JP2011084569A - Method of stimulating prosaposin receptor activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide medicament compositions for treating neurodegenerative or myelination disorders. <P>SOLUTION: Medicament compositions containing isolated DNA or RNA molecule encoding as an effective form, one selected from the group consisting of prosaposin, saposin C, peptide containing amino acids 18 to 29 from saposin C, and amino acid sequence shown in SEQ ID NO: 3, are prepared. By transfecting DNA or RNA molecule encoding prosaposin or prosaposin receptor agonist into cells, prosaposin receptor activity of the cells can be stimulated. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method of stimulating prosaposin receptor activity by transfecting cells with DNA or RNA encoding prosaposin or a prosaposin receptor agonist.

Prosaposin, a 70-kilodalton glycoprotein, is a precursor to a group (four types) of thermostable glycoproteins required for the hydrolysis of glycosphingolipids by lysosomal hydrolases (Kishimoto et al., J. Lipid Res., 33: 1255-1267, 1992). Prosaposin is proteolytically processed in lysosomes to produce saposins A, B, C and D that exist as four tandem domains in prosaposin (O'Brien et al., FASEB J., 5: 301-308, 1991 ). All four saposins are structurally similar to each other, including the arrangement of six cysteines, glycosylation sites, and conserved proline residues.

Various peptides derived from prosaposin, saposin C and saposin C (18-mer and 22-mer peptides) induce neurite outgrowth, as described in US Pat. No. 5,571,787 and international patent application PCT / US94 / 08453 Prevent nerve cell death and stimulate myelination. These proteins and peptides that are members of the “prosaposin receptor agonist” group also promote neuroprotection and can be used to treat various neurological disorders, including diabetic and taxol-induced neuropathies. Neurotrophic and myelinotropic activity is further restricted to the 12-mer region of saposin C (amino acids 18-29) (LIDNNKTEKEIL, SEQ ID NO: 1). Immunohistochemical studies have shown that prosaposin is localized in a large population of neurons, including upper and lower motor neurons. Prosaposin binds to cell surface receptors and stimulates ³² P incorporation into several proteins.

The use of neurotrophic peptides as therapeutics has inherent limitations, such as being susceptible to proteolysis. In the nervous system, it is desirable for therapeutic agents to cross the blood brain barrier. Although the above 18-mer can cross the blood-brain barrier, increased production of prosaposin, saposin C or related peptides by peripheral and / or central nervous system cells is associated with neurodegenerative and myelination disorders. Would be beneficial in preventing and treating.

Prosaposin receptors are described in US Pat. No. 5,571,787. This receptor also binds prosaposin receptor agonists including saposin C and the 12 mer described above. Methods for identifying prosaposin receptor agonists are described in US patent application Ser. No. 08 / 896,181. Since the discovery of neurotrophic, neuroprotective and myelinotropic activity of prosaposin receptor agonists, various researchers have demonstrated the in vitro and in vivo utility of such various agonists (O'Brien et al., Proc. Natl. Acad. Sci. USA 91: 9593-9396, 1994; O'Brien et al., FASEB J. 9: 681-685, 1994; Sano et al., Biochem. Biophys. Res. Commun. 204: 994-1000, Kotani et al., J. Neurochem. 66: 2197-2200, 1996; Kotani et al., J. Neurochem. 66: 2019-2025; Qi et al., J. Biol Chem. 271: 6874-6880, 1996).

Therapeutic treatment of disease by gene therapy involves the transient or stable insertion of new genetic information into the cell (see Crystal et al., Science, 270: 404-410, 1995 for an overview) ). Correction of genetic defects has been achieved by reintroduction of normal alleles encoding the desired function (Rosenberg et al., New Engl. J. Med., 323: 570, 1990; Boris-Lawrie et al., Ann. NY) Acad. Sci., 716: 59, 1994; Wivel et al., Science, 262: 533, 1993).

In order to be therapeutically effective in the treatment of neurodegenerative or myelination disorders, prosaposin and prosaposin receptor agonists need to be delivered transiently or stably to nerve cells. The present invention provides such a delivery method.

US Pat. No. 5,571,787 International Patent Application PCT / US94 / 08453 US patent application Ser. No. 08 / 896,181

Kishimoto et al., J. Lipid Res., 33: 1255-1267, 1992) O'Brien et al., Proc. Natl. Acad. Sci. USA 91: 9593-9396, 1994 O'Brien et al., FASEB J. 9: 681-685, 1994 Sano et al., Biochem. Biophys. Res. Commun. 204: 994-1000, 1994 Kotani et al., J. Neurochem. 66: 2197-2200, 1996 Kotani et al., J. Neurochem. 66: 2019-2025 Qi et al., J. Biol Chem. 271: 6874-6880, 1996. Crystal et al., Science 270: 404-410, 1995 Rosenberg et al., New Engl. J. Med., 323: 570, 1990 Boris-Lawrie et al., Ann. N.Y. Acad. Sci., 716: 59, 1994 Wivel et al., Science 262: 533, 1993 O'Brien et al., FASEB J. 5: 301-308, 1991

One aspect of the invention is the use of an isolated DNA or RNA molecule that effectively encodes a prosaposin or prosaposin receptor agonist in the treatment of a neurodegenerative disorder or a myelination disorder. The prosaposin receptor agonist is preferably selected from the group consisting of saposin C, a peptide comprising amino acids 18-29 of saposin C and a peptide comprising the amino acid sequence shown in SEQ ID NO: 3. In one aspect of this preferred embodiment, the DNA or RNA molecule is contained in an expression vector. The expression vector is preferably selected from the group consisting of adenoviral vectors, retroviral vectors, plasmid vectors and plasmid-liposome vectors. The disorder may be selected from the group consisting of multiple sclerosis, spinal cord injury, macular degeneration, amyotrophic lateral sclerosis, spinal muscular atrophy, post-polio syndrome, muscular dystrophy, peripheral neuropathy, stroke and peripheral nerve injury It is advantageous. In another aspect of this preferred embodiment, the disorder results from a disorder resulting from apoptosis induced by pro-inflammatory cytokines. The disorder is preferably cerebral infarction or myocardial infarction. In another aspect of this preferred embodiment, the medicament is selected from the group consisting of intravenous, intracerebral spinal, intramuscular, intradermal, subcutaneous, intracranial, epidural, topical, intranasal, transmucosal and oral. The shape is suitable for the route of administration. This medicament is preferably for human use. In another aspect of this preferred embodiment, the DNA or RNA molecule is transfected or infected into a neuronal cell obtained from a mammal. Advantageously, the DNA or RNA molecule is contained in an expression vector. The expression vector is preferably selected from the group selected from the group consisting of adenoviral vectors, retroviral vectors, plasmid vectors and plasmid-liposome vectors. In another aspect of this preferred embodiment, the cells are encapsulated. The encapsulated cells are preferably suitable for intrathecal or intracranial transplantation. In another aspect of this preferred embodiment, the cell is a neural stem cell. This stem cell is preferably a progenitor cell of a cell selected from the group consisting of neurons, astrocytes and oligodendrocytes. The medicament preferably contains a DNA molecule that encodes a prosaposin receptor agonist in an effective manner.

The present invention also provides viral vectors containing DNA or RNA molecules that effectively encode a prosaposin receptor agonist.

Another aspect of the present invention includes the steps of administering to a mammal an isolated DNA or RNA molecule that effectively encodes a prosaposin or a prosaposin receptor agonist, isolating a body fluid from the mammal, and said body fluid A method for producing a recombinant prosaposin or prosaposin receptor agonist, comprising the step of isolating the prosaposin or prosaposin receptor agonist from The body fluid is preferably selected from the group consisting of blood, milk, cerebrospinal fluid and semen.

The present invention relates to the delivery of DNA or RNA molecules encoding prosaposin, prosaposin receptor agonists (such as peptides containing amino acids 8 to 29 of saposin C and saposin C) or prosaposin receptor in vivo or ex vivo to neurons. Including methods of promoting neuroprotection and treating neurodegenerative or myelinating disorders. In the method of the present invention, the level of prosaposin / prosaposin receptor agonist is increased or the level of prosaposin receptor is increased so that the prosaposin receptor binds and enhances more circulating prosaposin / receptor agonists. A neuroprotective and / or neurite forming effect. A receptor agonist is defined as a compound that has an affinity for a cellular receptor that is normally stimulated by an endogenous substance and stimulates a physiological activity at that cellular receptor. Thus, receptor agonists bind to the receptor and stimulate its activity. In another preferred embodiment, a DNA or RNA molecule encoding a prosaposin receptor is delivered to a neuronal cell.

The natural 15-mer (TKLIDNNKTEKEILD, SEQ ID NO: 2) contained in human saposin C and encompassing the active neurite promoting region of SEQ ID NO: 1 was modified as follows to reduce its sensitivity to proteolysis in vivo. . Lys2 was replaced with D-ala to increase resistance to exopeptidase, lys8 was replaced with ala to increase resistance to trypsin digestion, and lys11 was deleted to increase resistance to trypsin digestion. In addition, asp15 was replaced with tyr to provide an iodination site. Thus, the resulting peptide TX14 (A) does not contain a trypsin or chymotrypsin cleavage site.

SEQ ID NO: 1 can be modified as follows and still retain neurotrophic and myelinotropic activity. Leu1 and Ile2 are essential, Asp3 is any amino acid, Asn4 and Asn5 are essential, Lys6 is any amino acid (preferably neither lysine nor arginine), Thr7 is essential, Glu8 is a charged amino acid, Lys9 is absent or charged amino acid , Glu10 is any charged amino acid, and Ile11 and Leu12 are any amino acid. With these guidelines, a consensus sequence of LIX ₁ NNX ₂ TX ₃ X ₄ X ₅ X ₆ X ₇ (SEQ ID NO: 3) is obtained.

DNA or RNA molecules encoding prosaposin or prosaposin receptor agonists are used for transient or stable transfection or infection of neuronal populations, which are either prosaposin or prosaposin when under the control of a constitutive promoter Receptor agonists are produced continuously, and prosaposin or a prosaposin receptor agonist is produced transiently when under the control of an inducible promoter. Increased intracellular production of prosaposin or a prosaposin receptor agonist stimulates prosaposin receptors and, inter alia, initiates a cascade of events that leads to neuroprotection, inhibition of neurodegeneration and inhibition of myelination, thereby increasing the level of prosaposin activity Increase.

In a preferred embodiment of the present invention, ex vivo expression of a neuronal cell obtained from an individual having a neurodegenerative disorder or a myelination disorder by placing DNA or RNA encoding a prosaposin or a prosaposin receptor agonist in an expression vector for eukaryotic cells. Perform transfection or infection. Such transfected or infected cells are then reintroduced into the patient. Such cells include Schwann cells, oligodendrocytes, glial cells, astrocytes and dendrocytes. These transfected cells can be transplanted into appropriate nerve sites including the brain, cerebrospinal fluid and peripheral nerves.

Neurons and glia can be derived from common fetal progenitor cells (McKay, Science 276: 66-71, 1997). The adult nervous system also contains pluripotent progenitors of neurons, astrocytes and oligodendrocytes (Reynolds et al., Science 255: 1707, 1992; Gritti et al., J. Neurosci. 16: 1091, 1995; Johe Et al., Genes Dev. 10: 3129, 1996). Adult CNS and fetal CNS cultured cells grown in vitro can both differentiate to display neuronal specific morphological and electrophysiological features, ie, regenerative synaptic structures (Gritti et al., Supra; Vicario-Abejon et al., Neuron 15: 105, 1995; McKay et al., Supra).

In another preferred embodiment, the pluripotent neural stem cell is obtained from a mammal (preferably human), cultured ex vivo, and transfected or infected with an expression vector encoding prosaposin or a prosaposin receptor agonist, Reintroduced into the mammal. The stem cells containing the protein or peptide are then differentiated into specific neuronal cell types, producing the peptide continuously.

Many expression vectors for eukaryotic cells as described above are known and commercially available. Standard techniques for constructing these expression vectors are well known and include references such as Sambrook et al. “Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) It can be found in any of the widely available experiments on recombinant DNA technology. Various methods can be used for ligating DNA fragments, and the selection depends on the nature and ends of the DNA fragments and can be easily determined by those having ordinary knowledge in the art.

Preferred expression vectors include viral vectors such as retroviral vectors, adenoviral vectors and adeno-associated viral vectors. Herpes virus vectors can also be used. Although these viruses do not integrate their genes into host DNA, they are attracted to neurons, and some of these neurons retain their viruses and the exogenous DNA sequences they contain in an almost harmless state. Therefore, the use of herpes virus vectors is desirable for treatment directed at neurological disorders. These vectors commonly used for gene therapy are discussed in detail by Miller et al. (FASEB J., 9: 190-199, 1995).

Expression vectors typically also contain a selectable marker, such as antibiotic resistance, to select cells expressing the therapeutic protein or peptide. The preferred method of ex vivo cell transfection is electroporation, but other methods such as calcium phosphate precipitation, microinjection and cell fusion are also contemplated. Gene delivery systems are described by Felgner et al. (Hum. Gene Ther. 8: 511-512, 1997). These gene delivery systems include lipid-based delivery systems (lipoplexes) and polycation-based delivery systems (polyesters). Plexes) and combinations thereof (lipopolyplexes), all of which are contemplated for use in the present invention.

Expression vector constructs containing DNA or RNA encoding prosaposin, saposin C, a neurotrophic peptide derived therefrom or a prosaposin receptor agonist can be administered to neuronal cells in vivo by two methods. In the first method, expression cassettes are introduced into cells obtained from individuals with neuropathy or myelination disorders by standard transfection or infection methods in the laboratory, and then the modified cells are introduced into the individual. Gene therapy is performed ex vivo, with the procedure of being returned. Alternatively, gene transfer can be performed in vivo by directly introducing the expression cassette into cells within the individual. In either case, the introduction process is usually facilitated by a vector that assists in delivering the cassette to an intracellular site that can function properly. Vector systems for gene therapy are discussed in detail by Hodgson (Exp. Opin. Ther. Patents, 5: 459-468, 1995). Expression cassettes usually contain a suitable heterologous promoter to drive expression of the gene. Such promoters are well known in the art and include, for example, the SV40 promoter and the cytomegalovirus (CMV) promoter. Any use of constitutive promoters, inducible promoters and tissue specific promoters is within the scope of the present invention. The expression vector may incorporate other nucleotide sequence elements to facilitate integration of the DNA into the chromosome, expression of the DNA and cloning of the vector. For example, if an enhancer is present upstream of the promoter or a terminator is present downstream of the coding region, the expression of DNA or RNA contained in the expression vector can be promoted.

In one embodiment of the invention, an expression vector containing the DNA or RNA of interest is injected directly into the blood. In another embodiment, the expression vector is administered by direct intracranial injection or cerebrospinal fluid injection. In either case, a pharmaceutically acceptable carrier such as phosphate buffered saline (PBS) or lactated Ringer's solution is used. Rather than an expression vector containing an appropriately encoded DNA segment, the DNA segment of pure DNA (“naked DNA”) in a pharmaceutically acceptable carrier may be injected. Alternatively, the composition can be administered to peripheral nerve tissue by direct local injection or systemic administration. Various conventional administration methods are contemplated, including intravenous, intramuscular, intradermal, subcutaneous, intracerebral spinal, intracranial, epidural, topical, intranasal, transmucosal and oral.

Transfected or infected cells that express prosaposin or a prosaposin receptor agonist can also be encapsulated within a biocompatible polymer membrane. Some examples of these materials include polyacrylonitrile vinyl chloride (PAN / PVC) acrylic copolymers, hydrogels such as alginate and agarose, mixed esters, cellulose, polytetrafluoroethylene / polypropylene (Lum et al., Diabetes, 40 : 1511-1516, 1991; Aebischer et al., Exp. Neurol., 111: 269-275, 1991; Liu et al., Hum. Gene Ther., 4: 291-301, 1993; Hill et al., Cell Transplantation, 1: 168, 1992) and polyethylene glycol (PEG) similar coating structures (US Pat. No. 5,529,914). Encapsulated cells are transplanted into animals with neurodegenerative or myelinating disorders. These permselective membranes allow the inflow of oxygen and other essential nutrients, but exclude antibodies and immune system cells, thus preventing the cells from being recognized as foreign and the transplanted cells to be neurotrophic proteins or peptides Makes it possible to produce constantly. For an overview of this technique, see Lanza et al., Surgery, 121: 1-9, 1997. For example, encapsulated cells are transplanted into the lumbar subarachnoid space of patients with amyotrophic lateral sclerosis and into the brain gap to treat Parkinson's disease. Encapsulation of genetically engineered cells and transplantation into mammals has been reported by several groups (Sagot et al., Eur. J. Neurosci., 7: 1313-1322, 1995; Sagen et al., J. Neurosci., 13: 2415-2423, 1993; Aebischer et al., Nature Medicine, 2: 696-699, 1996).

The composition can be packaged and administered as a unit dosage, such as an injectable composition or topical formulation, or as a sustained release composition at a dose corresponding to the daily dose administered to a patient. A septum vial containing a daily dose of active ingredient in PBS or lyophilized is an example of a unit dose.

In another preferred embodiment of the present invention, an expression vector containing the DNA or RNA of interest is locally administered to nerve cells in vivo by transplantation of the substance. For example, polylactic acid, polygalactic acid, regenerated collagen, multilamellar liposomes and many other conventional depots consist of bioerodible or biodegradable substances that can be formulated with bioactive compositions. When implanted, these substances gradually disintegrate and release the active substance to the surrounding tissue. In the present invention, bioerodible, biodegradable and other depots are particularly conceivable. Infusion pumps, matrix capture systems and transdermal delivery devices are also contemplated.

It may also be advantageous to encapsulate the DNA or RNA construct of the present invention in a micelle or liposome vector. Liposome encapsulation technology is well known. Liposomes can be directed to specific tissues, such as neural tissue, by using receptors, ligands or antibodies capable of binding the target tissue and can promote fusion with the plasma membrane. The manufacture of these formulations is well known in the art (Radin et al., Methods Enzymol, 98: 613-618, 1983). Another method for producing liposomes uses, for example, Lipofectin ^™ and Lipofectamin ^™ reagents (GIBCO BRL, Gaithersburg, MD). DNA or RNA encoding prosaposin or a prosaposin receptor agonist is conjugated to a receptor ligand such as transferrin that transports the gene to the cell surface and / or facilitates translocation into the cell by receptor-mediated endocytosis You can also

The gene therapy method of the present invention can be used for treatment of disorders of the central nervous system and treatment of disorders of the peripheral nervous system. Post-polio syndrome is characterized by muscle fatigue and decreased endurance, with muscle weakness and muscle atrophy. One cause of this disease is thought to be a type of spinal motor neuron injury similar to that occurring in amyotrophic lateral sclerosis. Peripheral nerve injury and peripheral neuropathy, such as those caused by diabetes or chemotherapy, constitute the most common peripheral neuropathy and can be treated using the methods of the present invention. Such neuropathies include spinal cord injury, macular degeneration, amyotrophic lateral sclerosis, spinal muscular atrophy, post-polio syndrome, muscular dystrophy, peripheral neuropathy, stroke and peripheral nerve injury. Any traumatic or ischemic damage to the central or peripheral nervous system can be treated using the methods of the present invention.

Cells can be treated as described above either in vivo or ex vivo to promote myelination or to prevent demyelination. In the ex vivo method, the transfected neuronal cells are returned to the individual and continually express the encoded prosaposin or prosaposin receptor agonist. There are several diseases of the central nervous system that cause demyelination of nerve fibers, such as multiple sclerosis, acute diffuse leukoencephalitis, progressive multifocal leukoencephalitis, metachromatic leukotrophy, and adrenoleukodystrophy. An example of a demyelinating disease of the peripheral nervous system is Guillain-Barre syndrome. These diseases can be treated by administration of expression vectors encoding cDNAs encoding prosaposin, saposin C, neurotrophic peptides derived from saposin C or prosaposin receptor agonists, slowing or stopping the progression of demyelination be able to.

Anoxia is not the final event that destroys heart tissue. This process initiates apoptosis promoted by pro-inflammatory cytokines. The method can also be used to suppress apoptosis that occurs during cerebral infarction, myocardial infarction and congestive heart failure. Prosaposin and prosaposin receptor agonists can be used to inhibit this apoptosis, as described in US Provisional Patent Application No. 60 / 058,352.

In another preferred embodiment, mammals transfected with expression vectors encoding recombinant prosaposin, saposin C or other prosaposin receptor agonists are used as a source of these substances. Prosaposin is an integral membrane protein and secreted protein found in various body fluids such as milk, cerebrospinal fluid and seminal plasma. Thus, in vivo produced prosaposin, saposin C or other prosaposin receptor agonists will be present in these body fluids, and those body fluids could be used as a source of these molecules. Prosaposin is purified as described in US Pat. No. 5,571,787. Prosaposin receptor agonists are purified using antibodies raised against the agonist by standard affinity chromatography methods.

Claims (2)

A non-human isolated DNA or RNA molecule that effectively encodes one selected from the group consisting of prosaposin, saposin C, a peptide of amino acids 18-29 of saposin C, and the amino acid sequence shown in SEQ ID NO: 3 A group comprising the steps of administering to a mammal, isolating a body fluid from the mammal, and the prosaposin, saposin C, a peptide of amino acids 18-29 of saposin C from the body fluid, and the amino acid sequence shown in SEQ ID NO: 3 A method for producing a pharmaceutical composition for the treatment of a neurodegenerative disorder or a myelination disorder, comprising the step of isolating one selected from more. The method according to claim 1, wherein the body fluid is selected from the group consisting of blood, milk, cerebrospinal fluid, and semen.