CA2361034A1 - Proteins related to neuronal regeneration and uses thereof - Google Patents

Abstract

The invention provides a method for stimulating nerve growth, which also includes nerve regeneration, by contacting nerve cells with human Neural Plakophilin Related Armadillo Protein (hNPRAP). In a specific embodiment, hNPRAP causes the development of numerous long, cellular extensions, which a re similar to axonal sprouting observed during neuronal regeneration and synaps e formation. The invention further relates to pharmaceutical compositions comprising an hNPRAP, or alternatively a gene therapy vector that expresses an hNPRAP. Also provided are methods for identifying substances that modulate expression of hNPRAP.

Description

PROTEINS RELATED TO NEURONAL REGENERATION
AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates generally to the treatment of neurological injury and dysfunction associated with central nervous system trauma. In particular, the invention is directed to the identification of proteins which induce neuronal regeneration.
BACKGROUND OF THE INVENTION
The peripheral nervous system (PNS) comprises highly organized groups of axon fibers or nerves external to the brain and spinal cord, such as the nerves in the limbs. In response to nerve damage, the peripheral nervous system often attempts to repair itself. While the return of lost functions is usually incomplete, generally the injured organism can adapt and function.
By contrast, damage to the central nervous system (CNS), comprising the brain and spinal cord, is generally more serious, usually causing permanent severe disability or even death.
A number of conditions are known to affect both growth and spontaneous regeneration in nerves, but the underlying mechanisms are not well understood (Gibson et al., In Compered. Coretire. Educ. Pract. Yet., vol. 1 l, pp., 1989, 938-945; and Daniloff et al., J. Cell Bio., 1986, 103:929-945). These conditions include the location of injury, the type of injury, the severity of injury, and the age and general health of the patient.
It has been reported that minor prior recoveries somehow prime the nerve for greater recovery in secondary lesions, for example, recovery from an earlier compression injury.
There are no previous reports of an effective treatment for injuries to neurons of the central nervous system, i. e. ; the brain and spinal cord (see, M. Walker, New Engl. J. Med., 1991, 324:1885-1887.
The lack of effective treatments for nervous system injuries may be due to an insufficient understanding both of the formation of the nervous system and of its responses to injuries. Several attempts have been made to electrically stimulate injured nerves to try to cause regrowth; recovery was highly variable and inadequate (see, B.
Sisken et al., Restorative Neurology and Neuroscience, 1990, 1:303-309; see generally J. Daniloff et al., "The Molecular Bases of Nerve Regeneration," in S.
Malhotra (ed.), Advances in Neural Science, vol. 2, 1993). The method that is currently used most often to close gaps in severed nerves uses grafts of the patient's own sensory nerves, typically taken from the ankle; a minimal degree of recovery and permanent analgesia of the donor foot are the usual results.
Because an injured spinal cord has very limited ability to recover spontaneously, and because the consequences of spinal cord injuries can be so serious, there is a particular need for an effective treatment of spinal cord injuries.
Paralytic spinal cord injuries in the United States alone occur at the rate of about 10,000 per year. Although the mortality rate is less than 10%, approximately 720 Americans per million population are permanently disabled as a result of spinal cord injuries. Most of the injured are young people in the most productive stage of life.
Following injury to neuronal cells in the central nervous system, there is often an abortive attempt by injured neural cells to generate new cellular extensions (dendrites and axons) in order to reestablish inter-neural contacts. In the central nervous system, these nerve sprouting and regeneration activities are often modest and only poorly sustained such that regeneration following stroke, trauma, spinal cord injury, etc., does not usually occur.
Thus, there is a need in the art for material and methods for treating neuronal injury.
SUMMARY OF THE INVENTION
The present invention addresses this need. Applicants have surprisingly discovered that a neuron-specific armadillo protein -- Neural Plakophilin Related Armadillo Protein (NPRAP) -- causes the development of numerous long, cellular extensions, which are similar to axonal sprouting observed during neuronal regeneration and synapse formation.
One aspect of the invention is directed to a method of stimulating growth of nerve cells, which method comprises contacting the nerve cells with an hNPRAP having nerve growth stimulating activity in an amount effective to cause nerve cell growth.
In a specific embodiment related to a method of stimulating growth of nerve cells, the method comprises contacting nerve cells with an hNPRAP
stimulating agent in an amount sufficient to induce the expression of an hNPRAP and cause nerve cell growth.
A further related aspect of the invention is directed to a method of stimulating neuronal regeneration in a mammal, which method comprises administering to the mammal in need thereof an effective amount of an hNPRAP
or an effective amount of an hNPRAP expression stimulating agent as set forth above.
A further aspect of the invention is related to a pharmaceutical composition comprising an hNPRAP having nerve growth stimulating activity, and a pharmaceutically acceptable carrier.
Yet another aspect of the invention is related to a pharmaceutical 1 S composition comprising an hNPRAP expression stimulating agent and a pharmaceutically acceptable carrier.
In a specific embodiment, the invention provides a pharmaceutical composition comprising an hNPRAP gene therapy vector, which vector comprises a polynucleotid encoding hNPRAP and a promoter for expressing hNPRAP, and a carrier. Naturally, such gene therarpy vectors are also part of the invention as well.
A further aspect of the invention relates to a method for identifying substances that modulate the expression of hNPRAP, which method comprises contacting cultured cells that express hNPRAP with a test substance measuring levels of hNPRAP, as compared to a control in which the same cells that express hNPRAP
are not contacted with the test substance, as an indication of modulatory activity of said test substance.
These and other aspects of the invention are disclosed more fully in the accompanying detailed description.

DETAILED DESCRIPTION
The human Neural Plakophilin Related Armadillo Protein ("hNPRAP") (also described as GT24) consensus cDNA (SEQ ID N0:3) encodes a protein (SEQ
ID N0:4) of 1084 amino acid residues with a unique N-terminus, but with homology to proteins with armadillo (arm) repeat motifs at its C-terminus.
Applicants have now discovered that over-expression of hNPRAP, or functional derivatives thereof containing one or more armadillo repeats, causes the development of numerous long, dendritic processes which typically terminate upon distantly located cells. These target cells need not necessarily be expressing hNPRAP. The hNPRAP induced cellular extensions are highly similar to the axonal sprouting seen during neuronal regeneration and synapse formation.
Nucleotides 2920-2997 of the hNPRAP cDNA overlap the anonymous microsatellite locus DSS478, therefore placing the hNPRAP gene on chromosome SplS near the Cri-du-Chat deletion locus, a syndrome associated with congenital malformation and gross mental retardation. hNPRAP is described in detail in copending commonly assigned U.S. Application Serial Nos. 08/888,077, filed July 3, 1997 (PCT/CA97/00051), and 09/227,725, filed January 8, 1999 (PCT/CA99/00018), both of which are incorporated herein by reference.
As described in U.S. Application Serial Nos. 08/888,077 (PCT/CA97/00051) and 09/227,725 (PCT/CA99/00018), hNPRAP is known to interact with Presenilin I ("PS 1 ") and Presenilin II ("PS2") by direct protein:protein interaction studies. The domain of the PS 1 protein that interacts with hNPRAP
has also been shown to interact with other proteins, such as armadillo repeat proteins p0071 and $-catenin.
On Northern blots, the hNPRAP gene is expressed as a range of transcripts of 3.9 to S.0 kb in several regions of adult human brain, but is expressed at only very low levels in most non-neurologic tissues. Studies have shown that and hIVPRAP are both expressed in the same cell types and in adjacent/contiguous subcellular compartments.
In situ hybridization studies indicate that the transcriptional pattern of S
PS 1 and NPRAP overlap both in the brain of 4 month old mice, and in the neural tube and dorsal root ganglia of murine embryos. Both genes are expressed at high levels in dentate and hippocampal neurons, in scattered neocortical neurons, and in cerebellar Purkinje cells in adult mouse brain (Lee et al., J. Neurosci., 1996, 16:7513-7525;
S Paffenholz and Franke, Differentiation, 1997, 61:293-304).
Immunocytochemical studies show that PS 1 and hNPRAP have overlapping intracellular distributions.
Thus, in non-confluent transfected cell cultures, hNPRAP has a predominantly perinuclear cytoplasmic distribution contiguous with that of PS 1. In contrast, in confluent cells with abundant cell:cell contacts, hNPRAP is predominantly located near the cell membrane close to inter-cellular contact zones while PS 1 retains its predominantly perinuclear distribution.
The invention is directed to the use of an hNPRAP to stimulate neuronal regeneration and axon sprouting following a wide variety of insults and injuries. An "hNPRAP" is defined herein as a biologically active polypeptide that 1 S contains a sequence of hNPRAP that mediate its nerve cell growth stimulating activity, e.g., the armadillo repeats. Thus, hNPRAP includes full-length (naturally occurring) hNPRAP, as well as biologically active analogues thereof. By "analogues"
it is meant modifications such as point mutations, amino acid substitutions, additions or deletions, or other mammalian homologues, such as mouse (SEQ ID NO:S and SEQ ID N0:6), which have similar activity to hNPRAP, the identification and selection of which are well-known to those skilled in the art. In addition to hNPRAP, the use of recombinant proteins such as p120cas and chimeric proteins having all or parts of the C-terminal armadillo-like repeat and C-terminal unique sequences of hNPRAP may also be utilized in the practice of this invention. Analogues of these 2S proteins which replicate the effects thereof may also be utilized in the practice of this invention.
In a first embodiment, the invention provides a method of stimulating growth of nerve cells, comprising contacting nerve cells with an hNPRAP.
A second embodiment is directed to a method of stimulating growth of nerve cells, comprising contacting nerve cells with an hNPRAP stimulating agent in an amount sufficient to induce the expression of hNPRAP. Such agents may induce the expression of hNPRAP by positively binding to the hNPRAP gene to induce expression, or may alter the interaction of hNPRAP with an inhibitor of hNPRAP
expression, e.g., by binding to the inhibitor itself or to hNPRAP such that the inhibitor no longer modulates the expression of hNPRAP.
Alternatively, the expression of hNPRAP may be induced by the use of an appropriate viral vector system, or by the administration of recombinant proteins, biological molecules or small molecules which simulate or resemble either the armadillo binding domain of the presenilins or the armadillo repeats of hNPRAP.
Another embodiment is directed to a method for identifying substances that simulate or resemble (mimic) either the armadillo binding domain of the presenilins or the armadillo repeats of hNPRAP, and which substances cause neural growth.
Candidate compounds which are shown to mimic either the armadillo binding domain of the presenilins or the armadillo repeats of hNPRAP may be produced in pharmaceutically useful quantities for use in the treatment of neurological injury and dysfunction 1 S associated with central nervous system trauma. Candidate compounds include endogenous cellular components which interact with the presenilins in vivo and which, therefore, provide new targets for pharmaceutical and therapeutic interventions, as well as recombinant, synthetic and otherwise exogenous compounds which may have presenilin binding capacity and, therefore, may be candidates for pharmaceutical agents. Thus, in one procedure, cell lysates or tissue homogenates (e.g., human brain homogenates, lymphocyte lysates) may be screened for proteins or other compounds which bind to one of the normal or mutant presenilins.
Alternatively, any of a variety of exogenous compounds, both naturally occurring and/or synthetic (e.g., libraries of small molecules or peptides), may be screened for presenilin binding capacity. In each of these embodiments, an assay is conducted to detect binding between a presenilin component containing at least the interacting domain of a presenilin protein described herein and some other moiety.
As described in U.S. Application Serial No. 09/227,725, the presenilin domain that interacts with PS-interacting proteins, such as armadillo repeat proteins hNPRAP, p0071 and $-catenin, has been identified as including or being contained in the sequence of amino acid residues from about 260 to about 409 of PS1 or corresponding residues from about 260 to about 390 in PS2. More preferably, the interacting domain contains or is contained in amino acid residues from about 372 to about 399 of PS1 or corresponding residues from about 350 to about 380 in PS2.
The amino acid sequences of wild-type human PS 1 and PS2 are shown in SEQ ID NO:1 and SEQ ID N0:2, respectively.
Binding may be detected by indirect functional measures reflecting the functional consequences of the interaction (e.g., changes in intracellular Ca2+, Na+, KT, or GTP/GDP ratio, changes in apoptosis or microtubule associated protein phosphorylation, changes in A$ peptide production or changes in the expression of other downstream genes which can be monitored by differential display, 2D gel electrophoresis, differential hybridization, or SAGE methods) or by direct measures such as immunoprecipitation, the Biomolecular Interaction Assay (BIAcore) or alteration of protein gel electrophoresis. The preferred methods involve variations on the following techniques: (1) direct extraction by affinity chromatography;
(2) co-isolation of presenilin components and bound proteins or other compounds by immunoprecipitation; (3) BIAcore analysis; and (4) the yeast two-hybrid systems.
Other procedures include methods which detect abnormal processing of PS 1, PS2, APP, or proteins reacting with PSl, PS2, or APP (e.g., abnormal phosphorylation, glycosylation, glycation amidation or proteolytic cleavage) alterations in presenilin transcription, translation, and post-translational modification; alterations in the intracellular and extracellular trafficking of presenilin gene products; or abnormal intracellular localization of the presenilins.
The proteins or other compounds identified by these methods may then be assayed for their ability to promote sprouting in axons of neuronal cultures or dendrite formation in non-neurological cells using morphometric analyses which are well-known to those skilled in the art of neuronal regeneration.
Alternatively, assays for regeneration following sectioning of the optic nerve, spinal cord, etc. in animals may be performed. Such assays are well-known to those in the field of neuronal regeneration.
The proteins or other compounds identified by these methods may be purified and characterized by any of the standard methods known in the art.
Proteins may, for example, be purified and separated using electrophoretic (e.g., SDS-PAGE, 2D PAGE) or chromatographic (e.g., HPLC) techniques and may then be microsequenced. For proteins with a blocked N-terminus, cleavage (e.g., by CNBr and/or trypsin) of the particular binding protein is used to release peptide fragments.
Further purification/characterization by HPLC and microsequencing and/or mass spectrometry by conventional methods provides internal sequence data on such blocked proteins. For non-protein compounds, standard organic chemical analysis techniques (e.g., IR, NMR and mass spectrometry; functional group analysis; X-ray crystallography) may be employed to determine their structure and identity.
These hNPRAPs, and compounds which activate hNPRAP, may be employed in combination with a suitable pharmaceutical, physiologically acceptable carrier. Administration of hNPRAP of this invention can be through the administration of hNPRAP peptides agonists or antagonists synthesized from recombinant constructs of hNPRAP DNA or from peptide chemical synthesis (Woo, et al., Protein Engineering, 1989, 3:29-37) or in the form of gene therapy (Goldspiel et al., Clin. Pharm., 1993, 12:488; Wu and Wu, Biotherapy, 1991, 3:87;
Mulligan, Science, 1993, 260:926; Morgan and Anderson, Ann. Rev. Biochem., 1993, 62:191;
and, May TIBTECH, 1993, 11:155).
Generally, hNPRPA and/or activating agents) are administered as pharmaceutical compositions comprising an effective amount of hNPRAP and/or activating agents) in a pharmaceutical carrier. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients. A pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the invention to a patient.
The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Animal testing of effective doses for treatment of particular injuries will provide further predicative indication of human dosage. Various considerations are described, e.g., in Gilman et al.
(eds.) (1990) Goodman and Gilman's; The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. ( 1990), Mack Publishing Co., Easton, PA. Methods for administration are discussed therein and below, e.g., for intravenous, intraperitoneal, or intramuscular administration, transfermal diffusion, and others. Pharmaceutically acceptable carriers include water, saline, buffers and other compounds described, e.g., in the Merck Index, Merck &
Co., Rahway, New Jersey, and in Remington, supra. Slow release formulations, or a slow release apparatus, may be used for continuous administration.
Dosage range$ for hNPRAP and/or activating agents) would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ~M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM
(femtomolar), with an appropriate carrier. Generally, treatment is initiated with I S smaller dosages which are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small increments until the optimum effect under the circumstance is reached. Determination of the proper dosage and administration regime for a particular situation is within the skill of the art.
Polypeptides and other compounds of the present invention which activate or inhibit hNPRAP may be employed alone or in conjunction with other compounds, such as therapeutic compounds. Once identified by the methods described above, the candidate compounds may then be produced in quantities sufficient for pharmaceutical .administration or testing (e.gJ., mg or mg or greater quantities), and formulated in a pharmaceutically acceptable carrier (see, e.g., Remington's, supra).
Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
5 The compositions of the present invention may be administered orally, parenterally, by spray inhalation, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

10 Preferably, the compositions are administered orally, intraperitoneally or intravenously.
Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.
Topically-transdermal patches may also be used.
For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, ~ propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably., as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
The methods and compositions of this invention may be used to treat nerve damage caused by a wide variety of diseases or physical traumas. These include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, stroke and ischemia associated with stroke, neural paropathy, other neural degenerative diseases, motor neuron diseases, sciatic crush, peripheral neuropathy, particularly neuropathy associated with diabetes, spinal cord injuries and facial nerve crush.
The hNPRAP polynucleotides, polypeptides, agonists and antagonists that are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in treatment modalities often referred to as "gene therapy". Thus, for example, cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo. The engineered cells can then be provided to a patient to be treated with the polypeptide.
In this embodiment, cells may be engineered ex vivo, for example, by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. Such methods are well-known in the art and their use in the present invention will be apparent from the teachings herein.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral or viral vector, as discussed above. The retroviral expression construct may then be isolated. A packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention, such that the packaging cell now produces infectious viral particles containing the gene of interest. These viral particles may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention should be apparent to those skilled in the art from the teachings of the present invention.
Retroviruses or. viruses from which the plasmid vectors hereinabove-mentioned may be derived include, but are not limited to, SimiForest Virus, Lenti-virus, Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Rous Sarcoma Virus, Harvey Sarcoma Virus, Avian Leukosis Virus, Gibbon Ape Leukemia Virus, Human Immunodeficiency Virus, Adenovirus, Myeloproliferative Sarcoma Virus, and Mammary Tumor Virus. In a preferred embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
Such vectors will include one or more promoters for expressing the polypeptide. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques, 1989, 7:980-990. Cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III, and $-actin promoters, can also be used.
Additional viral promoters which may be employed include, but are not limited to, adenovirus promoters such as the adenoviral major late promoter, thymidine kinase (TK) promoters such as the Herpes Simplex thymidine kinase promoters; the respiratory syncytial virus (RSV) promoters; and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention may be placed under the control of an inducible promoter. Suitable inducible promoters which may be employed include, but are not limited to, the MMT
promoter, the metallothionein promoter; heat shock promoters; the albumin promoter;
the ApoAI promoter; human globin promoters; viral thymidine kinase promoters;
and human growth hormone promoters. The promoter may also be the native promoter which controls the gene encoding the polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, .PSL-2, .omega.-AM, PA12, T19-14X, VT-19-17-H2, .omega.CRE, .omega.CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, A., Human Gene Therapy, 1990, 1:5-14. The vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPOa precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host. The producer cell line will generate infectious retroviral vector particles, which include the nucleic acid sequences) encoding the polypeptides. Such retroviral vector particles may'then be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequences) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.
Each patent, patent application, publication, and procedure disclosed in this application is specifically incorporated by reference in its entirety.

SEQUENCE LISTING
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<213> human <400> 1 Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg Ala Leu His Ser Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr Asp Leu Val A1a Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr SUBSTITUTE S~iE~T (RULE 26) Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln Phe Tyr Ile <210> 2 <211> 448 <212> PRT
<213>. human <400> 2 Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln Glu Gly Arg Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg Ser Gln Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val Cys Ser Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr Thr Glu Lys Asn Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Pro Ser Val Gly Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu Ile Met Ile Ser Val Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser Leu Met Leu Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val Leu SUBSTITUTE SHEET (RULE 26) Lys Thr Tyr Asn Vai Ala Met Asp Tyr Pro Thr Leu Leu Leu Thr Val Trp Asn Phe Gly Ala Val Gly Met Val Cys Ile His Trp Lys Gly Pro Leu Val Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu Gly Ala Ile Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Pro Ile Phe Pro Ala Leu Ile Tyr Ser Ser Ala Met Val Trp Thr Val Gly Met Ala Lys Leu Asp Pro Ser Ser Gln Gly Ala Leu Gln Leu Pro Tyr Asp Pro Glu Met Glu Glu Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr Pro Glu Val Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu Glu Glu Glu Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ala Ala Thr Gly Ser Gly Asp Trp Asn Thr Thr Leu Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Val Phe Lys Lys Ala Leu Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn Leu Val Arg Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr Ile <210> 3 <211> 4746 <212> DNA
<213> human <400> 3 gccagcatcc cttgtcccgc ggccggctca gacaacaaaa gcggaagatg ctgcagttgg gcaaggtcag gaccttgcct tgaaagccgg gcggcgccgc gcaacgcctc ttcccggact gaggagctgt cgccggcgga gggtgcatgt ttgcgaggaa gccgccgggc gccgcgcctt tgggagctat gcctgttcca gaccagcctt catcagcctc agagaagacg agttccctga gccccggctt aaacacctcc aacggggatg gctctgaaac agaaaccacc tctgccatcc tcgcctcagt caaagaacag gaattacagt ttgaaaggct gacccgagag ctggaggctg aacggcagat cgtagccagc cagctggagc gatgcaagct cggatccgag actggcagca tgagcagcat gagttcagca gaagagcagt ttcagtggca gtcacaagat ggtcaaaaag SUSSTITUT~ SHEET (RULE 26) atatcgaaga tgagcttaca acaggtctcg agctggtgga ctcctgtatt aggtcactac aggaatcagg aatacttgac ccacaggatt attctacagg tgaaaggccc agcctgctct cccagagtgc acttcagctc aattccaaac ctgaagggtc tttccagtat ccggccagct accatagcaa ccagaccctg gccctggggg aaaccacccc ttcacagctc ccggcccgag gcacacaagc ccgagctacg ggccagagct tcagccaggg cacgaccagc cgcgccggcc acctggcggg gcccgagccc gcgccgccgc cgccgccgcc gccgcgggag ccgttcgcgc ccagcctggg cagcgccttc cacctgcccg acgcgccgcc cgccgccgcc gccgccgcgc tctactactc cagctccacg ctgcccgcgc cgccgcgcgg gggctccccg ctggccgcgc cccagggcgg ttcgcccacc aagctgcagc gcggcggctc ggcccccgag ggcgccacct acgccgcgcc gcgcggctcc tcgcccaagc agtcgcccag ccgcctggcc aagtcctaca gcaccagctc gcccatcaac atcgtcgtgt cctcggccgg cctgtccccg atccgcgtga cctcgccccc caccgtgcag tccaccatct cctcctcgcc catccaccag ctgagctcca ccatcggcac gtacgccacc ctgtcgccca ccaagcgcct ggtccacgcg tccgagcagt acagcaagca ctcgcaggag ctgtatgcca cggccaccct ccagaggccg ggcagcctgg cagctggttc ccgagcctca tacagcagcc agcatgggca cctgggccca gagttgcggg ccctgcagtc cccagaacac cacatagatc ccatctatga agaccgcgtc tatcagaagc cccctatgag gagtctcagc cagagccagg gggaccctct gccgccagca cacaccggca cctaccgcac gagcacagcc ccatcttccc ctggtgtcga ctccgtcccc ttgcagcgca caggcagcca gcacggccca cagaatgccg ccgcggccac cttccagagg gccagctatg ccgccggccc agcctccaat tacgcggacc cctaccgaca gctgcagtat tgtccctctg ttgagtctcc atacagcaaa tccggccctg ctctcccgcc tgaaggcacc ttggccaggt ccccgtccat tgatagcatt cagaaagatc ccagagaatt tggatggaga gacccggaac tgccggaagt gattcagatg ttgcagcacc agtttccctc ggtccagtct aacgcggcag cctacttgca acacctctgt tttggagaca acaaaattaa agccgagata aggagacaag gaggcatcca gctcctggtg gacctgttgg atcatcggat gaccgaagtc caccgtagtg cctgtggagc tctgagaaac ctggtgtatg ggaaggccaa cgatgataac aaaattgccc tgaaaaactg tggtggcatc ccagcactgg tgaggttact ccgcaagacg actgacctgg agatccggga gctggtcaca ggagtccttt ggaacctctc ctcatgcgat gcactcaaaa SUBSTITUTE SHEET (RULE 26) tgccaatcat ccaggatgcc ctagcagtac Lgaccaacgc ggtgattatc ccccactcag gctgggaaaa ttcgcctctt caggatgatc ggaaaataca gctgcattca tcacaggtgc tgcgtaacgc caccgggtgc ctaaggaatg ttagttcggc cggagaggag gcccgcagaa ggatgagaga gtgtgatggg cttacggatg ccttgctgta cgtgatccag tctgcgctgg ggagcagtga gatcgatagc aagaccgttg aaaactgtgt gtgcatttta aggaacctct cgtaccggct ggcggcagaa acgtctcagg gacagcacat gggcacggac gagctggacg ggctactctg tggcgaggcc aatggcaagg atgctgagag ctctgggtgc tggggcaaga agaagaagaa aaagaaatcc caagatcagt gggatggagt aggacctctt ccagactgtg ctgaaccacc aaaagggatc cagatgctgt ggcacccatc aatagtcaaa ccctacctca cactgctctc tgagtgctca aatccagaca cgctggaagg ggcggcaggc gccctgcaga acttggctgc agggagctgg aagtggtcag tatatatccg agccgctgtc cgaaaagaga aaggcctgcc catcctcgtg gagctgctcc gaatagacaa tgaccgtgtg gtgtgcgcgg tggccactgc gctgcggaac atggccttgg acgtcagaaa taaggagctc atcggcaaat acgccatgcg agacctagtc cacaggcttc caggagggaa caacagcaac aacactgcaa gcaaggccat gtcggatgac acagtgacag ctgtctgctg cacactgcac gaagtgatta ccaagaacat ggagaacgcc aaggccttac gggatgccgg tggcatcgag aagttggtcg gcatctccaa aagcaaagga gataaacact ctccaaaagt ggtcaaggct gcatctcagg tcctcaacag catgtggcag taccgagatc tgaggagtct ctacaaaaag gatggatggt cacaatacca ctttgtagcc tcgtcttcaa ccatcgagag ggaccggcaa aggccctact cctcctcccg cacgccctcc atctcccctg tgcgcgtgtc tcccaacaac cgctcagcaa gtgccccagc ttcacctcgg gaaatgatca gcctcaaaga aaggaaaaca gactacgagt gcaccggcag caacgccacc taccacggag ctaaaggcga acacacttcc aggaaagatg ccatgacagc tcaaaacact ggaatttcaa ctttgtatag gaattcttat ggtgcgcccg ctgaagacat caaacacaac caggtttcag cacagccagt cccacaggag cccagcagaa aagattacga gacctaccag ccatttcaga attccacaag aaattacgat gagtccttct tcgaggacca ggtccaccat cgccctcccg ccagcgagta caccatgcac ctgggtctca agtccaccgg caactacgtt gacttctact cagctgcccg tccctacagt gaactgaact atgaaacgag ccactacccg gcctcccccg actcctgggt gtgaggagca gggcacaggc SUBSTITUTE SHEET (RULE 26) gctccgggaa cagtgcatgt gcatgcatac cacaagacat ttctttctgt tttgtttttt tctcctgcaa atttagtttg ttaaagcctg ttccatagga aggctgtgat aaccagtaag gaaatattaa gagctatttt agaaagctaa atgaatcgca agttaacttg gaaatcagta gaaagctaaa gtgatcctaa atatgacagt gggcagcacc tttctagcgt gagctgtaaa gtaacgagaa gtgctttata ctgaacgtgg ttgatgggag gagagacgag gcattcgggc cggtggggcg taagggttat cgttaagcac aagacacaga atagtttaca cactgtgtgg gggacggctt ctcacgcttt gtttactctc ttcatccgtt gtgactctag gcttcaggtt gcattggggt tcctctgtac agcaagatgt ttcttgcctt ttgttaatgc attgttgtaa agtatttgat gtacattaca gattaaagaa gaaaagcgcg ttgtgtatat tacaccaatg ccgccgtgtt tcctcatcta tggttctaaa tattgcttca atttcaaact tttgaaagat gtatggattt ccagtttttc tttactttct cccagtatgt tttaacaaaa aaaaaaaaaa gcaggaaaaa aggaatattt agcagtattg ttcgttctga tatgtgaatt tgtttgtgac aactaaacaa ggcattcagc agtttctgac aattaacata catcattcca cactccttgt caacaaagtg ctttttcact gcctaaaatt ttagatgtag atatttgaaa tagatttttt catttatacc agttttcttt atgatgatac agtgttaaaa gaaaataaat tacaattgat ctgtca <210> 4 <211> 1225 <212> PRT
<213> human <400> 4 Met Phe Ala Arg Lys Pro Pro Gly Ala Ala Pro Leu Gly Ala Met Pro Val Pro Asp Gln Pro Ser Ser Ala Ser Glu Lys Thr Ser Ser Leu Ser Pro Gly Leu Asn Thr Ser Asn Gly Asp Gly Ser Glu Thr Glu Thr Thr Ser Ala Ile Leu Ala Ser Va1 Lys Glu Gln Glu Leu Gln Phe Glu Arg Leu Thr Arg Glu Leu Glu Ala Glu Arg Gln Ile Val Ala Ser Gln Leu Glu Arg Cys Lys Leu Gly Ser Glu Thr Gly Ser Met Ser Ser Met Ser Ser Ala Glu Glu Gln Phe Gln Trp Gln Ser Gln Asp Gly Gln Lys Asp Ile Glu Asp Glu Leu Thr Thr Gly Leu Glu Leu Val Asp Ser Cys Ile SUBSTITUTE SHEET (RULE 26) Arg Ser Leu Gln Giu Ser Gly Ile Leu Asp Pro Gln Asp Tyr 5er Thr Gly Glu Arg Pro Ser Leu Leu Ser Gln Ser Ala Leu Gln Leu Asn Ser Lys Pro Glu Gly Ser Phe Gln Tyr Pro Ala Ser Tyr His Ser Asn Gln Thr Leu Ala Leu Gly Glu Thr Thr Pro Ser Gln Leu Pro Ala Arg Gly Thr Gln Ala Arg Ala Thr Gly Gln Ser Phe Ser Gln Gly Thr Thr Ser Arg Ala Gly His Leu Ala Gly Pro Glu Pro Ala Pro Pro Pro Pro Pro Pro Pro Arg Glu Pro Phe Ala Pro Ser Leu Gly Ser Ala Phe His Leu Pro Asp Ala Pro Pro Ala Ala Ala Ala Ala Ala Leu Tyr Tyr Ser Ser Ser Thr Leu Pro Ala Pro Pro Arg Gly Gly Ser Pro Leu Ala Ala Pro Gln Gly Gly Ser Pro Thr Lys Leu Gln Arg Gly Gly Ser Ala Pro Glu Gly Ala Thr Tyr Ala Ala Pro Arg Gly Ser Ser Pro Lys Gln Ser Pro Ser Arg Leu Ala Lys Ser Tyr Ser Thr Ser Ser Pro Ile Asn Ile Val Val Ser Ser Ala Gly Leu Ser Pro Ile Arg Val Thr Ser Pro Pro Thr Val Gln Ser Thr Ile Ser Ser Ser Pro Ile His Gln Leu Ser Ser Thr Ile Gly Thr Tyr Ala Thr Leu Ser Pro Thr Lys Arg Leu Val His Ala Ser Glu Gln Tyr Ser Lys His Ser Gln Glu Leu Tyr Ala Thr Ala Thr Leu Gln Arg Pro Gly Ser Leu Ala Ala Gly Ser Arg Ala Ser Tyr Ser Ser Gln His Gly His Leu Gly Pro Glu Leu Arg Ala Leu Gln Ser Pro Glu His His Ile Asp Pro Ile Tyr Glu Asp Arg Val Tyr Gln Lys Pro Pro Met Arg Ser Leu Ser Gln Ser Gln Gly Asp Pro Leu Pro Pro Ala His Thr Gly Thr Tyr Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp Ser Val Pro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gln Asn Ala Ala Ala Ala Thr Phe Gln Arg Ala Ser Tyr Ala Ala Gly Pro Ala Ser Asn Tyr Ala Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Pro Ser Val Glu Ser Pro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala Arg Ser Pro Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu Phe Gly Trp Arg Asp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln His Gln Phe Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His SUBSTITUTE SHEET (RULE 26) Leu Cys Phe Gly Asp Asn Lys Ile Lys Aia Glu Ile Arg Arg Gln Gly Gly Ile Gln Leu Leu Val Asp Leu Leu Asp His Arg Met Thr Glu Val His Arg Ser Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala Asn Asp Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro Ala Leu Val Arg Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Leu Lys Met Pro Ile Ile Gln Asp Ala Leu Ala Val Leu Thr Asn Ala Val Ile Ile Pro His Ser Gly Trp Glu Asn Ser Pro Leu Gln Asp Asp Arg Lys Ile Gln Leu His Ser Ser Gln Val Leu Arg Asn Ala Thr Gly Cys Leu Arg Asn Val Ser Ser Ala Gly Glu Glu Ala Arg Arg Arg Met Arg Glu Cys Asp Gly Leu Thr Asp Ala Leu Leu Tyr Val Ile Gln Ser Ala Leu Gly Ser Ser Glu Ile Asp Ser Lys Thr Val Glu Asn Cys Val Cys Ile Leu Arg Asn Leu Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His Met Gly Thr Asp Glu Leu Asp Gly Leu Leu Cys Gly Glu Ala Asn Gly Lys Asp Ala Glu Ser Ser Gly Cys Trp Gly Lys Lys Lys Lys Lys Lys Lys Ser Gln Asp Gln Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro Lys Gly Ile Gln Met Leu Trp His Pro Ser Ile Val Lys Pro Tyr Leu Thr Leu Leu Ser Glu Cys Ser Asn Pro Asp Thr Leu Glu Gly Ala Ala Gly Ala Leu Gln Asn Leu Ala Ala Gly Ser Trp Lys Trp Ser Val Tyr Ile Arg Ala Ala Val Arg Lys Glu Lys Gly Leu Pro Ile Leu Val Glu Leu Leu Arg Ile Asp Asn Asp Arg Val Val Cys Ala Val Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr Ala Met Arg Asp Leu Val His Arg Leu Pro Gly Gly Asn Asn Ser Asn Asn Thr Ala Ser Lys Ala Met Ser Asp Asp Thr Val Thr Ala Val Cys Cys Thr Leu His Glu Val Ile Thr Lys Asn Met Glu Asn Ala Lys Ala Leu Arg Asp Ala Gly Gly Ile Glu Lys Leu Val Gly Ile Ser Lys Ser Lys Gly Asp Lys His Ser Pro Lys Val Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met Trp Gln Tyr Arg Asp Leu Arg Ser SUBSTITUTE SNEET (RULE 26) Leu Tyr Lys Lys Asp Gly Trp Ser Gln Tyr His Phe Val Ala Ser Ser Ser Thr Ile Glu Arg Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser Ala Pro Ala Ser Pro Arg Glu Met Ile Ser Leu Lys Glu Arg Lys Thr Asp Tyr Glu Cys Thr Gly Ser Asn Ala Thr Tyr His Gly Ala Lys Gly Glu His Thr Ser Arg Lys Asp Ala Met Thr Ala Gln Asn Thr Gly Ile Ser Thr Leu Tyr Arg Asn Ser Tyr Gly Ala Pro Ala Glu Asp Ile Lys His Asn Gln Val Ser Ala Gln Pro Val Pro Gln Glu Pro Ser Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe Gln Asn Ser Thr Arg Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val His His Arg Pro Pro Ala Ser Glu Tyr Thr Met His Leu Gly Leu Lys Ser Thr Gly Asn Tyr Val Asp Phe Tyr Ser Ala Ala Arg Pro Tyr Ser Glu Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp Val <210> 5 <211> 4998 <212> DNA
<213> mouse <400> 5 aagcgccgga gccggccgcc gcggctgagc cggaggctga gctgcggcgc gcggcgggag gagcctcgct ctcggcggcg gcggcggcgg cggcgacaca ggtggcgcgg gcggcgcgca gggcgcagct cgagagcgct cggcgccggg cgccagggcg gcccaggctc gcgcccgcgg cggcaaccgg ccgagcggag cggcgggcgc ggcggctcgg tagcccggcc cgagcccggg gagccccgcg gaaccctgag catcccgcgg cgcccgccga gtcgggcagg gggcgctacg ctcgccgcgc tcggaggggc ggccgggccg ggcgctgcgc actcgcgtcg ggagccgcct ctcgcctgcc gcgctcgccc ctgctccccg ccagcatcac ttgtcccgcg gccgcgctcc gacaacaaaa gcggaggatg ctgcagctgg gcaaggtcag gaccttgctc tgaagccggg cggcggcgcg cacgcctttc ccccgactga ggagctgtct ttggcggcgg gtgcatgttc gccaggaagc agtcgggcgc cgcgccgttc ggagctatgc ctgtcccaga ccagcctcca tcagcctcag agaagaacag ctccttgagc ccaggcttaa acacctccaa tggtgatggc SUBSTITUTE SHEET (RULE 26) tctgagacgg aaaccacctc tgctatcctt gcctccgtca aagaacagga attacagttt gaaaggctga cccgagagct ggaggctgaa cgccagatcg tagccagcca gctggagcga tgcaagcttg gctcggagac aggaagcatg agcagtatca gttcagcagg agagcagttt cactggcaga cacaagatgg ccaaaaagat atcgaagatg aacttacaac gggccttgag ctggtggact cctgtatccg ctctctgcag gagtcaggca ttctggaccc acaggattac tccacaagtg aaaggcctag cctgctctcc cagagtgcac ttcagctcaa ttctaaacct gaagggtctt tccagtatcc ggccagctac catagcaacc agaccctggc cctgggtgac acagcccctt ctcagctccc agcacgcagc acgcaagccc gagctgccgg ccagagcttc agccagggca cgaccggccg cgcggggcac ctggcgggct ccgagcctgc gccaccgcct ccgcctccgc gggaaccgtt cgcgcccagc ctgggcagcg ccttccacct gcccgacgcg ccgcccgccg ccgcggcgct ctactactcc agctccacgc tgcccgcgcc gccgcgcggg ggctccccgc tgaccaccac gcagggcggc tcacccacca agctgcagcg cggaggctcg gcccccgagg gtgccgccta cgccgcgccg cgcggctcct cgcccaagca gtcgcccagc cgcctggcta agtcctacag caccagctcg cccatcaaca tcgtcgtgtc ctcggccggc ctgtccccga tccgcgtgac ctcgcccccc accgtgcagt ccaccatctc ctcttcgccc atccaccagc tgagctccac catcggcacc tacgccaccc tgtcgcccac caagcgcctg gtccacgcgt ctgagcagta cagcaagcat tcgcaggagc tgtatgccac cgccaccctc cagaggccgg gcagcctggc agctggatcc cgagcctcgt atagcagcca gcatgggcac ctggcccctg agctgcgggc cctgcagtcc ccagagcacc acatagaccc catctatgaa gaccgtgtct atcagaagcc ccctatgagg agtctcagcc agagccaggg ggatcctctg ccgccagcac ataccggcac cttccgcacg agcacagccc cgtcctcccc tggtgtcgac tccgtcccct tgcagcgcac aggcagccaa cacgggccac agaatgccgc cgcagccacc ttccagaggg ccagctatgc tgccggccca gcctccaact acgcagaccc ctaccgacag ctgcagtatt gtgcctccgt tgactctccg tacagcaaat ctggccctgc cctcccaccc gaaggcacct tggccagatc cccatccatc gacagcattc agaaagaccc cagggagttt ggatggagag acccggagct gcctgaagtg atacagatgt tacagcacca gttcccttca gtccagtcca atgctgcagc ttacctgcaa cacctctgtt ttggagacaa taaaattaag gcagagataa ggagacaagg agggatacag ctcctggtgg acctgctgga tcaccgaatg SUBSTITUTE SHEET (RULE 26) acagaagtcc accgtagtgc ctgtggggct ctgaggaacc tggtgtatgg gaaggccaat gatgataaca aaatcgccct gaaaaactgt ggtggtatcc cagcgctggt gagactcctt cgcaagacca cagacctgga gatccgggag ctggtcacag gagtcctttg gaacctctca tcatgtgatg cactcaaaat gccaatcatc caggacgccc tggcagtgct gaccaatgcg gtgattatcc ctcactcggg ctgggagaat tcacctcttc aggatgatcg gaaaatacag ctgcattcat cacaggtgct gcgcaacgcc actgggtgcc taaggaatgt aagttcagct ggagaggagg cccgccgaag gatgcgggag tgtgatgggc tcacggatgc cttgctgtac gtgatccagt ctgcactggg gagcagtgag atcgatagca agaccgttga aaactgtgtg tgcatcttga ggaacctctc ctaccggcta gcagcagaaa cgtctcaggg acagcacatg ggcacagacg agctggacgg gctgctctgc ggggagacca acggcaaaga cacagagagt tctgggtgct ggggcaagaa gaagaagaaa aagaaatccc aggaccagtg ggatggagta ggacctcttc cagactgtgc agagccacca aaagggatcc agatgctgtg gcacccgtcc atagtcaaac cctacctcac actgctctct gagtgctcaa acccagacac gctggaaggg gcagcgggcg ccctgcagaa cttggctgca gggagctgga agggctgggc tgaggatgtg gcaggcatgg cgtatgccct acgttcactg ccagaggggg ctccctgcct gccacagtgg tccgtgtata tccgagctgc tgtccggaaa gagaaaggcc tgcccattct tgtggagctc ctccgaatag acaatgaccg tgtagtgtgt gcagtggcca cagcacttcg gaacatggcc ctcgatgtca gaaacaagga actcattggc aagtatgcca tgcgagacct ggtccaccgg cttcctggtg ggaacaacag caacaactcg gggagcaagg ccatgtcaga tgacaccgtg acggccgtgt gctgcaccct gcatgaagtg atcaccaaga acatggagaa tgccaaggcc ttacgggatg ctggtggcat cgagaagttg gtcggcatct ctaaaagcaa aggagacaag cactctccaa aggtggtcaa ggctgcttct caggtcctaa acagcatgtg gcagtatcgc gatctgagga gtctctacaa gaaggatgga tggtcacaat atcactttgt agcctcatct tcaaccatcg agagggatcg acaaaggccc tactcctcct cccgcacacc ctccatctct cccgtgcgtg tgtctcccaa caaccgctca gcaagtgccc cagcttcacc tcgggaaatg atcagcctca aagaaaggaa gacggactac gagtccgctg gcaacaacgc cacttaccac ggaactaaag gagaacacac ctccagaaaa gacaccatga cagctcaaaa cactggagtt tcaactttgt acaggaattc atacggtgcg cccgctgaag acatcaaaca gaaccaggtt SUBSTITUTE SHEET (RULE 26) tccacacagc ctgtccctca ggagcccagc aggaaagact acgagaccta ccagcccttt ccgaattcca cacgaaatta tgatgagtcc ttctttgagg accaggtcca ccaccgccct ccagccagcg agtacaccat gcacctgggc ctcaagtcca ctggcaacta tgtcgacttc tactctgcag cccgtcctta cagtgaactg aactatgaaa cgagccacta cccggcctcg cccgactcct gggtgtaagg agccaggaca cgaggcactc cggggacagt gcatgtgcat gcatacacca caggacattt tgtttctttt tttcttttct tttcttttgt tttttttttt ttttctttcc ctgcaaattt agtttgttaa agcctgttcc gtaggaaggc tgtgataacc aggaagaaat actcagagct attttagaaa gctaaaatga atcaagagtt aactgggaaa tcgataggaa gctaaacgca atcctaattg tgaccgcatt caacaccttt ctagtttgaa ctatagcatt ttgaaagtgc tttatagtcc ggtgaggctg aaggtaggag agaggagaca gtcagggtgg tgggcgtggt tatcgctaag cacaagacag actagtttac acactgtggg gacggcttct cacgctttgt ttactctctt catccgtgtg actctaggct tcaagttgca ttggggttcc tctgtacagc aagacgtctc ttgccttttg ttaatgcatt gttgtaaagt attcgatgta cattacagat taaagacgaa gagtgcattg tgtatattac accaatgcca ctgtgtttcc tcatcaatgg ttctaaatat tgcttcaatt tcaaactttt gaaagatgta tgggtttcca attttctttt tttttttctt tctcccagta tgttttaaca aaaaaggaaa aaaaaaacag gaaaaaaa <210> 6 <211> 1247 <212> PRT
<213> mouse <400> 6 Met Phe Ala Arg Lys Gln Ser Gly Ala Ala Pro Phe Gly Ala Met Pro Val Pro Asp Gln Pro Pro Ser Ala Ser Glu Lys Asn Ser Ser Leu Ser Pro Gly Leu Asn Thr Ser Asn Gly Asp Gly Ser Glu Thr Glu Thr Thr Ser Ala Ile Leu Ala Ser Val Lys Glu Gln Glu Leu Gln Phe Glu Arg Leu Thr Arg Glu Leu Glu Ala Glu Arg Gln Ile Val Ala Ser Gln Leu Glu Arg Cys Lys Leu Gly Ser Glu Thr Gly Ser Met Ser Ser Ile Ser Ser Ala Gly Glu Gln Phe His Trp Gln Thr Gln Asp Gly Gln Lys Asp SUBSTITUTE SHEET (RULE 26) Ile Glu Asp Glu Leu Thr Thr Gly Leu Glu Leu Val Asp Ser Cys Ile Arg Ser Leu Gln Glu Ser Gly Ile Leu Asp Pro Gln Asp Tyr Ser Thr Ser Glu Arg Pro Ser Leu Leu Ser Gln Ser Ala Leu Gln Leu Asn Ser Lys Pro Glu Gly Ser Phe Gln Tyr Pro Ala Ser Tyr His Ser Asn Gln Thr Leu Ala Leu Gly Asp Thr Ala Pro Ser Gln Leu Pro Ala Arg Ser Thr Gln Ala Arg Ala Ala Gly Gln Ser Phe Ser Gln Gly Thr Thr Gly Arg Ala Gly His Leu Ala Gly Ser Glu Pro Ala Pro Pro Pro Pro Pro Pro Arg Glu Pro Phe Ala Pro Ser Leu Gly Ser Ala Phe His Leu Pro Asp Ala Pro Pro Ala Ala Ala Ala Leu Tyr Tyr Ser Ser Ser Thr Leu Pro Ala Pro Pro Arg Gly Gly Ser Pro Leu Thr Thr Thr Gln Gly Gly Ser Pro Thr Lys Leu Gln Arg Gly Gly Ser Ala Pro Glu Gly Ala Ala Tyr Ala Ala Pro Arg Gly Ser Ser Pro Lys Gln Ser Pro Ser Arg Leu Ala Lys Ser Tyr Ser Thr Ser Ser Pro Ile Asn Ile Val Val Ser 5er Ala Gly Leu Ser Pro Ile Arg Val Thr Ser Pro Pro Thr Val Gln Ser Thr Ile Ser Ser Ser Pro Ile His Gln Leu Ser Ser Thr Ile Gly Thr Tyr Ala Thr Leu Ser Pro Thr Lys Arg Leu Val His Ala Ser Glu Gln Tyr Ser Lys His Ser Gln Glu Leu Tyr Ala Thr Ala Thr Leu Gln Arg Pro Gly Ser Leu Ala Ala Gly Ser Arg Ala Ser Tyr Ser Ser Gln His Gly His Leu Ala Pro Glu Leu Arg Ala Leu Gln Ser Pro Glu His His Ile Asp Pro Ile Tyr Glu Asp Arg Val Tyr Gln Lys Pro Pro Met Arg Ser Leu Ser Gln Ser Gln Gly Asp Pro Leu Pro Pro Ala His Thr Gly Thr Phe Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp Ser Val Pro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gln Asn Ala Ala Ala Ala Thr Phe Gln Arg Ala 5er Tyr Ala Ala Gly Pro Ala Ser Asn Tyr Ala Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Ala Ser Val Asp Ser Pro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala Arg Ser Pro Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu Phe Gly Trp Arg Asp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln His Gln Phe SUBSTITUTE SHiEET (RUL.E 28) Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His Lea Cys Phe Gly Asp Asn Lys Ile Lys Ala Glu Ile Arg Arg Gln Gly Gly Ile Gln Leu Leu Val Asp Leu Leu Asp His Arg Met Thr Glu Val His Arg Ser Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala Asn Asp Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro Ala Leu Val Arg Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Leu Lys Met Pro Ile Ile Gln Asp Ala Leu Ala Val Leu Thr Asn Ala Val Ile Ile Pro His Ser Gly Trp Glu Asn Ser Pro Leu Gln Asp Asp Arg Lys Ile Gln Leu His Ser Ser Gln Val Leu Arg Asn Ala Thr Gly Cys Leu Arg Asn Val Ser Ser Ala Gly Glu Glu Ala Arg Arg Arg Met Arg Glu Cys Asp Gly Leu Thr Asp Ala Leu Leu Tyr Val Ile Gln Ser Ala Leu Gly Ser Ser Glu Ile Asp Ser Lys Thr Val Glu Asn Cys Val Cys Ile Leu Arg Asn Leu Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His Met Gly Thr Asp Glu Leu Asp Gly Leu Leu Cys Gly Glu Thr Asn Gly Lys Asp Thr Glu Ser Ser Gly Cys Trp Gly Lys Lys Lys Lys Lys Lys Lys Ser Gln Asp Gln Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro Lys Gly Ile Gln Met Leu Trp His Pro Ser Ile Val Lys Pro Tyr Leu Thr Leu Leu Ser Glu Cys Ser Asn Pro Asp Thr Leu Glu Gly Ala Ala Gly Ala Leu Gln Asn Leu Ala Ala Gly Ser Trp Lys Gly Trp Ala Glu Asp Val Ala Gly Met Ala Tyr Ala Leu Arg Ser Leu Pro Glu Gly Ala Pro Cys Leu Pro Gln Trp Ser Val Tyr Ile Arg Ala Ala Val Arg Lys Glu Lys Gly Leu Pro Ile Leu Val Glu Leu Leu Arg Ile Asp Asn Asp Arg Val Val Cys Ala Val Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr Ala Met Arg Asp Leu Val His Arg Leu Pro Gly Gly Asn Asn Ser Asn Asn Ser Gly Ser Lys Ala Met Ser Asp Asp Thr Val Thr Ala Val Cys Cys Thr Leu His Glu Val Ile Thr Lys Asn Met Glu Asn Ala Lys Ala Leu Arg Asp Ala Gly Gly SUBSTITUTE Sh~EET (RULE 26) Ile Glu Lys Leu Val Gly Ile Ser Lys Ser Lys Gly Asp Lys His Ser Pro Lys Val Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met Trp Gln Tyr Arg Asp Leu Arg Ser Leu Tyr Lys Lys Asp Gly Trp Ser Gln Tyr His Phe Val Ala Ser Ser Ser Thr Ile Glu Arg Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser Ala Pro Ala Ser Pro Arg Glu Met Ile Ser Leu Lys Glu Arg Lys Thr Asp Tyr Glu Ser Ala Gly Asn Asn Ala Thr Tyr His Gly Thr Lys Gly Glu His Thr Ser Arg Lys Asp Thr Met Thr Ala Gln Asn Thr Gly Val Ser Thr Leu Tyr Arg Asn Ser Tyr Gly Ala Pro Ala Glu Asp Ile Lys Gln Asn Gln Val Ser Thr Gln Pro Val Pro Gln Glu Pro Ser Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe Pro Asn Ser Thr Arg Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val His His Arg Pro Pro Ala Ser Glu Tyr Thr Met His Leu Gly Leu Lys Ser Thr Gly Asn Tyr Val Asp Phe Tyr Ser Ala Ala Arg Pro Tyr Ser Glu Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp Val SUBSTITUTE SHEET (RULE 26)

Claims (21)

CLAIMS:

1. A method of stimulating growth of nerve cells, which method comprises contacting nerve cells with a human Neural Plakophilin Related Armadillo Protein (hNPRAP) polypeptide having nerve growth stimulating activity in an amount effective to cause nerve cell growth.

2. The method according to claim 1, wherein the hNPRAP is a full length hNPRAP.

3. The method according to claim 2, wherein the hNPRAP has an amino acid sequence as set forth in SEQ ID NO:4.

4. The method according to claim 1, wherein the growth of nerve cells results in neuronal regeneration.

5. The method according to claim 4, wherein the neuronal regeneration results in synapse formation.

6. The method according to claim 1, wherein the nerve cells are contacted with an hNPRAP induced by an hNPRAP expression stimulating agent.

7. The method according to claim 6, wherein the hNPRAP expression stimulating agent is a gene therapy vector comprising a polynucleotide encoding the hNPRAP and a promoter for expressing the hNPRAP.

8. The method according to claim 6, wherein the growth of nerve cells results in neuronal regeneration.

9. A pharmaceutical composition comprising an hNPRAP having nerve growth stimulating activity and a carrier.

10. The pharmaceutical composition of claim 9, wherein the hNPRAP is a full length hNPRAP.

11. The pharmaceutical composition of claim 10, wherein the hNPRAP has an amino acid sequence as set forth in SEQ ID NO:4.

12. A pharmaceutical composition comprising an hNPRAP gene therapy vector, which vector comprises a polynucleotide encoding the hNPRAP and a promoter for expressing the hNPRAP, and a carrier.

13. A method of identifying substances that modulate the expression of hNPRAP, which method comprises measuring the levels of hNPRAP
expressed in cultured cells that express hNPRAP contacted with a test substance as compared to a control, wherein a difference in the level of hNPRAP expression in the cells contacted with the test substance compared to the control indicates that the test substance modulates expression of hNPRAP.

14. Use of an hNPRAP polypeptide for the preparation of a pharmaceutical composition for stimulating growth of nerve cells.

15. Use according to claim 14, wherein the hNPRAP is a full length hNPRAP.

16. Use according to claim 75, wherein the hNPRAP has an amino acid sequence as set forth in SEQ ID NO:4.

17. Use according to claim 14, wherein the growth of nerve cells results in neuronal regeneration.

18. Use according to claim 17, wherein the neuronal regeneration results in synapse formation.

19. Use according to claim 14, wherein the nerve cells are contacted with an hNPRAP induced by an hNPRAP expression stimulating agent.

20. Use according to claim 19, wherein the hNPRAP expression stimulating agent is a gene therapy vector comprising a polynucleotide encoding the hNPRAP and a promoter for expressing the hNPRAP.

21. Use according to claim 19, wherein the growth of nerve cells results in neuronal regeneration.