WO1998050061A1 - Nouvelles therapies des maladies kystiques renales - Google Patents

Nouvelles therapies des maladies kystiques renales Download PDF

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Publication number
WO1998050061A1
WO1998050061A1 PCT/US1998/009268 US9809268W WO9850061A1 WO 1998050061 A1 WO1998050061 A1 WO 1998050061A1 US 9809268 W US9809268 W US 9809268W WO 9850061 A1 WO9850061 A1 WO 9850061A1
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xaa xaa
renal
renal therapeutic
isolated
protein
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PCT/US1998/009268
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English (en)
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Per Gjorstrup
Ray Harris
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Biogen, Inc.
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Priority to AU74730/98A priority Critical patent/AU7473098A/en
Publication of WO1998050061A1 publication Critical patent/WO1998050061A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the treatment of cystic renal disease in mammalian subjects.
  • the methods involve administering, to patients with cystic renal disease, certain proteins of the osteogenic protein bone morphogenetic protein (OP/BMP) family within the TGF- ⁇ superfamily of proteins.
  • OP/BMP osteogenic protein bone morphogenetic protein
  • Renal disease affects millions of people worldwide each year and adds substantially to health care costs.
  • treatment is usually effected by transplanting organs from donors, by surgical reconstruction, or with mechanical devices.
  • Each of these remedies has shortcomings. Transplantation is limited by donor shortage, surgical reconstruction can create other long-term problems, and mechanical devices cannot perform all the functions of a single organ, and therefore cannot prevent progressive deterioration.
  • Cystic renal disease of various presentations and etiologies, is of high prevalence, and causes much morbidity and mortality.
  • Autosomal recessive polycystic disease is characterized by the renal distal tubules and collecting ducts being dilated into cysts, making the kidneys huge, white, and smooth-surfaced. This is normally fatal in infancy or early childhood; typically, the enormous kidneys restrict the ability the lungs and gut to function. Many children also have congenital portal fibrosis of the liver. Multicystic dysplastic kidney, or "renal dysplasia”, is characterized by multiple cysts, usually unilateral. Uremic medullary cystic disease, or “nephronophthisis”, refers to a group of diseases (including an "autoimmune familial syndrome": Am. J. Kid. Dis.
  • ADPKD Autosomal dominant polycystic kidney disease
  • ADPKD usually does not symptomatically present until adult life.
  • cysts measuring up to 4 cm in diameter, develop from all levels of the nephron, including Bowman's capsule.
  • the cysts form, the surrounding normal kidney cells undergo apoptosis (N. Engl. J. Med. 333: 18 & 56, 1995).
  • the kidneys are often the size of footballs. Surprisingly, they may still be working.
  • Half of these patients are on dialysis or transplanted by the age of 70. Patients typically get high blood pressure as adults, years before renal failure develops. Many die of ruptured berry aneurysms. The disease has complete penetrance.
  • ADPKD is associated with a number of other complications. About 60% of patients have back, flank, or abdominal pain that can be severe and disabling. Other complications of the disease include hematuria, kidney stones, hypertension, urinary infections, and development of cysts in the liver, spleen, pancreas, lungs, gonads, thyroid, uterus and bladder. One tenth of patients die from an intracranial aneurism, possibly related to the hypertension. ADPKD is diagnosed by soundwave testing (ultrasound), radioactive scanning, or x-ray dye testing (intravenous pyelography or IVP). Recently testing for the associated gene has been described (Lancet 2: 6, 1986; Am. J. Kid. Dis. 13: 85, 1989). Treatment is aimed at complications, such as high blood pressure, pain, or infection of the cysts. The disease itself has no cure.
  • Autosomal dominant polycystic kidney disease is caused by mutations in at least three different genes: PKD1, PKD2, and PKD3, with the implicated mutation on chromosome 16pl.3 (PCKD 1 locus) where it codes for "polycystin", a tubular organizer (Proc. Natl. Acad. Sci. 93: 1524, 1996) Polycystin, a 460 kd protein with a host of domains implicating a potential role in cell-cell and cell-matrix regulation, is encoded by a 52 kb gene with a 14 kb mRNA. The PKD2 protein is also large (110 kd) and is thought to interact with polycystin.
  • ADPKD is caused by mutated DNA that encodes an abnormal form of polycystin.
  • Polycystin appears to have a normal role in the differentiation of epithelial cells, and when defective, these cells fail to maturate fully. These incompletely differentiated cells proliferate abnormally and express altered amounts of otherwise normal electrolyte transport proteins that result in excessive secretion of solute and fluid into the cysts.
  • the proliferation of the cells and the associated apoptosis, and the secretion of the fluid into the cysts created by the enlarging tubule segments appear to be regulated by growth factors, hormones, and cytokines that can alter the extent to which the disease is clinically expressed among individuals.
  • cysts The formation of the cysts is associated with complex changes in the extracellular matrix of the kidneys and other organs that may be directly or indirectly tied to mutated polycystin.
  • the summation of these pathogenetic elements leads to renal interstitial infiltration, with monocytes, macrophages, and fibroblasts culminating in fibrosis and progressive loss of renal function.
  • kidney therapeutic agents defined herein as renal therapeutic agents, and including members of the osteogenic protein/bone morphogenetic protein (OP/BMP) family of proteins, may be used in the treatment of subjects at risk, as defined herein, of cystic kidney disease or the need for renal replacement therapy.
  • OP/BMP osteogenic protein/bone morphogenetic protein
  • Useful renal therapeutic agents include polypeptides, or functional variants of polypeptides, comprising at least the C-terminal six- or seven-cysteine domain of a mammalian protein selected from the group consisting of OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, and proteins, at least a portion of which, exhibit at least 60% amino acid sequence homology with the amino acid sequence of the six- or seven-cysteine domain of human OP-1; and which are (a) capable of inducing chondrogenesis in the Reddi-Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci.
  • One aspect of the invention is a method of treating cystic kidney disease comprising administering to a subject having, or at risk for, cystic kidney disease, an isolated, renal therapeutic agent or an isolated polynucleotide sequence encoding, upon expression, an isolated, renal therapeutic agent.
  • the isolated, renal therapeutic agent is selected from a member of an osteogenic protein/bone mo ⁇ hogenic protein (OP/BMP) family within a transforming growth factor-beta superfamily of proteins.
  • the isolated, renal therapeutic agent includes a portion of which has at least 60% amino acid sequence homology with amino acid sequences of SEQ ID NOS: 1-5.
  • the step of administering also includes administering an isolated polypeptide selected from the group consisting of those polypeptides listed in Table 1 and biologically active homologs thereof.
  • Preferred methods include the step of administering via systemic or local administration.
  • a further method of the invention comprises the step of administering to a subject having, or at risk for, cystic kidney disease, a therapeutically effective composition selected from the group consisting of: (i) an isolated, renal therapeutic agent; (ii) an isolated polynucleotide capable of encoding, upon expression, an isolated renal therapeutic agent; and (iii) an isolated polynucleotide sequence capable of hybridizing under stringent conditions to the isolated polynucleotide sequence of (ii).
  • the diseases treatable by this method include autosomal recessive ("infantile") polycystic disease, multicystic dysplastic kidney disease, uremic medullary cystic disease, and autosomal dominant polycystic kidney disease.
  • the method includes administration of a renal therapeutic agent is a polypeptide that is soluble under physiological conditions and in physiological solutions.
  • the method of the present invention includes prophylactic prevention of a renal condition (i.e., dysfunction) in a mammal at risk for a cystic kidney disease and/or treatment in order to arrest the development and retard the progression of the cystic kidney disease in a mammal having clinical manifestations of the cystic kidney disease.
  • the methods are used for promoting, among other things, the restoration of a functioning renal system. As such they are of great value in preventing or delaying the onset of chronic renal failure, with the resultant need for chronic dialysis or renal replacement therapy. As such, they are useful in prolonging the lives, and in maintaining the quality of life, of subjects at risk of, or already afflicted with, cystic kidney disease.
  • N-terminus including a conserved cysteine residue
  • SEQ ID NO: 5 is a composite sequence (beginning at residue 38 of Fig. 24 from U.S. Patent 5,266,683), and includes both the specific amino acid sequence created by the amino acid identity shared by the four OPl, OP2 species, as well as alternative residues for the variable positions within the sequence.
  • Cystic kidney disease includes any disease characterized by the presence of cysts in the kidney.
  • the group specifically includes, but is not limited to, autosomal recessive ("infantile") polycystic disease, multicystic dysplastic kidney, uremic medullary cystic disease, and autosomal dominant polycystic kidney disease.
  • autosomal recessive infantile
  • multicystic dysplastic kidney uremic medullary cystic disease
  • uremic medullary cystic disease uremic medullary cystic disease
  • autosomal dominant polycystic kidney disease can be made on the basis of clinical observations, or by genetic testing for implicated genes.
  • Ring therapeutic agent means a polypeptide, or a functional variant of a polypeptide, comprising at least the C-terminal six- or seven-cysteine domain of a mammalian protein selected from the group consisting of OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, and proteins, at least a portion of which exhibit at least 60% amino acid sequence homology with the amino acid sequence of the six- or seven-cysteine domain of human OP-1 ; and which is (a) capable of inducing chondrogenesis in the Reddi-Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci.
  • a percentage "homology" between two amino acid sequences indicates the percentage of amino acid residues which are identical or similar between the sequences and, as used herein, "similar” residues are "conservative substitutions” which fulfill the criteria defined for an "accepted point mutation” in Dayhoff et al. (1978), Atlas of Protein Sequence and Structure Vol. 5 (Suppl. 3), pp. 354-352, Natl. Biomed. Res. Found., Washington, D.C.
  • proteins are defined in Section IIB, below.
  • “Prophylactic efficacy" of a renal therapeutic agent of the invention and an amount of the agent is said to be “prophylactically effective,” if administration of that amount of the agent is sufficient to cause a significant increase in the average patient age at which the disease progresses to the point at which the patient's GFR is 50% or less of normal, at which time the patient's disease is considered to be “clinically significant.”
  • “Therapeutic efficacy' of a renal therapeutic agent of the invention, and an amount of the agent is said to be “therapeutically effective,” if administration of that amount of the agent is sufficient to cause a clinically significant improvement in a standard marker of renal function when administered to a human patient who has been diagnosed with cystic renal disease.
  • markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium (Na+), urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like. See, for example, Brenner and Lazarus in Harrison's Principles of Internal Medicine, 13th ed., Isselbacher et al., (eds), McGraw Hill Text, NY.
  • a subject is said to be "at risk" of developing clinically significant cystic renal disease if the subject is reasonably expected to suffer a progressive loss of renal function associated with progressive loss of functioning nephron units associated with cystic renal disease.
  • Subjects at risk of cystic renal disease include those having a demonstrated mutation in a gene associated with developing cystic kidney disease, and also having a GFR which is chronically less than about 50%, and more particularly less than about 40%, 30% or 20%, of the expected GFR for the subject.
  • amino acid a monomeric unit of a peptide, polypeptide, or protein. There are twenty L-isomers of amino acids. The term also includes analogs of the amino acids and D-isomers of the protein amino acids and their analogs.
  • protein any polymer consisting essentially of any of the 20 protein amino acids, regardless of its size.
  • polypeptide is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied.
  • protein refers to peptides, proteins and polypeptides, unless otherwise noted.
  • “genetic fusion”- refers to a co-linear, covalent linkage of two or more proteins via their individual peptide backbones, through genetic expression of a polynucleotide molecule encoding those proteins.
  • mutant any change in quantity or structure of genetic material of an organism, in particular any change (i.e., deletion, substitution, addition, or alteration) in a wild type renal therapeutic agent polynucleotide sequence or any change in a wild type renal therapeutic agent protein.
  • wild type - the naturally-occurring polynucleotide sequence of a renal therapeutic agent exon, or a portion thereof, or renal therapeutic agent protein, or portion thereof, respectively, as it exists in vivo.
  • standard hybridization conditions - salt and temperature conditions substantially equivalent to 0.5 X SSC to about 5 X SSC and 65 degrees C for both hybridization and wash.
  • standard hybridization conditions as used herein is therefore an operational definition and encompasses a range of hybridization.
  • Higher stringency conditions may, for example, include hybridizing with plaque screen buffer (0.2% polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serum albumin, 50 mM Tris-HCl (pH 7.5); 1 M NaCl; 0.1% sodium pyrophosphate; 1 % SDS); 10% dextran sulphate, and 100 ug/ml denatured, sonicated salmon sperm DNA at 65 degrees C for 12-20 hours, and washing with 75 mM NaCl/7.5 mM sodium citrate (0.5 x SSC)/1% SDS at 65 degrees C.
  • plaque screen buffer (0.2% polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serum albumin, 50 mM Tris-HCl (pH 7.5); 1 M NaCl; 0.1% sodium pyrophosphate; 1 % SDS); 10% dextran sulphate, and 100 ug/ml denatured, sonicated salmon sperm DNA at 65
  • Lower stringency conditions may, for example, include hybridizing with plaque screen buffer, 10% dextran sulphate and 110 ug/ml denatured, sonicated salmon sperm DNA at 55 degrees C for 12-20 hours, and washing with 300 mM NaCl/30mM sodium citrate (2.0 X SSC)/1% SDS at 55 degrees C. See also Current Protocols in Molecular Biology, John Wiley & Sons, Inc. New York, Sections 6.3.1-6.3.6, (1989).
  • expression control sequence a sequence of polynucleotides that controls and regulates expression of genes when operatively linked to those genes.
  • operatively linked a polynucleotide sequence (DNA, RNA) is operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence and production of the desired renal therapeutic polypeptide encoded by the polynucleotide sequence.
  • expression vector a polynucleotide, most commonly a DNA plasmid, which allows expression of at least one gene when the expression vector is introduced into a host cell.
  • the vector may, or may not, be able to replicate in a cell.
  • isolated when applied to nucleic acid i.e., polynucleotide sequences, that encode renal therapeutic agent polypeptides, means an RNA or DNA polynucleotide, portion of genomic polynucleotide, cDNA or synthetic polynucleotide which, by virtue of its origin or manipulation: (i) is not associated with all of a polynucleotide with which it is associated in nature (e.g., is present in a host cell as an expression vector, or a portion thereof); or (ii) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature.
  • isolated it is further meant a polynucleotide sequence: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) chemically synthesized; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
  • substantially pure nucleic acid is a nucleic acid which is not immediately contiguous with one or both of the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid is derived.
  • Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional renal therapeutic agent sequences.
  • isolated when applied to a renal therapeutic agent polypeptides means a polypeptide or a portion thereof which, by virtue of its origin or manipulation: (i) is present in a host cell as the expression product of a portion of an expression vector; or (ii) is linked to a protein or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature.
  • isolated it is further meant a protein that is : (i) chemically synthesized; or (ii) expressed in a host cell and purified away from associated proteins, as by gel chromatography.
  • the term generally means a polypeptide that has been separated from other proteins, lipids and nucleic acids with which it naturally occurs.
  • the polypeptide is also separated from substances such as antibodies or gel matrices (polyacrylamide) which are used to purify it. It is preferred that an isolated polypeptide constitute at least 10 % dry weight of a purified preparation, although it may constitute higher percentages e.g., 20,50,70,80 or 95%.
  • the purified preparation also preferably contains sufficient polypeptide to allow protein sequencing.
  • heterologous promoter is a promoter which is not naturally associated with a gene or a purified nucleic acid.
  • Homologous is synonymous with the term “identity” and refers to the sequence similarity between two polypeptides, molecules or between two polynucleotides.
  • identity refers to the sequence similarity between two polypeptides, molecules or between two polynucleotides.
  • a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit (for instance, if a position in each of the two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by a lysine)
  • the percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100.
  • the two sequences are 60% homologous.
  • the DNA sequences CTGACT and CAGGTT share 50% homology (3 of the 6 total positions are matched).
  • a comparison is made when two sequences are aligned to give maximum homology.
  • Such alignment can be provided using, for instance, the method of Needleman et al., J. Mol Biol. 48: 443-453 (1970), implemented conveniently by computer programs such as the Align program (DNAstar, Inc.).
  • Homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence.
  • a "conservative substitution" of a residue in a reference sequence are those substitutions that are physically or functionally similar to the corresponding reference residues, e.g., that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like.
  • Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" in Dayhoff et al., 5: Atlas of Protein Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed. Res. Foundation, Washington, D.C. (1978).
  • a "renal therapeutic agent homolog” includes all: (i) polypeptides derived from a renal therapeutic agent protein having similar biological activity to, e.g., OP-1, the homologs preferably including biologically active variants of mammalian origin; (ii) protein molecules having biological activity produced by screening with a renal therapeutic agent probe; (iii) ligands of renal therapeutic agent polypeptides; (iv) non- polypeptide molecules having the identical biological activity as, for example, OP-1; and (v) all renal therapeutic agent homologs.
  • peptide peptide
  • proteins proteins
  • polypeptides are used interchangeably herein.
  • polynucleotide sequence and “nucleotide sequence” are also used interchangeably herein.
  • renal therapeutic fragment is used interchangeably with “renal therapeutic agent”.
  • a “purified preparation of cells” refers to an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and preferably 50% of the subject cells.
  • transgene means a nucleic acid sequence encoding one or more polypeptides which is partly or entirely heterologous (i.e., foreign) to the transgenic animal or cell into which it is introduced, or is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted or is inserted into the animal or cell genome in such a way as to alter the genome of the cell into which it is inserted by being inserted at a location which differs from that of the natural gene or which, by its insertion, results in a "knockout”.
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acids, such as introns, that may be needed for optimal expression of the selected 1 nucleic acids, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
  • transgenic cell refers to a cell containing a transgene.
  • a "transgenic animal” is any animal into which one or more and preferably essentially all, of the cells of the animal includes a transgene.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by micro injection or by infection with a recombinant virus.
  • the molecule may be integrated within a chromosome or it may be extrachromosally replicating DNA.
  • the renal therapeutic agents of the present invention can prevent, inhibit or delay the development or progression of cystic renal disease, which usually leads to the need for renal replacement therapy (i.e., renal transplant or chronic dialysis) or death.
  • the therapeutic agents of the invention are members of the osteogenic protein/bone mo ⁇ hogenetic protein (OP/BMP) family within the TGF- ⁇ superfamily of proteins.
  • OP/BMP osteogenic protein/bone mo ⁇ hogenetic protein
  • the renal therapeutic agents of the present invention are naturally occurring proteins, or functional variants of naturally occurring proteins, in the osteogenic protein/bone mo ⁇ hogenetic protein (OP/BMP) family within the TGF- ⁇ superfamily of proteins. That is, these proteins form a distinct subgroup, referred to herein as the "OP/BMP family," within the loose evolutionary grouping of sequence-related proteins known as the TGF- ⁇ superfamily.
  • OP/BMP family within the loose evolutionary grouping of sequence-related proteins known as the TGF- ⁇ superfamily.
  • Members of this protein family comprise secreted polypeptides that share common structural features, and that are similarly processed from a pro-protein to yield a carboxy-terminal mature protein.
  • the 30 kDa protein When reduced, the 30 kDa protein gives rise to two glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa.
  • the unglycosylated protein has an apparent molecular weight of about 27 kDa.
  • the 27 kDa protein gives rise to two unglycosylated polypeptide chains, having molecular weights of about 14 kDa to 16 kDa.
  • the naturally occurring OP/BMP proteins are translated as a precursor, having an N-terminal signal peptide sequence, a "pro" domain, and a "mature" protein domain.
  • the signal peptide is typically less than 30 residues, and is cleaved rapidly upon translation at a cleavage site that can be predicted using the method of Von Heijne (1986), Nucleic Acids Research 14:4683-4691.
  • the "pro" domain is variable both in sequence and in length, ranging from approximately 200 to over 400 residues.
  • the pro domain is cleaved to yield the "mature" C-terminal domain of approximately 115-180 residues, which includes the conserved six- or seven-cysteine C-terminal domain of 97-106 residues.
  • the "pro form" of an OP/BMP family member refers to a protein comprising a folded pair of polypeptides, each comprising a pro domain in either covalent or noncovalent association with the mature domains of the OP BMP polypeptide.
  • the pro form of the protein is more soluble than the mature form under physiological conditions (e.g., in vivo) and in physiological solutions (e.g., biocompatible solutions such as blood, saline or pharmaceutical carriers that are capable of being introduced into a living subject, it being understood by workers of ordinary skill in the art that the biocompatibility of a given solution is not a fixed quantity and may depend in large part upon the dosage.)
  • the pro form appears to be the primary form secreted from cultured mammalian cells.
  • the "mature form” of the protein refers to mature C-terminal domain which is not associated, either covalently or noncovalently, with the pro domain. Any preparation of OP-1 is considered to contain mature form when the amount of pro domain in the preparation is no more than 5% of the amount of "mature" C-terminal domain.
  • OP/BMP family members useful herein include any of the known naturally- occurring native proteins including allelic, phylogenetic counte ⁇ art and other variants thereof, whether naturally-sourced or biosynthetically produced (e.g., including “muteins” or “mutant proteins”), as well as new, active members of the OP/BMP family of proteins.
  • Particularly useful sequences include those comprising the C-terminal seven cysteine domains of mammalian, preferably human, human OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP8 and BMP9.
  • Other proteins useful in the practice of the invention include active forms of GDF-5, GDF-6, GDF-7, DPP, Vg 1 , Vgr- 1 ,
  • the renal therapeutic agents of the invention are selected from any one of: OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, and BMP9. Publications disclosing these sequences, as well as their chemical and physical properties, include: OP-1 and OP-2: U.S. Patent No. 5,011,691, U.S. Patent No. 5,266,683, and Ozkaynak et al. (1990), EMBO J.
  • useful proteins include biologically active biosynthetic constructs, including novel biosynthetic proteins and chimeric proteins designed using sequences from two or more known OP/BMP family proteins. See also the biosynthetic constructs disclosed in U.S. Patent No. 5,011,691, the disclosure of which is inco ⁇ orated herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, COP-7, and COP- 16).
  • the invention comprises methods of using renal therapeutic proteins encoded by nucleic acids which hybridize to DNA or RNA sequences encoding the above-identified proteins; more particularly which hybridize under standard conditions to DNA and RNA sequences encoding the active region of OPl or OP2 (see, for example amino acid SEQ ID NOS: 1 -5, herein.
  • the invention further comprises nucleic acids and the therapeutically effective polypeptide chains encoded by these nucleic acids which hybridize to the "pro" region of the OPl or OP2 proteins under standard hybridization conditions.
  • the invention provides a substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a polypeptide, the amino acid sequence of which includes, or is, the sequence of a renal therapeutic agent.
  • the subject renal therapeutic nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the renal therapeutic gene sequence, e.g., to render the renal therapeutic gene sequence suitable for use as an expression vector.
  • the nucleic acid which encodes a renal therapeutic polypeptide of the invention hybridizes under standard conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from a polynucleotide sequence that encodes, for example, SEQ ID NOS: 1 or 2, more preferably to at least 20 consecutive nucleotides from a polynucleotide sequence that encodes, for example, SEQ ID NOS: l or 2.
  • the nucleic acid differs by at least one nucleotide from a nucleotide sequence which encodes amino acids 330 to 431 from a region of human OP-1
  • hOPl-PP immature translation product
  • PP prepro form
  • prepro form an immature translation product
  • OPl-18Ser an immature translation product
  • the "active region” (also called the “functional domain") of the protein includes the C-terminal 97 amino acids of the OPl sequence ("OPS”: see SEQ ID NO: 2).
  • a longer active sequence is OP7 (comprising the C-terminal 102 amino acids: SEQ ID NO: 1).
  • analogs of the active region e.g., non-native forms never before known in nature, designed based on the observed homologies and known structure and properties of the native protein can be capable of having therapeutic efficacy.
  • the amino acid sequences of the renal therapeutic proteins disclosed herein share significant homology with various regulatory proteins on which a consensus probe can be modeled.
  • these proteins share a conserved six or seven cysteine skeleton in this region (e.g., the linear arrangement of these C-terminal cysteine residues is conserved in the different proteins.) See, for example, SEQ ID NO: 1, the sequence which defines the seven cysteine skeleton, or SEQ ID NO: 2, the sequence defines the six cysteine skeleton.
  • the OP2 proteins contain an additional cysteine residue within this region.
  • OPS Human OPl protein species defining the conserved 6 cysteine skeleton in the active region (97 amino acids, Seq. ED No. 2, residues 335-431, described in U.S. Patent No. 5,266,683 and as the amino acid residues in SEQ ED NO: 2, herein).
  • N-terminal amino acid is methionine OPl-16Ala N-terminally truncated mature human OPl protein species, Seq. ED No. 1, residue 316-431, described in U.S. Patent No. 5,266,683.
  • N-terminal amino acid is alanine
  • N-terminal amino acid is valine.
  • Patent No. 5,266,683. mOPl-PP Prepro form of mouse protein, Seq. ED No. 24, residues 1-430, described in U.S. Patent No. 5,266,683.
  • hOP2-PP Prepro form of human OP2 protein, Seq. ED No. 28, res. 1-402, described in U.S. Patent No. 5,266,683.
  • hOP2-Ala Possible mature human OP2 protein species: Seq. ED No. 28, residues 264-402, described in U.S. Patent No. 5,266,683.
  • hOP2-Pro Possible mature human OP2 protein species Seq. ED NO. 28, residues 267-402, described in U.S. Patent No. 5,266,683. N-terminal amino acid is proline.
  • hOP2-Arg Possible mature human OP2 protein species: Seq. ED No. 28, res. 270- 402, described in U.S. Patent No. 5,266,683. N-terminal amino acid is arginine.
  • hOP2-Ser Possible mature human OP2 protein species: Seq. ED No. 28, res. 243- 402, described in U.S. Patent No. 5,266,683. N-terminal amino acid is serine.
  • the invention comprises methods using renal therapeutic proteins wherein the proteins comprise a polypeptide chain having an amino acid sequence sufficiently duplicative of the encoded amino acid sequence of the Seq No:. 1 (hOPl) that described in U.S. Patent No. 5,266,683 or the Seq No: 28 (hOP2) that is also described in U.S. Patent No. 5,266,683.
  • the term "sufficiently duplicative" is understood to encompass all renal therapeutic agents whose amino acid sequence comprises at least the conserved six- or seven- cysteine skeleton and shares greater than 60% amino acid sequence identity in its active region with SEQ ED NO: 1 (seven cysteine region) and SEQ ED NO: 2 (six cysteine region) described herein.
  • SEQ ED NO: 5 is a composite sequence (beginning at residue 38 of Fig. 24 from U.S. Patent 5,266,683), and includes both the specific amino acid sequence created by the amino acid identity shared by the four OPl, OP2 species, as well as alternative residues for the variable positions within the sequence.
  • the invention comprises methods of using species of polypeptide chains comprising the conserved six cysteine skeleton plus additional cysteine residues identified in the OP2 proteins, and also comprising the conserved seven cysteine skeleton plus additional cysteine residues identified in the OP2 proteins.
  • the identification of renal therapeutic agents can thus be patterned after either of the following template amino acid sequence.
  • the template sequences contemplated are SEQ ED NO: 3, described below (and also as Seq. No. 31 of U.S. Patent 5,266,683) comprising the conserved six cysteine skeleton plus the additional cysteine residue identified in the OP2 proteins, and SEQ ED NO: 4, described below and as Seq No: 32 of U.S.
  • Patent 5,266, 683 comprising the conserved seven cysteine skeleton plus the additional cysteine residue identified in the OP2 proteins.
  • Each Xaa in these template sequences independently represents one of the 20 naturally-occurring L-isomer, alpha - amino acids, or a derivative thereof.
  • Biosynthetic constructs patterned after this template readily are constructed using conventional DNA synthesis or peptide synthesis techniques well known in the art.. Once constructed, renal therapeutic agents comprising these polypeptide chains can be tested as disclosed herein so that the agents define a polypeptide chain that have a conformation capable of being therapeutically effective when introduced into a subject with cystic kidney disease.
  • N-terminus including a conserved cysteine residue
  • One preferred isolated renal therapeutic polypeptide comprises a polypeptide with an overall architecture and sequence identical to those sequences of SEQ ED NOS: 1-5. More preferably, the isolated polypeptide contains an amino acid sequence at least 60%, 80%, 90%, 95%, 98% or 99% homologous to an amino acid sequence from SEQ ED NOS: 1-5.
  • the isolated polypeptide is at least 5, 10, 20, 50, or 90 amino acids long.
  • the isolated polypeptide includes at least 5, preferably at least 20, more preferably at least 50, most preferably at least 90 contiguous amino acids from SEQ ED NOS: 1-5.
  • the invention also pertains to an renal therapeutic polypeptide which includes an amino acid sequence described herein as well as other N-terminal or C-terminal amino acid sequences.
  • the polypeptide may include all or a fragment of an amino acid sequence from those in Table 1, including a portion such as SEQ ED NOS: 1-5 fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from those in Table 1.
  • the renal therapeutic agent may also be a recombinant fusion protein having a first renal therapeutic portion and a second polypeptide portion having an amino acid sequence unrelated to the renal therapeutic polypeptide.
  • the second polypeptide may be any of, for instance, glutathione-S-transferase, a DNA binding domain, or a polymerase activation domain.
  • the first renal therapeutic polypeptide includes amino acid residues from those in Table 1, inlucding a portion such as SEQ EDS NOS: 1-5.
  • the renal therapeutic polypeptide may also be a renal therapeutic fragment of a naturally occurring renal therapeutic agent which has biological activity.
  • the renal therapeutic polypeptides described herein can be produced by any suitable method known in the art. Such methods include constructing a DNA sequence encoding renal therapeutic polypeptide sequences and expressing those sequences in a suitable transformed host. This method will produce isolated, renal therapeutic polypeptide.
  • cDNA may be isolated by screening a human cDNA library with a labeled DNA fragment encoding any one of SEQ ED NOS: 1-5 and identifying positive clones by autoradiography. Further rounds of plaque purification and hybridization are performed using conventional methods.
  • the renal therapeutic polypeptide may also be produced, albeit less preferably, by chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.
  • a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding wild type OPl.
  • the sequence may be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g., Zoeller et al., "Site-specific Mutagenesis Of The Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci. USA, 81, pp.
  • Another method of constructing a DNA sequence encoding renal therapeutic polypeptide would be by chemical synthesis using an oligonucleotide synthesizer.
  • Such oligonucleotides may be preferably designed based on the amino acid sequence of the desired renal therapeutic polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant renal therapeutic polypeptide will be produced.
  • the DNA sequence encoding renal therapeutic polypeptides may or may not also include DNA sequences that encode a signal sequence.
  • the inclusion of a signal sequence depends on whether it is desired to secrete the renal therapeutic polypeptide from the recombinant cells in which it is made. If the cells chosen to provide the expression host are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence.
  • Standard methods may be applied to synthesize a polynucleotide sequence encoding a renal therapeutic polypeptide. For example, a complete amino acid sequence may be used to construct a back-translated gene. See Maniatis et al., supra. Further, a DNA oligomer containing a nucleotide sequence coding for the particular renal therapeutic polypeptide may be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the mutant DNA sequences encoding a particular renal therapeutic polypeptide will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host. The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations may be employed.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalo virus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including coli ⁇ 1 , pCR 1 , pBR322, pMB9 and their derivatives, wider host range plasmids, such as Ml 3 and filamentous single stranded DNA phages.
  • Preferred ⁇ . coli vectors include pL vectors containing the lambda phage pL promoter (U.S.
  • Patent 4,874,702 p ⁇ T vectors containing the T7 polymerase promoter (Studier et al., Methods in Enzymology 185: 60-89, 1990 ) and the pSP72 vector (Kaelin et al., supra).
  • Useful vectors for insect cells include pVL 941.
  • any of a wide variety of expression control sequences may be used in these vectors. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors.
  • useful expression control sequences include, for example, the early and late promoters of S V40 or adenovirus, the lac system, the trjj system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example pL, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating system and other sequences known to control the expression of genes or prokaryotic or eukaryotic cells and their viruses, and various combinations thereof.
  • Any suitable host may be used to produce in quantity the renal therapeutic polypeptides described herein, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. More particularly, these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), and animal cells such as Chinese hamster ovary (CHO) and mouse cells such as NS/O, African green monkey cells such as COS1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well as plant cells in tissue culture. For animal cell expression, we prefer CHO cells and COS 7 cells in cultures.
  • Fragments of a renal therapeutic protein can also be produced efficiently by recombinant methods, by proteolytic digestion, or by chemical synthesis, using methods known to those of skill in the art.
  • internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes for the renal therapeutic polypeptide.
  • Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end nibbling" endonucleases can also generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination.
  • Fragments can also be chemically synthesized using techniques known in the art such as the Merrifield solid phase f-Moc or t-Boc chemistry.
  • peptides of the present invention may be arbitrarily divided into fragments of a desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length. 98/50061 _ 30 _
  • Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular portion thereof. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences.
  • PCR Mutagenesis Briefly, Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA. See Leung et al., Technique 1:11-15 (1989). PCR conditions are chosen so that the fidelity of DNA synthesis is reduced by Taq DNA polymers using, for instance, a dGTP/dATP ratio of five and adding Mn +2 to the PCR reaction. The pool of amplified DNA fragments is inserted into appropriate cloning vectors to provide random mutant libraries.
  • Saturation Mutagenesis The method is described generally in Mayers et al., Science 229: 242 (1985). Briefly, the technique includes generation of mutations by chemical treatment or irradiation of single stranded DNA in vitro, and synthesis of a cDNA strand. The mutation frequency is modulated by the severity of the treatment and essentially all possible base substitutions can be obtained.
  • a library of homologous peptides can be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of degenerate sequences can by performed in an automatic DNA synthesizer, and the synthetic genes than ligated into an appropriate expression vector. See, Harang, S.A. f Tetrahedron 39: 3 (1983); Itakura et al., Recombinant DNA, Proc. 3rd Cleveland Symposium on Macromolecules, pp. 273-289 (A.G. Walton, ed.), Elsevier, Amsterdam, 1981; Itakura et al., Ann. Rev. Biochem., 53: 323 (1984).
  • Non-random, or directed, mutagenesis provides specific sequences or mutations in specific portions of a polynucleotide sequence that encodes an renal therapeutic polypeptide, to provide variants which include deletions, insertion or substitutions or residues of the known amino acid sequence of the renal therapeutic polypeptide.
  • the mutation sites may be modified individually or in series, for instance by: (i) substituting first with conserved amino acids and them with more radical choices depending on the results achieved; (ii) deleting the target residue; or (iii) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
  • Alanine scanning Mutagenesis This method locates those residues or regions of a desired protein that are preferred locations or sites for mutagenesis. See Cunningham and Wells, Science 244: 1081-1085 (1989).
  • a residue or group of target residues are identified (e.g, charged residues such as Arg, Asp, His, Lys and Glu) and replaced by a neutral or a negatively charged amino acid such as alanine or polyalanine. This replacement can affect the interaction of the amino acids with the surrounding aqueous media in or outside the cell.
  • Those s having functional sensitivity to the substitutions are then refined by introducing further or other variants at, or for, the sites of substitution.
  • Oligonucleotide-Mediated Mutagenesis This method may be used to prepare substitution, deletion, and insertion variants of DNA. See, Adelman et al., DNA 2: 183 (1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide primer encoding a DNA mutation to a DNA template which typically is the single stranded form of a plasmid or phage containing the unaltered or wild-type DNA sequence template of the desired protein (e.g., the Renal therapeutic peptide).
  • a DNA template typically is the single stranded form of a plasmid or phage containing the unaltered or wild-type DNA sequence template of the desired protein (e.g., the Renal therapeutic peptide).
  • a DNA polymerase is used to make an entire second cDNA of the template that will inco ⁇ orate the oligonucleotide primer, and will code for the selected alteration in the desired protein's DNA.
  • oligonucleotide of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • This method requires a plasmid or other vector that includes the protein subunit DNA to be mutated.
  • the codon(s) in the protein subunit DNA are identified and there is inserted a unique restriction endonuclease site on each side of the identified mutation site(s), using the above-described oligonucleotide directed mutagenesis method.
  • the plasmid is then cut at these sites to linearize it.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures.
  • the two strands are synthesized separately and then hybridized together using standard methods.
  • This double-stranded oligonucleotide is the "cassette" and it has 3' and 5' ends that are compatible with the ends of the linearized plasmid so that it can be directly ligated therein.
  • the plasmid now contains the mutated desired protein subunit DNA sequence.
  • Combinatorial Mutagenesis In this method (Ladner et al., WO 88/06630), the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to created a degenerate set of combinatorial sequences.
  • the variegated library is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequence such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins contains the set of degenerate sequences.
  • Renal therapeutic Polypeptides Included in the invention are methods for treating renal dysfunction using renal therapeutic molecules that are: allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize under high or low stringency conditions to a nucleic acid which encodes a polypeptide comprising a sequence such as SEQ ED NOS: 1-5 and polypeptides specifically bound by antisera to renal therapeutic peptides, especially to antisera to an active site or binding region of renal therapeutic.
  • the methods of the invention also feature uses of fragments, preferably biologically active fragments, or analogs of a renal therapeutic peptide.
  • a biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of the peptides shown herein or of other naturally occurring renal therapeutic peptide.
  • fragments which exist in vivo such as fragments which arise from post-translational processing as the result of the removal of an amino-terminal signal sequences, as well as those made in expression systems such as in CHO cells.
  • Useful renal therapeutic analogs possess biological activity.
  • the fragment or analog has 10%, preferably 40%, or at least 90% of the activity of a renal therapeutic agent in any in vivo or in vitro assay.
  • Analogs can differ from naturally occurring renal therapeutics in amino acid sequence or in ways that do not involve sequence, or both.
  • Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatization as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycolsylation.
  • Particularly preferred analogs include renal therapeutics or their biologically active fragments whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions or by one or more non conservative amino acid substitutions, deletions or insertions which do not abolish the renal therapeutic biological activity.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid.glutamic acid; asparagine,glutamine; serine,threonine;lysine, arginine; and phenylalanine,tyrosine.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • substitutions that may be expected to induce changes in the functional properties of renal therapeutic polypeptides are those in which: (i) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative charge, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine.
  • a hydrophilic residue e.g.
  • analogs used within the methods of the invention are those with modifications which increase peptide stability. Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic analogs. Inco ⁇ oration of D- instead of L- amino acids into the renal therapeutic polypeptide may increase its resistance to proteases. See, U.S. Patent 5,219,990 supra.
  • fragment as applied to an renal therapeutic analog, will usually be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit renal therapeutic biological activity can be also assessed by methods known to those skilled in the art as described herein.
  • the invention also provides for use of renal therapeutic agents that are peptide mimetics, i.e., peptide or non-peptide agents that have the same biological activity as isolated renal therapeutic agent.
  • scanning mutagenesis is used to map the amino acid residues of a particular renal therapeutic polypeptide involved in binding its cell surface receptor.
  • Peptide mimetics are generated (e.g, diazepine or isoquinoline derivatives) which mimic those residues that bind to the receptor.
  • non-hydrolyzable peptide analogs of such residues can be generated using non-peptides described in Peptides: Chemistry and Biology (G.R. Marshall ed.).
  • benzodiazepone (Freidinger et al.); azepine (Huffman et al.,) ; substituted gamma lactam rings (Garvey et al., id).
  • Other mimetics include keto-methylene pseudopeptides (Ewenson et al.,J. Med. Chem. 29: 295 (1986) and beta-amino alcohols (Gordon et al., Biochem. Biophys. Res. Comm. 126: 419 (1986).
  • Another approach that does not require knowledge of the receptor is to use the renal therapeutic molecule itself to generate peptide mimetics using similar methods as described herein.
  • the renal therapeutic agents of the present invention also may be tested in animal models of cystic kidney disease. See Section XI, below.
  • the renal therapeutic agents of the present invention may be evaluated for their therapeutic efficacy in causing a clinically significant improvement in a standard marker of renal function when administered to a human patient with or likely to develop cystic kidney disease.
  • markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium (Na+), urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like.
  • the methods of the present invention may be utilized for any subject at risk of developing cystic kidney disease, or who has already been diagnosed as having clinically significant cystic kidney disease.
  • the compound is useful in delaying onset of loss of renal function.
  • Patients at risk of developing a disease of the invention may be treated periodically starting at the time of identification of their "at risk" status. For example, an infant with a genetic mutation, detected in utero by ultrasonography or by amniocentesis and chromosomal analysis, may be periodically treated from the time of birth, or even in utero, and continuing for life.
  • a child or adult with pre-symptomatic disease might also be beneficially treated periodically for life.
  • Mammalian subjects which may be treated according to the methods of the invention include, but are not limited to, human subjects or patients.
  • the invention may be employed in the treatment of domesticated mammals, including dogs, cats, horses, cattle, and laboratory animals.
  • the subjects for treatment with the methods of the present invention need not present indications for treatment with the renal therapeutic agents of the invention other than those indications associated with risk of developing cystic kidney disease. That is, the subjects for treatment are expected to be otherwise free of indications for treatment with the renal therapeutic agents of the invention.
  • the subjects may present with other symptoms (e.g., osteodystrophy) for which treatment with the agents of the present invention would be indicated. In such cases, the treatment should be adjusted accordingly so to avoid excessive dosing.
  • a mammalian subject may be regarded as being at risk of developing cystic kidney disease if the subject has been found to carry a genetic " mutation associated with a hereditary cystic kidney disease, or if the presence of such a gene is suggested by presence of hereditary cystic kidney disease in close relatives of the patient.
  • the success of a particular treatment may be measured by a variety of invasive and non-invasive procedures including biopsies, blood and urine chemical workup, MRI, CAT, ultrasonographic, or other imaging techniques.
  • GFR glomerular flow rate
  • Renal therapeutic agents in therapeutically effective amounts may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorasulfonate, cyclopentanepropionate, digluconate, dodecylsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, 2-hydroethanesulfonate, lactate, maleate, methensulfone, 3-na
  • Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine and so forth.
  • the basic nitrogen- containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides
  • dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as de
  • the compounds described herein may be formulated into pharmaceutical compositions that may preferably be adminstered to a subject having, or at risk for, a renal dysfunction.
  • Administration is systemic or local. Systemic administration may be done parenterally, or alternately by topical application, inhalation spray, nasally, buccally or via implanted reservoir.
  • parenteral as used herein includes oral administration, subcutaneous, intravenous, intramuscular, intra-arterial, intra-synovial, intrastemal, intrahepatic, intrarenal, and intracranial injection or infusion techniques. Administration may be by periodic injections of a bolus of a renal therapeutic agent
  • Local administration (which may also include the above injection or infusion techniques) is preferably perfomed within the lumen at, or near, a site of renal injury or lesion.
  • medicament-dispensing devices such as sustained release devices, grafts, catheters and stents (collectively referred to as "intra-luminal" devices) can be manufactured and used to locally deliver the compounds described herein.
  • intra-luminal devices such as sustained release devices, grafts, catheters and stents
  • Biodegradable drug delivery vascular stent - Buscemi, et al
  • a biodegradable stent may comprise polylactic acid, polyglycolic acid (PGA), collagen or other connective proteins or natural materials, polycaprolactone, hylauric acid, adhesive proteins, co-polymers of these materials as well as composites and combinations thereof and combinations of other biodegradable polymers.
  • the stent itself therefore mediates slow local release of a drug into the lumen of a blood vessel. See, also, U.S. Patent 5,370,681 ("Polylumenal implantable organ"), U.S. Patent 5,328,470 (" ...site-specific instillation of cells”).
  • Such devices may be conveniently impregnated (i.e. surface coated and/or intersititially filled) with renal therapeutic agents described herein, and delivered locally to a site (i.e., a site of renal dysfunction) using methods available to persons having ordinary skill in the art.
  • a prefened method of the present invention thus uses renal therapeutic polypeptide and polynucleotides at an local, intrarenal site of renal failure.
  • Other methods of the invention contemplate use of vesicles such as micelles or liposomes into, or onto which, is inco ⁇ orated renal therapeutic agents.
  • the vesicle may be a naturally occurring biological membrane, purified away from natural material or the vesicle may be a synthetic construction.
  • Prefened vesicles are substantially spherical structures made of amphiphiles, e.g., surfactants or phospholipids.
  • the lipids of these spherical vesicles are generally organized in the form of lipids having one or more structural layers, e.g., multilamellar vesicles (multiple onion-like shells of lipid bilayers which encompass an aqueous volume between the bilayers) or micelles.
  • liposomes are small, spherical vesicles composed primarily of various types of lipids, phospholipids and secondary lipophilic components. These components are normally arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • the polar end of a component lipid or lipid-like molecule is in contact with the surrounding solution, usually an aqueous solution, while the non-polar, hydrophobic end of the lipid or lipid-like molecule is in contact with the non-polar, hydrophobic end of another lipid or lipid-like molecule.
  • the resulting bilayer membrane i.e., vesicle
  • Most vesicles are lipid or lipid-like in nature, although alternative liposome bilayer formulations, comprising a surfactant with either a lipid or a cholesterol exist.
  • Liposome vesicles may be particularly preferred in that they find many therapeutic, diagnostic, industrial and commercial applications. They are used to deliver molecules which are not readily soluble in water, or when directed timed release is desired. Because of their selective permeability to many chemical compounds, liposomes are useful as delivery vehicles for renal therapeutic agents. Upon reaching the target site, the liposomes may be degraded or they may fuse with the membranes of cells.
  • Several methods of preparing vesicles such as liposomes are known. Among the more common of these are (1) sonication of a solution containing lipids sometimes followed by evaporation/lyophilization and rehydration (see, e.g. Stryer, Biochemistry, pp. 290-291, Freeman & Co., New York, (1988), and (55); (2) homogenization of lipid solution, sometimes at high pressure or high shearing force (see e.g. U.S. Pat. No.
  • Surfactants form micelles when added to aqueous solution above the surfactant's phase transition temperature.
  • the surfactants may be composed of one or more aliphatic chains. These aliphatic chains may be saturated, unsaturated, or substituted in other ways, such as by ethoxylation; typically the aliphatic chain contains greater than about 12 carbons.
  • Additional suitable surfactants include the following: lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl, myristyl-, or cetyl- betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.
  • lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT series (Mona Industries, Inc., Paterson, N.J.).
  • the agent preferably comprises part of an aqueous solution.
  • the solution is physiologically acceptable (i.e., it is a physiological solution that is biocompatible) so that in addition to delivery of the desired agent to the subject, the solution does not otherwise adversely affect the subject's electrolyte and/or volume balance.
  • the aqueous medium for the agent thus may comprise normal physiologic saline (e.g., 9.85% NaCl, 0.15M, pH 7- 7.4).
  • the compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension.
  • the suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents (see above discussion of vesicles).
  • the sterile injectable preparation may also be a sterile injectable solution or a suspension in a non- toixc parenterally-acceptable diluent or solvent, for example, as a solution in 1,3- butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this pu ⁇ ose, any bland fixed oil may be used including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in the polyoxyethylated versions.
  • oils such as olive oil or castor oil, especially in the polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
  • compositions comprise any of the above-described renal therapeutic compounds and homologs in therapeutically effective concentrations, together with any pharmaceutically acceptable carrier.
  • carrier includes acceptable adjuvants and vehicles.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hyudrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pynolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers and wool fat.
  • Compositions containing renal therapeutic agents may also comprise one or more additional agents selected from the group consisiting of corticosteroids, antiinflammatories (e.g., theophylline, sulfasalazine, aminosalicylates), antirheumatics, immunosuppressants (e.g., cyclosporin, rapamycin), antimetabolites (e.g., cyclophosphamide, methotrexate), immunomodulators (e.g., interferons).
  • additional agents selected from the group consisiting of corticosteroids, antiinflammatories (e.g., theophylline, sulfasalazine, aminosalicylates), antirheumatics, immunosuppressants (e.g., cyclosporin, rapamycin), antimetabolites (e.g., cyclophosphamide, methotrexate), immunomodulators (e.g., interferons).
  • a given renal therapeutic agent or other agent may be made more soluble by association with a suitable molecule.
  • association of a mature OP/BMP dimer with an OP/BMP pro domain results in the pro form of the renal therapeutic agent which typically is more soluble or dispersible in physiological solutions than the corresponding mature form.
  • endogenous members of the OP/BMP family are thought to be transported (e.g., secreted and circulated) in the mammalian body in this form.
  • This soluble form of the protein can be obtained from culture medium of OP/BMP-secreting mammalian cells, e.g., cells transfected with nucleic acid encoding and competent to express the protein.
  • a soluble species can be formulated by complexing the mature dimer (or an active fragment thereof) with a pro domain or a solubility-enhancing fragment thereof (described more fully below).
  • a solubility-enhancing fragment thereof Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 by 80%.
  • Other components found in milk and/or various serum proteins also may be useful.
  • the agents described herein may be administered orally.
  • Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream.
  • the renal therapeutic agents described herein typically are acid stable and protease-resistant (see, for example, U.S. Patent No. 4,968,590).
  • at least one of these renal therapeutic agents, OP-1 has been identified in mammary gland extract, colostrum and 57-day milk.
  • the OP-1 purified from mammary gland extract is therapeutically efficacious and also is detected in the bloodstream.
  • soluble form OP-1 e.g., mature OP-1 associated with the pro domain
  • oral and parenteral administration are viable means for administering the renal therapeutic agents of the invention to an individual.
  • the mature forms of certain renal therapeutic agents described herein typically are sparingly soluble
  • the form found in milk (and mammary gland extract and colostrum) is readily soluble, probably by association of the mature, therapeutically efficacious form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components.
  • the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.
  • the compounds provided herein also may be associated with molecules capable of targeting the renal therapeutic agent to the desired tissue.
  • an antibody, antibody fragment, or other binding protein that interacts specifically with a surface molecule on cells of the desired tissue may be used.
  • Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Patent No. 5,091,513.
  • the amount of renal therapeutic agent as the active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, the judgment of the treating physician and severity of the renal dysfunction.
  • the dosage and dose rate of the renal therapeutic agents described herein that are effective to prevent, suppress, or inhibit cystic kidney disease will depend on a variey of factors such as patient size, goal of treatment, specific pharmaceutical composition and judgment of the treating physician.
  • daily or weekly dosage levels of between about 0.00001 and about 1000 mg are sufficient with, 0.0001-100 mg being preferable and 0.001 to 10 mg being even more preferable.
  • a daily or weekly dosage of 0.01-1000 ug/kg body weight, more preferably between about 10-700 ug/kg body weight, of active renal therapeutic agent are useful. Dosages can be administered continuously, but daily, multi-weekly, weekly or monthly dosages may also be employed.
  • dosages are given prior to clinical manifestations of the renal dysfunction at a level and frequency which will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, the judgment of the treating physician and susceptibility to the renal dysfunction.
  • the formulated compositions contain therapeutically effective amounts of the renal therapeutic agent. That is, they contain amounts which provide appropriate concentrations of the agent to the renal tissues for a time sufficient to prevent, inhibit, delay or alleviate permanent or progressive loss of renal function, or otherwise provide therapeutic efficacy.
  • concentration of the compounds described in a therapeutic composition of the present invention will vary depending upon a number of factors, including the biological efficacy of the selected agent, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, the formulation of the compound excipients, the administration route, and the treatment envisioned, including whether the active ingredient will be administered directly into a kidney or renal capsule, or whether it will be administered systemically.
  • the preferred dosage to be administered also is likely to depend on such variables such as the condition of the renal tissues, extent of renal function loss, and the overall health status of the particular subject. Dosages may be administered continuously, or daily, but it is currently preferred that dosages be administered once, twice or three times per week for as long as satisfactory response persists (as measured, for example, by stabilization and/or improvement of renal function by appropriate medical markers and/or quality of life indices) Less frequent dosages, for example monthly dosages, may also be employed. For subjects which would otherwise require continuous, bi-weekly or tri-weekly hemodialysis sessions, continuous, bi-weekly or tri-weekly intravenous or intraperitoneal infusions are not considered unduly inconvenient. In addition, in order to facilitate frequent infusions, implantation of a semi-permanent stent (e.g., intravenous, intraperitoneal or intracapsular) may be advisable.
  • a semi-permanent stent e.g., intravenous, intraperi
  • a general method of the invention involves administering to a subject (preferably a mammal such as a human) having, or at risk for, cystic kidney disease, a renal therapeutic agent selected from the group consisting of: (i) an isolated, renal therapeutic agent (e.g., protein, fragment, antibody, fusion); (ii) an isolated polynucleotide (e.g., a polynucleotide encoding an amino acid sequence comprising one of SEQ ED NOS: 1-5) capable of encoding, upon expression, a renal therapeutic agent ; and (iii) an isolated polynucleotide sequence capable of hybridizing under standard conditions to an isolated polynucleotide (e.g., a polynucleotide encoding an amino acid sequence comprising one of SEQ ED NOS: 1-5) capable of encoding, upon expression, a renal therapeutic agent.
  • a renal therapeutic agent selected from the group consisting of: (i) an isolated, renal therapeutic agent (e.g., protein,
  • the methods of the present invention may be used for any mammalian subject in, or at risk of, cystic kidney disease.
  • Mammalian subjects include, but are not limited to humans since the methods may be employed in the treatment of domesticated mammals which are maintained as human companions (e.g., dogs, cats, horses), which have significant commercial value (e.g., dairy cows, beef cattle, sporting animals) or which have scientific value (e.g., captive or free specimens of endangered species).
  • human companions e.g., dogs, cats, horses
  • scientific value e.g., captive or free specimens of endangered species.
  • One of ordinary skill in the medical or veterinary arts is trained to recognize subjects which may be suitable for the methods of the invention.
  • clinical and non-clinical trials, as well as accumulated experience, relating to the presently disclosed and other treatment methods are expected to inform the skilled practitioner in deciding whether a given subject is in, or at risk of, renal dysfunction treatable using the present invention.
  • Gene constructs can also be used as part of a gene therapy protocol to deliver nucleic acid encoding a form of renal therapeutic polypeptide.
  • the invention features expression vectors for in vivo transfection and expression of renal therapeutic polypeptide in particular cell types so as to either: (i) antagonize the function of an renal therapeutic molecule such as renal therapeutic in an environment (e.g., a renal dysfunction) in which that protein is misexpressed; (ii) agonize the function of a renal therapeutic agent.
  • Expression constructs of renal therapeutic polypeptides may be administered in any biologically effective carrier that is capable of effectively delivery a polynucleotide sequence encoding the renal therapeutic polypeptide to cells in vivo.
  • Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus and he ⁇ es simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors transfect cells directly, plasmid DNA can be delivered with the help of, for , example, cationic liposomes or derivatized (e.g., antibody conjugated) polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo.
  • Promoter activation may be tissue specific or inducible by a metabolic product or administered substance.
  • Such promoters/enhancers include, but are not limited to, the native E2F promoter, the cytomegalovirus immediate-early promoter/enhancer (Karasuyama et al., J. Exp. Med., 169: 13 (1989)); the human beta- actin promoter (Gunning et al., Proc. Natl. Acad. Sci. USA, 84: 4831 (1987); the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al., Mol. Cell.
  • MMTV LTR mouse mammary tumor virus long terminal repeat
  • Rous sarcoma virus (Yamamoto et al., Cell, 22:787 (1980)); the he ⁇ es simplex virus (HSV) thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA, 78: 1441 (1981)); the adenovirus promoter (Yamada et al., Proc. Natl. Acad. Sci. USA, 82: 3567 (1985)).
  • RSV Rous sarcoma virus
  • HSV he ⁇ es simplex virus
  • adenovirus promoter Yamada et al., Proc. Natl. Acad. Sci. USA, 82: 3567 (1985)
  • Expression vectors compatible with mammalian host cells for use in gene therapy of tumor cells include, for example, plasmids; avian, murine and human retroviral vectors; adenovirus vectors; he ⁇ es viral vectors; and non-replicative pox viruses.
  • replication-defective recombinant viruses can be generated in packaging cell lines that produce only replication-defective viruses. See Current Protocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al., eds.), Greene Publishing Associcates, 1989. Specific viral vectors for use in gene transfer systems are now well established.
  • Preferred vectors are DNA viruses that include adenoviruses (preferably Ad-2 or
  • Ad-5 based vectors Ad-5 based vectors
  • he ⁇ es viruses preferably he ⁇ es simplex virus based vectors
  • parvoviruses preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors.
  • a particular vector system for transferring, for instance, an Renal therapeutic polynucleotide sequence will depend on a variety of factors.
  • One important factor is the nature of the target cell population.
  • retroviral vectors have been extensively studied and used in a number of gene therapy applications, they are generally unsuited for infecting cells that are not dividing but may be useful in cancer therapy since they only integrate and express their genes in replicating cells. They are useful for ex vivo approaches and are attractive in this regard due to their stable integration into the target cell genome.
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types.
  • the general adenoviruses types 2 and 5 (Ad2 and Ad5, respectively), which cause respiratory disease in humans, are cunently being developed for gene therapy of Duchenne Muscular Dystrophy (DMD)and Cystic Fibrosis (CF).
  • Ad2 and Ad5 belong to a subclass of adenovirus that are not associated with human malignancies.
  • Adenovirus vectors are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state.
  • High titers (10 13 plaque forming units/ml) of recombinant virus can be easily generated in 293 cells (an adenovirus-transformed, complementation human embryonic kidney cell line: ATCC CRL1573) and cryo-stored for extended periods without appreciable losses.
  • the efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders. See Y. Watanabe,
  • Adeno-associated viruses have also been employed as vectors for somatic gene therapy.
  • AAV is a small, single-stranded (ss) DNA virus with a simple genomic organization (4.7 kb) that makes it an ideal substrate for genetic engineering.
  • Rep polypeptides (rep78, rep68, rep 62 and rep 40) are involved in replication, rescue and integration of the AAV genome.
  • the cap proteins (VPl, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures.
  • ITRs inverted terminal repeats
  • the entire rep and cap s can be excised and replaced with a therapeutic or reporter transgene. See B.J. Carter, in Handbook of Parvoviruses, ed., P. Tijsser, CRC Press, pp. 155-168 (1990). It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
  • Adeno-associated viruses have significant potential in gene therapy.
  • the viral particles are very stable and recombinant AAVs (rAAV) have "drug-like" characteristics in that rAAV can be purified by pelleting or by CsCl gradient banding. They are heat stable and can be lyophilized to a powder and rehydrated to full activity.
  • rAAV recombinant AAVs
  • Their DNA stably integrates into host chromosomes so expression is long-term. Their host range is broad and AAV causes no known disease so that the recombinant vectors are non-toxic.
  • renal therapeutic polypeptides may also be introduced into a target cell using a variety of well-known methods that use non-viral based strategies that include electroporation, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection, and direct micro-injection into single cells.
  • an renal therapeutic polynucleotide encoding a renal therapeutic agent may be introduced into a endothelial cell by calcium phosphate coprecipitation (Pillicer et al., Science, 209: 1414-1422 (1980); mechanical microinjection and/or particle acceleration (Anderson et al., Proc. Natl. Acad.
  • liposome based DNA transfer e.g., LIPOFECTIN-mediated transfection- Fefgner et al., Proc. Natl. Acad. Sci. USA, 84: 471-477 (1987), Gao and Huang, Biochem. Biophys. Res. Comm., 179: 280-285, 1991
  • DEAE Dextran-mediated transfection electroporation (U.S.
  • Patent 4,956,288) or polylysine-based methods in which DNA is conjugated to deliver DNA preferentially to liver hepatocytes (Wolff et al., Science, 247: 465-468 (1990), Curiel et al., Human Gene Therapy 3: 147-154 (1992).
  • plasmids containing isolated polynucleotide sequences encoding renal therapeutic polypeptide may placed into, for example, endothelial cells using many of these same methods. Renal therapeutic polypeptide itself may also be chemically modified to facilitate its delivery to a target cell.
  • One such modification involves increasing the lipophilicity of the renal therapeutic polypeptide in order to increase cell surface binding and stimulate non-specific endocytosis of the polypeptide.
  • a wide variety of lipopeptides, fatty acids, and basic polymers e.g., tripalmitoyl-S-glycerylcysteil-seryl- serine; palmitic acid; polyarginine
  • tripalmitoyl-S-glycerylcysteil-seryl- serine; palmitic acid; polyarginine may be linked to an renal therapeutic polypeptide to accomplish this. See U.S. Patent 5, 219,990, inco ⁇ orated herein by reference.
  • Delivery may also be effected by using carrier moieties known to cross cell membranes.
  • an renal therapeutic agent that is a polypeptide such as one containing an amino acid sequence that includes SEQ ED NOS: 1-5 may be fused to a carrier moiety, preferably by genetic fusion, and the fused construct may be expressed in bacteria or yeast using standard techniques.
  • polynucleotide sequences encoding Renal therapeutic polypeptide useful in the present invention, operatively linked to regulatory sequences may be constructed and introduced into appropriate expression systems using conventional recombinant DNA techniques. The resulting fusion protein may then be purified and tested for its capacity to enter intact target cells and inhibit growth of the target cells once inside the target.
  • recombinant methods may be used to attach a carrier moiety to renal therapeutic polynucleotide sequences by joining the polynucleotide sequence encoding for Renal therapeutic polypeptide with the polynucleotide sequence encoding a carrier moiety and introducing the resulting construct into a cell capable of expressing the conjugate.
  • Two separate sequences may be synthesized, either by recombinant means or chemically, and subsequently joined using known methods.
  • the entire conjugate may be chemically synthesized as a single amino acid sequence.
  • Useful carrier moieties include, for example, bacterial hemolysins or "blending agents" such as alamethicin or sulfhydryl activated lysins.
  • carrier moieties include cell entry components of bacterial toxins such as Pseudomonas exotoxin, tetanus toxin, ricin toxin and diphtheria toxin.
  • Other useful carrier moieties include proteins which are viral receptors, cell receptors or cell ligands for specific receptors that are internalized and cross mammalian cell membranes via specific interaction with cell surface receptors.
  • Such cell ligands include epidermal growth factor, fibroblast growth factor, transferrin and platelet derived growth factor.
  • the carrier moiety may also include bacterial immunogens, parasitic immunogens, viral immunogens, immunoglobulins, cytokines.
  • purified human immunodeficiency virus type-1 (HIV) tat protein is the carrier moiety.
  • Purified human immunodeficiency virus type-1 (HIV) tat protein is taken up from the sunounding medium by human cells growing in culture. See Frankel et al., Cell 55: 1189-1193, (1988); Fawell et al., Proc. Natl. Acad. Sci. USA, 91: 664-668 (1994)( use of tat conjugate); and Pepinsky et al., DNA and Cell Biology, 13: 1011-1019 (1994)(use of tat genetic fusion construct), all of which are inco ⁇ orated herein by reference.
  • tat protein can deliver, for example, renal therapeutic polypeptide and polynucleotide sequences into cells, either in vitro or in vivo.
  • delivery can be carried out in vitro by adding a genetic fusion encoding an renal therapeutic-tat conjugate to cultured cells to produce cells that synthesize the tat conjugate or by combining a sample (e.g., blood, bone marrow, tumor cell) from an individual directly with the conjugate, under appropriate conditions.
  • the target cells may be in vitro cells such as cultured animal cells, human cells or microorganisms. Delivery may be carried out in vivo by administering the renal therapeutic agent and tat protein to an individual in which it is to be used.
  • the target may be in vivo cells, i.e., cells composing the organs or tissue of living animals or humans, or microorganisms found in living animals or humans.
  • the ADP ribosylation from Pseudomonas exotoxin ("PE") and pancreatic ribonuclease have been conjugated to tat to confirm cytoplasmic delivery of a protein.
  • the ADP phosphorylation is incapable of entering cells so that cytoplasmic delivery of this molecule would be confirmed if cell death occurs.
  • ribonuclease itself is incapable of entering cells so that inhibition of protein synthesis would be a hallmark of intracellular delivery using a tat conjugate.
  • renal therapeutic polynucleotide sequences can be identified by conventional methods such as nucleic acid hybridization using probes comprising sequences that are homologous/complementary to the inserted renal therapeutic sequences of the vector.
  • sequence(s) may be identified by the presence or absence of a "marker" gene function (e.g, thymidine kinase activity, antibiotic resistance, and the like) caused by introduction of the expression vector into the target cell.
  • a polynucleotide encoding a renal therapeutic agent is inserted into a vector having a dominant selectable marker gene such as a neomycin phosphotransferase gene under separate control of an SV40 early promoter, the sequence can be identified by the presence of the marker gene function (Geneticin resistance).
  • a dominant selectable marker gene such as a neomycin phosphotransferase gene
  • Other methods of detecting the appropriate vector e.g., PCR methods will be readily available to perons having ordinary skill in the art.
  • the delivery systems for the renal therapeutic polynucleotide sequence can be introduced into a patient by any number of methods, each of which is familiar to persons of ordinary skill. Specific inco ⁇ oration of the delivery system in the target cells occurs primarily from specificity of transfection provided by the gene delivery vehicle, cell type or tissue type expression due to the transcriptional regulatory sequences controlling expression of the polynucleotide, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being localized by, for example, catheter (U.S. Patent
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Where the complete gene delivery system can be produced intact from recombinant cells such as retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • An effective amount of renal therapeutic polynucleotide sequence or polypeptide sequence may be administered intraocularly, parenterally, orally, intranasally, intravenously, intramuscularly, subcutaneously, or by other means.
  • pharmaceutically acceptable carrier means one or more organic or inorganic ingredients, natural or synthetic, with which the renal therapeutic molecule is combined to facilitate its application.
  • a suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.
  • the term "carrier” encompasses liposomes and the HIV-1 tat protein (See Pepinsky et al., supra) as well as any plasmid and viral expression vectors.
  • An "effective amount” refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • an effective amount of the renal therapeutic renal therapeutic polynucleotide sequence contained within its attendant vector may be directly administered to a target cell or tissue via direct injection with a needle or via a catheter of other delivery tube placed into the cell or tissue. Dosages will depend primarily on factors such as the condition being treated, the selected polynucleotide, the age, weight, and health of the subject, and may thus vary among subjects.
  • An effective amount for a human subject is believed to be in the range of about 0.1 to about 50 ml of saline solution containing from about 1 x 10 to about 1 x 10 plaque forming units (pfu)/ml renal therapeutic renal therapeutic polynucleotide containing, viral expression vectors.
  • Target cells treated by renal therapeutic polynucleotide sequences may be administered topically, intraocularly, parenterally, intranasally, intratracheal, intrabronchially, intramuscularly, subcutaneously or by any other means.
  • Target cells to be treated by renal therapeutic protein may be administered topically, intraocularly, parenterally, intranasally, intratracheal, intrabronchially, intramuscularly, subcutaneously or by any other means.
  • the effect of a gene therapy vector carrying the renal therapeutic sequences can be determined by kidney biopsy or non-invasive kidney function tests such as described above.
  • cystic kidney disease Appropriate animal models for cystic kidney disease are well known and well characterized. For instance, several mutations (either spontaneous or induced) in mice give rise to cystic kidney disease, and these have developed into the cpk, pcy, jck, and cy mutation models.
  • cystic kidney in so-called "CFW w a" mice resembles human cystic kidney disease not only in terms of cyst localization and extrarenal manifestations but also with regard to onset of the disease and inheritance. See Schieren et al., Nephrol. Dial. Transplant., 11 Suppl. 6: 38-45 (1996) and Aziz, BioEssays 17: 703-712 (1995).
  • cystic disease animal models are based on transgenic manupulations in which the c-myc gene or large T antigen is overexpressed and models in which cystic disease is induced by various chemicals such as corticoids. See Aziz, supra, Schieren et al., supra, and Sommardahl et al., Pediatric Nephrology 11: 728-733 (1997). Persons having ordinary skill in the art can conveniently test methods of treating cystic kidney disease in one or more of these animal models by assaying, for example, serum concentrations of alkaline phosphatase and alanine transaminase to assess liver damage, urine and serum osmolalities to assess renal concentrating ability and by performing various histochemical analyses of kidney cyst formation. See, for example, the methods described in Sommardahl et al., supra.. Equivalents

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Abstract

Cette invention se rapporte à une méthode de traitement des maladies kystiques rénales. Cette méthode consiste à administrer à un sujet ayant contracté, ou susceptible de contracter, la maladie, une quantité efficace d'un agent thérapeutique rénal ou d'un polynucléotide codant la protéine correspondante. On isole des agents thérapeutiques rénaux, qui sont de préférence des molécules thérapeutiques rénales sélectionnées à partir d'un élément d'une famille de protéines ostéogéniques / de protéines morphogéniques osseuses (OP/BMP) appartenant à une superfamille de protéines du facteur de croissance transformant bêta. Un agent particulièrement préféré comporte au minimum 60 % d'homologie d'une séquence d'acide aminé avec le groupe constitué de la séquence SEQ ID NOS:1-5.
PCT/US1998/009268 1997-05-07 1998-05-06 Nouvelles therapies des maladies kystiques renales WO1998050061A1 (fr)

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WO2000029012A2 (fr) * 1998-11-13 2000-05-25 Curis, Inc. Procedes servant a soulager les symptomes du cancer
WO2000041713A2 (fr) * 1998-12-29 2000-07-20 Washington University Composition et methode pouvant ameliorer le fonctionnement de transplants renaux embryonnaires
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US7074762B2 (en) 1998-01-05 2006-07-11 Washington University Composition and method for improving function of embryonic kidney transplants
WO2000029012A2 (fr) * 1998-11-13 2000-05-25 Curis, Inc. Procedes servant a soulager les symptomes du cancer
WO2000029012A3 (fr) * 1998-11-13 2000-11-16 Creative Biomolecules Inc Procedes servant a soulager les symptomes du cancer
EP1435243A2 (fr) * 1998-11-13 2004-07-07 Curis, Inc. Procédés servant à soulager les symptomes du cancer
EP1435243A3 (fr) * 1998-11-13 2005-02-09 Curis, Inc. Procédés servant à soulager les symptomes du cancer
WO2000041713A2 (fr) * 1998-12-29 2000-07-20 Washington University Composition et methode pouvant ameliorer le fonctionnement de transplants renaux embryonnaires
WO2000041713A3 (fr) * 1998-12-29 2000-11-09 Univ Washington Composition et methode pouvant ameliorer le fonctionnement de transplants renaux embryonnaires
JP2009515529A (ja) * 2005-11-14 2009-04-16 メリアル リミテッド 腎不全のための遺伝子療法
EP3147296A1 (fr) * 2005-11-14 2017-03-29 Merial, Inc. Thérapie génique pour traiter l'insuffisance rénale
EP1951751B1 (fr) * 2005-11-14 2017-11-01 Merial, Inc. Thérapie génique pour insuffisance rénale
EP3536704A1 (fr) * 2005-11-14 2019-09-11 Boehringer Ingelheim Animal Health USA Inc. Thérapie génique pour traiter l'insuffisance rénale

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