Therapeutic acid ceramidase compositions and methods of making and using them

Abstract

To provide therapeutic acid ceramidase compositions and methods of producing and using them.SOLUTION: A method of producing a therapeutic composition includes: providing a medium containing an inactive acid ceramidase precursor; incubating the medium under conditions effective to transform a portion of the inactive acid ceramidase precursor to active acid ceramidase; and recovering the incubated medium as a ceramidase mixture comprising the inactive acid ceramidase precursor and an active acid ceramidase.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 784,594, filed March 14, 2013, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to therapeutic acid ceramidase compositions and methods of making and using same. [Background technology]

[0003] 2. Background of the Invention Acid ceramidase ("AC"; N-acylsphingosine deacylase, IUBMB Enzyme No. EC 3.5.1.23) is the most extensively studied member of the ceramidase enzyme family due to its involvement in the human genetic disorder Farber lipogranulomatosis ("FD"). The protein has been purified from multiple sources, and human and mouse cDNAs and genes have been obtained (Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270:11098-102 (1995) (Non-Patent Document 1); Koch et al., "Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase. Identification of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 2711:33110-5 (1996) (Non-Patent Document 2); Li et al., "Cloning and Characterization of the Full-length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase," Genomics 50:267-74 (1998) (Non-Patent Document 3); Li et al., "The Human Acid Ceramidase Gene (ASAH): Chromosomal Location, Mutation Analysis, and Expression," Genomics 62:223-31 (1999) (Non-Patent Document 4)). The growing interest in the biology of this and other ceramidases arises from the fact that these enzymes play a central role in ceramide metabolism.

[0004] Ceramide is a signaling lipid produced in response to a variety of stimuli and external factors, including serum withdrawal and treatment with many chemotherapeutic agents, as well as in many human diseases (Hannun, "Function of Ceramide in Coordinating Cellular Responses to Stress," Science 274:1855-9 (1996); Spiegel et al., "Signal Transduction Through Lipid Second Messengers," Curr. Opin. Cell. Biol. 8:159-67 (1996)). Inside the cell, ceramide can affect proliferation and differentiation, regulate protein secretion, induce DNA fragmentation and apoptosis, and increase cytokine synthesis and secretion. Ceramide is normally present in low amounts and, in response to these factors, is rapidly produced at the cell surface, leading to downstream signaling that results in membrane remodeling and apoptosis. After stimulation, AC and / or other ceramidases can then hydrolyze ceramide into its respective fatty acid and sphingosine components (Gatt, "Enzymic Hydrolysis and Synthesis of Ceramide," J. Biol. Chem. 238:3131-3 (1963) (Non-Patent Document 7); Gatt, "Enzymatic Hydrolysis of Sphingolipids. 1. Hydrolysis and Synthesis of Ceramides by an Enzyme from Rat Brain," J. Biol. Chem. 241:3724-31 (1966) (Non-Patent Document 8); Hassler & Bell, "Ceramidase: Enzymology and Metabolic Roles," Adv. Lip. Res. 26:49-57 (1993) (Non-Patent Document 9)).Because ceramide degradation is the only source of intracellular sphingosine (Rother et al., "Biosynthesis of Sphingolipids: Dihydroceramide and Not Sphinganine Is Desaturated by Cultured Cells," Biochem. Biophys. Res. Commun. 189:14-20 (1992)), these enzymes may also be the rate-limiting step in determining the intracellular levels of this compound. Importantly, a derivative of sphingosine, sphingosine-1-phosphate ("S1P"), can counteract the apoptotic effects of ceramide (Cuvillier et al., "Suppression of Ceramide-mediated Programmed Cell Death by Sphingosine-1-phosphate," Nature 381:800-3 (1996) (Non-Patent Document 11)), leading to the suggestion that ceramidase may be a "rheostat" that maintains the appropriate balance between cell proliferation and cell death (Spiegel & Merrill, "Sphingolipids Metabolism and Cell Growth Regulation," FASEB J. 10:1388-97 (1996) (Non-Patent Document 12)).

[0005] AC hydrolyzes the amide bond that links the sphingosine and fatty acid moieties of the lipid ceramide (Park and Schuchman, "Acid Ceramidase and Human Disease," Biochim. Biophys. Acta. 1758(12): 2133-2138 (2006)). Ceramide, sphingosine (and its phosphorylated derivative S1P) are bioactive lipids, and therefore the activity of AC must be carefully regulated in cells (Young et al., "Sphingolipids: Regulators of Crosstalk Between Apoptosis and Autophagy," J. Lipid Res. 54:5-19 (2013)). One important mechanism that regulates AC activity is the cleavage of an inactive precursor polypeptide into an active enzyme consisting of α and β subunits linked via disulfide bonds (Shtraizent et al., "Autoproteolytic Cleavage and Activation of Human Acid Ceramidase," J. Biol. Chem. 283:11253-11259 (2008)). It has previously been shown that recombinant AC produced in Chinese Hamster Ovary ("CHO") cells and secreted into the medium is a mixture of inactive precursor and active (cleaved) enzyme (He et al., "Purification and Characterization of Recombinant, Human Acid Ceramidase," J. Biol. Chem. 278:32978-32986 (2003)).

[0006] The present invention seeks to overcome these and other deficiencies in the art. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270:11098-102 (1995) [Non-Patent Document 2] Koch et al., "Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase. Identification of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 2711:33110-5 (1996) [Non-Patent Document 3] Li et al., "Cloning and Characterization of the Full-length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase," Genomics 50:267-74 (1998) [Non-Patent Document 4] Li et al., "The Human Acid Ceramidase Gene (ASAH): Chromosomal Location, Mutation Analysis, and Expression," Genomics 62:223-31 (1999) [Non-Patent Document 5] Hannun, "Function of Ceramide in Coordinating Cellular Responses to Stress," Science 274:1855-9 (1996) [Non-Patent Document 6] Spiegel et al., "Signal Transduction Through Lipid Second Messengers," Curr. Opin. Cell. Biol. 8:159-67 (1996) [Non-Patent Document 7] Gatt, "Enzymic Hydrolysis and Synthesis of Ceramide," J. Biol. Chem. 238:3131-3 (1963) [Non-Patent Document 8] Gatt, "Enzymatic Hydrolysis of Sphingolipids. 1. Hydrolysis and Synthesis of Ceramides by an Enzyme from Rat Brain," J. Biol. Chem. 241:3724-31 (1966) [Non-Patent Document 9] Hassler & Bell, "Ceramidase: Enzymology and Metabolic Roles," Adv. Lip. Res. 26:49-57 (1993) [Non-Patent Document 10] Rother et al., "Biosynthesis of Sphingolipids: Dihydroceramide and Not Sphinganine Is Desaturated by Cultured Cells," Biochem. Biophys. Res. Commun. 189:14-20 (1992) [Non-Patent Document 11] Cuvillier et al., "Suppression of Ceramide-mediated Programmed Cell Death by Sphingosine-1-phosphate," Nature 381:800-3 (1996) [Non-Patent Document 12] Spiegel & Merrill, "Sphingolipids Metabolism and Cell Growth Regulation," FASEB J. 10:1388-97 (1996) [Non-Patent Document 13] Park and Schuchman, "Acid Ceramidase and Human Disease," Biochim. Biophys. Acta. 1758(12): 2133-2138 (2006) [Non-Patent Document 14] Young et al., "Sphingolipids: Regulations of Crosstalk Between Apoptosis and Autophagy," J. Lipid Res. 54:5-19 (2013) [Non-Patent Document 15] Shtraizent et al., "Autoproteolytic Cleavage and Activation of Human Acid Ceramidase," J. Biol. Chem. 283:11253-11259 (2008) [Non-Patent Document 16] He et al., "Purification and Characterization of Recombinant, Human Acid Ceramidase," J. Biol. Chem. 278:32978-32986 (2003) Summary of the Invention

[0008] A first aspect of the present invention relates to a therapeutic composition comprising a ceramidase mixture, the ceramidase mixture including an inactive acid ceramidase precursor and an active acid ceramidase, and a pharma- ceutically acceptable carrier.

[0009] A second aspect of the present invention relates to a method of acid ceramidase treatment comprising formulating the acid ceramidase used in the acid ceramidase treatment as a ceramidase mixture comprising an inactive acid ceramidase precursor and an active acid ceramidase.

[0010] A third aspect of the present invention relates to a method for producing a therapeutic composition comprising the steps of providing a medium containing an inactive acid ceramidase precursor and incubating the medium under conditions effective to convert a portion of the inactive acid ceramidase precursor to active acid ceramidase, and recovering the incubated medium as a ceramidase mixture comprising the inactive acid ceramidase precursor and the active acid ceramidase.

[0011] The present invention describes an optimal composition of recombinant AC (rAC). The present invention further describes the novel finding that, contrary to expectations, the fully active enzyme is not the best form for promoting cell survival. Rather, purified rAC preparations containing a higher amount of inactive acid ceramidase (AC) precursor relative to the processed active AC are more effective in promoting cell survival and / or improving cell phenotype. Two recombinant AC preparations containing different ratios of precursor to active enzyme were obtained. They were then used to evaluate the effect on oocyte survival in culture. Contrary to expectations, the preparation containing a higher ratio of inactive precursor had a better effect on cell survival. This is hypothesized to be due to the fact that the fully active enzyme has a shorter half-life in cells and cell culture medium. The same two preparations were tested with cultured primary chondrocytes. As with oocytes, the less active recombinant AC preparation had a better effect on the expression of collagen 2, a marker of chondrogenesis.

[0012] rAC has been experimentally used in several cell lines and animal models to delay ceramide-associated cell death and / or improve the phenotype of cells used in cell transplantation.It has also been studied in several disease models.The present invention describes optimal rAC preparations for use in these purposes, with many potential practical implications (e.g., in vitro fertilization, cartilage repair, and treatment of cystic fibrosis).

[0013] In another derivative of the present invention, a new method for the purification of recombinant AC was developed. In this method, heat inactivation was used to remove acid sphingomyelinase and other contaminating proteins from recombinant AC preparations. Previous studies have shown that a related lipid hydrolase, acid sphingomyelinase, binds tightly to AC and co-purifies with AC (Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270:11098-11102 (1995), which is incorporated herein by reference in its entirety). Now, unlike many proteins, AC activity has been found to be sufficiently stable when heated to 60°C. Thus, heat inactivation can be used to remove acid sphingomyelinase activity from recombinant AC preparations after column chromatography.

[0014] Overall, these two new findings regarding (i) the importance of maintaining an optimal ratio of precursor to active AC and (ii) the use of heat inactivation to remove acid sphingomyelinase activity and other contaminating proteins from the preparation constitute unique and important observations regarding the composition of recombinant AC. [The present invention 1001] providing a medium containing an inactive acid ceramidase precursor; incubating the medium under conditions effective to convert a portion of the inactive acid ceramidase precursor to active acid ceramidase; and recovering the incubated medium as a ceramidase mixture containing inactive acid ceramidase precursor and active acid ceramidase. A method for producing a therapeutic composition comprising: [The present invention 1002] The method of the present invention 1001, wherein the incubating step is carried out under conditions effective to reduce the conversion rate of inactive acid ceramidase precursor to active acid ceramidase compared to the conversion rate achieved when the incubating step is carried out for 24 hours, at pH 4 and at a temperature of 4°C or 37°C, under otherwise consistent conditions. [The present invention 1003] The method of claim 1002, wherein the pH of the ceramidase mixture during the incubating step is greater than 4.0 and less than or equal to 6.5. [The present invention 1004] The method of claim 1002, wherein the temperature of the ceramidase mixture during the incubating step is at least -30°C and less than 37°C. [The present invention 1005] The method of claim 1001, wherein the incubating step comprises heating the medium under conditions effective to remove acid sphingomyelinase activity. [The present invention 1006] Providing a culture medium comprises: Providing a cell transformed with DNA encoding acid ceramidase; culturing the transformed cells under conditions effective to produce a medium containing an inactive acid ceramidase precursor. The method of the present invention 1001, comprising: [The present invention 1007] The method of the present invention 1001, wherein the incubating step is carried out under conditions effective to increase the conversion rate of inactive acid ceramidase precursor to active acid ceramidase compared to the conversion rate achieved when the incubating step is carried out for 24 hours, at pH 4 and at a temperature of 4°C or 37°C, under otherwise consistent conditions. [The present invention 1008] an inactive acid ceramidase precursor, and Active acid ceramidase a ceramidase mixture comprising: A pharma- ceutically acceptable carrier; A therapeutic composition comprising: [The present invention 1009] A therapeutic composition according to the present invention, wherein the amount of inactive acid ceramidase precursor is greater than the amount of active acid ceramidase in the ceramidase mixture. [The present invention 1010] A therapeutic composition according to the present invention, wherein the amount of inactive acid ceramidase precursor in the ceramidase mixture is less than the amount of active acid ceramidase. [The present invention 1011] 1008. A therapeutic composition according to the present invention, wherein the ceramidase mixture comprises 5-95% by weight of inactive acid ceramidase precursor and 95-5% by weight of active acid ceramidase. [The present invention 1012] 10. The therapeutic composition of the present invention, wherein the ceramidase mixture comprises 20-80% by weight of an inactive acid ceramidase precursor and 80-20% by weight of an active acid ceramidase. [The present invention 1013] 10. The therapeutic composition of the present invention, wherein the ceramidase mixture comprises 30-70% by weight of an inactive acid ceramidase precursor and 70-30% by weight of an active acid ceramidase. [The present invention 1014] 10. The therapeutic composition of the present invention, wherein the ceramidase mixture comprises 40-60% by weight of inactive acid ceramidase precursor and 60-40% by weight of active acid ceramidase. [The present invention 1015] 10. The therapeutic composition of the present invention, wherein the ceramidase mixture comprises 55-95% by weight of inactive acid ceramidase precursor and 45-5% by weight of active acid ceramidase. [The present invention 1016] 10. The therapeutic composition of the present invention, wherein the ceramidase mixture comprises 70-95% by weight of inactive acid ceramidase precursor and 30-5% by weight of active acid ceramidase. [The present invention 1017] 10. The therapeutic composition of the present invention, wherein the ceramidase mixture comprises 80-90% by weight of an inactive acid ceramidase precursor and 20-10% by weight of an active acid ceramidase. [The present invention 1018] A therapeutic composition of the present invention 1008 further comprising an adjuvant. [The present invention 1019] The therapeutic composition of the present invention 1018, wherein the adjuvant is selected from the group consisting of flagellin, Freund's complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, iscomatrix, and liposomal polycation DNA particles. [The present invention 1020] An improvement in a method of acid ceramidase treatment comprising formulating the acid ceramidase used in the treatment as a ceramidase mixture comprising an inactive acid ceramidase precursor and an active acid ceramidase. [The present invention 1021] The method of claim 1020, further comprising a step of generating chondrocytes using the ceramidase mixture. [The present invention 1022] selecting a population of cells that have the potential to differentiate into chondrocytes; and treating a cell population of selected cells with said ceramidase mixture to convert one or more cells in the selected population into chondrocytes. The method of the present invention 1021 further comprises: [The present invention 1023] The method of claim 1022, wherein the cells in the selected population are mammalian cells. [The present invention 1024] The method of any one of claims 10 to 13, wherein the selected cell population comprises a population of bone marrow cells, mesenchymal stem cells and / or fibroblasts. [The present invention 1025] The method of claim 1020, further comprising a step of promoting chondrogenesis with said ceramidase mixture. [The present invention 1026] selecting a population of stem cells that need to be differentiated into chondrocytes; treating the population of stem cells with the ceramidase mixture to enrich for mesenchymal stem cells within the stem cell population; and treating the enriched population of mesenchymal stem cells with the ceramidase mixture to promote differentiation of the mesenchymal stem cells into chondrocytes. The method of the present invention 1025, comprising: [The present invention 1027] The method of claim 1026, wherein the mesenchymal stem cells are mammalian. [The present invention 1028] The method of the present invention 1020, comprising administering the ceramidase mixture to a subject to treat the subject for a joint disease or disorder. [The present invention 1029] The method of claim 1028, wherein the joint disease or disorder is selected from the group consisting of osteoarthritis, rheumatoid arthritis, mucopolysaccharidoses, degenerative joint disease, joint injury, and Farber lipogranulomatosis. [The present invention 1030] The method of the present invention 1020, comprising administering the ceramidase mixture to a subject to treat the subject for a neurodegenerative disease or neurodegenerative disorder. [The present invention 1031] The method of the present invention 1030, wherein the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, prion disease, Parkinson's disease, Huntington's disease, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, amyotrophic lateral sclerosis, inclusion body myositis, degenerative myopathy, spinocerebellar atrophy, metabolic neuropathies, diabetic neuropathies, endocrine neuropathies, orthostatic hypotension, brain injury, spinal cord injury, stroke, and motor neuron diseases such as spinal muscular atrophy. [The present invention 1032] The method of the present invention 1020, comprising administering the ceramidase mixture to a subject to treat the subject for a cardiac disease or disorder. [The present invention 1033] The method of claim 1032, wherein the cardiac disease or disorder is selected from the group consisting of cardiac disease, cardiac trauma, atherosclerosis, thrombosis, and cardiomyocyte apoptosis. [The present invention 1034] The method of claim 1020, comprising administering said ceramidase mixture to a subject to treat the subject for diabetes. [The present invention 1035] The method of claim 1020, comprising administering said ceramidase mixture to a subject to treat the subject for pathogen infection in a subject having cystic fibrosis, COPD and / or open wounds. [The present invention 1036] The method of claim 1035, wherein the pathogen infection is a viral infection, a fungal infection, a prion infection, or a bacterial infection. [The present invention 1037] The method of claim 1020, comprising administering the ceramidase mixture to a subject to treat the subject for a ceramide-accumulating infection. [The present invention 1038] The method of claim 1020, comprising administering the ceramidase mixture to a subject to treat the subject for Farber disease. [The present invention 1039] The method of the present invention 1020, wherein the treatment comprises administering the ceramidase mixture to a subject orally, by inhalation, by intranasal instillation, topically, transdermally, parenterally, subcutaneously, intravenously, intraarterially, intramuscularly, intrapleurally, intraperitoneally, intrathecally, or by application to a mucous membrane. [The present invention 1040] The method of claim 1039, further comprising repeating said administration. [The present invention 1041] The method of claim 1020, wherein said treatment is performed in vivo. [The present invention 1042] The method of claim 1020, wherein said treatment is performed in vitro. [The present invention 1043] The method of claim 1020, further comprising administering one or more additional agents that reduce ceramide levels. [Brief description of the drawings]

[0015] [Figure 1]rAC preparations with less active ceramidase are shown to be superior to preparations with more active ceramidase. Figure 1A is a Western blot analysis showing the relative amount of active (α / β) rAC to inactive precursor rAC in two different bioreactor cultures (Lot 6 and Lot 7). Figure 1B summarizes the results showing normal mouse embryogenesis potential of Lot 6 and Lot 7. Figure 1C shows the results of Lot 6 and Lot 7 after testing with cultured rat chondrocytes. At 2 weeks, the amount of collagen 2 expression was analyzed using Western blotting. [Diagram 2] Figure 1 illustrates the time-response curves of AC activity and acid sphingomyelinase activity of Lot 7. Acid sphingomyelinase activity was removed without affecting AC activity. [Diagram 3] A plot of acid ceramidase activity (nmol / ml / hr) versus incubation time (days). [Figure 4] 1 is a Western blot showing the conversion of inactive acid ceramidase to active acid ceramidase. [Diagram 5] 1 is a Western blot showing the conversion of inactive acid ceramidase to active acid ceramidase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention A first aspect of the present invention relates to a therapeutic composition comprising a ceramidase mixture, the ceramidase mixture including an inactive AC precursor and an active AC, and a pharma- ceutically acceptable carrier.

[0017] Ceramidases hydrolyze the amide bonds of ceramides to produce free fatty acids and sphingoid bases (Nikolova-Karakashian et al., "Ceramidases," Methods Enzymol. 311:194-201 (2000); Hassler et al., "Ceramidases: Enzymology and Metabolic Roles," Adv. Lipid Res. 26:49-57 (1993), which are incorporated herein by reference in their entireties). Three types of ceramidases have been described to date (Nikolova-Karakashian et al., "Ceramidases," Methods Enzymol. 311:194-201 (2000), which are incorporated herein by reference in their entireties). These are classified into acid ceramidases, neutral ceramidases, and alkaline ceramidases, depending on the optimum pH for their enzymatic activity.

[0018] AC has optimal enzymatic activity at pH<5. Human AC was the first ceramidase to be cloned (Koch et al., "Molecular Cloning and Characterization of a Full-Length Complementary DNA Encoding Human Acid Ceramidase. Identification Of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 271:33110-33115 (1996), which is incorporated herein by reference in its entirety). It is localized in lysosomes and is primarily responsible for the catabolism of ceramide. Dysfunction of this enzyme due to genetic abnormalities leads to a sphingolipidosis disease called lipogranulomatosis or Farber disease (Koch et al., "Molecular Cloning and Characterization of a Full-Length Complementary DNA Encoding Human Acid Ceramidase. Identification Of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 271:33110-33115 (1996); Young et al., "Sphingolipids: Regulators of Crosstalk Between Apoptosis and Autophagy," J. Lipid. Res. 54:5-19 (2013), which are incorporated by reference in their entireties).

[0019] The inactive AC precursor and active AC suitable for use in the ceramidase mixture of this and all aspects of the present invention can be homologous (i.e., from the same species) or heterologous (i.e., from a different species) to the tissue, cell and / or subject to be treated. The ceramidase (e.g., AC) precursor protein undergoes autoproteolytic cleavage to become active (composed of α and β subunits). The mechanism of cleavage and activation of human AC is reported in Shtraizent et al., "Autoproteolytic Cleavage and Activation of Human Acid Ceramidase," J. Biol. Chem. 283(17):11253-11259 (2008), the entirety of which is incorporated herein by reference. This is expected to occur in most, if not all, cell types, based on the sequence of the cleavage site of the ceramidase precursor protein, which is promoted by the intracellular environment and is highly conserved among species. Thus, ceramidase as used herein includes both active ceramidase and ceramidase precursor protein, where ceramidase precursor protein is converted to active ceramidase protein via autoproteolytic cleavage.Both embodiments are contemplated, in which precursor protein is taken up by cells of interest and thereby converted to active ceramidase, and in which precursor protein is converted to active ceramidase by different cells or agents (e.g., present in culture medium).

[0020] AC (N-acylsphingosine deacylase, IUBMB Enzyme No. EC 3.5.1.23) protein has been purified from multiple sources, and human and mouse cDNAs and genes are available. See Bernardo et al., "Purification, Characterization, and Biosynthesis of Human Acid Ceramidase," J. Biol. Chem. 270:11098-102 (1995); Koch et al., "Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase. Identification of the First Molecular Lesion Causing Farber Disease," J. Biol. Chem. 2711:33110-5 (1996); Li et al., "Cloning and Characterization of the Full-length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase," Genomics 50:267-74 (1998); Li et al., "The Human Acid Ceramidase Gene (ASAH): Chromosomal Location, Mutation Analysis, and Expression," Genomics 62:223-31 (1999), all of which are incorporated by reference in their entireties. (1999).It is produced via cleavage of the AC precursor protein (see Ferlinz et al., "Human Acid Ceramidase: Processing, Glycosylation, and Lysosomal Targeting," J. Biol. Chem. 276(38):35352-60 (2001), which is incorporated by reference in its entirety), which is the product of the Asah1 gene (NCBI UniGene GeneID No. 427, which is incorporated by reference in its entirety). The AC protein [Homo sapiens] (Accession No. AAC50907) is shown below in SEQ ID NO: 1. TIFF2025000947000001.tif27146The AC α subunit begins at amino acid position 22 and continues to position 142 (shown in bold in SEQ ID NO: 1), while the AC β subunit begins at amino acid position 143 and continues to position 395 (shown in italics in SEQ ID NO: 1).

[0021] Active AC and inactive AC precursor proteins that can be used in this and all aspects of the invention include, but are not limited to, those set forth in Table 1 below.

[0022] (Table 1) Representative members of the acid ceramidase family TIFF2025000947000002.tif133157

[0023] In some embodiments, the ceramidase mixture of the therapeutic composition may contain a greater amount of inactive AC precursor than active AC. Alternatively, in some instances, the ceramidase mixture of the therapeutic composition may contain a lesser amount of inactive AC precursor than active AC.

[0024] In some embodiments, the effective amount of inactive AC precursor compared to active AC in the mixture can be in the range of about 5-95% by weight inactive AC precursor and 95-5% by weight active AC; 20-80% by weight inactive AC precursor and 80-20% by weight active AC; 30-70% by weight inactive AC precursor and 70-30% by weight active AC; 40-60% by weight inactive AC precursor and 60-40% by weight active AC; 55-95% by weight inactive AC precursor and 45-5% by weight active AC; 70-95% by weight inactive AC precursor and 30-5% by weight active AC, or 80-90% by weight inactive AC precursor and 20-10% by weight active AC. Of the mixture, the effective amount of inactive AC precursor is 90% by weight and active ceramidase is 10% by weight. Alternative embodiments can include 80% by weight inactive ceramidase precursor and 20% by weight active AC in the ceramidase mixture. In still further embodiments, the ceramidase mixture may contain 60% by weight inactive ceramidase precursor and 40% by weight active ceramidase.

[0025] The therapeutic composition may also include pharma- ceutically acceptable adjuvants, excipients and / or stabilizers, and the therapeutic composition may be in solid or liquid form, such as a tablet, capsule, powder, solution, suspension or emulsion, etc. Suitable adjuvants include, but are not limited to, flagellin, Freund's complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, iscomatrix, and liposomal polycation DNA particles.

[0026] A second aspect of the present invention relates to a method of treating AC comprising formulating the AC used in the treatment as a ceramidase mixture comprising an inactive AC precursor and an active AC.

[0027] Treatment according to this aspect of the invention is carried out using methods that will be apparent to those skilled in the art. For a discussion of AC in the context of human disease, see Park et al., "Acid Ceramidase and Human Disease," Biochim. Phyiophys. Act. 1758:2133-2138 (2006) and Zeidan et al., "Molecular Targeting of Acid Ceramidase: Implications to Cancer Therapy," Curr. Drug Targets 9(8):653-661 (2008), both of which are incorporated herein by reference in their entirety.

[0028] In some embodiments, the treatment is carried out by introducing ceramidase protein into cells. Means for the delivery of protein or polypeptide agents (e.g., active ceramidase, inactive ceramidase precursor protein) include binding of the desired protein or polypeptide to a stabilized polymer to avoid enzymatic degradation of the complex protein or complex polypeptide. This type of complex protein or complex peptide is described in U.S. Patent No. 5,681,811 to Ekwuribe, which is incorporated herein by reference in its entirety.

[0029] Yet another means for the delivery of protein or polypeptide agents includes the preparation of chimeric proteins according to US Patent No. 5,817,789 to Heartlein et al., the entirety of which is incorporated herein by reference. The chimeric protein can include a ligand domain and a polypeptide agent (e.g., rAC, active AC, other ceramidases, inactive AC precursor proteins, other ceramidase precursor proteins). The ligand domain is specific for a receptor located on a target cell. Thus, when the chimeric protein is delivered to a cell, it is adsorbed to the target cell, and the target cell internalizes the chimeric protein.

[0030] Further embodiments of this aspect relate to methods of treatment for certain diseases or disorders, comprising formulating the AC used in the treatment as a ceramidase mixture comprising an inactive ceramidase precursor and an active AC.

[0031] In one embodiment, the disease or disorder is a joint disease or disorder, and the ceramidase mixture according to the method of the present invention is administered to the subject to treat the subject for the joint disease or disorder.Representative types of joint disease or disorder include, but are not limited to, osteoarthritis, rheumatoid arthritis, mucopolysaccharidosis, degenerative joint disease, joint injury, and Farber lipogranulomatosis.

[0032] In another embodiment, the disease or disorder is a neurodegenerative disease or disorder, and the ceramidase mixture according to the method of the present invention is administered to the subject to treat the subject for the neurodegenerative disease or disorder.Representative types of neurodegenerative disease or disorder include, but are not limited to, Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, prion disease, Parkinson's disease, Huntington's disease, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, amyotrophic lateral sclerosis, inclusion body myositis, degenerative myopathy, spinocerebellar atrophy, metabolic neuropathy, diabetic neuropathy, endocrine neuropathy, orthostatic hypotension, brain injury, spinal cord injury, stroke, and motor neuron disease such as spinal muscular atrophy.

[0033] In another embodiment, the disease or disorder is a cardiac disease or disorder, and the ceramidase mixture according to the method of the present invention is administered to the subject to treat the subject for the cardiac disease or disorder.Representative types of cardiac disease or disorder include, but are not limited to, cardiac disease, cardiac trauma, atherosclerosis, thrombosis, cardiomyocyte apoptosis, cardiac hypertrophy, myocardial infarction, mitral regurgitation, aortic regurgitation, septal defect, and tachycardia bradycardia syndrome.

[0034] In another embodiment, the disease or disorder is diabetes, and a ceramidase mixture according to the methods of the present invention is administered to the subject to treat the subject for diabetes.

[0035] In another embodiment, the disease or disorder is a pathogen infection in a patient with cystic fibrosis, chronic obstructive pulmonary disease (COPD), and / or open wounds, and the ceramidase mixture according to the method of the present invention is administered to the subject to treat the subject for the pathogen infection. Representative types of pathogen infection include, but are not limited to, viral, fungal, prion, and bacterial.

[0036] Subjects suffering from cystic fibrosis, COPD, and / or open wounds may have an increased susceptibility to acquiring acute and / or chronic pathogen infections, such as, for example, bacterial, viral, fungal, protozoal and / or prion pathogen infections. Bacterial pathogens include, but are not limited to, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Corynebacterium dipththeriae, Escherichia coli, enterohaemorrhagic E. coli, enterotoxigenic E. coli, Haemophilus influenzae type B and nontypeable, Helicobacter pylori, Legionella pneumophila, and the like. pneumophila, Listeria monocytogenes, Mycobacterium spp., Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus spp., Pseudomonas aeruginosa, Rickettsia, Salmonella spp., Shigella spp., Staphylococcus spp., Staphylococcus aureus, Streptococcus spp.), Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus B, Group A beta hemolytic Streptococcus, Streptococcus mutans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis. In some embodiments, the pathogen infection is a Pseudomonas infection.

[0037] Viral pathogens include, but are not limited to, RNA viruses, DNA viruses, adenovirdiae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus type 1, herpes simplex virus type 2, herpes simplex virus type 5, and herpes simplex virus type 6), leviviridae (e.g., levivirus, enterobacteria phage MS2, enterobacteria phage MS3, enterobacteria phage MS4, enterobacteria phage MS5, enterobacteria phage MS6, enterobacteria phage MS7, enterobacteria phage MS8, enterobacteria phage MS9, enterobacteria phage MS10, enterobacteria phage MS11, enterobacteria phage MS12, enterobacteria phage MS13, enterobacteria phage MS14, enterobacteria phage MS15, enterobacteria phage MS16, enterobacteria phage MS17, enterobacteria phage MS18, enterobacteria phage MS19, enterobacteria phage MS21, enterobacteria phage MS22, enterobacteria phage MS23, enterobacteria phage MS24, enterobacteria phage MS25, enterobacteria phage MS26, enterobacteria phage MS27, enterobacteria phage MS28, enterobacteria phage MS29, enterobacteria phage MS31, enterobacteria phage MS32, enterobacteria phage MS33, enterobacteria phage MS34, enterobacteria phage MS35, enterobacteria phage MS36, enterobacteria phage MS37, enterobacteria phage MS38, enterobacteria phage MS39, enterobacteria phage MS41, enterobacteria ph MS2), allolevirus), poxyiridae (e.g., chordopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, molluscipox virus, and entomopoxyirinae), papovaviridae (e.g., polyomavirus, and papillomavirus), paramyxoviridae (e.g., paramyxovirus, parainfluenza virus type 1, measles virus, and rabies virus), Morbilliviruses (e.g., Morbillivirus, Rubulavirus, Mumps virus, etc.), Pneumonoviridae (e.g., Pneumovirus, Human respiratory syncytial virus), Metapneumovirus (e.g., Avian pneumovirus,pneumovirus and human metapneumo virus), Picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus such as human hepatitis A virus, cardiovirus, and apthovirus), Reoviridae (e.g., orthoreo virus, orbivirus, rotavirus, cypo virus, fijivirus, phytoreovirus, virus, and oryzavirus), retroviridae (e.g., mammalian type B retrovirus, mammalian type C retrovirus, avian type C retrovirus, D type retrovirus group, BLV-HTLV retrovirus, lentivirus (e.g., human immunodeficiency virus type 1 and human immunodeficiency virus type 2); and spuma virus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus-sindbis virus, and rubivirus, such as rubella virus), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemera virus, and the like),virus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and Lassa virus), and coronaviridae (e.g., coronavirus and torovirus), cytomegalovirus (mononucleosis), Dengue virus (dengue fever, shock syndrome), Epstein-Barr virus (mononucleosis, Burkitt's leukemia), human T-cell lymphotropic virus type 1 (T-cell leukemia), influenza A, B, and C (respiratory disease), Japanese encephalitis These include: Poliovirus (pneumonia, encephalopathy), Rhinovirus (common cold), Rubella virus (fetal abnormalities), Vaccinia virus (systemic infection), Yellow fever virus (jaundice, kidney and liver failure), and Varicella zoster virus (chickenpox).

[0038] Pathogenic bacteria include, but are not limited to, Aspergillus (e.g., Aspergillus fumigates), Blastomyces, Candida (e.g., Candida albicans), Coccidiodes, Cryptococcus, Histoplasma, Phycomyces, Tinea corporis, Tinea unguis, Sporothrix schenckii, and Pneumocystis carinii. Pathogenic protozoa include, but are not limited to, Trypanosome spp., Leishmania spp., Plasmodium spp., Entamoeba spp., and Giardia spp., such as Giardia lamblia.

[0039] "Open wound" as described herein refers to the type of injury in which the epidermal layer, i.e., skin, is torn, cut, and / or punctured.In some embodiments, open wound refers to a sharp injury that damages the dermis of skin, and at the same time increases the chance of infection.The term "open wound" also includes burns.

[0040] In another embodiment, the disease or disorder is an infection caused by ceramide accumulation, and a ceramidase mixture according to the methods of the present invention is administered to the subject to treat the subject for a ceramide-accumulating infection.

[0041] In another embodiment, the present invention can be used to treat Farber's disease.

[0042] In at least one embodiment, the treatment is performed in vitro. In this embodiment, the ceramidase mixture can be taken from the subject or a second subject and then administered to the first subject (e.g., by injecting the mixture into the first subject). In at least one embodiment, the treatment is performed in vivo.

[0043] Mammalian subjects according to these aspects of the invention include, for example, human subjects, equine subjects, porcine subjects, feline subjects, and canine subjects. Human subjects are particularly preferred.

[0044] In all embodiments involving administration of a ceramidase mixture to a subject, any combination of active ceramidase, ceramidase precursor protein, and / or nucleic acid encoding ceramidase / ceramidase precursor protein can be administered. Administration can be accomplished either through systemic administration to the subject or targeted administration to the affected tissue, organ, and / or cell. The ceramidase mixture can be administered to a non-targeted area along with one or more agents that facilitate the transfer of the ceramidase mixture to (and / or uptake by) the targeted tissue, organ, or cell. Additionally and / or alternatively, the ceramidase mixture itself can be modified to facilitate its transport to (and uptake by) the desired tissue, organ, or cell, as will be apparent to those skilled in the art.

[0045] Typically, the ceramidase mixture is administered to the subject in a vehicle that delivers ceramidase to the target cell, tissue or organ.Exemplary routes of administration include, but are not limited to, oral, inhalation, intratracheal inoculation, aspiration, airway instillation, aerosolization, spraying, intranasal instillation, oral or nasogastric instillation, intraperitoneal injection, intravascular injection, topical, transdermal, parenteral, subcutaneous, intravenous injection, intraarterial injection (such as via the pulmonary artery), intramuscular injection, intrapleural injection, intraventricular, intralesional, intrathecal, application to mucosa (such as mucosa of nose, throat, trachea, genitals, and / or anus), or implantation of a sustained release vehicle.

[0046] In some embodiments, the ceramidase mixture is administered orally, topically, intranasally, intraperitoneally, intravenously, subcutaneously, or by aerosol inhalation. In some embodiments, the ceramidase mixture is administered via aerosol inhalation. In some embodiments, the ceramidase mixture can be incorporated into a pharmaceutical composition suitable for administration as described herein.

[0047] The ceramidase mixture may be administered orally, for example, with an inert diluent or an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be taken directly with food. For oral therapeutic administration, the ceramidase mixture may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% ceramidase. The percentage of the ceramidase mixture in these compositions may of course vary, and may conveniently be about 2% to about 60% by weight of the unit amount. The amount of the ceramidase mixture in such therapeutically useful compositions is such that a suitable dosage is obtained.

[0048] Tablets and capsules and the like may also contain binders such as gum tragacanth, gum arabic, corn starch or gelatin, excipients such as dicalcium phosphate, disintegrating agents such as corn starch, potato starch or alginic acid, lubricants such as magnesium stearate, and sweeteners such as sucrose, lactose or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

[0049] The ceramidase mixture can also be administered parenterally. A solution or suspension of ceramidase can be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oil. Exemplary oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0050] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0051] The ceramidase mixture can also be administered directly to the airways in the form of an aerosol. For use as an aerosol, the ceramidase in solution or suspension can be packaged in a pressurized aerosol container together with a suitable propellant, for example, a hydrocarbon propellant such as propane, butane or isobutane, together with a conventional adjuvant. The ceramidase mixture can also be administered in a non-pressurized form.

[0052] Representative delivery devices include, but are not limited to, nebulizers, atomizers, liposomes (including both active and passive drug delivery techniques) (see Wang et al., "pH-Sensitive Immunoliposomes Mediate Target-cell-specific Delivery and Controlled Expression of a Foreign Gene in Mouse," Proc. Nat'l Acad. Sci. USA 84:7851-5 (1987); Bangham et al., "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids," J. Mol. Biol. 13:238-52 (1965); U.S. Patent No. 5,653,996 to Hsu; U.S. Patent No. 5,643,599 to Lee et al.; U.S. Patent No. 5,885,613 to Holland et al.; U.S. Patent No. 5,631,237 to Dzau et al.; and Loughrey et al., each of which is incorporated by reference in its entirety. No. 5,059,421 to Wolff et al.; "The Use of Monoclonal Anti-Thy1 IgG1 for the Targeting of Liposomes to AKR-A Cells in Vitro and in Vivo," Biochim. Biophys. Acta 802:259-73 (1984)), transdermal patches, implants, implantable or injectable protein depot compositions, and syringes. Other delivery systems known to those skilled in the art can also be utilized to achieve the desired delivery of ceramidase to the desired organ, tissue, or cell.

[0053] Administration can be as frequent as needed and for a suitable period of time to provide effective treatment, for example, using a single sustained release dosage formulation or in multiple doses per day.

[0054] Treatment according to this and all aspects of the invention can be performed in vitro or in vivo. In vivo treatment includes, for example, embodiments in which the population of cells is present in a mammalian subject. In such embodiments, the population of cells can be either autologous (produced by the subject), allogeneic, or xenogeneic. Suitable subjects according to these embodiments include mammals, for example, human subjects, equine subjects, porcine subjects, feline subjects, and canine subjects.

[0055] In one embodiment, one or more additional agents that reduce ceramide levels can be administered with the ceramidase mixture, including, but not limited to, inhibitors of acid sphingomyelinase (e.g., amitriptyline (Becker et al., "Acid Sphingomyelinase Inhibitors Normalize Pulmonary Ceramide and Inflammation in Cystic Fibrosis," Am. J. Respir. Cell. Mol. Biol. 42:716-724 (2010)), which is incorporated herein by reference in its entirety), and inhibitors of ceramide synthase (e.g., Shiffmann et al., "Inhibitors of Specific Ceramide Synthases," Biochimie 94:558-565 (2012)), which is incorporated herein by reference in its entirety).

[0056] The effective amount of the therapeutic agent / cell population of the present invention administered to a subject depends on the type and severity of the disease or disorder, as well as individual characteristics such as general health, age, sex, weight, and drug tolerance. It also depends on the extent, severity, and type of the disease or disorder. Those skilled in the art can determine the appropriate dosage depending on these and other factors.

[0057] In one embodiment of the invention, the method comprises treating one or more mammalian cells ex vivo with said ceramidase mixture to promote cell survival. Cells whose survival may be promoted by this aspect of the invention include, but are not limited to, a wide range of cells (Obeid et al., "Programmed Cell Death Induced by Ceramide," Science 259:1769-71 (1993), which is incorporated by reference in its entirety), such as hepatocytes (Arora et al., "Ceramide Induces Hepatocyte Cell Death Through Disruption of Mitochondrial Function in the Rat," Hepatol. 25:958-63 (1997), which is incorporated by reference in its entirety), skin fibroblasts (Mizushima et al., "Ceramide, a Mediator of Interleukin 1, Tumour Necrosis Factor α, as Well as Fas Receptor Signalling, Induces Apoptosis of Rheumatoid Arthritis Synovial Cells," Ann. Rheum. Dis. 57:495-9 (1998), which is incorporated by reference in its entirety), and fibroblasts (Mizushima et al., "Ceramide, a Mediator of Interleukin 1, Tumour Necrosis Factor α, as Well as Fas Receptor Signalling, Induces Apoptosis of Rheumatoid Arthritis Synovial Cells," Ann. Rheum. Dis. 57:495-9 (1998), which is incorporated by reference in its entirety). (1998)), chondrocytes (MacRae et al., "Ceramide Inhibition of Chondrocyte Proliferation and Bone Growth Is IGF-I Independent," J. Endocrinol. 191(2):369-77 (2006) which is incorporated by reference in its entirety), lung epithelium (Chan & Goldkorn, "Ceramide Path in Human Lung Cell Death," Am. J. Respir. Cell Mol. Biol. 22(4):460-8 (2000) which is incorporated by reference in its entirety), red blood cells (Lang et al., "Ceramide Inhibition of Chondrocyte Proliferation and Bone Growth Is IGF-I Independent," J. Endocrinol. 191(2):369-77 (2006) which is incorporated by reference in its entirety),, "Mechanisms of Suicidal Erythrocyte Death," Cell. Physiol. Biochem. 15:195-202 (2005)), cardiomyocytes (Parra et al., "Changes in Mitochondrial Dynamics During Ceramide-induced Cardiomyocyte Early Apoptosis," Cardiovasc. Res. (2007), which is incorporated by reference in its entirety), and lymphocytes (Gombos et al., "Cholesterol and Sphingolipids as Lipid Organizers of the Immune Cells' Plasma Membrane: Their Impact on the Functions of MHC Molecules, Effector T-lymphocytes and T-cell Death," Immunol. Lett. 104(1-2):59-69 (2006), which is incorporated by reference in its entirety). The cell types that utilize the ceramidase apoptosis pathway include cells, eggs, embryos, neurons, sperm, synovial fibroblasts, and embryonic stem cells, including ceramide-induced cell death pathway (TRAIL and Ceramide), ...(2007) (rat); see also de Castro E Paula et al., "Ceramide Inhibits Development and Cytokinesis and Induces Apoptosis in Preimplantation Bovine Embryos," Mol. Reprod. Devel., DOI No. 10.1002 / mrd.20841 (2007) (bovine), each of which is incorporated herein by reference in its entirety). Thus, for each of the above cell types, suitable cells are expected to include those of human, monkey, mouse, rat, guinea pig, cow, horse, sheep, pig, dog and cat. The method may also extend the survival of eggs and / or embryos during in vitro fertilization procedures and facilitate the identification and selection of normal embryos for reimplantation, particularly for older human females and veterinary reproductive methods.

[0058] The cell according to this aspect of the present invention can be provided by a method that is clear to those skilled in the art.By way of example, the cell can be obtained from animals or existing ex vivo sources (e.g., tissue samples, cell cultures, etc.) using standard techniques.Treatment of cells ex vivo includes treating cells that exist in homogeneous cultures as well as cells that exist in heterogeneous cultures (e.g., tissue samples).

[0059] Inactive AC precursors and active ACs that can be used to prepare ceramidase mixtures in this and all aspects of the invention include, but are not limited to, those set forth above in Table 1. In this and all aspects of the invention (including the in vivo methods discussed below), the AC can be allogeneic (i.e., derived from the same species) or xenogenic (i.e., derived from a different species) to the cell or cells being treated.

[0060] One embodiment of this aspect of AC treatment relates to a method of generating chondrocytes with a ceramidase mixture, the method comprising the steps of selecting a population of cells having the potential to differentiate into chondrocytes, and treating the selected cell population with a ceramidase mixture to convert one or more of the cells in the selected population into chondrocytes.

[0061] Cells that have the potential to differentiate into chondrocytes include bone marrow cells, fibroblasts, mesenchymal stem cells, and / or fibroblasts (see Mizushima et al., "Ceramide, a Mediator of Interleukin 1, Tumour Necrosis Factor α, as Well as Fas Receptor Signaling, Induces Apoptosis of Rheumatoid Arthritis Synovial Cells," Ann. Rheum. Dis. 57:495-9 (1998), which is incorporated by reference in its entirety).

[0062] Chondrocytes according to this aspect of the invention include, but are not limited to, articular chondrocytes, nasal chondrocytes, tracheal chondrocytes, meniscal chondrocytes, and ear chondrocytes. These include, for example, mammalian chondrocytes, such as human chondrocytes, equine chondrocytes, porcine chondrocytes, feline chondrocytes, and canine chondrocytes. Preferably, the chondrocytes are primary chondrocytes.

[0063] Suitable cells according to this and all aspects of the invention include mammalian cells, such as human cells, equine cells, porcine cells, feline cells, and / or canine cells. Human cells are particularly preferred.

[0064] In this and all other aspects of the invention involving cell populations, both embodiments in which the cells are all of one cell type, as well as embodiments in which the population is a mixture of two or more cell types, are contemplated.

[0065] The ceramidase mixtures and methods of treating populations of cells with the ceramidase mixtures include all of those described above.

[0066] Another embodiment of this aspect of AC treatment relates to a method of promoting chondrogenesis with a ceramidase mixture, hi one embodiment, the method further comprises the steps of selecting a population of stem cells in need of differentiation into chondrocytes, treating the population of stem cells with a ceramidase mixture to enrich for mesenchymal stem cells in the stem cell population, and treating the enriched population of mesenchymal stem cells with the ceramidase mixture to promote differentiation of the mesenchymal stem cells into chondrocytes.

[0067] Suitable cell populations according to this aspect of the invention include mammalian cell populations, such as human cell populations, equine cell populations, porcine cell populations, feline cell populations, and / or canine cell populations. Human cell populations are particularly preferred.

[0068] Suitable stem cells according to this and all other aspects of the invention include bone marrow cells, fat cells, and skin cells. Additional stem cells according to this aspect of the invention include, but are not limited to, embryonic stem cells, somatic stem cells, induced pluripotent stem cells, totipotent stem cells, multipotent stem cells, and multipotent stem cells. Representative stem cells include, for example, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, endothelial progenitor cells, epithelial stem cells, epidermal stem cells, adipocytes, and cardiac stem cells. Suitable stem cells include, but are not limited to, mammalian cells, such as human, equine, porcine, feline, and canine bone marrow cells, fat cells, and skin cells. Human cells are particularly preferred.

[0069] Suitable chondrocytes are consistent with those described above. Alternatively, the differentiated mesenchymal stem cells can be primary cells such as, but not limited to, neural cells, hepatocytes, bone cells, lung cells and cardiomyocytes.

[0070] In at least one embodiment, the number of differentiated cells in the cell population is maintained. In at least one embodiment, the number of differentiated cells in the cell population is increased. As will be apparent to one of skill in the art, maintaining or increasing the overall number of differentiated cells in a population can be achieved by reducing or preventing dedifferentiation of already differentiated cells in the population, stimulating differentiation of undifferentiated cells in the population, or both.

[0071] The ceramidase mixtures and methods of treating populations of cells with the ceramidase mixtures include all of those described above.

[0072] A third aspect of the present invention relates to a method for producing a therapeutic composition, the method including the steps of providing a medium containing inactive AC precursors, incubating the medium under conditions effective to convert a portion of the inactive AC precursors to active AC, and recovering the incubated medium as a ceramidase mixture comprising inactive AC precursors and active AC.

[0073] The therapeutic composition of the present invention contains a recombinant protein that contains both an inactive AC precursor and an active AC. The recombinant protein of the present invention can be prepared for use in the above-mentioned method of the present invention using standard synthetic methods known in the art, including solid-phase peptide synthesis (Fmoc or Boc method) or solution-phase peptide synthesis. Alternatively, the protein of the present invention can be prepared using a recombinant expression system.

[0074] In general, the use of a recombinant expression system involves inserting a nucleic acid molecule encoding the amino acid sequence of a desired peptide into an expression system that is heterologous (i.e., not normally present) to the molecule. One or more desired nucleic acid molecules encoding the peptides of the invention may be inserted into a vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may encode the same or different peptides. The heterologous nucleic acid molecule is inserted into the expression system or vector in the sense forward orientation (5'→3') and in the correct reading frame relative to the promoter and any other 5' regulatory molecules.

[0075] Preparation of nucleic acid constructs can be performed using standard cloning techniques well known to those skilled in the art, as described in Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989). U.S. Patent No. 4,237,224 to Cohen and Boyer, incorporated herein by reference in its entirety, describes the generation of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced into suitable host cells by transformation.

[0076] Various genetic signals and processing events that control the level of many gene expression (e.g., DNA transcription and messenger RNA ("mRNA") transcription) can be incorporated into the nucleic acid construct to maximize peptide production. In order to express the cloned nucleic acid sequence encoding the desired recombinant protein, it is advantageous to obtain high levels of transcription using a strong promoter. Depending on the host system utilized, any one of a number of suitable promoters can be used. For example, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoters, including but not limited to lacUV5, ompF, bla, and lpp, as well as the P promoter of E. coli phage lambda. R and P LA promoter such as the promoter can be used to direct high levels of transcription of adjacent DNA segments. In addition, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA technology or other synthetic DNA technology can also be used to effect transcription of the inserted gene. Common promoters suitable for directing expression in mammalian cells include, but are not limited to, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR. Mammalian cells that can be used for the production of the recombinant proteins of the present invention include, for example, Chinese hamster ovary (CHO) cells, plant cells, chicken eggs, and human fibroblasts.

[0077] There are other specific initiation signals required for efficient gene transcription and translation in prokaryotic cells that can be included in nucleic acid constructs to maximize peptide production.Depending on the vector system and host utilized, any number of suitable transcription and / or translation elements can be used, including constitutive promoters, inducible promoters and repressible promoters, as well as minimal 5' promoter elements, enhancers or leader sequences.For a discussion of maximizing gene expression, see Roberts and Lauer, "Maximizing Gene Expression On a Plasmid Using Recombination In Vitro," Methods in Enzymology 68:473-82 (1979), which is incorporated herein by reference in its entirety.

[0078] The nucleic acid molecule encoding the recombinant protein of the invention, a selected promoter molecule, including but not limited to enhancer and leader sequences; an appropriate 3' regulatory region enabling transcription in the host, and any additional desired components, such as a reporter or marker gene, are cloned into the selected vector using cloning methods standard in the art, such as those described in Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley 1999), and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are incorporated by reference in their entireties.

[0079] Once the nucleic acid molecule encoding the peptide is cloned into an expression vector, it is ready to be integrated into the host. Recombinant molecules can be introduced into cells using standard cloning methods known in the art, such as those described in JOSEPH SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Harbor 1989), which is incorporated herein by reference in its entirety, including, but not limited to, transfection (if the host is eukaryotic), transduction, conjugation, mobilization, or via electroporation, lipofection, protoplast fusion, mobilization, or particle bombardment.

[0080] A variety of suitable host-vector systems can be utilized to express recombinant proteins or polypeptides. First, the vector system must be compatible with the host used. Host-vector systems include, but are not limited to, bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (e.g., baculovirus); and plant cells infected with bacteria.

[0081] Purified peptides may be obtained by a number of methods readily known in the art, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration, and reverse phase chromatography. The peptides are preferably produced in purified form (preferably at least about 80% or 85% pure, more preferably at least about 90% or 95% pure) by conventional techniques. Depending on whether the recombinant host cells have been made to secrete the peptide into the growth medium (see U.S. Patent No. 6,596,509 to Bauer et al., which is incorporated herein by reference in its entirety), the peptides may be isolated and purified by centrifugation (to separate cellular components from the supernatant containing the secreted peptide), followed by successive ammonium sulfate precipitation of the supernatant. In one embodiment of the invention, cells may be transformed with DNA encoding an AC and then cultured under conditions effective to produce a medium containing an inactive AC precursor. The fraction containing the peptide is subjected to gel filtration on an appropriately sized dextran or polyacrylamide column to separate the peptide from other proteins. If necessary, the peptide fraction can be further purified by other chromatographic procedures.

[0082] In one embodiment of the invention, incubation is carried out under conditions effective to reduce the rate of conversion of inactive AC precursors to active AC compared to the rate of conversion achieved when the incubation is carried out for 24 hours at pH 4 and at a temperature of 4° C. or 37° C. under otherwise matched conditions. Alternatively, incubation can be carried out under conditions effective to increase the rate of conversion of inactive AC precursors to active AC compared to the same conditions.

[0083] In some embodiments, the ceramidase mixture during incubation may have a pH greater than 4.0 and less than or equal to 6.5. The mixture may have a pH of, for example, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. In other embodiments, the temperature of the ceramidase mixture during incubation may be at least -30°C and less than 37°C. The temperature of the mixture may be, for example, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C. Alternatively, the mixture may be incubated under conditions of -30°C at pH 4.0, 4°C at pH 4.0 or 6.5, 25°C at pH 4.0, or 37°C at pH 4.0. The mixture may be incubated for a period of time, such as, but not limited to, approximately 30 minutes, 1 hour, 3 hours, 30 hours, or 300 hours.

[0084] During incubation in this aspect of the invention, the medium may be heated under conditions effective to eliminate acid sphingomyelinase activity, hi this embodiment, the medium may be heated to 60° C. for a period of time including, but not limited to, less than 20 minutes, 20-40 minutes, 40-60 minutes, or more than 60 minutes.

[0085] It should be understood that certain aspects, embodiments, aspects, variations and features of the present invention are described in various levels of detail to provide a substantial understanding of the present technology. Definitions of certain terms used in this specification are also provided. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. EXAMPLES

[0086] The following examples provide illustrative embodiments of the invention but are not intended to limit its scope.

[0087] Example 1 - Materials and Methods Preparation of rAC (Lot 6 and Lot 7) - Chinese hamster ovary cells overexpressing the human Asah1 gene were generated and rAC was purified from the culture medium (He et al., "Purification and Characterization of Recombinant, Human Acid Ceramidase. Catalytic Reactions and Interactions With Sphingomyelinase," J. Biol. Chem. 278:32978-32986 (2003), which is incorporated by reference in its entirety). No in vitro manipulations were performed after purification of Lot 6 (higher amount of inactive AC precursor). After purification of Lot 7 enzyme, rAC was incubated in citrate phosphate buffer at pH 4 for 3 hours at 37°C.

[0088] Comparison of Lot 6 and Lot 7 for Mouse Embryo Production - Methods for using rAC for mouse embryo production are described in Eliyahu et al., "Acid Ceramidase Improves the Quality of Oocytes and Embryos and the Outcome of In Vitro Fertilization," FASEB J. 24:1229-1238 (2010), which is incorporated by reference in its entirety. Sperm and mature oocytes were obtained from C57 Black mice and in vitro fertilization was performed using equal amounts of Lot 6 and Lot 7 enzyme.

[0089] Comparison of Lot 6 and Lot 7 for Improvement of Chondrocyte Phenotype in Rat Articular Chondrocytes - Equal amounts of Lot 6 and Lot 7 rAC were added to the culture medium of primary articular chondrocytes isolated from femurs. Cartilage was digested and cells were placed in culture medium supplemented and unsupplemented with rAC. After 3 days, the medium was replaced with medium without rAC. Cells were grown for an additional 2 weeks and levels of collagen 2 (a marker of mature articular chondrocytes) were measured by Western blotting.

[0090] Example 2 - rAC with less active AC is superior to rAC with more active AC rAC preparations with less active ceramidase are superior to those with more active ceramidase (Figures 1A-1C). As shown in Figure 1A, Western blot analysis showed the relative amount of active (α / β) rAC to inactive precursor rAC in two different bioreactor cultures (Lot 6 and Lot 7). Lot 7 had more active rAC than Lot 6. In vitro examples (IVF and chondrocytes) compared two rAC preparations with ratios of approximately 90:10 (inactive:active) (Lot 6) and approximately 80:20 (inactive:active) (Lot 7) (Figure 1A). Apoptosis was measured at 24 hours using standard morphological methods (e.g., membrane integrity, etc.) (Eliyahu et al., "Acid Ceramidase Improves the Quality of Oocytes and Embryos and the Outcome of In Vitro Fertilization," FASEB J. 24:1229-1238 (2010) which is incorporated by reference in its entirety).

[0091] Figure 1B summarizes the results for the formation potential of normal mouse embryos in Lot 6 and Lot 7. Lot 7 (containing more active rAC) produced more apoptotic embryos than Lot 6 (containing less active rAC). As can be seen in Figure 1B, the preparation with less active enzyme (Lot 6) produced better results in IVF (fewer apoptotic embryos). This was unexpected.

[0092] Lots 6 and 7 were further tested with cultured rat chondrocytes (Figure 1C). At 2 weeks, the amount of collagen 2 expression was analyzed using Western blotting. Cells cultured with lot 7 (higher active rAC) produced less collagen 2. Lot 6 was superior in maintaining the chondrocyte phenotype after expansion (Figure 1C, based on collagen 2 expression). This was unexpected.

[0093] The improved potency of rAC containing less active AC is hypothesized to be due to the shorter half-life of the active enzyme in cultured cells (and conversely, the longer half-life of the precursor).

[0094] To manipulate the ratio of inactive to active enzyme, the pH was adjusted to 4.0 and the preparation was incubated at 37° C. Under these conditions, an increase in active enzyme of approximately 10% was observed for every 3 hours of incubation. Thus, to convert the 90:10 inactive:active preparation to 100% activity, the preparation was incubated for 27 hours.

[0095] The key variable here is temperature. If the preparation is kept frozen (with pH adjusted), no conversion occurs. If the preparation is kept at 4°C (in a refrigerator), conversion proceeds, but at 1% of the efficiency at 37°C (300 hours required for a 10% increase in active enzyme versus 3 hours). If the preparation is kept at room temperature (25°C), it proceeds at 10% efficiency (30 hours required for a 10% increase). If the pH is not acidified, no conversion occurs at 4°C, with only 1% conversion at room temperature (300 hours required for a 10% increase).

[0096] Example 3 - Removal of contaminating acid sphingomyelinase activity from rAC We developed a method to remove contaminating acid sphingomyelinase activity (ASM) from rAC preparations, which requires incubation of the final rAC preparation at 60 °C for 10-20 min. This incubation does not affect the rAC (activity, or the ratio of active to inactive), but it does remove all ASM activity, which is essential for manufacturing (Figure 2).

[0097] Example 4 - Incubation of media containing recombinant human acid ceramidase at 37°C for various lengths of time Conditioned medium (DMEM, pH 6.8, containing 10% fetal bovine serum) was collected from Chinese hamster ovary cells overexpressing and secreting recombinant human acid ceramidase (rhAC) (He et al., "Purification and Characterization of Recombinant, Human Acid Ceramidase. Catalytic Reactions and Interactions With Acid Sphingomyelinase," J. Biol. Chem. 278:32978-32986 (2003), incorporated herein by reference in its entirety). Cells were grown to approximately 100% confluency in T-75 mm flasks, and then the medium was harvested after an additional 4 days of growth. The harvested medium was filtered through a 0.22 mm membrane to remove debris and placed in a 37°C incubator for various lengths of time. At the end of the incubation period, the medium was frozen at -20°C prior to assay. AC activity (Figure 3) was determined as previously described (He et al., Anal Biochem, 274:264 (1999), incorporated herein by reference in its entirety): reaction mixtures were incubated at 37°C for 1 hour. AC Western blot (Figure 4): 6.5 μl / lane was developed with mouse anti-human AC monoclonal antibody against the α subunit (1:300, #SC136275, Santa Cruz). The data show that in vitro incubation of medium containing rhAC at 37°C for 3 to 17 days results in the conversion of the inactive precursor to the active enzyme (represented by an increase in the α subunit and enzyme activity).

[0098] Example 5 - In vitro conversion of purified recombinant human acid ceramidase at 37°C Purified recombinant human AC (rhAC; 4 ug / ul in EMEM, pH 6.8) was isolated from the medium of overexpressing Chinese Hamster Ovary cells as previously described (He et al., "Purification and Characterization of Recombinant, Human Acid Ceramidase. Catalytic Reactions and Interactions With Acid Sphingomyelinase," J. Biol. Chem. 278:32978-32986 (2003), which is incorporated herein by reference in its entirety). AC Western Blot (Figure 5): 6.5 μl / lane was developed with mouse anti-human AC monoclonal antibody against the α subunit (1:300, #SC136275, Santa Cruz). The data show that in vitro incubation of purified rhAC at 37°C for 24 hours (Figure 5A) resulted in complete conversion of the precursor to its active form. Incubation for 1 to 8 hours (Figure 5B) showed a linear progression of conversion.

[0099] Although the invention has been described in detail for purposes of illustration, it will be understood that such details are for the purposes of illustration only and that modifications may be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

[0100] Sequence information SEQUENCE LISTING <110> Icahn School of Medicine at Mount Sinai <120> THERAPEUTIC ACID CERAMIDASE COMPOSITIONS AND METHODS OF MAKING AND USING THEM <150> US 61 / 784,594 <151> 2013-03-14 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 395 <212> PRT <213> Homo sapiens <400> 1 Met Pro Gly Arg Ser Cys Val Ala Leu Val Leu Leu Ala Ala Ala Val 1 5 10 15 Ser Cys Ala Val Ala Gln His Ala Pro Pro Trp Thr Glu Asp Cys Arg 20 25 30 Lys Ser Thr Tyr Pro Pro Ser Gly Pro Thr Tyr Arg Gly Ala Val Pro 35 40 45 Trp Tyr Thr Ile Asn Leu Asp Leu Pro Pro Tyr Lys Arg Trp His Glu 50 55 60 Leu Met Leu Asp Lys Ala Pro Met Leu Lys Val Ile Val Asn Ser Leu 65 70 75 80 Lys Asn Met Ile Asn Thr Phe Val Pro Ser Gly Lys Val Met Gln Val 85 90 95 Val Asp Glu Lys Leu Pro Gly Leu Leu Gly Asn Phe Pro Gly Pro Phe 100 105 110 Glu Glu Glu Met Lys Gly Ile Ala Ala Val Thr Asp Ile Pro Leu Gly 115 120 125 Glu Ile Ile Ser Phe Asn Ile Phe Tyr Glu Leu Phe Thr Ile Cys Thr 130 135 140 Ser Ile Val Ala Glu Asp Lys Lys Gly His Leu Ile His Gly Arg Asn 145 150 155 160 Met Asp Phe Gly Val Phe Leu Gly Trp Asn Ile Asn Asn Asp Thr Trp 165 170 175 Val Ile Thr Glu Gln Leu Lys Pro Leu Thr Val Asn Leu Asp Phe Gln 180 185 190 Arg Asn Asn Lys Thr Val Phe Lys Ala Ser Ser Phe Ala Gly Tyr Val 195 200 205 Gly Met Leu Thr Gly Phe Lys Pro Gly Leu Phe Ser Leu Thr Leu Asn 210 215 220 Glu Arg Phe Ser Ile Asn Gly Gly Tyr Leu Gly Ile Leu Glu Trp Ile 225 230 235 240 Leu Gly Lys Lys Asp Ala Met Trp Ile Gly Phe Leu Thr Arg Thr Val 245 250 255 Leu Glu Asn Ser Thr Ser Tyr Glu Glu Ala Lys Asn Leu Leu Thr Lys 260 265 270 Thr Lys Ile Leu Ala Pro Ala Tyr Phe Ile Leu Gly Gly Asn Gln Ser 275 280 285 Gly Glu Gly Cys Val Ile Thr Arg Asp Arg Lys Glu Ser Leu Asp Val 290 295 300 Tyr Glu Leu Asp Ala Lys Gln Gly Arg Trp Tyr Val Val Gln Thr Asn 305 310 315 320 Tyr Asp Arg Trp Lys His Pro Phe Phe Leu Asp Asp Arg Arg Thr Pro 325 330 335 Ala Lys Met Cys Leu Asn Arg Thr Ser Gln Glu Asn Ile Ser Phe Glu 340 345 350 Thr Met Tyr Asp Val Leu Ser Thr Lys Pro Val Leu Asn Lys Leu Thr 355 360 365 Val Tyr Thr Thr Leu Ile Asp Val Thr Lys Gly Gln Phe Glu Thr Tyr 370 375 380 Leu Arg Asp Cys Pro Asp Pro Cys Ile Gly Trp 385 390 395

Claims

1. providing a medium containing an inactive acid ceramidase precursor; incubating the medium under conditions effective to convert a portion of the inactive acid ceramidase precursor to active acid ceramidase; and recovering the incubated medium as a ceramidase mixture containing inactive acid ceramidase precursor and active acid ceramidase. A method for producing a therapeutic composition comprising:

2. The method of claim 1, wherein the incubating step is carried out under conditions effective to reduce the conversion rate of inactive acid ceramidase precursor to active acid ceramidase compared to the conversion rate achieved when the incubating step is carried out for 24 hours at pH 4 and at a temperature of 4°C or 37°C under otherwise consistent conditions.

3. The method of claim 2, wherein the pH of the ceramidase mixture during the incubating step is greater than 4.0 and less than or equal to 6.

5.

4. The method of claim 2, wherein the temperature of the ceramidase mixture during the incubating step is at least -30°C and less than 37°C.

5. 10. The method of claim 1, wherein the incubating step comprises heating the medium under conditions effective to remove acid sphingomyelinase activity.

6. Providing a culture medium comprises: Providing a cell transformed with DNA encoding acid ceramidase; culturing the transformed cells under conditions effective to produce a medium containing an inactive acid ceramidase precursor.

2. The method of claim 1, comprising:

7. The method of claim 1, wherein the incubating step is carried out under conditions effective to increase the conversion rate of inactive acid ceramidase precursor to active acid ceramidase compared to the conversion rate achieved when the incubating step is carried out for 24 hours at pH 4 and at a temperature of 4°C or 37°C under otherwise consistent conditions.

8. an inactive acid ceramidase precursor, and Active acid ceramidase a ceramidase mixture comprising: A pharma- ceutically acceptable carrier; A therapeutic composition comprising:

9. The therapeutic composition of claim 8, wherein in the ceramidase mixture, the amount of inactive acid ceramidase precursor is greater than the amount of active acid ceramidase.

10. The therapeutic composition of claim 8, wherein in the ceramidase mixture, the amount of inactive acid ceramidase precursor is less than the amount of active acid ceramidase.

11. 9. The therapeutic composition of claim 8, wherein the ceramidase mixture comprises 5-95% by weight of inactive acid ceramidase precursor and 95-5% by weight of active acid ceramidase.

12. 12. The therapeutic composition of claim 11, wherein the ceramidase mixture comprises 20-80% by weight of inactive acid ceramidase precursor and 80-20% by weight of active acid ceramidase.

13. 12. The therapeutic composition of claim 11, wherein the ceramidase mixture comprises 30-70% by weight of inactive acid ceramidase precursor and 70-30% by weight of active acid ceramidase.

14. 12. The therapeutic composition of claim 11, wherein the ceramidase mixture comprises 40-60% by weight of inactive acid ceramidase precursor and 60-40% by weight of active acid ceramidase.

15. 12. The therapeutic composition of claim 11, wherein the ceramidase mixture comprises 55-95% by weight of inactive acid ceramidase precursor and 45-5% by weight of active acid ceramidase.

16. 12. The therapeutic composition of claim 11, wherein the ceramidase mixture comprises 70-95% by weight of inactive acid ceramidase precursor and 30-5% by weight of active acid ceramidase.

17. 12. The therapeutic composition of claim 11, wherein the ceramidase mixture comprises 80-90% by weight of inactive acid ceramidase precursor and 20-10% by weight of active acid ceramidase.

18. 9. The therapeutic composition of claim 8, further comprising an adjuvant.

19. 19. The therapeutic composition of claim 18, wherein the adjuvant is selected from the group consisting of flagellin, Freund's complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, iscomatrix, and liposomal polycation DNA particles.

20. An improvement in a method of acid ceramidase treatment comprising formulating the acid ceramidase used in the treatment as a ceramidase mixture comprising an inactive acid ceramidase precursor and an active acid ceramidase.

21. The method of claim 20, comprising generating chondrocytes with the ceramidase mixture.

22. selecting a population of cells that have the potential to differentiate into chondrocytes; and treating a cell population of selected cells with said ceramidase mixture to convert one or more cells in the selected population into chondrocytes.

22. The method of claim 21, further comprising:

23. 23. The method of claim 22, wherein the cells in the selected population are mammalian cells.

24. 24. The method of claim 23, wherein the selected cell population comprises a population of bone marrow cells, mesenchymal stem cells and / or fibroblasts.

25. The method of claim 20, comprising promoting chondrogenesis with the ceramidase mixture.

26. selecting a population of stem cells in need of differentiation into chondrocytes; treating said population of stem cells with said ceramidase mixture to enrich for mesenchymal stem cells within said stem cell population; and treating the enriched population of mesenchymal stem cells with the ceramidase mixture to promote differentiation of the mesenchymal stem cells into chondrocytes.

26. The method of claim 25, comprising:

27. 27. The method of claim 26, wherein the mesenchymal stem cells are mammalian.

28. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for a joint disease or disorder.

29. 29. The method of claim 28, wherein the joint disease or disorder is selected from the group consisting of osteoarthritis, rheumatoid arthritis, mucopolysaccharidoses, degenerative joint diseases, joint injuries, and Farber lipogranulomatosis.

30. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for a neurodegenerative disease or disorder.

31. 31. The method of claim 30, wherein the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, prion diseases, Parkinson's disease, Huntington's disease, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, amyotrophic lateral sclerosis, inclusion body myositis, degenerative myopathy, spinocerebellar atrophy, metabolic neuropathies, diabetic neuropathies, endocrine neuropathies, orthostatic hypotension, brain injury, spinal cord injury, stroke, and motor neuron diseases such as spinal muscular atrophy.

32. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for a cardiac disease or disorder.

33. 33. The method of claim 32, wherein the cardiac disease or disorder is selected from the group consisting of cardiac disease, cardiac trauma, atherosclerosis, thrombosis, and cardiomyocyte apoptosis.

34. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for diabetes.

35. 21. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for pathogen infection in a subject having cystic fibrosis, COPD and / or open wounds.

36. 36. The method of claim 35, wherein the pathogen infection is a viral infection, a fungal infection, a prion infection, or a bacterial infection.

37. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for a ceramide-accumulating infection.

38. 21. The method of claim 20, comprising administering the ceramidase mixture to a subject to treat the subject for Farber disease.

39. The method of claim 20, wherein the treatment comprises administering the ceramidase mixture to the subject orally, by inhalation, by intranasal drops, topically, transdermally, parenterally, subcutaneously, intravenously, intraarterially, intramuscularly, intrapleurally, intraperitoneally, intrathecally, or by application to a mucous membrane.

40. 40. The method of claim 39, further comprising repeating said administration.

41. The method of claim 20, wherein the treatment is performed in vivo.

42. The method of claim 20, wherein the treatment is performed in vitro.

43. 21. The method of claim 20, further comprising administering one or more additional agents that reduce ceramide levels.

Images