance of FoxO in bone, the interaction of FoxO with -catenin, along with mechanical stress-induced inactivation of FoxO via PI3K/Akt.
Development of these various types of atrophy requires the transcription of a common set of atrophy-related genes (“atrogenes”) by FoxO transcription factors, whose activation is sufficient to cause accelerated proteolysis and atrophy. In atrophying muscles, multiple components of the ubiquitin-proteasome pathway (UPS), such as the muscle-specific ubiquitin ligases, MuRF1 and Atrogin1/MAFbx, are induced and their induction is essential for rapid wasting. Another ubiquitin ligase that appears to be critical for atrophy is Trim32. Like MuRF1, Trim32 contains a tripartite motif (RING; B-box; coiled-coil), but also has six NHL repeats with putative protein binding properties, and mutations in the third repeat causes Limb Girdle Muscular Dystrophy 2H. We demonstrated that during muscle wasting, MuRF1 is essential for the ubiquitin-dependent degradation of proteins comprising the thick filament, while Trim32 catalyzes the linked disassembly and degradation of the desmin cytoskeleton, Z-band, and thin filament proteins, which are linked processes.
In one embodiment, the present invention provides a method for inducing glucose uptake in a muscle cell, comprising the step of inhibiting Trim32 protein in said cell, thereby inducing glucose uptake in a muscle cell. In some embodiments, the cell is a skeletal muscle cell. In some embodiments, inducing glucose uptake in a muscle cell further includes muscle fiber growth. In some embodiments, inducing glucose uptake in a muscle cell is inducing the phosphorylation of insulin receptor in the cell.
In a further embodiment, the present invention provides a method for inducing glucose uptake in a muscle cell, comprising the step of increasing the abundance of plakoglobin protein in the cell, thereby inducing glucose uptake in a muscle cell. In some embodiments, the cell is a skeletal muscle cell. In some embodiments, the cell is a skeletal muscle cell. In some embodiments, increasing the abundance of plakoglobin protein in the cell results in muscle fiber growth. In some embodiments, increasing the abundance of plakoglobin protein in the cell results in inducing the phosphorylation of insulin receptor in the cell.
In one embodiment, the present invention provides a method for inducing glucose uptake in a muscle cell, comprising the step of inhibiting Trim32 protein in the cell, thereby inducing glucose uptake in a muscle cell. In another embodiment, a muscle cell is a cell within an atrophying muscle. In another embodiment, a muscle cell is a cell within a muscle exposed to fasting conditions. In another embodiment, a muscle cell is a cell of a subject afflicted with diabetes. In another embodiment, a muscle cell is a cell of a subject afflicted with type I diabetes. In another embodiment, a muscle cell is a cell of a subject afflicted with type II diabetes.
In another embodiment, a Trim32 protein of the invention comprises or consists the amino acid sequence: MAAAAAASHLNLDALREVLECPICMESFTEEQLRPKLLHCGHTICRQCLEKLLASSINGV RCPFCSKITRITSLTQLTDNLTVLKIIDTAGLSEAVGLLMCRGCGRRLPRQFCRSCGVVLC EPCREADHQPPGHCTLPVKEAAEERRRDFGEKLTRLRELTGELQRRKAALEGVSRDLQA RYKAVLQEYGHEERRIQEELARSRKFFTGSLAEVEKSNSQVVEEQSYLLNIAEVQAVSRC DYFLAKIKQADVALLEETADEEEPELTASLPRELTLQDVELLKVGHVGPLQIGQAVKKPR TVNMEDSWAGEEGAASSASASVTFREMDMSPEEVAPSPRASPAKQRSSEAASGIQQCLF LKKMGAKGSTPGMFNLPVSLYVTSQSEVLVADRGNYRIQVFNRKGFLKEIRRSPSGIDSF VLSFLGADLPNLTPLSVAMNCHGLIGVTDSYDNSLKVYTMDGHCVACHRSQLSKPWGIT ALPSGQFVVTDVEGGKLWCFTVDRGAGVVKYSCLCSAVRPKFVTCDAEGTVYFTQGLG LNVENRQNEHHLEGGFSIGSVGPDGQLGRQISHFFSENEDFRCIAGMCVDARGDLIVADS SRKEILHFPKGGGYSVLIREGLTCPVGIALTPKGQLLVLDCWDHCVKIYSYHLRRYSTP (SEQ ID NO: 1). In another embodiment, a Trim32 protein is a fragment of SEQ ID NO: 1 having a Trim32 protein activity as described herein. In another embodiment, a Trim32 protein is a mutant of SEQ ID NO: 1 having a Trim32 protein activity as described herein.
In another embodiment, inducing glucose uptake is enhancing glucose uptake. In another embodiment, inducing glucose uptake is increasing glucose uptake. In another embodiment, inducing glucose uptake is increasing the concentration of glucose within a muscle cell. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 20%. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 30%. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 40%. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 50%. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 1.5 folds. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 2 folds. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 3 folds. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 4 folds. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 5 folds. In another embodiment, inducing glucose uptake is increasing intracellular glucose by at least 10 folds.
In another embodiment, inhibiting Trim32 protein is inhibiting the activity of the Trim32 protein. In another embodiment, inhibiting Trim32 protein is inhibiting the translation of the mRNA molecule encoding Trim32 protein. In another embodiment, inhibiting Trim32 protein is inhibiting the transcription of Trim32 mRNA. In another embodiment, a synthetic mRNA molecule encodes a dominant negative Trim 32 protein. In another embodiment, a synthetic mRNA molecule comprises a 5-methylcytidine base, a pseudouridine base, or a combination thereof.
In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with an antisense oligonucleotide (AS-ODNs) directed against Trim32. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a ribozyme directed against Trim32. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a small interfering RNAs (siRNAs) directed against Trim32. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a RNA interference (RNAi) molecule directed against Trim32. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with an antagomir antisense directed against Trim32. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a microRNA designed to silence Trim32.
In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a mutant protein Trim32 protein lacking Trim 32 activity. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a dominant negative (DN) Trim32 protein. In another embodiment, a dominant negative (DN) Trim32 protein of the invention comprises or consists the amino acid sequence: MAAAAAASHLNLDALREVLSKITRITGLTQLTDNLTVLKIIDTAGLSEAVGLLMCRGCGR RLPRQFCRSCGVVLCEPCREADHQPPGHCTLPVKEAAEERRRDFGEKLTRLRELTGELQR RKAALEGVSRDLQARYKAVLQEYGHEERRIQEELARSRKFFTGSLAEVEKSNSQVVEEQ SYLLNIAEVQAVSRCDYFLAKIKQADVALLEETADEEEPELTASLPRELTLQDVELLKVG HVGPLQIGQAVKKPRTVNMEDSWAGEEGAASSASASVTFREMDMSPEEVAPSPRASPAK QRSSEAASGIQQCLFLKKMGAKGSTPGMFNLPVSLYVTSQSEVLVADRGNYRIQVFNRK GFLKEIRRSPSGIDSFVLSFLGADLPNLTPLSVAMNCHGLIGVTDSYDNSLKVYTMDGHC VACHRSQLSKPWGITALPSGQFVVTDVEGGKLWCFTVDRGAGVVKYSCLCSAVRPKFV TCDAEGTVYFTQGLGLNVENRQNEHHLEGGFSIGSVGPDGQLGRQISHFFSENEDFRCIA GMCVDARGDLIVADSSRKEILHFPKGGGYSVLIREGLTCPVGIALTPKGQLLVLDCWDHC VKIYSYHLRRYSTP (SEQ ID NO: 2). In another embodiment, a DN Trim32 protein is a fragment of SEQ ID NO: 2 having a DN Trim32 protein activity as described herein. In another embodiment, a DN Trim32 protein is a mutant of SEQ ID NO: 2 having a Trim32 protein activity as described herein. In another embodiment, a DN Trim32 protein is a fragment of SEQ ID NO: 1 which abolishes a Trim32 protein activity as described herein. In another embodiment, a DN Trim32 protein is a mutant of SEQ ID NO: 1 which abolishes a Trim32 protein activity as described herein.
In another embodiment, a dominant negative (DN) Trim32 protein of the invention is encoded by a DNA sequence which comprises or consists the nucleic acid sequence: ACGCCGCCGCCATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTC GAGAATTCTGCCGAGGCTGTGGTGTGGTGTTGTGTGAACCCTGCCGGGAGGCAGATC ACCAACCCCCTGGCCACTGCACACTTCCGGTCAAGGAGGCAGCTGAGGAGCGGCGG AGGGACTTCGGGGAGAAGTTGACTCGTCTAAGGGAACTTACTGGAGAGCTGCAGAG GAGGAAGGTAGCCTTGGAGGGCGTCTCCAGGGATCTTCAGGCAAGGTATAAGGCTGT TCTTCAAGAATATGGCCATGAGGAACGCCGCATCCAGGAAGAGCTAGCCCGCTCTCG GAAGTTCTTCACAGGCTCCTTGGCTGAGGTTGAGAAGTCCAACAGTCAAGTGGTAGA GGAGCAGAGCTACCTACTCAACATTGCTGAGGTGCAGGCCGTGTCTCGCTGTGACTA CTTTCTAGCGAAGATCAAGCAAGCTGATGTAGCCCTCCTGGAGGAGACAGCGGATGA GGAGGAGCCCGAGCTCACTGCCAGCCTACCCCGGGAGCTTACCCTGCAAGATGTGGA GCTCCTTAAGGTAGGACACGTTGGTCCTCTGCAAATTGGCCAGGCTGTTAAGAAGCC CCGGACAGTTAACATGGAAGATTNCTGGGCAGGGGANGAGGGANCAGCATCTTCTG CCTCAGCCTCGGTAACCTTTAGAGAGATGGACATGAGCCCTGAGGAATAACTTCCCA CCCCTANGGCTTCCCCGCGAAACACGGAGTTCTTGAGGCAGCTTCCGGTATCCAACA GTGTCTGTTTCTCAANAAAATGGGGGCGAAAGGCAACCANTTCCCGGCANTGGTTCA ATCNTTCCANTCCANNCTTCTNTGGGAACNANNCCAAAATGGANGGGGTTGGTTTGC CCACCGGGGCAAATTTTCNAATCCCAAGNGGTTCAACCCCNAAAGGGTTTTTTTAAG GGAAATCNCCNNNNCCCCCNGGGGGNNTTGAAAACNTTCNGGNNAAANNTTCCCTT NGGGNNCNANTTGNCCAAANTNNCCNNCCCTTTTT (SEQ ID NO: 4).
In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a mutant protein Trim32 protein lacking Trim 32 muscle activity. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a small molecule that abolishes Trim32 muscle activity. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a Trim32 competitive inhibitor. In another embodiment, inhibiting Trim32 protein is contacting a muscle cell with a Trim32 antibody which renders Trim32 protein-inactive. In another embodiment, the antibody is a polyclonal antibody. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a single-chain variable fragment (scFv) directed against the Trim32 protein.
In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in increase in the concentration of plakoglobin in a muscle cell. In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in the induction of insulin receptor. In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in the induction of phosphorylation of insulin receptor in the cell. In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in increase of PI3K/Akt/FoxO signaling in a muscle cell. In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in enhancing glucose uptake in a muscle cell. In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in muscle fiber growth. In another embodiment, inhibiting Trim32 protein in a muscle cell according to the present invention results in induction of mitosis in a muscle cell.
In another embodiment, a muscle cell is a skeletal muscle cell. In another embodiment, a muscle cell is a cardiac muscle cell. In another embodiment, a muscle cell is a smooth muscle cell. In another embodiment, a muscle cell is a cell within an atrophying muscle. In another embodiment, a muscle cell is a cell of a subject afflicted with a wasting disease. In another embodiment, a muscle cell is a cell of a subject afflicted with a muscle denervation disease. In another embodiment, a muscle cell is a cell of a fasting subject. In another embodiment, a muscle cell is a cell of a subject afflicted with cachexia. In another embodiment, a muscle cell is a cell of a subject afflicted with cancer. In another embodiment, a muscle cell is a cell of a subject afflicted with sepsis. In another embodiment, a muscle cell is a cell of a subject afflicted with diabetes.
In another embodiment, a muscle cell is a cell of a subject suffering from loss of muscle mass. In another embodiment, a muscle cell is a cell of a subject suffering from accelerated degradation of myofibrillar and soluble proteins, and/or a catabolic state. In another embodiment, a muscle cell is a cell of a subject suffering from low levels of IGF-I. In another embodiment, a muscle cell is a cell of a subject suffering from low levels of insulin. In another embodiment, the method of the invention inhibits and/or downregulates Trim32 and thereby reduces muscle atrophy.
In another embodiment, the present invention further provides a method for inducing glucose uptake in a muscle cell, comprising the step of increasing the abundance of plakoglobin protein in the cell, thereby inducing glucose uptake in a muscle cell. In another embodiment, increasing the abundance of plakoglobin protein in the cell further results in inducing muscle fiber growth. In another embodiment, increasing the abundance of plakoglobin protein in a cell results in inducing the phosphorylation of insulin receptor in the cell. In another embodiment, increasing the abundance of plakoglobin protein in a cell is transfecting the cell with a vector comprising a nucleic acid molecule encoding the plakoglobin protein. In another embodiment, increasing the abundance is increasing the concentration. In another embodiment, increasing the abundance is increasing the activity.
In another embodiment, activation of the PI3K/Akt/FoxO pathway according to the invention induces cell growth, while its inhibition reduces cell survival and in muscle, causes atrophy. In another embodiment, Trim32 inhibition induces the accumulation of plakoglobin, which in turn binds to the insulin receptor and the PI3K subunit, p85, and promotes PI3K/Akt/FoxO signaling. In another embodiment, the invention further provides stabilizing plakoglobin for enhancing PI3K/Akt/FoxO signaling.
In another embodiment, plakoglobin comprises or consists the amino acid sequence: MEVMNLIEQPIKVTEWQQTYTYDSGIHSGVNTCVPSVSSKGIMDEDDACGRQYTLKKTT TYTQGVPQNQGDLEYQMSTTARAKRVREAMCPGVSGEDSSLLLATQVEGQTTNLQRL AEPSQLLKSAIVHLINYQDDAELATRALPELTKLLNDEDPVVVTKAAMIVNQLSKKEAS RRALMGSPQLVAAVVRTMQNTSDLDTARCTTSILHNLSHHREGLLAIFKSGGIPALVRM LSSPVESVLFYAITTLHNLLLYQEGAKMAVRLADGLQKMVPLLNKNNPKFLAITTDCLQ LLAYGNQESKLIILANGGPQGLVQIMRNYSYEKLLWTTSRVLKVLSVCPSNKPAIVEAG GMQALGKHLTSNSPRLVQNCLWTLRNLSDVATKQEGLESVLKILVNQLSVDDVNVLTC ATGTLSNLTCNNSKNKTLVTQNSGVEALIHAILRAGDKDDITEPAVCALRHLTSRHPEAE MAQNSVRLNYGIPAIVKLLNQPNQWPLVKATIGLIRNLALCPANHAPLQEAAVIPRLVQ LLVKAHQDAQRHVAAGTQQPYTDGVRMEEIVEGCTGALHILARDPMNRMEIFRLNTIPL FVQLLYSSVENIQRVAAGVLCELAQDKEAADAIDAEGASAPLMELLHSRNEGTATYAA AVLFRISEDKNPDYRKRVSVELTNSLFKHDPAAWEAAQSMIPINEPYADDMDATYRPM YSSDVPLDPLDMHMDLDGDYPMDTYSDGLRPPYPTADHMLA (SEQ ID NO: 3). In another embodiment, plakoglobin is a fragment of SEQ ID NO: 3 having a plakoglobin activity as described herein. In another embodiment, plakoglobin is a mutant of SEQ ID NO: 3 having a plakoglobin activity as described herein.
In another embodiment, the invention surprisingly provides that plakoglobin is an important constituent of skeletal muscle where it binds to both the insulin receptor and the p85 regulatory subunit of PI3K, to enhance signaling by the PI3K/Akt/FoxO cascade. Thus, in some embodiments, changes in plakoglobin levels alone influence PI3K/Akt/FoxO pathway in muscle and thereby causes muscle growth or block atrophy. In another embodiment, the invention surprisingly provides that Trim32 functions as a novel inhibitor of PI3K/Akt/FoxO signaling by promoting the degradation of plakoglobin, which is important for activation of this pathway in a cell.
In another embodiment, downregulation of Trim32 reduces muscle atrophy. In another embodiment, downregulation of Trim32 is achieved by electroporation of a dominant negative Trim32 (Trim32-DN), which lacks the catalytic RING domain. In another embodiment, overexpression of Trim32-DN results in increase in weight over that of muscles electroporated (
In some embodiments, any protein or a polypeptide described herein encompasses native polypeptides (either degradation products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (typically, synthetically synthesized polypeptides), as well as peptoids and semipeptoids which are polypeptide analogs, which have, in some embodiments, modifications rendering the polypeptides even more stable while in a body or more capable of penetrating into cells. In some embodiments, the terms “protein” and “polypeptide” are used interchangeably.
In some embodiments, modifications to a protein of the invention include, but are not limited to N terminus modification, C terminus modification, polypeptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
In some embodiments, polypeptide bonds (—CO—NH—) within the polypeptide are substituted. In some embodiments, the polypeptide bonds are substituted by N-methylated bonds (—N(CH3)-CO—). In some embodiments, the polypeptide bonds are substituted by ester bonds (—C(R)H—C—O—O—C(R)—N—). In some embodiments, the polypeptide bonds are substituted by ketomethylen bonds (—CO—CH2-). In some embodiments, the polypeptide bonds are substituted by α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds (—CH2-NH—). In some embodiments, the polypeptide bonds are substituted by hydroxyethylene bonds (—CH(OH)—CH2-). In some embodiments, the polypeptide bonds are substituted by thioamide bonds (—CS—NH—). In some embodiments, the polypeptide bonds are substituted by olefinic double bonds (—CH═CH—). In some embodiments, the polypeptide bonds are substituted by retro amide bonds (—NH—CO—). In some embodiments, the polypeptide bonds are substituted by polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presented on the carbon atom. In some embodiments, these modifications occur at any of the bonds along the polypeptide chain and even at several (2-3 bonds) at the same time.
In some embodiments, natural aromatic amino acids of the polypeptide such as Trp, Tyr and Phe, be substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In some embodiments, the polypeptides of the present invention include one or more modified amino acid or one or more non-amino acid monomers (e.g. fatty acid, complex carbohydrates etc).
In one embodiment, “amino acid” or “amino acid” is understood to include the 20 naturally occurring amino acid; those amino acid often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acid including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment, “amino acid” includes both D- and L-amino acid.
In some embodiments, the polypeptides of the present invention are utilized in therapeutics which requires the polypeptides to be in a soluble form. In some embodiments, the polypeptides of the present invention include one or more non-natural or natural polar amino acid, including but not limited to serine and threonine which are capable of increasing polypeptide solubility due to their hydroxyl-containing side chain.
In some embodiments, the polypeptides of the present invention are utilized in a linear form, although it will be appreciated by one skilled in the art that in cases where cyclicization does not severely interfere with polypeptide characteristics, cyclic forms of the polypeptide can also be utilized.
In some embodiments, the polypeptides of present invention are biochemically synthesized such as by using standard solid phase techniques. In some embodiments, these biochemical methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. In some embodiments, these methods are used when the polypeptide is relatively short (about 5-15 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
In some embodiments, solid phase polypeptide synthesis procedures are well known to one skilled in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic polypeptides are purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.] and the composition of which can be confirmed via amino acid sequencing by methods known to one skilled in the art.
In some embodiments, recombinant protein techniques are used to generate the polypeptides of the present invention. In some embodiments, recombinant protein techniques are used for generation of relatively long polypeptides (e.g., longer than 18-25 amino acid). In some embodiments, recombinant protein techniques are used for the generation of large amounts of the polypeptide of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al, (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
In one embodiment, a polypeptide of the present invention is synthesized using a polynucleotide encoding a polypeptide of the present invention. In some embodiments, the polynucleotide encoding a polypeptide of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue specific expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention. In some embodiments, the vector is a cationic vector.
In some embodiment, tissue-specific promoters such as muscle specific promoters suitable for use with the present invention include sequences which are functional in specific cell population, such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477]. Inducible promoters suitable for use with the present invention include for example the tetracycline-inducible promoter (Srour, M. A., et al., 2003. Thromb. Haemost. 90: 398-405).
In one embodiment, the phrase “a polynucleotide” refers to a single or double stranded nucleic acid sequence which be isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above). In another embodiment, a cDNA of the invention encodes a TRIM 32 protein. In another embodiment, a cDNA of the invention encodes a TRIM 32 protein having SEQ ID NO: 1. In another embodiment, a cDNA of the invention encodes a DN TRIM 32 protein. In another embodiment, a cDNA of the invention encodes a DN TRIM 32 protein having SEQ ID NO: 2.
In one embodiment, “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. In one embodiment, the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase. In another embodiment, a cDNA of the invention encodes a plakoglobin. In another embodiment, a cDNA of the invention encodes a plakoglobin having SEQ ID NO: 3.
In one embodiment, “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
In one embodiment, “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements.
In one embodiment, the polynucleotides of the present invention further comprise a signal sequence encoding a signal peptide for the secretion of the polypeptides of the present invention. In some embodiments, signal sequences include, but are not limited to the endogenous signal sequence. In one embodiment, following expression and secretion, the signal peptides are cleaved from the precursor proteins resulting in the mature proteins. In one embodiment, the polynucleotides of the present invention are transferred into the target tissue/muscle. In one embodiment, the polynucleotides of the present invention are electro-transferred into the target tissue/muscle.
In some embodiments, polynucleotides of the present invention are prepared using PCR techniques, or any other method or procedure known to one skilled in the art. In some embodiments, the procedure involves the legation of two different DNA sequences (See, for example, “Current Protocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons, 1992).
In one embodiment, polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators (e.g., polyadenylation signals).
In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.
In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems such as CHO cells) to express the polypeptide of the present invention. In one embodiment, the expression vector used to express polynucleotides of the present invention in mammalian cells is pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene. Construction of the pCI-DHFR vector is described, according to one embodiment, in Example 1.
In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].
In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.
In one embodiment, the expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
In some embodiments, recombinant viral vectors are useful for in vivo expression of the polypeptides of the present invention since they offer advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
In one embodiment, various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
In some embodiments, introduction of nucleic acid by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
In one embodiment, it will be appreciated that the polypeptides of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in-vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).
In one embodiment, in vivo gene therapy using EPO has been attempted in animal models such as rodents [Bohl et al., Blood. 2000; 95:2793-2798], primates [Gao et al., Blood, 2004, Volume 103, Number 9] and has proven successful in human clinical trials for patients with chronic renal failure [Lippin et al Blood 2005, 106, Number 7].
In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.
Various methods, in some embodiments, can be used to introduce the expression vector of the present invention into the host cell system. In some embodiments, such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.
In some embodiments, depending on the vector and host system used for production, resultant polypeptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.
In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is effected.
In one embodiment, the phrase “recovering the recombinant polypeptide” used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.
In one embodiment, polypeptides of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].
In one embodiment, the polypeptide of the present invention is retrieved in “substantially pure” form.
In one embodiment, the phrase “substantially pure” refers to a purity that allows for the effective use of the protein in the applications described herein.
In one embodiment, the polypeptide of the present invention can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.
In one embodiment, a protein of the present invention can be provided to an individual/subject per se. In one embodiment, the protein of the present invention can be provided to the individual as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. In another embodiment, the protein of the present invention can be provided to the individual as part of a pharmaceutical composition where it is mixed with at least one additional anti-inflammatory agent and a pharmaceutically acceptable carrier.
In one embodiment, a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
In one embodiment, “active ingredient” refers to the protein of interest, which is accountable for the biological effect.
In one embodiment, the present invention provides combined preparations. In one embodiment, “a combined preparation” defines especially a “kit of parts” in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation. In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.
In one embodiment, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. In one embodiment, one of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
In one embodiment, “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs are found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
In one embodiment, suitable routes of administration, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
In one embodiment, the preparation is administered in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body.
Peroral compositions, in some embodiments, comprise liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. In some embodiments, liquid oral compositions comprise from about 0.012% to about 0.933% of the desired compound or compounds, or in another embodiment, from about 0.033% to about 0.7%.
In some embodiments, compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of the compounds of the present invention and optionally, other compounds, intended for topical intranasal administration. In some embodiments, h compositions comprise from about 0.01% to about 10.0% w/v of a subject compound, more preferably from about 0.1% to about 2.0, which is used for systemic delivery of the compounds by the intranasal route.
In another embodiment, the pharmaceutical compositions are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment, the pharmaceutical compositions are administered intravenously, and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra-arterially, and are thus formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly, and are thus formulated in a form suitable for intramuscular administration.
Further, in another embodiment, the pharmaceutical compositions are administered topically to body surfaces, and are thus formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops and the like. For topical administration, the compounds of the present invention are combined with an additional appropriate therapeutic agent or agents, prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.
In one embodiment, pharmaceutical compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
In one embodiment, pharmaceutical compositions for use in accordance with the present invention is formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. In one embodiment, formulation is dependent upon the route of administration chosen.
In one embodiment, injectables, of the invention are formulated in aqueous solutions. In one embodiment, injectables, of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodiments, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In one embodiment, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
The compositions also comprise, in some embodiments, preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.
In some embodiments, pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients, in some embodiments, are prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include, in some embodiments, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions contain, in some embodiments, substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In another embodiment, the suspension also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
In another embodiment, the pharmaceutical composition delivered in a controlled release system is formulated for intravenous infusion, implantable osmotic pump, transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).
In some embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. Compositions are formulated, in some embodiments, for atomization and inhalation administration. In another embodiment, compositions are contained in a container with attached atomizing means.
In one embodiment, the preparation of the present invention is formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
In some embodiments, pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
In one embodiment, determination of a therapeutically effective amount is well within the capability of those skilled in the art.
The compositions also comprise preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like.
In some embodiments, preparation of effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1].
In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
In one embodiment, the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosage forms containing the active ingredient. In one embodiment, the pack, for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
In one embodiment, it will be appreciated that the proteins of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
Materials and Methods
In Vivo Transfection
Animal experiments were conducted according to the ethical guidelines of the NIH Guide for the Care and Use of Laboratory Animals. Animal care was provided by specialized personnel in the Institutional Animal Care facility. Experiments were performed in adult CD-1 male mice (27-28 g). In vivo electroporation was performed by the injection of 20 μg of plasmid DNA into adult mouse tibialis anterior muscle and application in of electric pulses (12V, 5 pulses, 200-ms intervals). In fasting experiments food was removed from cages 4 days after electroporation for 48 hours. Fiber size was determined by measurements of cross-sectional area of 500 transfected (express GFP) and 500 adjacent nontransfected fibers in the same muscle section (10 m), using MetaMorph (Molecular Devices).
Antibodies and Constructs.
The Trim32 and control shRNAs were previously described (shTrim32-1, GGCTGATTGGTGTCACTGATA (SEQ ID NO: 5); shTrim32-2, AGCTGCTGGTCTTGGACTGTT (SEQ ID NO: 6); shLacz, AAATCGCTGATTTGTGTAGTC (SEQ ID NO: 7), and the plakoglobin shRNA was designed using Invitrogen's BLOCK-iT RNAi expression vector kit with the pcDNA 6.2-GW/EmGFP-miR vector. Anti plakoglobin, Laminin and GAPDH are from Sigma. Anti-tubulin from Invitrogen and anti GFP from abcam. Anti Akt, P-Akt, PI3K-p85, P-PI3K-p85, FOXO3 and P-FOXO3 are from Cell Signaling.
Protein Analysis
Immunoblotting and immunoprecipitation were performed as follows: the cytosolic fraction from tibialis anterior muscle was used for immunoblotting or immunoprecipitation and was resolved by SDS-PAGE and immunoblotting with specific antibodies and secondary antibodies conjugated to alkaline phosphatase. Immunoprecipitation assays of plakoglobin or PI3K-p85 from the soluble fraction of muscle were performed overnight at 4° C. and then protein A/G agaroze was added for 4 hours. Phosphatase inhibitors were not added to extraction buffer except for the immunoprecipitation experiments and blots in
Quantitative Real-Time PCR
Total RNA was isolated from muscle and cDNA synthesized by reverse transcription. Real-time qPCR was performed on mouse target genes using specific primers (Table 1) and DyNAmo HS SYBR Green qPCR kit (F-410S; Finnzymes) according to the manufacturers' protocol.
Immunofluorescence
Immunofluorescence of paraffin-embedded muscle sections was performed as follows: muscle cross or longitudinal sections from fed and fasted mice were embedded in paraffin and then gradually rehydrated in ethanol/PBS. For immunofluorescence of rehydrated samples 1:50 dilution of primary antibody and 1:1000 dilution of Alexa 555 conjugated secondary antibody were used. In
Glucose Uptake Assay
C2C12 cells were plated on a 6-well plate coated with 5 ug/ml fibronectin (f1141, Sigma). Forty two hours after transfection (lipofectoamine 2000), cells were washed in warm PBS, starved in DMEM/0.1% BSA for 6 hours and washed again. Following treatment with 200 nM insulin/PBS (Sigma, I0516) for 30 min at 37° C., cells were washed and incubated for 10 min at 37° C. with 1 uCi/ml 2-deoxy-D-[3H]glucose (NET328A250UC, PerkinElmer) and 0.1 mM cold 2-deoxy-D-glucose (D8375, Sigma). 20 nM of cytochalasin B (glucose transport inhibitor) (C6762, Sigma) were added to control wells. Then, cells were washed in cold PBS, lyzed in 0.2N NaOH for 2 hours at room temperature, and radioactivity determined using scintillation fluid.
Fractionation of Muscle Tissue.
Work was performed at 4° C. Mouse tibialis anterior muscles were homogenized on ice for 30 s in 19 volumes of buffer C (20 mM Tris-HCl, pH 7.6, 5 mM EDTA/NaOH pH 7.4, 100 mM KCl, 1 mM DTT and 1 mM sodium OrthoVanadate), and spun at 2900×g for 20 min to pellet nuclei and unbroken tissue.
The supernatant was centrifuged at 180,000×g for 90 min and the supernatant (i.e., cytosolic fraction) stored at −80° C. The pellet was resuspended in 10 volumes of buffer M (20 mM Tris-HCl, pH 7.6, 5 mM EDTA/NaOH pH 7.4, 100 mM KCl, 1 mM DTT, 0.25% sodium deoxycholate, 1% NP-40 and 1 mM sodium OrthoVanadate), rotated at 4° C. for 20 min and centrifuged at 100,000×g for 30 min. The supernatant (i.e., membrane fraction) was then collected and stored at −80° C. All buffers contained protease inhibitors (10 g/ml leupeptin, 3 mM benzamidine, 1 g/ml trypsin inhibitor, and 1 mM PMSF). 0.25% of membrane fraction and 0.01% of cytosolic fraction were separated on SDS-PAGE for Western Blot analysis.
Statistical Analysis and Image Acquisition
Data are presented as means±SEM. The statistical significance was determined with one-tailed paired Student's t test. Alpha level was set to 0.05. Muscle sections were imaged at room temperature with an upright fluorescent microscope (Nikon 80i) and a monochrome camera (Hamamatsu C8484-03), and C2C12 myotubes with Nikon Ti-E inverted motorized microscope and a Hamamatsu ORCA-R2 cooled CCD camera. Image acquisition and processing was performed using MetaMorph software. Black and white images were processed with Adobe Photoshop CS3, version 10.0.1. software.
In Vitro Ubiquitination
A plasmid DNA encoding His-plakoglobin was electroporated into tibialis anterior muscle and 4 d later the food was removed from cages for 2 days. His-plakoglobin was purified from muscle homogenates using Ni-Column and subjected to ubiquitination reaction containing 22.5 nM E1, 0.75 M E2, 0.4 M Trim32, 0.75 M UbcH5 and 59 M ubiquitin in reaction buffer (2 mM ATP, 20 mM Tris-HCl, pH 7.6, 20 mM KCl, 5 mM MgCl2, and 1 mM DTT). Plakoglobin ubiquitination was analyzed by SDS-PAGE and immunoblotting with plakoglobin antibody (Sigma).
Immunofluorescence
A vector encoding GFP-plakoglobin was electroporated into tibialis anterior of adult wild-type mouse for 6 d. Muscle cross sections were paraffin-embedded as reported and images were collected at room temperature using an upright epifluorescence microscope (model 80i; Nikon) with a Plan Fluor 40×1.4 NA objective lens, a 545/30 excitation filter and 620/60 emission filter (Chroma Technology Corp.) and a cooled CCD camera (model C8484-03; Hamamatsu Photonics).
DNA Constructs
The desmoplakin shRNA was designed using Invitrogen's RNAi esigner tool, and cloned into pcDNA 6.2-GW/EmGFP-miR vector using Invitrogen's BLOCK-iT RNAi expression vector kit.
Because downregulation of Trim32 reduces muscle atrophy, the experiment also determined whether it also affects normal muscle mass by electroporation of a dominant negative Trim32 (Trim32-DN), which lacks the catalytic RING domain (Kano et al., 2008), into normal tibialis anterior muscle (TA). Overexpression of Trim32-DN for 6 days resulted in a 15% increase in weight over that of muscles electroporated with a control vector (
The present findings suggest that Trim32 substrates accumulate upon its downregulation and reduce atrophy or induce growth. Using immobilized GST-Trim32 and Mass Spectrometry, several Trim32 substrates were identified in muscle extracts, which were bound and could be ubiquitinated by Trim32, including thin filament and Z-band components, plus the cytoskeletal protein desmin. Surprisingly, the immobilized Trim32 also bound plakoglobin, which, in other tissues, is a component of the desmosome complex. Its presence was unexpected because there had been no prior reports of desmosomes or its components in skeletal muscle. To determine whether it is ubiquitinated by Trim32, GST-Trim32 precipitates were incubated with a ubiquitination system containing E1, E2 (UbcH5), ATP, and 6His-ubiquitin. The 6His-tagged ubiquitinated proteins were then purified with a nickel column. Mass spectrometric analysis revealed that plakoglobin was polyubiquitinated by Trim32 (as documented by the presence of 13 unique peptides). Furthermore, recombinant Trim32 also polyubiquitinated 6His-plakoglobin, which was expressed and purified from normal muscle (
In epithelial cells, binding of PI3K to plakoglobin was proposed to enhance PI3K/Akt signaling (Calautti et al., 2005; Woodfield et al., 2001). Because this pathway is the primary regulator of muscle mass. However, it was important initially to confirm that plakoglobin is actually present in the satellite cells and muscle fibers. Therefore, its spatial distribution in normal tibialis anterior (TA) was analyzed by immunofluorescence staining of paraffin-embedded cross and longitudinal sections. In epithelia, plakoglobin has a punctate distribution throughout the cell (Chen et al., 2002; Nathke et al., 1994). A similar spot-like distribution of plakoglobin was found within the muscle fiber, and in the plane of the fiber membrane (FIG. 2A). Thus, this protein, unlike myofibrillar components or desmin, did not show a specific periodic distribution along the sarcomere. A similar diffuse distribution of plakoglobin was observed in C2C12 myotubes (
To determine whether plakoglobin loss influences the activity of the PI3K/Akt/FoxO pathway, as had been suggested in keratinocytes (Calautti et al., 2005), the question of whether plakoglobin interacts with p85/PI3K in muscle was tested. Plakoglobin co-precipitated with p85/PI3K from normal muscle, but not during fasting (
Similar interactions between p85/PI3K and plakoglobin were demonstrated by co-immunoprecipitation in the heart and liver (
Further studies determined whether the interaction of plakoglobin with p85/PI3K, in fact, influences PI3K/Akt/FoxO signaling. By two days after food deprivation, phosphorylation of PI3K, Akt, and its target FoxO3 was markedly reduced (
However, Trim32 inhibition by overexpression of the Trim32-DN resulted in a marked decrease in MuRF1 and Atrogin1 expression in the TA muscles during fasting (
To learn whether this reduction in PI3K/Akt/FoxO signaling upon fasting is dependent on the presence of plakoglobin, plakoglobin was downregulated by transfection of shRNA (shJUP) into normal muscle (
Although expressing the Trim32-DN prevented the decrease in PI3K/Akt/FoxO signaling in the atrophying muscles, this effect was markedly attenuated by simultaneously downregulating plakoglobin (
In addition to regulating cell size and protein balance, the PI3K/Akt/FoxO pathway also mediates insulin's stimulation of glucose-uptake into muscle and adipose tissue. As predicted, plakoglobin downregulation in myoblasts reduced the stimulation by insulin of glucose uptake (
In light of these effects in fasting, it was determined whether Trim32 may also influence normal muscle mass (
These studies have uncovered a novel mechanism regulating the PI3K/Akt/FoxO/mTOR pathway that involves the desmosomal component plakoglobin and the ubiquitin ligase, Trim32 (
In normal muscle, plakoglobin downregulation reduces PI3K/Akt/FoxO signaling and causes atrophy (
However, it is noteworthy that Trim32 overexpression alone is not sufficient to reduce plakoglobin levels or to cause atrophy (
This study also provides the first evidence for the presence of the desmosomal component plakoglobin in skeletal muscle. In related studies, it was found that other components of this complex, such as desmoplakin, are present within the muscle fiber, although desmoplakin levels, unlike plakoglobin's, did not alter PI3K/Akt/FoxO signaling (
In addition to causing Limb-Girdle Muscular Dystrophy, Trim32 mutations can lead to Bardet-Biedl syndrome, which is characterized by cardiac hypertrophy and dilated cardiomyopathy (Elbedour et al., 1994). The present findings would predict that a deficiency of Trim32 could lead to excessive tissue growth and possibly inappropriate activation of PI3K/Akt/FoxO signaling. Surprisingly, Kudryashova et al. recently described a Trim32 null mouse that exhibited mild myopathies, neurogenic defects, cellular disorganization, and reduced muscle growth (Kudryashova et al., 2012; Kudryashova et al., 2011), and upon fasting or disuse (Kudryashova et al., 2012), muscles from these mice atrophy as in WT mice. These surprising observations differ markedly from the present findings on the effects of selective downregulation of Trim32 in adult muscle. Presumably, the complete deficiency of Trim32 during development causes multiple systemic defects and elicits compensatory responses, but the ability of these Trim32-deficient muscles to undergo atrophy suggest that additional ubiquitin ligases may replace Trim32 in many reactions. Conversely, expression of Trim32 rises in multiple diseases, including brains of Alzheimer patients (Yokota et al., 2006), psoriasis lesions (Liu et al., 2010) and various cancers, where it enhances invasion and metastasis (Horn et al., 2004; Kano et al., 2008). These effects may also involve Trim32-mediated degradation of plakoglobin since the loss of this protein reduces cell adhesion and increases cell migration and invasion (Gosavi et al., 2011; Kundu et al., 2008; Yin et al., 2005).
Together, these observations indicate close coupling between changes in cytoskeletal and myofibrillar components during atrophy and the cell's major growth regulatory system, which could be important in other pathological states (
This application is a U.S. national phase of International Application No. PCT/IL2014/050810, filed Sep. 11, 2014, which claims priority to U.S. Provisional Application No. 61/876,929, filed Sep. 12, 2013, the disclosures of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2014/050810 | 9/11/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/037001 | 3/19/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5627159 | Shih | May 1997 | A |
| Entry |
|---|
| Cohen et al., J Cell Biol. Aug. 20, 2012;198(4):575-589. |
| Kariko et al., Nucleic Acids Res. Nov. 2011;39(21):e142. |
| Number | Date | Country | |
|---|---|---|---|
| 20160230171 A1 | Aug 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61876929 | Sep 2013 | US |
