Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
  • C12N5/0619—Neurons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system

Definitions

• the invention relates to compositions and methods for increasing the survival of neurons.
• neurotrophic support is provided by co-culturing with astrocytes or by providing conditioned medium (CM) prepared from astrocytes.
• CM conditioned medium
• Astrocytes of ventral mesencephalic origin exert much greater efficacy in promoting the survival of ventral, mesencephalic dopaminergic neurons, compared with astrocytes from other regions of the CNS, such as the neostriatum and cerebral cortex.
• DIV chronic, mesencephalic cultures of 21 days i vitro (DIV) or longer, the percentage of dopaminergic neurons increases from 20% to 60%, coincident with proliferation of a monolayer of astrocytes.
• DIV did not GABAergic neurons
• VMCL-1 ventral mesencephalic cell line-1
• VA ATCC Accession No: , deposit date, September 18, 2000
• the CM prepared from these cells contains one or more neuronal survival factors that increase the survival of mesencephalic dopaminergic neurons at least 3-fold, and promotes their development as well.
• the potency of this neurotrophic activity and its low degree of toxicity on dopaminergic neurons in vitro are distinguishing features of the activity of VMCL-1 CM.
• using size fractionation techniques we have identified activities that elute at about 14-16 kilodaltons, 18-21 kilodaltons, and 25-35 kilodaltons.
• the VMCL-1 immortalized cell line does not require serum for its growth and thus allows us to identify the VMCL- 1 CM neuronal survival-promoting polypeptides.
• arginine-rich protein having a molecular weight of approximately 20 kilodaltons
• this protein is one of the factors in the VMCL-1 CM having the desired neuronal survival-promoting activity.
• the invention features methods for increasing the survival of neurons (e.g., dopaminergic neurons), as well as new polypeptides exhibiting such neuronal survival-promoting activity.
• the invention features a pharmaceutical composition that includes, as an active polypeptide, a substantially pure arginine-rich protein, and a pharmaceutically acceptable carrier.
• the arginine-rich protein is human arginine-rich protein (SEQ ID NO: 1).
• the invention features a substantially pure polypeptide having a molecular weight of about 14-16 kilodaltons that increases the survival of dopaminergic neurons.
• the invention features a substantially pure polypeptide having a molecular weight of about 18-21 kilodaltons that increases the survival of dopaminergic neurons.
• the invention features a substantially pure polypeptide having a molecular weight of about 25-35 kilodaltons that increases the survival of dopaminergic neurons.
• the polypeptides of the present invention can be obtained from a glial cell line, such as VMCL-1 or another immortalized type-1 astrocyte cell line.
• the survival of dopaminergic neurons is increased at least three-fold. More preferably, survival is increased at least four-fold, while most preferably, survival is increased at least five-fold.
• the invention features a method for increasing dopaminergic neuronal survival. The method includes contacting a dopaminergic neuron (either in vitro or in vivo) with a polypeptide of the first, second, third, or fourth aspect.
• a preferred polypeptide is human arginine-rich protein.
• the survival of dopaminergic neurons is increased at least three-fold, more preferably at least four-fold, and most preferably at least fivefold.
• the invention features a method for growing dopaminergic neurons for transplantation, including the step of culturing the neurons, or progenitor cells thereof, with an effective amount of a polypeptide of the first, second, third, or fourth aspect.
• a preferred polypeptide is human arginine-rich protein.
• the amount is sufficient to increase the survival of dopaminergic neurons by at least threefold, by at least four-fold, or even by at least five-fold.
• the invention features a method of treating a patient having a disease or disorder of the nervous system, this method includes the step of administering to the patient a survival-promoting amount of a substantially purified arginine-rich protein.
• the invention features another method for preventing dopaminergic neuronal cell death in a mammal.
• This method includes administering to the mammal a dopaminergic neuron survival- promoting amount of a substantially purified arginine-rich protein.
• a preferred mammal is a human.
• the invention also features a method of transplanting cells into the nervous system of a mammal, including (i) transplanting cells into the nervous system of the mammal; and (ii) administering a dopaminergic neuronal survival-promoting amount of arginine-rich protein (e.g., human arginine-rich protein) to the mammal (e.g., a human) in a time window from four hours before transplanting of the cells to four hours after transplantation of the cells.
• the time window is from two hours before transplantation of the cells to two hours after transplantation of the cells.
• the invention features another method of transplanting cells into the nervous system of a mammal.
• the cells are contacted with arginine-rich protein; and then transplanted into the nervous system of the mammal.
• these two steps are performed within four hours of each other.
• the invention features a method for the preparation of a dopaminergic neuronal survival-promoting polypeptide of the present invention, including culturing an immortalized type-1 astrocyte cell line under conditions permitting expression of the polypeptide.
• the invention features a substantially pure composition that includes a polypeptide that increases the survival of dopaminergic neurons, the polypeptide having a molecular weight of about 14- 16 kilodaltons, about 18-21 kilodaltons, or about 25-35 kilodaltons.
• Methods for treatment of diseases and disorders using the polypeptides or compositions of the invention are also features of the invention.
• a method of treatment of a disease or disorder of the nervous system e.g., Parkinson's disease
• the invention also features a method for preventing dopaminergic neuronal cell death by administering an effective amount of a polypeptide of the invention.
• Such a medicament is made by administering the polypeptide with a pharmaceutically acceptable carrier.
• the invention features the use of a polypeptide of the first, second, third, or fourth aspect in the manufacture of a medicament.
• the invention further features the use of a polypeptide as defined herein: (1) to immunize a mammal for producing antibodies, which can optionally be used for therapeutic or diagnostic purposes; (2) in a competitive assay to identify or quantify molecules having receptor binding characteristics corresponding to those of the polypeptide; (3) for contacting a sample with a polypeptide, as mentioned above, along with a receptor capable of binding specifically to the polypeptide for the purpose of detecting competitive inhibition of binding to the polypeptide; and (4) in an affinity isolation process, optionally affinity chromatography, for the separation of a corresponding receptor.
• the invention provides, from mammalian sources, new dopaminergic neuronal survival factors (e.g., arginine-rich protein) that are distinguishable from known factors. These factors promote the survival of dopaminergic neurons.
• the invention also provides processes for the preparation of these factors, and a method for defining activity of these and other factors. Therapeutic application of the factors is a further significant aspect of the invention.
• the invention features a polypeptide that increases the survival of dopaminergic neurons, the polypeptide having a molecular weight of about 14-16 kilodaltons or 25-35 kilodaltons (relative to proteins of known molecular weights, ranging from 15-102 kDa, run under the same conditions), as determined using a heparin sepharose CL-6B column (Sigma Chemicals, St. Louis, MO), and which has survival-promoting activity for dopaminergic neurons.
• heparin sepharose CL-6B column Sigma Chemicals, St. Louis, MO
• the molecular weight range limits quoted are not exact, but are subject to slight variations depending upon the source of the particular polypeptide factor. A variation of about 10% would not, for example, be impossible for material from another source.
• the invention features a pharmaceutical formulation that includes a polypeptide of the present invention formulated for pharmaceutical use, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
• a pharmaceutical formulation that includes a polypeptide of the present invention formulated for pharmaceutical use, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
• conventional pharmaceutical practice may be employed to provide suitable formulations or compositions.
• the pharmaceutical formulation includes cells (e.g., dopaminergic neurons or their progenitors) for transplantation.
• the invention features a method of transplanting cells (e.g., dopaminergic neurons or their progenitors) into the nervous system of a mammal.
• the method includes administering a polypeptide or composition of the present invention, in a pharmaceutically acceptable carrier to the mammal before, during, or after the cell transplantation.
• the polypeptide or composition is administered to the mammal in a time window from four hours before transplantation to four hours after transplantation. More preferably, the time window is from two hours before transplantation to two hours after transplantation. It is understood that the polypeptide or composition does not have to be present during the entire time window for it to prevent or decrease cell death.
• the invention features another method of transplanting cells into the nervous system of a mammal.
• the method includes contacting the cells to be transplanted with a polypeptide or composition of the present invention, in a pharmaceutically acceptable carrier before cell transplantation.
• the cells to be transplanted are contacted with the polypeptide or composition within four hours of transplantation, and, more preferably, within two hours of transplantation. It is understood that the polypeptide or composition does not have to be present for the entire time in order to prevent or decrease cell death following transplantation.
• Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
• Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors.
• Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
• the present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.
• the concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.
• the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v polypeptide for parenteral administration.
• General dose ranges are from about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day.
• the preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of the patient, the make up of the formulation, and the route of administration.
• dopaminergic neurons are, in large part, prevented from dying in the presence of the factors of the invention.
• Dopaminergic neurons of the mesencephalon die in patients having Parkinson's disease.
• the invention thus provides a treatment of Parkinson's disease.
• the use of the present factors in the treatment of disorders or diseases of the nervous system in which the loss of dopaminergic neurons is present or anticipated is included in the invention.
• the invention also features screening methods for identifying factors that potentiate or mimic arginine-rich neuronal survival-promoting activity.
• screening methods for potentiators the ability of candidate compounds to increase arginine-rich protein expression, stability, or biological activity is tested using standard techniques.
• a candidate compound that binds to arginine- rich protein may act as a potentiating agent.
• a mimetic e.g., a compound that binds the arginine-rich protein receptor
• substantially pure is meant that a polypeptide (e.g., arginine-rich protein) has been separated from the components that naturally accompany it.
• the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
• the polypeptide is an arginine-rich protein that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure.
• a substantially pure arginine-rich protein may be obtained, for example, by extraction from a natural source (e.g., a neuronal cell), by expression of a recombinant nucleic acid encoding an arginine-rich protein, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
• a natural source e.g., a neuronal cell
• Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
• polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state.
• a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
• substantially pure polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes.
• polypeptide or protein is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
• An arginine-rich protein that is a part of the invention includes a protein having dopaminergic neuronal survival-promoting activity and encoded by a nucleic acid that hybridizes at high stringency to the cDNA encoding human arginine-rich protein.
• a preferred arginine-rich protein is represented by the amino acid sequence of SEQ ID NO: 1.
• Nucleic acids that are a part of the invention include those nucleic acids encoding proteins having dopaminergic neuronal survival-promoting activity and that hybridize at high stringency to the one of the strands of the cDNA encoding human arginine-rich protein (SEQ ID NO: 5).
• a preferred nucleic acid is represented by the nucleotide sequence of SEQ ID NO: 5.
• substantially identical is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence.
• the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
• the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
• Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin
• Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
• high stringency conditions hybridization in 2X SSC at
• polypeptide or "factor” is meant a molecule having an activity that promotes the survival (or, conversely, prevents the death) of dopaminergic neurons in a standard cell survival assay.
• Compounds of the present invention have a molecular weight of about 14-16 kilodaltons, about 18-21 kilodaltons, or, alternatively, about 25-35 kilodaltons.
• GDNF glial cell-derived neurotrophic factor
• composition is meant a collection of polypeptides, including a polypeptide of the present invention.
• pharmaceutically acceptable carrier is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the polypeptide with which it is administered.
• physiological saline solution is physiological saline solution.
• physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy. (19th ed.) ed. A.R. Gennaro AR., 1995, Mack Publishing Company, Easton, PA. It will be understood that viral pathogens and toxic compounds that may inadvertently be included with a polypeptide or composition if the present invention may be inactivated or removed using any suitable method known in the art.
• a compound having "dopaminergic neuronal survival-promoting activity" is the presence of the compound increases survival of dopaminergic neurons by at least two-fold in a neuronal survival assay (such as the one described herein) relative to survival of dopaminergic neurons in the absence of the compound.
• the increase in the survival of dopaminergic neurons is by at least three-fold, more preferably by at least four-fold, and most preferably by at least five-fold.
• the assay can be an in vitro assay or an in vivo assay.
• the assay is an in vitro assay (see the section entitled "cell viability assay," infra)
• the present invention provides new methods and reagents for the prevention of neuronal cell death.
• the invention also provides pharmaceutical compositions for the treatment of neurological diseases or disorders of which aberrant neuronal cell death is one of the causes.
• Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
• VMCL-1 mesencephalic origin
• CM prepared from these cells contains one or more neuronal survival factors that increase the survival of mesencephalic dopaminergic neurons at least 3-fold, and promotes their development as well.
• VMCL-1 cell line a protein that we identified to be arginine-rich protein.
• This protein was purified as follows. A 3 L volume of VMCL-1 conditioned medium was prepared, and subjected to five sequential steps of column chromatography. At each purification step, each column fraction was tested for biological activity in the bioassay referred to above. An estimate of the effect of each fraction on neuronal survival was done at 24 hour intervals, over a period of five days, and rated on a scale of 1-10. After the fifth purification step, the biologically active fraction and an adjacent inactive fraction were analyzed by SDS-PAGE.
• the results of the SDS-PAGE analysis revealed a distinctive protein band in the 20 kDa range in the lane from the active fraction.
• the "active" band was excised and subjected to tryptic digest, and the molecular mass and sequence of each peptide above background were determined by mass spectrometry analysis.
• the following two peptide sequences were identified: DVTFSPATIE (SEQ ID NO: 3) and QIDLSTVDL (SEQ ID NO: 4).
• a search of the database identified a match for human arginine-rich protein (SEQ ID NO: 1) and its mouse orthologue (SEQ ID NO: 2).
• the predicted protein encoded by the mouse EST sequence is about 95% identical to the predicted human protein.
• arginine-rich protein is useful as a neurotrophic factor for the treatment of a neurodegenerative disease and for improving neuronal survival during or following transplantation into a human. Arginine-rich protein can also be used to improve the in vitro production of neurons for transplantation. In another use, arginine-rich protein allows for the identification of compounds that modulate or mimic its dopaminergic neuronal survival-promoting activity. The protein can also be used to identify its cognate receptor. Each of these uses is described in greater detail below.
• the effect of candidate molecules on arginine-rich protein-mediated regulation of neuronal survival may be measured at the level of translation by using standard protein detection techniques, such as western blotting or immunoprecipitation with an arginine-rich protein-specific antibody.
• Compounds that modulate the level of arginine-rich protein may be purified, or substantially purified, or may be one component of a mixture of compounds such as an extract or supernatant obtained from cells (Ausubel et al., supra).
• arginine-rich protein expression is measured in cells administered progressively smaller subsets of the compound pool (e.g., produced by standard purification techniques such as HPLC or FPLC) until a single compound or minimal number of effective compounds is demonstrated to arginine-rich protein expression.
• Compounds may also be directly screened for their ability to modulate arginine-rich protein-mediated neuronal survival.
• the amount of neuronal survival in the presence of a candidate compound is compared to the amount of neuronal survival in its absence, under equivalent conditions.
• the screen may begin with a pool of candidate compounds, from which one or more useful modulator compounds are isolated in a step-wise fashion. Survival-promoting activity may be measured by any standard assay.
• Another method for detecting compounds that modulate the activity of arginine-rich protein is to screen for compounds that interact physically with arginine-rich protein. These compounds may be detected by adapting interaction trap expression systems known in the art. These systems detect protein interactions using a transcriptional activation assay and are generally described by Gyuris et al. (Cell 75:791-803, 1993) and Field et al., (Nature 340:245-246, 1989). Alternatively, arginine-rich protein or biologically active fragments thereof can be labeled with 125 I Bolton-Hunter reagent (Bolton et al. Biochem. J. 133: 529, 1973).
• Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled arginine-rich protein, washed and any wells with labeled arginine-rich protein complex are assayed. Data obtained using different concentrations of arginine-rich protein are used to calculate values for the number, affinity, and association of arginine-rich protein with the candidate molecules.
• Compounds or molecules that function as modulators of arginine-rich protein neuronal survival-promoting activity may include peptide and non- peptide molecules such as those present in cell extracts, mammalian serum, or growth medium in which mammalian cells have been cultured.
• a molecule that modulates arginine-rich protein expression or arginine- rich protein-mediated biological activity such that there is an increase in neuronal cell survival is considered useful in the invention; such a molecule may be used, for example, as a therapeutic agent, as described below.
• arginine-rich protein as a neurotrophic factor that promotes the survival of dopaminergic neurons allows for its use for the therapeutic treatment of neurodegenerative diseases such as Parkinson's disease.
• arginine-rich protein to cells in order to prevent neuronal death, it is preferable to obtain sufficient amounts of pure recombinant arginine-rich protein from cultured cell systems that can express the protein.
• Preferred arginine-rich protein is human arginine-rich protein, but arginine-rich protein derived from other animals (e.g., pig, rat, mouse, dog, baboon, cow, and the like) can also be used. Delivery of the protein to the affected tissue can then be accomplished using appropriate packaging or administrating systems. Alternatively, small molecule analogs may be used and administered to act as arginine-rich protein agonists and in this manner produce a desired physiological effect.
• Gene therapy is another potential therapeutic approach in which normal copies of the gene encoding arginine-rich protein (or nucleic acid encoding arginine-rich protein sense RNA) is introduced into cells to successfully produce arginine-rich protein.
• the gene must be delivered to those cells in a form in which it can be taken up and encode for sufficient protein to provide effective neuronal survival-promoting activity.
• Retroviral vectors, adenoviral vectors, adenovirus-associated viral vectors, or other viral vectors with the appropriate tropism for neural cells may be used as a gene transfer delivery system for a therapeutic arginine-rich protein gene construct.
• Numerous vectors useful for this purpose are generally known (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev and Anderson, Curr. Opin. Biotech. 1:55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al, Nucl. Acid Res. and Mol. Biol.
• Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370, 1990; Anderson et al., U.S. Patent No. 5,399,346). Non-viral approaches may also be employed for the introduction of therapeutic DNA into the desired cells.
• arginine-rich protein may be introduced into a cell by lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al., Neurosci. Lett. 117: 259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Meth. Enzymol. 101:512, 1983), asialorosonucoid-polylysine conjugation (Wu et al., J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem. 264: 16985, 1989); or, less preferably, micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990).
• Gene transfer could also be achieved using non-viral means requiring infection in vitro. This would include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for delivery of DNA into a cell. Although these methods are available, many of these are of lower efficiency.
• vectors may be introduced into neural stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods which are well known in the art. Transplantation of normal genes into the affected cells of a patient can also be useful therapy. In this procedure, a normal arginine-rich protein gene is transferred into neurons or glia, either exogenously or endogenously to the patient. These cells are then injected into the targeted tissue(s).
• arginine-rich protein cDNA expression can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element.
• CMV human cytomegalovirus
• SV40 simian virus 40
• metallothionein promoters e.g., metallothionein promoters
• enhancers known to preferentially direct gene expression in neural cells may be used to direct arginine-rich protein expression.
• the enhancers used could include, without limitation, those that are characterized as tissue- or cell- specific in their expression.
• regulation may be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
• RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
• nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, th
• Another therapeutic approach within the invention involves administration of recombinant arginine-rich protein, either directly to the site of a potential or actual cell loss (for example, by injection) or systemically (for example, by any conventional recombinant protein administration technique).
• An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
• Such pharmaceutical compositions may consist of arginine-rich protein, antibodies to arginine-rich protein, mimetics, or agonists of arginine-rich protein.
• compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
• agent such as stabilizing compound
• the compositions may be administered to a patient alone, or in combination with other agents, drags or hormones.
• arginine-rich protein is administered to a subject at the site that cells are transplanted.
• the administration of arginine-rich protein can be performed before or after the transplantation of the cells. Preferably, the two steps are within about four hours of each other.
• arginine-rich protein can be repeatedly administered to the subject at various intervals before and/or after cell transplantation. This protective administration of arginine-rich protein may occur months or even years after the cell transplantation.
• arginine- rich protein can also be used in culture to improve the survival of neurons during their production any time prior to transplantation.
• the cells to be transplanted are suspended in a pharmaceutical carrier that also includes a survival-promoting amount of arginine-rich protein.
• Arginine-rich protein can also be administered to the cultures earlier in the process (e.g., as the neurons are first differentiating). It is understood that the neurons need not be primary dopaminergic neurons.
• Neurons e.g., dopaminergic neurons
• stem cells or any other cell capable of producing neurons can be cultured in the presence of arginine-rich protein during their production and maintenance.
• arginine-rich protein While human arginine-rich protein is preferred for use in the methods described herein, arginine-rich protein has been identified in numerous species, including rat, mouse, and cow. One in the art will recognize that the identification of arginine-rich protein from other animals can be readily performed using standard methods. Any protein having dopaminergic neuronal survival-promoting activity and encoded by a nucleic acid that hybridizes to the cDNA encoding human arginine-rich protein is considered part of the invention.
• Antibodies which specifically bind arginine-rich protein may be used for the diagnosis of conditions or diseases characterized by alterations in the levels of arginine-rich protein, or in assays to monitor patients being treated with arginine-rich protein.
• the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics.
• Diagnostic assays for arginine-rich protein include methods which utilize the antibody and a label to detect arginine-rich protein in human body fluids or extracts of cells or tissues.
• the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
• a wide variety of reporter molecules which are known in the art may be used, several of which are described herein.
• a variety of protocols including ELISA, RIA, and FACS are known in the art for measuring arginine-rich protein art and provide a basis for diagnosing altered or abnormal levels of arginine-rich protein expression.
• Normal or standard values for arginine-rich protein expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to arginine-rich protein under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric, means. Quantities of arginine-rich protein expressed in subject, control and disease, samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
• the nucleic acid sequences encoding arginine-rich protein may also be used for diagnostic purposes.
• the nucleic acid sequences which may be used include antisense RNA and DNA molecules, and oligonucleotide sequences.
• the nucleic acid sequences may be used to detect and quantitate gene expression in biopsied tissues in which expression of arginine-rich protein may be correlated with disease.
• the diagnostic assay may be used to distinguish between absence, presence, and excess expression of arginine-rich protein, and to monitor regulation of arginine-rich protein levels during therapeutic intervention.
• Nucleic acid sequences encoding arginine-rich protein may be used for the diagnosis of conditions or diseases which are associated with altered expression of arginine-rich protein.
• the nucleic acid sequences encoding arginine-rich protein may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pIN, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered arginine-rich protein expression. Such qualitative or quantitative methods are well known in the art.
• the nucleotide sequences encoding arginine-rich protein may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding arginine-rich protein in the sample indicates the presence of the associated disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
• a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes arginine-rich protein, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
• hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
• the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
• Example 1 Production and Analysis of VMCL-1 Cells
• the VMCL-1 cell line was made as follows. Rat E14 mesencephalic cells, approximately 2-3% of which are glioblasts, were incubated in medium containing 10% (v/v) fetal bovine serum for 12 hours and subsequently expanded in a serum-free medium, containing basic fibroblast growth factor (bFGF) as a mitogen. After more than 15 DIV, several islets of proliferating, glial-like cells were observed. Following isolation and passaging, the cells
• bFGF basic fibroblast growth factor
• VMCL-1 cells proliferated rapidly in either a serum-free or serum-containing growth medium. Subsequent immunocytochemical analysis showed that they stained positive for two astrocytic markers, GFAP and vimentin, and negative for markers of oligodendroglial or neuronal lineages, including A2B5, 04, GalC and MAP2.
• GFAP astrocytic markers
• vimentin markers of oligodendroglial or neuronal lineages
• A2B5 markers of oligodendroglial or neuronal lineages
• Serum-free CM prepared from the VMCL-1 cells, caused increased survival and differentiation of E14 mesencephalic dopaminergic neurons in culture. These actions are similar to those exerted by CM derived from primary, mesencephalic type-1 astrocytes.
• the expression of mesencephalic region-specific genes e.g., wnt-1, en-1, en-2, pax-2, pax-5 and pax-8, was similar between VMCL-1 cells and primary, type-1 astrocytes of E14 ventral mesencephalic origin. In both, wnt-1 was expressed strongly, and en-1 less strongly, supporting an expression pattern expected of their mesencephalic origin.
• a chromosomal analysis showed that 70% of the cells were heteroploid, and of these, 50% were tetraploid. No apparent decline in prohferative capacity has been observed after more than twenty-five passages. The properties of this cell line are consistent with those of an immortalized, type-1 astrocyte.
• the VMCL-1 cells have a distinctly non-neuronal, glial-like morphology, but lack the large, flattened shape that is typical of type-1 astrocytes in culture. Immunocytochemical analysis demonstrated that they stained positive for GFAP and vimentin, and negative for MAP2, A2B5 and 04. The cells were therefore not of the oligodendrocyte lineage. On the basis of a negative reaction to A2B5 and their morphological characteristics they were also not type-2 astrocytes. The classification that is supported by the immunocytochemical evidence is of type-1 astrocytes, although, as noted, these cells lack the classical morphological traits of primary type-1 astrocytes in culture.
• Example 2 Action of VMCL-1 CM on E14 Dopaminergic Neurons in Culture
• VMCL-1 CM was tested at 0, 5, 20 and 50% v/v, for its ability to influence survival and development of E14 mesencephalic dopaminergic neurons in culture.
• the cultures were primed with 10% fetal bovine serum (FBS) for 12 hours, then grown in a serum-free growth medium thereafter, until they were stained and analyzed after 7 DIV.
• FBS fetal bovine serum
• the CM increased survival by 5-fold. In contrast, there was no significant increase in non-dopaminergic neuronal survival.
• the profile of the biological action of this putative factor is quite different from that of CM derived from the B49 glioma cell line, the source of GDNF (Lin et al., Science 260: 1130-1132).
• en-1 The degree of expression of en-1 was similar in primary astrocytes and VMCL-1 cells, although at a lower level versus expression in E13 and E14 ventral mesencephalic tissue.
• en-2, pax-5 and pax-8 were not expressed in either primary astrocytes or VMCL-1.
• Pax-2 was expressed in E13 but not E14 ventral mesencephalon, and in primary astrocytes, but not in VMCL- 1.
• Chromosomes were counted in 34 cells. Of these, 9 had a count of 42, the diploid number for rat. Of the 25 cells that were heteroploid, 12/25 or 48% were in the tetraploid range. Hyperdiploid (counts of 43-48) and hypodiploid (counts of 39-41) cells each accounted for 20% of the population, while 12% of the cells had structurally rearranged chromosomes.
• VMCL-1 CM The selective action of VMCL-1 CM in increasing the survival of dopaminergic neurons in culture provides a potential clinical use for the molecule(s) produced by this cell line.
• the action exerted by VMCL-1 CM mirrors almost exactly that of CM prepared from mesencephalic, primary type-1 astrocytes (Takeshima et al., J. Neurosci. 14: 4769-4779, 1994).
• Example 5 Production of Type-1 Astrocyte-Conditioned Medium E16 type-1 astrocyte CM (10 L) was filtered and applied to a heparin sepharose CL-6B column (bed volume 80 mL) which had previously been equilibrated with 20 mM Tris-HCl (Mallinckrodt Chemical Co. Paris, KY) pH 7.6 containing 0.2 M NaCl. After washing with equilibration buffer, bound proteins were eluted from the column with a linear gradient of 0.2 M - 2 M NaCl in 20 mM Tris-HCl pH 7.6 (400 mL total volume, flow rate 100 mL/hr).
• Fractions were collected using a Pharmacia LKB fraction collector and absorbance was measured at 280 nm (Sargent- Welch PU 8600 UV/VIS Spectrophotometer). A 1 mL aliquot was taken from each fraction, pooled into groups of four (4 mL total volume) and desalted using Centricon-10® membrane concentrators (Millipore, Bedford, MA). Samples were diluted 1:4 in defined medium and bioassayed for dopaminergic activity. Active fractions were pooled (80 mL total volume) and then applied to a G-75 Sephadex® column (70 x 2.5 cm, Pharmacia Biotechnology Ltd., Cambridge, UK) which had been pre-equilibrated with 50 mM ammonium formate pH 7.4.
• Proteins were separated with the same buffer (flow rate, 75 mL/hr) and absorbance was measured at 280 nm. A 1 mL aliquot was taken from each fraction, pooled into groups of four (4 mL total volume), concentrated by lyopholyzation and reconstituted in 1 mL distilled water volume. Samples were then diluted 1:4 in defined medium for dopaminergic bioassay. Those with neurotrophic activity were further bioassayed as individual fractions.
• VMCL-1 CM An important distinguishing feature of VMCL-1 CM is that it promotes predominantly the survival of dopaminergic neurons, compared with the survival of GABAergic, serotonergic, and other neuronal phenotypes present in the culture. This claim of specificity is also made for GDNF. The results of extensive testing have demonstrated, however, that the VMCL-1 -derived compound is not GDNF.
• a pcDNA3-hARP expression construct containing the human arginine-rich protein cDNA under the control of the CMV promoter, is transiently transfected into COS cells and the conditioned medium tested for dopaminergic neuronal survival-promoting activity.
• a myc tag can be inserted to facilitate purification and immunodetection of the recombinant protein.
• Example 6 Isolation and Purification of a Protein having Dopaminergic Neuronal Survival-Promoting Activity
• the purification protocol was performed as follows. All salts used were of the highest purity and obtained from Sigma Chemical Co. All buffers were freshly prepared and filtered via 0.2 ⁇ M filter (GP Express vacuum-driven system from Millipore)
• VMCL-1 conditioned medium Three liters of VMCL-1 conditioned medium was diluted with an equal volume of 20 mM sodium phosphate buffer, pH 7.2 at room temperature, filtered, and concentrated to 550 mL volume with 5K PREP/SCALE-TFF 2.5 ft 2 cartridge (Millipore).
• the concentrated material was loaded onto a 10 mL Heparin-Sepharose column assembled from 2 x 5 mL HiTrap Heparin columns (Pharmacia Biotech) and pre-equilibrated with at least 100 mL of 10 mM sodium phosphate buffer, pH 7.2 (buffer A). After the loading was complete, the column was washed with 100 mL of buffer A. A total of 10 fractions were eluted with buffer B (buffer A plus 1 M sodium chloride) in about 3 mL volumes each. A 300 ⁇ L sample was withdrawn for analysis.
• the active protein was eluted in a 15 mL volume after 84 ml of GF buffer was passed through the column and corresponded to an approximately 20-30 kDa elution region based on the column calibration data obtained with protein standards (Bio-Rad).
• Step 3 Ceramic Hydroxyapatite column chromatography (room temperature; FPLC system)
• the active fractions from step 2 that corresponded to the 20-30 kDa elution region were pooled and concentrated to 7.5 mL, using a Centricon Plus-20 concentrator (5,000 MWCO), dialyzed overnight at 4°C against 2 L of 10 mM sodium phosphate buffer, pH 7.2 (buffer A) and loaded (via Superloop) onto a 1 mL pre-packed ceramic hydroxyapatite (Type I, Bio-Rad) column equiUbrated with buffer A.
• a Centricon Plus-20 concentrator 5,000 MWCO
• buffer A containing 1.0 M NaCl was applied from 0 to 100%.
• One milliliter fractions were collected and analyzed for activity.
• the active protein was eluted as a broad peak within the region of gradient corresponding to 0.4-0.8 M NaCl concentration.
• Step 4 Anion-exchange column chromatography (room temperature; FPLC system)
• the active protein fraction from Step 4 (7 mL of total volume) was concentrated down to nearly zero volume (about 1 ⁇ L) using Centricon Plus-20 concentrator (5,000 MWCO) and reconstituted in 0.6 mL of 10 mM sodium phosphate buffer, pH 7.2.
• the reconstituted material 70 ⁇ L, analytical run) was loaded onto BioSil 125 HPLC gel-filtration column (Bio-Rad) equilibrated with 20 mM sodium phosphate buffer, pH 7.2 (GF buffer).
• the chromatography was conducted using HP 1100 Series HPLC system (Hewlett-Packard). The eluate was collected in 120 ⁇ L fractions and analyzed for activity and protein content (SDS-PAGE).
• both P-l and P-2 were dried on SpeedVac, reconstituted (each) in 10 ⁇ L of freshly prepared SDS-PAGE reducing sample buffer (Bio-Rad), incubated for one minute in a boiling water bath and loaded onto a 12% SDS-PAGE gel. After electrophoresis was complete, the gel was fixed in methanol/acetic acid/water solution (50: 10:40) for 40 minutes at room temperature, washed three times with nanopure water, and stained overnight with GelCode Blue Stain Reagent (Pierce) at room temperature.
• SDS-PAGE reducing sample buffer Bio-Rad
• Example 7 Analysis of In-gel Digested Fragments bv nESI-MS/MS
• the protein gel bands were incubated with 100 mM ammonium bicarbonate in 30% acetonitrile (aq.) at room temperature for 1 hour in order to remove the colloidal Coomassie blue stain.
• the destaining solution was replaced a number of times until the dye was completely removed.
• the gel pieces were then covered with deionized water ( ⁇ 200 ⁇ L) and shaken for 10 minutes. The gel pieces were dehydrated in acetonitrile and, after removing the excess liquid, were dried completely on a centrifugal evaporator.
• the gel bands were rehydrated with 20 ⁇ L of 50 mM ammonium bicarbonate, pH 8.3, containing 200 ng of modified trypsin (Promega, Madison, WI).
• the gel pieces were covered with 50 mM ammonium bicarbonate, pH 8.3 (approximately 50 ⁇ L), and were incubated overnight at 37°C.
• the digest solutions were then transferred to clean eppendorf tubes and the gel pieces were sonicated for 30 minutes in 50-100 ⁇ L of 5% acetic acid (aq).
• the extract solutions were combined with the digest solutions and evaporated to dryness on a centrifugal evaporator.
• the in-gel digest extracts were first analyzed by matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOFMS) using a Voyager Elite STR MALDI-TOFMS instrument (Applied Biosystems Inc., Framingham, MA).
• the extracts were dissolved in 5 ⁇ L of 50% acetonitrile, 1% acetic acid. Dihydroxybenzoic acid was used as the matrix and spectra were acquired in positive ion, reflection mode. Approximately one fifth of each sample was used for this analysis. These spectra provided the masses of the peptides in the digest extracts which were then used to search an in-house, non-redundant protein sequence database, a process called peptide mass fingerprinting.
• nESI-MS/MS nanoelectrospray ionization - tandem mass spectrometry
• MS/MS spectra were typically acquired every second over a period of two minutes.
• the MS/MS spectra were used to search an in-house non-redundant protein sequence database using partial sequence tags (i.e., only the peptide mass and a few fragment ions are used to search the database). If the protein was not identified by this procedure then the amino acid sequences of two or more peptides were determined as fully as possible from the MS/MS spectra. These sequences were used to carry out BLAST searches on NCBFs protein, nucleotide and EST sequence databases.
• arginine-rich protein is likely to be a main protein that is responsible for activity observed in P-l, while the necessity of the presence of nexin for activity cannot be excluded.
• a type-1 astrocyte-derived cell line having the same or similar neuronal survival- promoting activity, from aborted human tissue.
• the corresponding gestational age of E14 is approximately 9-10 weeks, although other ages are also likely to be successful.
• the human compound is identified using standard protein purification techniques, as described herein.
• ventral mesencephalic tissue is dissected from human fetal brain.
• the dissection is preferably performed under sterile conditions in salt solution (e.g., Hank's balanced salt solution (HBSS)), at pH 7.4.
• salt solution e.g., Hank's balanced salt solution (HBSS)
• HBSS Hank's balanced salt solution
• VM ventral mesencephalon
• the ventral mesencephalon (VM) with the floor plate intact is localized, micro-dissected in a culture dish in fresh, salt solution, thoroughly cleared of non-neural tissue, and stored in salt solution.
• the salt solution is removed, and the tissue rinsed with two changes of growth medium (e.g., N2), then dispersed in 2.0 mL of growth medium, which is used in all subsequent procedures.
• the tissue is then triturated.
• the cells are centrifuged (1,000 rpm, 2 minutes), the medium aspirated, and the pellet dispersed in growth medium. The cells are counted using a hemocytometer, and the density adjusted approximately 2.5 x 10 5 cells/mL. The cells are then dispersed in cultures dishes previously coated polyornithine (15 mg/mL) and fibronectin (1.0 mg/mL), at a density of 5.0 x 10 4 cells/cm 2 . The dishes are transferred to the incubator (37°C, 5% C0 2 , 100% humidity). bFGF (10 ng/mL) is added daily, and the medium changed every second day.
• a human mesencephalic type-1 astrocyte cell line may be established from primary cultures by transforming the cells with a DNA construct containing the oncogenic early region of S V40, under the transcriptional control of a human GFAP promoter, and a selectable marker (e.g., pPGK-neo, which contains the murine phosphoglycerate kinase gene promoter). The transformants are selected with G418 and cloned.
• the dissected tissue was collected and pooled in oxygenated, cold (4°C), HBSS or medium containing 10% fetal bovine serum (Biofluids Laboratories, Rockville, MD), depending on the purpose of the experiment.
• Pregnant rats were killed by exposure to C0 2 on the fourteenth gestational day (i.e., E14), the abdominal region was cleaned with 70% EtOH, a laparotomy was performed, and the fetuses collected and pooled in cold Dulbecco's phosphate-buffered saline (DPBS), pH 7.4, without Ca 2+ or Mg 2+ .
• DPBS cold Dulbecco's phosphate-buffered saline
• the intact brain was then removed, a cut was made between the diencephalon and mesencephalon, and the tectum slit medially and spread out laterally.
• the ventral, medial 1.0 mm 3 block of tissue comprising the mesencephalic dopaminergic region was isolated. Dissected tissue blocks were pooled in cold (4°C), oxygenated medium. The tissue was triturated without prior digestion. Alternatively, the cells were incubated in L-15 growth medium containing papain (Sigma Chemical Co.), 10 U/mL, at 37°C, for 15 minutes, washed (3 x 2 mL) with medium, and triturated using only the needle and syringe.
• papain Sigma Chemical Co.
• the dispersed cells were transferred to 1.5 mL Eppendorf tubes (1.0 mL / tube), and centrifuged at -600 g for 2 minutes. The use of higher centrifugation speeds for longer periods results in contamination of the cultures with debris and, as a result, suboptimal growth of the cells.
• the medium was carefully aspirated, and the cells resuspended in fresh medium and counted using a hemocytometer. All procedures, from laparotomy to plating were completed within 2 hours. In a typical experiment, one litter of 10-15 fetuses yielded 1.0 x 10 5 cells/fetus, or 1.0 x 10 6 - 1.5 x 10 6 cells/litter.
• the cultures were incubated for 30 minutes at 37°C, in 5% C0 2 at 100% humidity, to allow the cells to attach, and 375 ⁇ L of growth medium was then added to each well.
• the medium was changed after the first 12 hours, and approximately half of the medium was changed every second day thereafter.
• a two-color fluorescence cell viability assay kit (Live/Dead Viability/Cytotoxicity Assay Kits, #L-3224, Molecular Probes, Inc., Eugene, OR) was used to identify live and dead cells prior to plating and in cultures. Live and dead cells fluoresced green and red, respectively, giving two positive indicators of viability. Ethidium homodimer and calcein-AM were diluted with DPBS to give final concentrations of 3.8 ⁇ M and 2.0 ⁇ M, respectively. Evaluation of cell viability was done before plating.
• a cell suspension was incubated for 15 minutes with an equal volume of dye (typically 20 ⁇ L) on glass slides at room temperature in a dark, humid chamber, coverslipped, and then examined with a fluorescent microscope using FITC optics. Cell viability just before plating was about 95%.
• dye typically 20 ⁇ L
• the serum-free medium used consisted of equal volumes of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 (Gibco, Grand Island, N.Y.;
• conditioned medium from the VMCL-1 cell line 2.0 x 10 6 cells were plated in a 15 cm uncoated culture dish, in 20 mL of growth medium containing 1.0% of FBS. At 80% confluence, the medium was aspirated and the cells washed once with serum-free medium. 20 mL of serum-free N2 medium without albumin was added, and conditioning allowed to continue for 48 hours. During this time, the cells usually expanded to 100% confluence. The medium was aspirated, pooled in 50 mL tubes, centrifuged (15,000 rpm for 20 minutes) and subsequently pooled in a 1.0 L plastic bottle.
• CM CM
• VMCL-1 CM can be made in large quantities using standard industrial cell culture techniques known to those in the art.
• Type-1 astrocytes were prepared as follows. E16 rat fetal brain stem was dissected in cold DPBS, and the mesencephalic region transferred to astrocyte culture medium (DMEM/Ham's F-12, 1 : 1, 15% FBS, 4.0 mM glutamine, 30 nM sodium selenite, penicillin, and streptomycin). Cells were dispersed by trituration in 2 mL of fresh medium using an 18-gauge needle fitted to a syringe. Cells were centrifuged for 5 minutes at 2,000 rpm in a centrifuge, re-suspended in medium, and triturated again.
• Cells were dispersed by trituration in 2 mL of fresh medium using an 18-gauge needle fitted to a syringe. Cell
• the final cell pellet was dispersed and plated at a density of 1 x 10 6 cells / 75 cm 2 flask in 15 mL of medium. Cells were incubated at 37°C in an atmosphere of 5% carbon dioxide and 95% air for 24 hours, and unattached cells were removed by aspiration. Cells were cultured for an additional nine days, and flasks were then shaken vigorously for 16 hours to remove any contaminating cell types. Astrocyte monolayers were washed three times with DPBS, trypsinized and replated (density of 1 x 10 6 cells/flask).
• conditioned medium was harvested and mixed with leupeptin (10 mM: Bachem, Torrance, CA) and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (1.0 mM: ICN Biochemicals, Aurora, OH) to inhibit proteolysis.
• leupeptin 10 mM: Bachem, Torrance, CA
• 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride 1.0 mM: ICN Biochemicals, Aurora, OH
• VMCL-1 In culturing VMCL-1 and preparing VMCL-1 CM, 2.0 x 10 6 cells were plated in a 15-cm uncoated culture dish, in 20 mL growth medium initially containing 10% FBS. At 80% confluence, the medium was aspirated and the cells washed once with serum-free medium. Usually 20 mL of serum-free medium without albumin was added, and conditioning allowed to continue for 48 hours. N2 medium proved to be particularly suitable for use to collect conditioned medium. During these 48 hours, the cells usually expanded to 100% confluence. The medium was aspirated, pooled in 50 mL tubes, centrifuged (15,000 rpm, 20 min) and pooled in a 1.0 L plastic bottle.
• CM Approximately 5 mL of each batch of CM was sterilized using a 0.22 mm filter, stored at aliquots of 0.5 mL, at -70°C, and used to determine neurotrophic potency, before being pooled with the larger store of CM.
• the VMCL-1 cell line has now been passaged greater than 50 times.
• MAP2 and TH immunocytochemistry the cultures were washed (2 x 250 ⁇ L) with cold DPBS, fixed with 4% formaldehyde in PBS for 10 minutes, permeabilized using 1% CH 3 COOH/95% EtOH at -20°C, for 5 minutes, and then washed (3 x 250 ⁇ L) with PBS. Non-specific binding was blocked with 1% bovine serum albumin in PBS (BSA-PBS) for 15 minutes.
• Anti-TH antibody 50 ⁇ L
• Boehringer-Mannheim Indianapolis, IN
• anti-MAP2 antibody Boehringer-Mannheim
• Control staining was done using mouse serum at the same dilution as the anti-TH antibody. After washing (2 x 250 ⁇ L) with PBS, anti-mouse IgG-FITC (50 ⁇ L) was applied, and the slides incubated for an additional 1 hour. After washing with PBS (2 x 250 ⁇ L), excess fluid was aspirated, the chamber walls removed, and a single drop of VectaShield mounting medium (Vector Laboratories, Burlingame, CA) applied, followed by a cover glass, which was sealed with nail polish.
• VectaShield mounting medium Vector Laboratories, Burlingame, CA
• TH was identified using biotinylated, secondary antibodies, and the nickel-enhanced, diaminobenzidine (DAB) reaction product was developed using the biotinylated peroxidase-avidin complex (ABC kit; Vector Laboratories).
• DAB diaminobenzidine
• glial fibrillary acidic protein For glial fibrillary acidic protein (GFAP, Boehringer-Mannheim, #814369), fixation and permeabilization were done in one step using 5% CH 3 COOH/95% C 2 H 5 OH at -20°C. The subsequent procedures were the same as those used to visualize TH.
• A2B5 and 04 the cultures were washed with cold DPBS (2 x 250 ⁇ L) and blocked with 1% BSA-PBS for 10 minutes.
• the A2B5 antibody 50 ⁇ L was applied to each well, and incubated for 1 hour. After washing with DPBS (2 x 250 ⁇ L), the secondary antibody, anti- IgM-FITC, was applied for 30 minutes.
• the cells were then washed with DPBS (2 x 250 ⁇ L).
• cells were incubated with 0.5 ⁇ g/mL of nucleic acid dye H33258 (Hoechst, Kansas City, MO) in 10 mM sodium bicarbonate for 15 minutes at room temperature, then rinsed in PBS for 2 x 10 minutes. After a final washing with cold DPBS (2 x 250 ⁇ L), they were mounted as described above.
• nucleic acid dye H33258 Hoechst, Kansas City, MO
• Reaction products were resolved by 2% agarose gel electrophoresis to determine size and relative abundance of fragments.
• PCR results for ⁇ -actin and GAPDH were included as controls to confirm equal loading of cDNA.
• the cells were grown in DMEM/F-12 1 : 1 medium supplemented with 2.5% FBS, D-glucose (2.5 g/L) and ITS supplement, diluted 1: 100. Twenty- four hours later, subcultures at metaphase stage were arrested with colchicine (10 ⁇ g/mL). The cells were trypsinized and subjected to hypotonic shock (75 mM KCl). The cells were then fixed in three changes of MeOH/CH 3 COOH, 3: 1, and air-dried. The cells were then stained using 4% Geisma, and microscopically examined. Deposit
• Applicant has made a deposit of at least 25 vials containing cell line VMCL-1 with the American Type Culture Collection, Manassas VA, 20110

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