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  Pharmaceutical Patents  


Title:  Combination therapy for treating protein deficiencies
United States Patent: 
November 4, 2008

Fan; Jian-Qiang (Demarest, NJ)
Mount Sinai School of Medicine of New York University (New York, NY)
Appl. No.: 
February 17, 2004


Covidien Pharmaceuticals Outsourcing


This application provides methods of improving gene therapy by combining gene therapy with active site-specific chaperones (ASSCs). The ASSC increases the stability and efficiency of the protein encoded by the recombinant gene that is administered.

Description of the Invention

The present invention improves the efficiency of gene therapy for protein deficiencies by combining standard gene therapy approaches with an active site-specific chaperone (ASSC), i.e., an agent capable of inducing the proper/native folding conformation of the protein, and stabilizing the protein encoded by the gene. The ASSC enhances the in vivo expression, efficiency, and stability of the expressed protein. The invention further provides formulations comprising a recombinant gene and an ASSC specific for the induction of the proper/native folding conformation of the protein and stabilization of the protein encoded by the gene. The invention is based on the discovery that ASSCs can be used as a combination therapy with gene therapy for the treatment of genetic disorders and other disorders. Although previous studies have demonstrated the ability of ASSCs to increase the level of expression of a normal, wild-type protein in tissue culture, modifying expression levels in artificial systems does not establish that one can achieve this result for a wild-type therapeutic protein in vivo. It has now been recognized that, instead, the in vivo methods for rescuing defective misfolded proteins can be modified as set forth herein to improve the efficiency of expression of a therapeutic (wild-type) protein delivered through gene therapy.

ASSCs can be screened and identified using methods known in the art. Once an ASSC useful for a particular disorder is identified, the chaperone can be administered to a patient receiving gene therapy. The ASSC can supplement endogenous molecular chaperones during high level expression of the therapeutic gene to increase the efficiency of expression by inhibiting aggregation in the ER. The chaperone can also as a stabilizer to prevent the degradation of the encoded protein being produced by the administered gene.

Disorders Characterized by Protein Deficiencies

There currently are about 1100 known inherited disorders characterized by protein deficiency or loss-of-function in a specific tissue. These disorders may be treatable by gene therapy in theory. The method of the present invention contemplates co-therapy for proteins currently suited for use in gene therapy that is available now or will be in the future. In such disorders, certain cells or all of the cells of an individual lack a sufficient functional protein, contain an inactive form of the protein or contain insufficient levels of the protein for biological function.

Further, the list of diseases identified as being conformational disorders, caused by mutations that alter protein folding and retardation of the mutant protein in the ER, resulting in protein deficiency, is increasing. These include cystic fibrosis, .alpha.1-antitrpsin deficiency, familial hypercholesterolemia, Alzheimer's disease (Selkoe, Annu. Rev. Neurosci. 1994; 17:489-517), osteogenesis imperfecta (Chessler et al., J. Biol. Chem. 1993; 268:18226-18233), carbohydrate-deficient glycoprotein syndrome (Marquardt et al., Eur. J. Cell. Biol. 1995; 66: 268-273), Maroteaux-Lamy syndrome (Bradford et al., Biochem. J. 1999; 341:193-201), hereditary blindness (Kaushal et al., Biochemistry 1994; 33:6121-8), Glanzmann thrombasthenia (Kato et al., Blood 1992; 79:3212-8), hereditary factor VII deficiency (Arbini et al., Blood 1996; 87:5085-94), oculocutaneous albinism (Halaban et al., Proc. Natl. Acad. Sci. USA. 2000; 97:5889-94) and protein C deficiency (Katsumi, et al., Blood 1996; 87:4164-75). Recently, one mutation in the X-linked disease adrenoleukodystrophy (ALD) resulted in misfolding of the defective peroxisome transporter, which could be rescued by low-temperature cultivation of affected cells (Walter et al., Am. J. Hum. Genet. 2001;69:35-48). It is generally accepted that mutations take place evenly over the entire sequence of a gene. Therefore, it is predictable that the phenotype resulting from protein deficiencies exists in many other genetic disorders.

Lysosomal Storage Disorders

Many of the inherited protein deficient disorders are enzyme deficiencies. As indicated above, a large class of inherited enzyme disorders involve mutations in lysosomal enzymes and are referred to as lysosomal storage disorders (LSDs). Lysosomal storage disorders are a group of diseases caused by the accumulation of glycosphingolipids, glycogen, mucopolysaccharides Examples of lysosomal disorders include Gaucher disease (Beutler et al., The Metabolic and Molecular Bases of Inherited Disease, 8th ed. 2001 Scriver et al., ed. pp. 3635-3668, McGraw-Hill, New York), GM1-gangliosidosis (id. at pp 3775-3810), fucosidosis (The Metabolic and Molecular Bases of Inherited Disease 1995. Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., ed pp. 2529-2561, McGraw-Hill, New York), mucopolysaccharidoses (id. at pp 3421-3452), Pompe disease (id. at pp. 3389-3420), Hurler-Scheie disease (Weismann et al., Science 1970; 169, 72-74), Niemann-Pick A and B diseases, (The Metabolic and Molecular Bases of Inherited Disease 8th ed. 2001. Scriver et al. ed., pp 3589-3610, McGraw-Hill, New York), and Fabry disease (id. at pp. 3733-3774). A list of LSDs and their associated deficient enzymes can be found in Table 1 (see Original Patent). Two are discussed specifically infra.

Fabry disease

Fabry disease is an X-linked inborn error of glycosphingolipid metabolism caused by deficient lysosomal .alpha.-galactosidase A (.alpha.-Gal A) activity (Desnick et al., The Metabolic and Molecular Bases of Inherited Disease, Edition Scriver et al. ed., pp. 3733-3774, McGraw-Hill, New York 2001; Brady et al., N. Engl. J. Med. 1967; 276, 1163-1167). This enzymatic defect leads to the progressive deposition of neutral glycosphingolipids with .alpha.-galactosyl residues, predominantly globotriaosylceramide (GL-3), in body fluids and tissue lysosomes. The frequency of the disease is estimated to be about 1:40,000 in males, and is reported throughout the world within different ethnic groups. In classically affected males, the clinical manifestations include angiokeratoma, acroparesthesias, hypohidrosis, and characteristic corneal and lenticular opacities (The Metabolic and Molecular Bases of Inherited Disease, Edition 2001, Scriver et al., ed., pp. 3733-3774, McGraw-Hill, New York). The affected male's life expectancy is reduced, and death usually occurs in the fourth or fifth decade as a result of vascular disease of the heart, brain, and/or kidneys. In contrast, patients with the milder "cardiac variant" normally have 5-15% of normal .alpha.-Gal A activity, and present with left ventricular hypertrophy or a cardiomyopathy. These cardiac variant patients remain essentially asymptomatic when their classically affected counterparts are severely compromised. Recently, cardiac variants were found in 11% of adult male patients with unexplained left ventricular hypertrophic cardiomyopathy, suggesting that Fabry disease may be more frequent than previously estimated (Nakao et al., N. Engl. J. Med. 1995; 333, 288-293). The .alpha.-Gal A gene has been mapped to Xq22, (Bishop et al., Am. J. Hum. Genet. 1985; 37: A144), and the full-length cDNA and entire 12-kb genomic sequences encoding .alpha.-Gal A have been reported (Calhoun et al., Proc. Natl. Acad. Sci. USA 1985; 82; 7364-7368; Bishop et al., Proc. Natl. Acad. Sci. USA 1986; 83: 4859-4863; Tsuji et al., Eur. J. Biochem. 1987; 165; 275-280; and Kornreich et al., Nucleic Acids Res. 1989; 17: 3301-3302). There is a marked genetic heterogeneity of mutations that cause Fabry disease (The Metabolic and Molecular Bases of Inherited Disease, Edition 2001, Scriver et al., ed., pp. 3733-3774, McGraw-Hill, New York.; Eng et al., Am. J. Hum. Genet. 1993; 53: 1186-1197; Eng et al., Mol. Med. 1997; 3: 174-182; and Davies et al., Eur. J. Hum. Genet. 1996; 4: 219-224). To date, a variety of missense, nonsense, and splicing mutations, in addition to small deletions and insertions, and larger gene rearrangements have been reported.

Gaucher Disease

Gaucher disease is a deficiency of the lysosomal enzyme .beta.-glucocerebrosidase that breaks down fatty glucocerebrosides. The fat then accumulates, mostly in the liver, spleen and bone marrow. Gaucher disease can result in pain, fatigue, jaundice, bone damage, anemia and even death. There are three clinical phenotypes of Gaucher disease. Patients with, Type 1 manifest either early in life or in young adulthood, bruise easily and experience fatigue due to anemia, low blood platelets, enlargement of the liver and spleen, weakening of the skeleton, and in some instances have lung and kidney impairment. There are no signs of brain involvement. In Type II, early-onset, liver and spleen enlargement occurs by 3 months of age and there is extensive brain involvement. There is a high mortality rate by age 2. Type III is characterized by liver and spleen enlargement and brain seizures. The .beta.-glucocerebrosidase gene is located on the human 1q21 chromosome. Its protein precursor contains 536 amino acids and its mature protein is 497 amino acids long.

Gaucher disease is considerably more common in the descendants of Jewish people from Eastern Europe (Ashkenazi), although individuals from any ethnic group may be affected. Among the Ashkenazi Jewish population, Gaucher disease is the most common genetic disorder, with an incidence of approximately 1 in 450 persons. In the general public, Gaucher disease affects approximately 1 in 100,000 persons. According to the National Gaucher Foundation, 2,500 Americans suffer from Gaucher disease.

Other Enzyme Deficiency Disorders

Glucose-6-phosphate dehydrogenase (G6PD) deficiency the most common X-linked human enzyme deficiency. The G6PD enzyme catalyzes an oxidation/reduction reaction that is essential for the production of ribose, which is an essential component of both DNA and RNA. G6PD also involved in maintaining adequate levels of NADPH inside the cell. NADPH is a required cofactor in many biosynthetic reactions. Individuals with this deficiency have clinical symptoms including neonatal jaundice, abdominal and/or back pain, dizziness, headache, dyspnea (irregular breathing), and palpitations.

One form of severe combined immunodeficiency (SCID) is due to lack of the enzyme adenosine deaminase (ADA), coded for by a gene on chromosome 20. This means that the substrates for this enzyme accumulate in cells. Immature lymphoid cells of the immune system are particularly sensitive to the toxic effects of these unused substrates, so fail to reach maturity. As a result, the immune system of the afflicted individual is severely compromised or completely lacking.

In addition to inherited disorders, other enzyme deficiencies arise from damage to a tissue or organ resulting from primary or secondary disorders. For example, damaged pancreatic tissue, or pancreatitis, is caused by alcoholism results in a deficiency in pancreatic enzymes necessary for digestion.

Other Disorders Treated Using Gene Therapy

There are numerous disorders involving defective genes other than enzymes involved in metabolic disorders that can be treated using gene therapy. Such disorder include but are not limed to severe combined immunodeficiency (SCID), phagocyte disorders such as Wiskott-Aldrich syndrome, bleeding disorders such as von Willebrand's disease and hemophilia, endocrine disorders such as growth hormone deficiency and hypothalamic diabetes insipidus, retinal degradation, cancers caused by inherited genetic defects such as heredetary non-polyposis colon cancer (HNPCC). Such disorders are listed in Table 2 (see Original Patent).

Treatment of Protein Deficiencies and Other Disorders

By overexpression of wild-type protein in suitable cells (e.g., stem cells or somatic tissue-specific cells) of an individual, using molecular biology techniques, the missing or deficient protein is produced in the cells, and in most cases circulates within the blood stream to the particular tissues. In order to achieve the therapeutic purpose, it is important to maintain high expression level of the protein for a sufficient time to confer a therapeutic benefit. Further, it is important to ensure specific delivery of the protein to the appropriate tissues.

Co-Therapy Using ASSCs and Gene Therapy

The present invention increases the effectiveness of gene therapy by increasing the folding and processing of the protein encoded by the gene administered during synthesis, and increasing the stability of the newly-synthesized protein in vivo by co-administration of an ASSC for the protein encoded by the administered gene. Screening for an appropriate ASSC for the target protein can be achieved by known methods in the art, e.g., as described in U.S. patent application Ser. No. 10/377,179, filed Feb. 28, 2003.

Gene Therapy

Disorders that can be treated using the method of the present invention include but are not limited to those mentioned above and those listed in Table 1. This method can be used in combination with any defective gene contemplated to be replaced using gene therapy. For example, the method can be used to provide secreted proteins, membrane proteins, or intracellular proteins.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 1993, 12:488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 1993, 32:573-596; Mulligan, Science 1993, 260:926-932; and Morgan and Anderson, Ann. Rev. Biochem. 1993, 62:191-217; May, TIBTECH 1993, 11:155-215. Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al., (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al., (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colosimo et al., Biotechniques 2000;29(2):314-8, 320-2, 324.

The gene to be administered for the methods of the present invention can be isolated and purified using ordinary molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. For example, nucleic acids encoding the target protein can be isolated using recombinant DNA expression as described in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning. A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. EHiggins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. EPerbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). The nucleic acid encoding the protein may be full-length or truncated, so long as the gene encodes a biologically active protein. For example, a biologically active, truncated form of .alpha.-Gal A, the defective enzyme associated with Fabry disease, has been described in U.S. Pat. No. 6,210,666 to Miyamura et al.

The identified and isolated gene can then be inserted into an appropriate cloning vector. Vectors suitable for gene therapy include viruses, such as adenoviruses, adeno-associated virus (AAV), vaccinia, herpesviruses, baculoviruses and retroviruses, parvovirus, lentivirus, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

In a preferred embodiment, the vector is a viral vector. Viral vectors, especially adenoviral vectors can be complexed with a cationic amphiphile, such as a cationic lipid, polyL-lysine (PLL), and diethylaminoethyldextran (DELAE-dextran), which provide increased efficiency of viral infection of target cells (See, e.g., PCT/US97/21496 filed Nov. 20, 1997, incorporated herein by reference). Preferred viral vectors for use in the present invention include vectors derived from vaccinia, herpesvirus, AAV and retroviruses. In particular, herpesviruses, especially herpes simplex virus (HSV), such as those disclosed in U.S. Pat. No. 5,672,344, the disclosure of which is incorporated herein by reference, are particularly useful for delivery of a transgene to a neuronal cell, which has importance for those lysosomal storage diseases in which the enzymatic defect manifests in neuronal cells, e.g, Hurler Scheie, Hunter's, and Tay-Sach's diseases. AAV vectors, such as those disclosed in U.S. Pat. Nos. 5,139,941, 5,252,479 and 5,753,500 and PCT publication WO 97/09441, the disclosures of which are incorporated herein, are also useful since these vectors integrate into host chromosomes, with a minimal need for repeat administration of vector. For a review of viral vectors in gene therapy, see Mah et al., Clin. Pharmacokinet. 2002; 41(12):901-11; Scott et al., Neuromuscul. Disord. 2002;12 Suppl 1:S23-9. In addition, see U.S. Pat. No. 5,670,488.

The coding sequences of the gene to be delivered are operably linked to expression control sequences, e.g., a promoter that directs expression of the gene. As used herein, the phrase "operatively linked" refers to the functional relationship of a polynucleotide/gene with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of a nucleic acid to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter, and wherein the promoter directs the transcription of RNA from the polynucleotide.

In one specific embodiment, a vector is used in which the coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for expression of the construct from a nucleic acid molecule that has integrated into the genome (Koller and Smithies, Proc. Natl. Acad. Sci. USA 1989, 86:8932-8935; Zijlstra et al., Nature 1989, 342:435-438; U.S. Pat. No. 6,244,113 to Zarling et al.; and U.S. Pat. No. 6,200,812 to Pati et al.)

Delivery of the vector into a patient may be either direct, in which case the patient is directly exposed to the vector or a delivery complex, or indirect, in which case, cells are first transformed with the vector in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo and ex vivo gene therapy.

Direct transfer. In a specific embodiment, the vector is directly administered in vivo, where it enters the cells of the organism and mediates expression of the gene. This can be accomplished by any of numerous methods known in the art and discussed above, e.g., by constructing it as part of an appropriate expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in biopolymers (e.g., poly-.beta.-1-64-N-acetylglucosamine polysaccharide; see U.S. Pat. No. 5,635,493), encapsulation in liposomes, microparticles, or microcapsules; by administering it in linkage to a peptide or other ligand known to enter the nucleus; or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 1987, 62:4429-4432), etc. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation, or cationic 12-mer peptides, e.g., derived from antennapedia, that can be used to transfer therapeutic DNA into cells (Mi et al., Mol. Therapy 2000, 2:339-47). In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO 92/22635, WO 92/20316 and WO 93/14188). Recently, a technique referred to as magnetofection has been used to deliver vectors to mammals. This technique associates the vectors with superparamagnetic nanoparticles for delivery under the influence of magnetic fields. This application reduces the delivery time and enhances vector efficacy (Scherer et al., Gene Therapy 2002; 9:102-9). Additional targeting and delivery methodologies are contemplated in the description of the vectors, below.

In a specific embodiment, the nucleic acid can be administered using a lipid carrier. Lipid carriers can be associated with naked nucleic acids (e.g., plasmid DNA) to facilitate passage through cellular membranes. Cationic, anionic, or neutral lipids can be used for this purpose. However, cationic lipids are preferred because they have been shown to associate better with DNA which, generally, has a negative charge. Cationic lipids have also been shown to mediate intracellular delivery of plasmid DNA (Felgner and Ringold, Nature 1989; 337:387). Intravenous injection of cationic lipid-plasmid complexes into mice has been shown to result in expression of the DNA in lung (Brigham et al., Am. J. Med. Sci. 1989; 298:278). See also, Osaka et al., J. Pharm. Sci. 1996; 85(6):612-618; San et al., Human Gene Therapy 1993; 4:781-788; Senior et al., Biochemica et Biophysica Acta 1991; 1070:173-179); Kabanov and Kabanov, Bioconjugate Chem. 1995; 6:7-20; Liu et al., Pharmaceut. Res. 1996; 13; Remy et al., Bioconjugate Chem. 1994; 5:647-654; Behr, J-P., Bioconjugate Chem 1994; 5:382-389; Wyman et al., Biochem. 1997; 36:3008-3017; U.S. Pat. No. 5,939,401 to Marshall et al; U.S. Pat. No. 6,331,524 to Scheule et al.

Representative cationic lipids include those disclosed, for example, in U.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099, the disclosures of which are incorporated herein by reference. In a preferred embodiment, the cationic lipid is N.sub.4-spermine cholesteryl carbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additional preferred lipids include N.sub.4-spermidine cholestryl carbamate (GL-53) and 1-(N.sub.4-spermine)-2,3-dilaurylglycerol carbamate (GL-89)

Preferably, for in vivo administration of viral vectors, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-.gamma. (IFN-.gamma.), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors. In that regard, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

Indirect transfer. Somatic cells may be engineered ex vivo with a construct encoding a wild-type protein using any of the methods described above, and re-implanted into an individual. This method is described generally in WO 93/09222 to Selden et al. In addition, this technology is used in Cell Based Delivery's proprietary ImPACT technology, described in Payumo et al., Clin. Orthopaed. and Related Res. 2002; 403S: S228-S242. In such a gene therapy system, somatic cells (e.g., fibroblasts, hepatocytes, or endothelial cells) are removed from the patient, cultured in vitro, transfected with the gene(s) of therapeutic interest, characterized, and reintroduced into the patient. Both primary cells (derived from an individual or tissue and engineered prior to passaging), and secondary cells (passaged in vitro prior to introduction in vivo) can be used, as well as immortalized cell lines known in the art. Somatic cells useful for the methods of the present invention include but are not limited to somatic cells, such as fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, other somatic cells that can be cultured, and somatic cell precursors. In a preferred embodiment, the cells are fibroblasts or mesenchymal stem cells.

Nucleic acid constructs, which include the exogenous gene and, optionally, nucleic acids encoding a selectable marker, along with additional sequences necessary for expression of the exogenous gene in recipient primary or secondary cells, are used to transfect primary or secondary cells in which the encoded product is to be produced. Such constructs include but are not limited to infectious vectors, such as retroviral, herpes, adenovirus, adenovirus-associated, mumps and poliovirus vectors, can be used for this purpose.

Transdermal delivery is especially suited for indirect transfer using cell types of the epidermis including keratinocytes, melanocytes, and dendritic cells (Pfutzner et al., Expert Opin. Investig. Drugs 2000; 9:2069-83).

Mesenchymal stem cells (MSCs) are non-blood-producing stem cells produced in the bone marrow. MSCs can be made to differentiate and proliferate into specialized non-blood tissues. Stem cells transfected with retroviruses are good candidates for the therapy due to their capacity for self-renewal. This ability precludes repetitive administration of the gene therapy. Another advantage is that if the injected stem cells reach the target organ and then differentiate, they can replace the damaged or malformed cells at the organ.

Gene Therapy in Lysosomal Storage Disorders. Recently, recombinant gene therapy methods are in clinical or pre-clinical development for the treatment of lysosomal storage disorders, see, e.g., U.S. Pat. No. 5,658,567 issued Aug. 19, 1997 for recombinant alpha-galactosidase A therapy for Fabry disease; U.S. Pat. No. 5,580,757 issued Dec. 3, 1996 for Cloning and Expression of Biologically Active .alpha.-galactosidase A as a Fusion Protein; U.S. Pat. No. 6,066,626, issued May 23, 2000 for Compositions and method for treating lysosomal storage disease; U.S. Pat. No. 6,083,725, issued Jul. 4, 2000 for Transfected human cells expressing human alph.alpha.-galactosidase A protein; U.S. Pat. No. 6,335,011, issued Jan. 1, 2002 for Methods for delivering DNA to muscle cells using recombinant adeno-associated virus virions to treat lysosomal storage disease; Bishop, D. F. et al., Proc. Natl. Acad. Sci., USA. 1986; 83:4859-4863; Medin, J. A. et al., Proc. Natl. Acad. Sci., USA. 1996; 93:7917-7922; Novo, F. J., Gene Therapy 1997; 4:488-492; Ohshima, T. et al., Proc. Natl. Acad. Sci., USA. 1997; 94:2540-2544; Sugimoto Y. et al., Human Gene Therapy 1995; 6:905-915; Sly et al., Proc. Natl. Acad. Sci. USA. 2002;99(9):5760-2; Raben et al., Curr. Mol. Med 0.2002; 2(2):145-66; Eto et al., Curr. Mol. Med. 2002; 2(1):83-9; Vogler et al., Pediatr. Dev. Pathol. 2001; 4(5):421-33; Barranger et al., Expert Opin. Biol. Ther. 2001; 1(5):857-67; Yew et al., Curr. Opin. Mol. Ther. 2001; 3(4):399-406; Caillaud et al., Biomed. Pharmacother. 2000; 54(10):505-12 and Ioannu et al., J. Am. Soc. Nephrol. 2000; 11(8):1542-7.

In 2002, Brooks et al. demonstrated that gene transfer of .beta.-glucuronidase into a mouse model of MPS VII using a feline leukemia virus, corrected associated CNS deficits (PNAS 2002; 99: 6216-6221). Indirect transfer of encapsulated Madin-Darby canine kidney cells that were genetically modified to express canine alpha-iduronidase, and implanted into dog brains under steoreotaxic guidance, was demonstrated to be efficacious in a dog model of MPS (Barsoum et al., J Lab Clin Med. 2003;142(6):399-413).

Active Site-Specific Chaperones

ASSCs contemplated by the present invention include but are not limited to small molecules (e.g., organic or inorganic molecules which are less than about 2 kD in molecular weight, are more preferably less than about 1 kD in molecular weight), including substrate or binding partner mimetics; small ligand-derived peptides or mimetics thereof; nucleic acids such as DNA, RNA; antibodies, including Fv and single chain antibodies, and Fab fragments; other macromolecules (e.g., molecules greater than about 2 kD in molecular weight) and members of libraries derived by combinatorial chemistry, such as molecular libraries of D- and/or L-configuration amino acids; phosphopeptides, such as members of random or partially degenerate, directed phosphopeptide libraries (see, e.g., Songyang et al., Cell 1993, 72:767-778).

Synthetic libraries (Needels et al., Proc. Natl. Acad. Sci. USA 1993, 90:10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993, 90:10922-10926; Lam et al., PCT Publication No. WO 92/00252; Kocis et al., PCT Publication No. WO 9428028) and the like provide a source of ASSCs according to the present invention. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available, e.g., from Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TIBTech 1996, 14:60).

U.S. patent application Ser. No. 10/377,179, filed Feb. 28, 2003 and incorporated herein by reference, describes screening methods for ASSC's for misfolded proteins.

In a preferred embodiment, small molecules useful for the present invention are inhibitors of lysosomal enzymes and include glucose and galactose imino sugar derivatives as described in Asano et al., J. Med. Chem 1994; 37:3701-06; Dale et al., Biochemistry 1985; 24:3530-39; Goldman et al., J. Nat. Prod. 1996; 59:1137-42; Legler et al, Carbohydrate Res. 1986; 155:119-29; and Okumiya et al., Biochem. Biophys. Res. Comm. 1995; 214:1219-240. Such derivatives include but are not limited those compound listed in Table 1.

Other ASSCs can be those mentioned above, e.g., small synthetic compounds, which were found to stabilize mutant forms of p53 (Foster et al., Science 1999; 286:2507-10); small molecule receptor antagonists and ligands, which were found to stabilize receptors (Morello et al., J. Clin. Invest. 2000; 105: 887-95; Petaja-Repo et al., EMBO J. 2002; 21:1628-37); and drugs or substrates, which were found to stabilize channel proteins and transporters (Rajamani et al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem. 1999; 274:31123-26; Loo et al., J. Biol. Chem. 1997; 272: 709-12).

In another embodiment, ASSC's useful in the method of the present invention are activators of cystic fibrosis transmembrane conductance regulator and, are identified using physical, and biochemical means (Blondelle et al., TIBTech 1996, 14:60).

In another preferred embodiment, ASSC's useful for the present invention are ligands of G protein-coupled receptors, such as .delta. opioid receptor, V2 vasopressin receptor, and photopigment rhodopsin, as described in Petaja-Repo et al., EMBO J. 2002; 21: 1628-37; Morello et al., J. Clin. Invest.2000; 105: 887-95; Saliba et al., J. Cell Sci. 2002; 115: 2907-18.

In yet another preferred embodiment, ASSC's useful for the present invention are blockers of ion channel proteins, such as HERG potassium channel in human Long QT syndrome, pancereatic ATP-sensitive potassium (KATP) channel in familial hyperinsulinism, as described in Zhou et al., J. Biol. Chem. 1999; 274: 31123-26; Taschenberger et al., J. Biol. Chem. 2002; 277: 17139-46.

Formulations and Administration

ASSCs. The ASSCs to be administered to an individual with a recombinant gene may be formulated for administration by, e.g., oral, parenteral, transdermal, or transmucosal routes, depending on whether the chaperone is a small molecule, synthetic compound, or protein or peptide.

For oral administration, e.g., for small molecules, the pharmaceutical compositions may take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the chaperones for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The ASSCs may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the ASSCs may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Recombinant gene. As described above, there are several methods known in the art for delivering naked DNA to individuals, including direct injection into the target tissue, e.g., intramuscular, use of cationic lipid carriers, by intravenous infusion or inhalation. See the gene therapy disclosure above.

For administration of somatic cells engineered to overexpress the recombinant gene product, the cells may be introduced into an individual, through various standardized routes of administration, so that they will reside in, for example, the renal subcapsule, a subcutaneous compartment, the central nervous system, the intrathecal space, the liver, the intraperiotoneal cavity, or within a muscle. The cells may also be injected intravenously or intra-arterially so that they circulate within the individual's bloodstream.

The cells may alternatively be embedded in a matrix or gel material, such as described in U.S. Pat. No. 5,965,125 to Mineau-Hanschke, which describes the use of hybrid matrix implants, or in Jain et al. (PCT application WO 95/19430), which describes macroencapsulation of secretory cells in a hydrophilic gel material (each of which is hereby incorporated by reference).

The number of genetically modified cells will depend on the individual's weight, age, and clinical status, and can be routinely determined by those skilled in the art. In one embodiment, about 1.times.10.sup.6 and 1.times.10.sup.9 cells/day will be used.

Timing. Administration of the ASSC according to the present invention will generally follow delivery of the gene, to allow for expression of the recombinant protein by the target cells/tissue. Since the expression of the gene will be sustained for a period of time, for as long as the gene is expressible, the ASSC will be remained effective as a chaperone and stabilizer for the recombinant protein. Therefore, administration of ASSC will be necessary for the same period as the gene is expressed.

In an embodiment where the ASSC has a short circulating half-life (e.g., a small molecule), the ASSC will be orally administered continuously, such as daily, in order to maintain a constant level in the circulation. Such a constant level will be one that has been determined to be non-toxic to the patient, and optimal regarding interaction with the protein, which will be continuously produced, to confer a non-inhibitory, therapeutic effect.

In the event that the therapeutic gene supplements inadequate activity of an endogenous mutant gene, the timing of chaperone delivery becomes less significant since the effective amount can enhance the activity of the endogenous mutant as well as increase the efficiency of the therapeutic gene.

In vivo stability. The presence of an ASSC for the protein encoded by the administered gene will have the benefit of improving the efficiency of protein processing during synthesis in the ER (i.e. by preventing aggregation), and prolonging in the circulation and tissue the half-life of the protein, thereby maintaining effective protein levels over longer time periods. This will result in increased expression in clinically affected tissues. This confers such beneficial effects to the patient as enhanced relief, reduction in the frequency of treatment, and/or reduction in the amount of gene administered. This will also reduce the cost of treatment.

In addition to stabilizing the expressed protein, the ASSC will also stabilize and enhance expression of any endogenous mutant proteins that are deficient as a result of mutations that prevent proper folding and processing in the ER, as in conformational disorders such as the LSDs.


The effective amount of ASSC to be administered with the recombinant gene will depend, in part, on the method of delivery, specific amount and typical expression level of the recombinant gene administered. The specific effective amount can be determined on a case-by-case basis, depending on the protein and corresponding ASSC, by those skilled in the art. The variation depends, for example, on the patient and the recombinant gene and ASSC used. Other factors to consider in determining doses are the individual's age, weight, sex, and clinical status. Pharmacokinetic and pharmacodynamics such as half-life (t.sub.1/2), peak plasma concentration (c.sub.max) time to peak plasma concentration (t.sub.max), exposure as measured by area under the curve (AUC) and tissue distribution for both the protein and the ASSC, as well as data for ASSC-replacement protein binding (affinity constants, association and dissociation constants, and valency), can be obtained using ordinary methods known in the art to determine compatible amounts required in a dosage form to confer a therapeutic effect.

Data obtained from cell culture assay or animal studies may be used to formulate a range of dosages for use in humans. The dosage of compounds used in therapeutic methods of the present invention preferably lie within a range of circulating concentrations that includes the ED.sub.50 concentration (effective for 50% of the tested population) but with little or no toxicity (e.g., below the LD.sub.50 concentration). The particular dosage used in any application may vary within this range, depending upon factors such as the particular dosage form employed, the route of administration utilized, the conditions of the individual (e.g., patient), and so forth.

A therapeutically effective dose may be initially estimated from cell culture assays and formulated in animal models to achieve a circulating concentration range that includes the IC.sub.50. The IC.sub.50 concentration of a compound is the concentration that achieves a half-maximal inhibition of symptoms (e.g., as determined from the cell culture assays). Appropriate dosages for use in a particular individual, for example in human patients, may then be more accurately determined using such information.

Measures of compounds in plasma may be routinely measured in an individual such as a patient by techniques such as high performance liquid chromatography (HPLC) or gas chromatography.

Toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures, for example in cell culture assays or using experimental animals to determine the LD.sub.50 and the ED.sub.50. The parameters LD.sub.50 and ED.sub.50 are well known in the art, and refer to the doses of a compound that is lethal to 50% of a population and therapeutically effective in 50% of a population, respectively. The dose ratio between toxic and therapeutic effects is referred to as the therapeutic index and may be expressed as the ratio: LD.sub.50/ED.sub.50. Chaperone compounds that exhibit large therapeutic indices are preferred.

The concentrations of the ASSC will be determined according to the amount required to stabilize the protein in vivo, in tissue or circulation, without preventing its activity. For example, where the ASSC is an enzyme inhibitor, the concentration of the inhibitor can be determined by calculating the IC.sub.50 value of the ASSC for the enzyme. Concentrations below the IC.sub.50 value can then be evaluated based on effects on enzyme activity, e.g., the amount of inhibitor needed to increase the amount of enzyme activity or prolong enzyme activity of the administered enzyme. The IC.sub.50 value of the compound deoxygalactonojiromycin (DGJ) for the .alpha.-Gal A enzyme is 0.04 .mu.M, indicating that DGJ is a potent inhibitor. Accordingly, it is expected that the concentration of .alpha.-Gal A would be much lower than that of the .alpha.-Gal A administered.

Claim 1 of 40 Claims

1. A method of improving gene therapy by increasing the level of expression of a recombinant protein corresponding to an individual's endogenous protein in vivo in cells of an individual, wherein the recombinant protein is expressed from an expression vector which has been introduced into the cells, which method comprises administering to the individual an active site-specific chaperone of the protein, with the proviso that the individual's endogenous protein is not a mutant protein that is deficient due to defective folding or processing in the endoplasmic reticulum.

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