NOVEL METHODS FOR TREATING NEURODEGENERATIVE DISORDERS

FIELD OF INVENTION This invention relates to a novel method for treating neurodegenerative diseases.

More specifically, the invention pertains to the use of synthetic compounds to dimerize and activate chimeric proteins within cells, specifically within neural cells. The invention also pertains to the use of adeno-associated virus as a vector to specifically deliver the gene for the chimeric protein to cells in a specific region of the brain or to neural progenitor and/or stem cells used for the treatment of neurodegenerative diseases.

BACKGROUND

Many biological process require highly specific interactions among proteins. This is exemplified by various signal transduction processes required for the normal functioning of cells. A majority of the signaling processes rely on the binding of a natural ligand to an extracellular receptor to promote dimerization or clustering of receptors. The extracellular association between ligand and receptor initiates a cascade of signaling events within cells responsible for the recruitment of proteins required for various cellular functions. Many neurological disorder such as Parkinson's disease, Huntington's disease,

Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Epilepsy Multiple Sclerosis, Senile Dementia, and Alzheimer's disease, are caused by the death of neurons in selected regions of the brain, and the inability of the diseased area to stimulate new neurons to compensate for those that have died. Thus far, such diseases have proved difficult to treat, and few, if any therapies, have proved effective in slowing or arresting the degenerative process associated with these diseases.

In recent years, there has been considerable research activity aimed at understanding various signaling cascades to identify critical steps (within the signaling process) responsible for causing the death of neurons associated with unwanted neurobiological events. Early work in this field has focused on the identification of natural ligands involved in controlling various steps of a signaling cascade, and many groups, both in academia and industry have focused on identifying inhibitors to disrupt ligand receptor interactions believed to play a role in disease and disorder.

Yet still, other research work has focused promoting dimerization or oligomerization events using either natural ligand analogs or highly selective small molecule analogs. This technique of regulating protein-protein interaction has potential utility in the pharmaceutical field, both as a diagnostic method for detecting disease as well as in the therapeutic arena. For example, small molecules could be used to dimerize and trigger a sequence of intracellular activity aimed at promoting the expression of a protein required to correct a particular cellular disorder.

A fundamental impediment in the treatment of neurodegenerative disorders, particularly those associated with the neuronal death or the reduced ability of a lesioned area to grow new neurons or promote new neuronal connections, is the current inability to deliver therapeutics agents selectively to a desired region of the brain. This lack of selectivity further results in deleterious and unwanted toxic side-effects. Thus, there has been an ongoing need for the development of methods to selectively deliver therapeutic agents to a designated area or tissue. Additionally, most of the earlier work has focused on the use of functional proteins, peptides and/or small molecules as inhibitors of ligand- receptor interactions, or as tools to interrupt normal ligand-mediated dimerization events that have been identified as participating in disease processes.

In a departure from those efforts, and from research exploring the dimerization of proteins using small molecules or the identification of such dimerizers, our work has focused on the use of chimerical growth factor receptors to regulate neuritogenesis and neuronal survival for the treatment of neurodegenerative disorders. The invention specifically relates to the use of a dimerizer to promote the association of two molecules of a chimeric growth factor receptor so as to initiate a cascade of signal transduction events essential for the expression growth factors required for neurite outgrowth and neuronal survival. A particularly important aspect of this invention relates to the use of viral vectors and in particular the use of recombinant adeno-associated viral vectors (AAVs) to deliver the modified growth factor receptors to a desired region of the brain. By controlling the exposure of the transfected cells expressing the chimeric protein to a dimerizing compound (ligand), one can limit the amount of growth factor expressed within the cells to a desired level. Another aspect of our current invention focuses on regulating the differentiation and increasing the survival of implanted stem and neural

progenitor cells used as treatments for a variety of neurodegenerative disorders. SUMMARY OF THE INVENTION

The present invention is drawn to the use of chimeric receptors, and the use of small dimerizer compounds to selectively activate the receptors. In one embodiment, the invention provides a method for delivering a modified protein such as a chimeric growth factor receptor to a desired area of the central nervous system. More specifically, the invention relates to providing a gene for expressing a chimeric protein. A favorable type of chimeric protein has a membrane targeting motif, dimerization domain and an intracellular signaling domain. Additionally, the invention relates to the incorporation of the gene for chimeric protein in a suitable vector, and transfecting the vector into cells so as to express the chimeric protein.

Accordingly, the invention is directed to chimeric proteins comprising a membrane targeting motif derived from human FYN, and includes a myristoylation and/or palmitoylation signal. In one embodiment, the chimeric protein has a dimerization domain comprising a ligand-binding domain of human FKBP 12, and an intracellular signaling domain comprising a tyrosine kinase domain of human TrkB. In one embodiment, the invention provides a method for using a viral or non- viral vector to deliver the gene for the chimeric receptor into cells of the brain. Preferably, the viral vector is selected from a group consisting of adeno-associated viral vector, herpes simplex viral vector, parovirus vector and lentivirus vectors. In a preferred embodiment, the viral vector is an adeno-associated viral vector (AAV).

In yet another aspect, this invention discloses the use of recombinant adeno- associated virions having a Cap-region from one type of AAV and a Rep-region from a second type of AAV distinct from the first AAV. Such recombinant AAVs have the advantage of exhibiting modified tropism, (i.e., being highly selective with respect to the tissues it infects), as well as having higher rate of transduction when compared to native AAV. A particularly favorable recombinant adeno-associated virion has a non-native capsid from AAV-I and a Rep-region from AAV-2. In a further embodiment, the vector is a non-viral vector. In a preferred embodiment, the non-viral vector is a liposome-mediated delivery vector.

In one embodiment, the vector is delivered to a desired region of the central nervous system using stereotaxic delivery. In a more preferred embodiment, the desired region of the central nervous system is a region of the brain. It is further advantageous to express the chimeric receptor proteins within a region of the brain that is associated with a particular disorder. In a preferred embodiment, the region of the brain is selected from the group consisting of basal ganglia, subthalamic nucleus (STN), substantia nigra (SN), thalamus, hippocampus, amygdala, hypothalamus, cortex, dopamine neurons, and combinations thereof. In yet another aspect, the expressed chimeric protein is a neurotrophic growth factor receptor. Particularly favorable, is the expression of chimeric proteins for brain derived neurotrophic factor receptor, glial neurotrophic factor receptor, receptors for EPO, G-CSF, TPO, GH, IL-2, interferon-alpha, interferon-beta, insulin and combinations thereof. Another object of the instant invention pertains to the treatment of neurological disorders by identifying a target site in the central nervous system that requires modification, and transfecting at least one cell at the target site with a vector expressing a chimeric growth factor receptor. In one aspect of the invention, the cells expressing the chimeric receptor are exposed to a dimerizing molecule. In yet another embodiment, dimerization of the chimeric receptors initiates a signaling cascade that allows the expression, recruitment and accumulation of a particular growth factor within a diseased cell, so as to effectively treat the neurodegenerative disorder.

In one preferred embodiment, the desired region of the central nervous system is a region of the brain. More preferably, the region of the brain is selected from the group consisting of basal ganglia, subthalamic nucleus (STN), substantia nigra (SN), thalamus, hippocampus, amygdala, hypothalamus, cortex, dopamine neurons, and combinations thereof.

In still another embodiment, the expressed growth factor receptors are selected from the group consisting of brain derived neurotrophic factor receptor, glial neurotrophic factor receptor, receptors for EPO, G-CSF, TPO, GH, IL-2, interferon- alpha, interferon-beta, insulin and combinations thereof.

In one embodiment the dimerizer is a bifunctional compound capable of associating more than one receptor. In another embodiment, the dimerizer is a molecule selected from the group consisting of a peptide ligand, peptidomimetic, organic compounds and combinations thereof. Preferably, the dimerizer is a cell permeant synthetic compound. By way of example, the dimerizer could potentially be the synthetic ligand AP20187 that binds FKBP-12.

In another preferred embodiment the dimerizer is administered either orally, bucally, intra-peritoneally, intrathecally, intravenously, or any combination thereof. In yet another embodiment, the neurodegenerative disease is selected from the group consisting of Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS or Lou Gerighs disease), Alzheimer's disease, senile dementia, multiple sclerosis and epilepsy.

Another embodiment of this invention is a method for regulating the differentiation of stem cells or neural progenitor cells used in the treatment of neurodegenerative disorders. Accordingly, an important object of this invention is the ability to regulate differentiation of stem and/or neural progenitor cells into neurons. In one exemplary embodiment, neural progenitor cells or stem cells are stably transfected with a gene for expressing the chimeric receptors. The transfected cells are then implanted into the diseased region of the brain. Following implantation, the cells are exposed to a synthetic dimerizer that facilitates the clustering of the chimeric receptors, thus initiating a signaling cascade responsible for the recruitment of proteins involved in the differentiation of neural progenitor cells or stem cells into neurons.

According to one aspect of the invention, the transfected stem or neural progenitor cells are implanted either into the substantia nigra or the region near the dopamine neurons in the brain.

In still another embodiment, this invention provides a method for increasing the survival of stem cells or neural progenitor cells following implantation into a diseased area of the brain. In yet another embodiment, the invention provides a method for the selective differentiation of these cells into neurons post implantation.

In another aspect, the invention provides a method for modulating the activity of a chimeric growth factor receptor expressed in neural cells. Accordingly, one object of the invention is transfecting at least one neural cell with a vector encoding a gene for a

chimeric growth factor receptor, expressing the chimeric protein and exposing the transfected neural cell to a dimerizing molecule. In a further embodiment, the dimerizer activates the chimeric receptor to initiate a cascade of intracellular signal transduction processes.

In one embodiment, the expressed chimeric receptors are selected from the group consisting of brain derived neurotrophic factor receptor, glial neurotrophic factor receptor, receptors for EPO, G-CSF, TPO, GH, IL-2, interferon-alpha, interferon-beta, insulin and combinations thereof. In still another aspect of this invention, the dimerizing molecule is a birunctional molecule capable of associating with more than one chimeric receptor. Preferably, the dimerizing molecule is selected from the group consisting of a peptide ligand, peptidomimetic, organic compounds and combinations thereof. Preferably, the dimerizer is a cell permeant synthetic compound. By way of example, the dimerizer could potentially be the synthetic ligand AP20187 that binds FKBP- 12.

In a further aspect of this invention the dimerizer can be administered orally, bucally, intraperitoneally, intravenously, intrathecally, or by any other appropriate route, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA depicts a fluorescence micrograph of rat pheochromocytoma PC 12 cells transiently transfected with pMP-GFP, a negative control comprising the myristoylation/ palmitoylation signal domains operably linked to the gene for green fluorescent protein (GFP).

Figure IB depicts a fluorescence micrograph of rat pheochromocytoma PC 12 cells transiently transfected with pMP-TrkB, control plasmid comprising the myristoylation/ palmitoylation signal domains operably linked to the gene for human TrkB domain.

Figure 1C depicts a fluorescence micrograph of rat pheochromocytoma PC 12 cells transiently transfected with pMP-F-TrkB, plasmid containing the

myristoylation/palmitoylation signal domains operably linked to the gene human FKBP 12 dimerization domain linked to the gene for human TrkB domain.

Figure 2 A shows a picture of rat pheochromocytoma PC 12 cells transiently transfected with (1) pMP-GFP, a negative control comprising the myristoylation/palmitoylation signal domains operably linked to the gene for green fluorescent protein (GFP); (2) pMP-TrkB, control plasmid comprising the myristoylation/palmitoylation signal domains operably linked to the gene for human TrkB domain; and (3) pMP-F-TrkB, plasmid containing the myristoylation/palmitoylation signal domains operably linked to the gene human

FKBP 12 dimerization domain linked to the gene for human TrkB domain in the absence of dimerizer AP20817.

Figure 2B shows a picture of rat pheochromocytoma PC 12 cells transiently transfected with (1) pMP-GFP, a negative control comprising the myristoylation/palmitoylation signal domains operably linked to the gene for green fluorescent protein (GFP); (2) pMP-TrkB, control plasmid comprising the myristoylation/palmitoylation signal domains operably linked to the gene for human TrkB domain; and (3) pMP-F-TrkB, plasmid containing the myristoylation/ palmitoylation signal domains operably linked to the gene human FKBP 12 dimerization domain linked to the gene for human TrkB domain in the presence of dimerizer

AP20817.

Figure 3 survival of rat pheochromocytoma PC 12 cells 24 hours after exposure to 6-OHDA (lOOμM) and in the presence of AP-20187. The cells were transiently transfected with (a) pMP-GFP, a negative control comprising the myristoylation/palmitoylation signal domains operably linked to the gene for green fluorescent protein (GFP); (b) pMP-TrkB, control plasmid comprising the myristoylation/palmitoylation signal domains operably linked to the gene for human TrkB domain; and (c) pMP-F-TrkB, plasmid containing the myristoylation/palmitoylation signal domains operably linked to the gene human FKBP 12 dimerization domain linked to the gene for human TrkB domain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel methods for treating neurodegenerative disorders. Growth factors, such as glial derived neurotrophic factor (GDNF), neural growth factor (NGF), and neuritin are currently being tested clinically as treatments for neurodegenerative disorders such as Parkinson's Disease (PD), Alzheimer's Disease, amyotrophic lateral sclerosis (ALS), Huntington's Disease (HD), and epilepsy. Early results from these studies have indicated that, while the growth factors ameliorate the deleterious effects of neurodegenerative conditions by promoting neuronal outgrowth, growth of new neurons and help in preventing neuronal death, these therapeutic proteins also display severe toxic side effects. For example, the delivery of such protein therapeutics via an intravenous, or intracranial route will result in exposing both normal as well lesioned parts of the brain to the growth factor, and may cause the unwanted growth of neuronal cells (neuritogenesis) in normal parts of the brain. Thus there exists a need for methods to selectively deliver protein therapeutics to diseased regions of the brain. Furthermore, there is also a need for methods to regulate the differentiation of stem and neural progenitor cells and to selectively express a protein of interest inside genetically modified stem or neural progenitor cells after implanting the genetically modified cells at the site of repair. The practice of the present invention employs, conventional techniques of virology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art. Such techniques are fully explained in the literature. (See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Homes & S. Higgins, eds., Current Edition); Transcription and Translation ( B. Homes & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.); Fundamental Virology, 2nd Edition, Vol. I & II ( B. N. Fields and D. M. Knipe, eds.)).

I. Definitions:

So that the invention is more clearly understood, the following terms are defined: The term "central nervous system" or "CNS" as used herein refers to the art recognized use of the term. The CNS pertains to the brain, cranial nerves and spinal

cord. The CNS also comprises the cerebrospinal fluid, which fills the ventricles of the brain and the central canal of the spinal cord.

The terms "neurodegenerative disorder" or a "neurological disorder" as used herein refers to a disorder which causes morphological and/or functional abnormality of a neural cell or a population of neural cells. The neurodegenerative disorder can result in an impairment or absence of a normal neurological function or presence of an abnormal neurological function in a subject. For example, neurodegenerative disorders can be the result of disease, injury, and/or aging. Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of neural cells, abnormal growth patterns of neural cells, abnormalities in the physical connection between neural cells, under- or over production of a substance or substances, e.g., a neurotransmitter, by neural cells, failure of neural cells to produce a substance or substances which it normally produces, production of substances, e.g., neurotransmitters, and/or transmission of electrical impulses in abnormal patterns or at abnormal times.

Neurodegeneration can occur in any area of the brain of a subject and is associated with many neurological disorders including, for example, head trauma, stroke, ALS, multiple sclerosis, senile dementia, Huntington's disease, Parkinson's disease, epilepsy, and Alzheimer's disease. As used herein, the terms "Parkinson's subject" and "a subject who has

Parkinson's" are intended to refer to subjects who have been diagnosed with Parkinson's or probable Parkinson's. The terms "non-Parkinson's subject" and "a subject who does not have Parkinson's" are intended to refer to a subject who has not been diagnosed with Parkinson's or probable Parkinson's. A non-Parkinson's subject may be healthy and have no other disease, or they may have a disease other than Parkinson's. The term

"subject," as used herein, refers to any living organism capable of eliciting an immune response. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, Parkinson's adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term "polypeptide" refers to a single amino acid or a polymer of amino acid residues. A polypeptide may be composed of two or more polypeptide chains. A polypeptide includes a protein, a peptide, an oligopeptide, and an amino acid. A polypeptide can be linear or branched. A polypeptide can comprise modified amino acid residues, amino acid analogs or non-naturally occurring amino acid residues and can be interrupted by non-amino acid residues. Included within the definition are amino acid polymers that have been modified, whether naturally or by intervention, e.g., formation of a disulfide bond, glycosylation, lipidation, methylation, acetylation, phosphorylation, or by manipulation, such as conjugation with a labeling component.

As used herein, the term "polynucleotide" refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition are nucleic acid polymers that have been modified, whether naturally or by intervention.

As used herein, the term "specifically binding," refers to the interaction between binding pairs (e.g., an antibody and an antigen) with an affinity constant of at most 10 ^"6 moles/liter, at most 10 ^"7 moles/liter, or at most 10 ^"8 moles/liter.

As used herein, the term "chimeric protein" refers to a protein that is encoded by a nucleotide sequence made by splicing two or more partial genes or c-DNA. The pieces spliced together may be from the same or different species. The gene that encodes for the chimeric protein is cloned into a plasmid that is able to express the chimeric protein in a cellular environment.

As used herein, the term "membrane targeting motif refers to a polypeptide or a region of the protein that specifically targets and binds to a receptor on the external surface of a cell. The phosphatidylinositol (Ptd-Ins) phospholipids, collectively called phosphoinositides (PIs), are major determinants in localizing proteins to their site of function. The importance of membrane targeting by PIs is exemplified by a number of

human diseases linked to defects in PI signaling (3-5), including cancer, immunodeficiency disorders (X-linked agammaglobulinemina and chronic granulomatous disease), myotubular myopathy, kidney and neurological diseases (oculocerebro-renal syndrome of Lowe), and faciogenital dysplasia (Aarskog-Scott syndrome).

As used herein, the term "dimerizer" refers to a bifunctional molecule capable of selectively and preferably simultaneously cross linking two (or more) polypeptides or proteins with a dissociation constant below 10 ^"6 preferably below 10 ^'7 , and even more preferably below 10 ^"8 , and in some cases below 10 ^"9 . Dimerizers can be peptides, peptidomimetics, small molecules or combinations of the above.

As used herein, the term "dimerization domain" refers to a region of a protein or a polypeptide that specifically binds a dimerizer. Dimerization domains are present in many signaling proteins including tyrosine kinase inhibitors, neurotrophic tyrosine kinases such as Trk family of proteins, the interferon family of receptors, G-protein coupled receptors, the TGF-β family of receptors to name a few.

Certain exemplary embodiments will now be described to provide an overall understanding of the methods for selective delivery of chimeric receptors to specific regions of the brain, methods for activating the chimeric receptors using highly selective dimerizing agents, and the use of such receptors to express neurotrophic growth factors for treating neurodegenerative disorders is disclosed herein. Those of ordinary skill in the art will understand that the methods specifically described herein and the results illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present invention.

II. Neurodegenerative Diseases Generally, the methods of the current invention can be used for the treatment of neurodegenerative disorders. More specifically, methods of the current invention can be used for the treatment of neurodegenerative disorders associated with the loss of neurons and neuronal function. This invention pertains to the delivery of chimeric growth factor

receptors and the activation of these receptors using membrane permeable dimerizer so as to secrete endogenous growth factors that at the site of repair, and result in an overall improvement of the neurological condition. The invention also provides for methods to selectively deliver genes that encode a chimeric receptor protein to the site of disease in the brain using viral and non-viral vectors, and particularly the use of recombinant adeno-associated viral vectors. The regions of the brain associated with a given neurological condition may vary, but are well know to the skilled artisan. For example, the region of the brain associated with Parkinson's disease can be the STN, while the region of the brain associated with epilepsy can be the hippocampus.

The invention also provides methods for regulating the differentiation of stem and/or neural progenitor cells so as to use these cells for the treatment of neurodegenerative disorders. In particular, the invention provides a way for replenishing diseased or dead neuronal cells in a diseased area. The methods of the invention also allow for increasing the survival of these cells in vivo. Some exemplary neurodegenerative conditions that can be treated using the methods of this invention are:

(a) Parkinson 's Disease

Parkinson's Disease (PD) is a movement disorder characterized by tremors, muscular rigidity, restricted limb movements and walking with small, slow steps. It typically onsets around middle age, and although it is not fatal it is an ongoing degenerative disease, the process of which cannot be reversed. Not much is known about the cause of PD, there may be genetic causality, but the main pathological process involves degeneration of the substantia nigra (SN), which is situated in the basal ganglia and is an area rich in dopamine cells. Dopamine is one of the major neurotransmitters, or naturally occurring chemicals, found in the brain. In PD there is a major depletion of dopamine, especially in the fiber projections from the substantia nigra to the corpus striatum. Dopamine is believed to be the main, but not the only, neurotransmitter involved in PD. Decreased dopamine leads to secondary effects like degeneration of GABA receptors, and there is believed to be an interaction of dopamine with another neurotransmitter - Acetylcholine (Ach). While dopamine is depleted, acetylcholine (activity and production) is increased. It is the over activity of Ach which causes the tremors and rigidity which are the trademark symptoms of PD.

Traditionally, the symptoms of PD have been treated via drug therapy, the principal theory behind which involves reducing the activity of Ach (via an anticholinesterase e.g. scopolamine) or increasing the amount of dopamine (via the drug L-Dopa). The major problem with drug therapies however is that there are many, and often severe, side effects including nausea, dizziness etc. This is because the drugs often do not work on the specific areas where they are required (e.g. in the substantia nigra) but also in other areas of the brain where they are not needed. The other drawback is that although the drugs 'contain' the disease they do not 'cure' it. Accordingly, the present invention provides a novel method for treating PD. In one embodiment it allows the implantation of stem or neural progenitor cells that can be induced to differentiate into dopamine producing neurons, i.e, the emphasis is on encouraging new neurons to 'sprout' or grow in the damaged areas, (because there is now considerable evidence that neurons can sprout new synapses and fibers to replace those that have been lost). In another embodiment of this invention, an adeno-associated viral vector can used to deliver a gene for GAD to excitatory glutaminergic neurons of the STN. The transduced neurons resulted in strong neuroprotection of nigral dopamine neurons and rescue of the parkinsonian behavioral phenotype.

Accordingly, a region of the brain associated with Parkinson's disease can be inhibited, reduced, treated, or altered using the methods and compositions of the invention. In particular, a vector comprising a therapeutic agent, e.g., a nucleotide sequence encoding GAD, can be delivered to the site of dopaminergic cell loss or other regions of the basal ganglia and output nuclei. In one embodiment, the vector comprising a therapeutic agent can be delivered to the subthalamic nucleus (SN). In another embodiment, the vector comprising a therapeutic agent can be delivered to the substantia nigra pars reticulata (SNPR).

(b) Alzheimer's Disease

Alzheimer's disease is characterized by the gradual loss of intellectual capabilities. Post-mortem examination of the brain shows a generalized atrophy. There are extensive histological changes in Alzheimer's disease dominated by the presence of intracellular amyloid plaques and neurofibrillary tangles. Plaques and tangles are rare, however, in the basal ganglia and substantia nigra. Many specimens from Alzheimer's

disease patients demonstrate a loss of pigmentation in the area of the locus ceruleus, which is a major source of noradrenergic synthesis in the brain. Accordingly, a region of the brain associated with Alzheimer's disease can be inhibited, reduced, treated, or altered using the methods and compositions of the invention.

(c) Epilepsy

Epileptic seizures are the outward manifestation of excessive and/or hypersynchronous abnormal activity of neurons in the cerebral cortex. Seizures are usually self limiting. Many types of seizures occur. The behavioral features of a seizure reflect function of the portion of the cortex where the hyper activity is occurring. Seizures can be generalized, appearing to involve the entire brain simultaneously. Generalized seizures can result in the loss of conscious awareness only and are then called absence seizures (previously referred to as "petit mal"). Alternatively, the generalized seizure may result in a convulsion with tonic-clonic contractions of the muscles ("grand mall" seizure). Some types of seizures, partial seizures, begin in one part of the brain and remain local. The person may remain conscious throughout the seizure. If the person loses consciousness the seizure is referred to as a complex partial seizure. Simple partial seizures include autonomic and mental symptoms and sensory symptoms such as olfaction, audition, or vision, sometimes concomitant with symptoms of experiences such as deja-vu and jamais-vu. Complex partial seizures often exhibit motion stopping followed by eating-function automatism, and are divided into amygdala-hippocampus seizures and lateral temporal lobe seizures according to localization. In the case of temporal lobe epilepsy, 70-80% of the seizures are hippocampus seizures, in which aura, motion stopping, lip automatism, and clouding of consciousness are successively developed to result in amnesia. When the focus is in the amygdala, there are caused autonomic symptoms such as dysphoria in the epigastrium; phobia; and olfactory hallucination. Lateral temporal lobe seizures include auditory illusion, hallucination, and a dreamy state, and disturbance of speech when the focus is in the dominant hemisphere. Temporal lobe epilepsy exhibits a long-term psychosis-like state in addition to other symptoms and recognition-and-memory disorder more frequently than do other epilepsies. Treatment of temporal lobe epilepsy is carried out

through pharmacotherapy employing a maximum dose of a combination of drugs, or through surgical treatment. A complex partial seizure is a partial seizure with impairment of consciousness, and is similar to a seizure that has conventionally been called a psycho-motor seizure or a seizure associated with temporal lobe epilepsy.

The neuromechanism responsible for seizures includes the amygdala, the hippocampus, the hypothalamus, the parolfactory cortex, etc., in addition to the frontal and temporal lobes. The seizures typically last 1-2 minutes or slightly longer, and the onset and cessation of the seizures are not abrupt but gradual. The existence of a system which can control the propagation and/or the generation of different kinds of seizures is known. The involvement of the substantia nigra, a particular portion of the brain considered to be part of neural circuitry referred to as the basal ganglia (See e.g., Depaulis, et al. (1994) Prog. Neurobiology, 42: 33-52) is known. The inhibition of the substantia nigra will increase the threshold for seizure. The neural connections that make up the basal ganglia are also important in epilepsy. These connections are reviewed by Alexander et. al. (Alexander, et al. Prog. Brain Res. 85: 119-146). The substantia nigra receives input from the subthalamic nucleus (STN) which is excitatory and involves glutamate as the neurotransmitter conveying information at the synapse. Bergman et al. have shown that a lesion of the subthalamic nucleus will reduce the inhibitory output of the internal segment of the globus pallidus and substantia nigra reticulata (SN) (Bergman, et al (1990), Science, 249: 1436-1438). The subthalamic nucleus receives input from the external segment of the globus pallidus (GPe). This input is inhibitory using GABA as a transmitter substance. Hence, increased activity of the neurons in GPe will increase inhibition of neurons in the subthalamic nucleus which will reduce the excitation of neurons in the substantia nigra.

Accordingly, a region of the brain associated with epilepsy can be inhibited, reduced, treated, or altered using the methods and compositions of the invention. The invention is intended to include all regions of the brain associated with epilepsy. The methods and compositions of the invention can be used to be used to inhibit, reduce, or treat seizures that include, but are not limited to, tonic seizures, tonic-clonic seizures, atypical absence seizures, atonic seizures, myoclonic seizures, clonic seizures, simple partial seizures, complex partial seizures, and secondary generalized seizures. In

one aspect, the invention provides a method for selectively transfecting cells in the subthalamic region of the brain with a gene that codes for GAD. Increase in GAD levels will decrease the glutamate concentration in the subthalamic region and help in controlling seizures.

(d) Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a universally fatal neurodegenerative condition in which patients progressively lose all motor function - unable to walk, speak, or breathe on their own, ALS patients die within two to five years of diagnosis.

The incidence of ALS increases substantially in the fifth decade of life. Evidence is accumulating that as a result of the normal aging process the body increasingly loses the ability to adequately degrade mutated or misfolded proteins. The proteasome is the piece of biological machinery responsible for most normal degradation of proteins inside cells. Age related loss of function or change of function of the proteasome is now thought to be at the heart of many neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and ALS.

The cardinal feature of ALS is the loss of spinal motor neurons, which causes the muscles under their control to weaken and waste away leading to paralysis. ALS has both familial (5-10%) and sporadic forms and the familial forms have now been linked to several distinct genetic loci (Deng, H.X., et al., "Two novel SODl mutations in patients with familial amyotrophic lateral sclerosis," Hum. MoI. Genet., 4(6): 1113-16 (1995); Siddique, T. and A. Hentati, "Familial amyotrophic lateral sclerosis," CHn. Neurosci., 3(6): 338-47(1995); Siddique, T., et al., "Familial amyotrophic lateral sclerosis," J. Neural Transm. SuppL, 49: 219-33(1997); Ben Hamida, et al., "Hereditary motor system diseases (chronic juvenile amyotrophic lateral sclerosis). Conditions combining a bilateral pyramidal syndrome with limb and bulbar amyotrophy," Brain, 113(2): 347-63 (1990); Yang, Y., et al., "The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis," Nat. Genet., 29(2): 160-65 (2001); Hadano, S., et al., "A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2 " Nat. Genet., 29(2): 166-73 (2001)). About 15-20% of familial cases are due to mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SODl) (Siddique, T.,

et al., "Linkage of a gene causing familial amyotrophic lateral sclerosis to chromosome 21 and evidence of genetic-locus heterogeneity," N. Engl. J. Med., 324(20): 1381-84 (1991); Rosen, D.R., et al., "Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis." Nature, 362(6415): 59-62

(1993)).

Although the etiology of the disease is unknown, the dominant theory is that neuronal cell death in ALS is the result of over-excitement of neuronal cells due to excess extracellular glutamate. Glutamate is a neurotransmitter that is released by glutaminergic neurons, and is taken up into glial cells where it is converted into glutamine by the enzyme glutamine synthetase, glutamine then re-enters the neurons and is hydrolyzed by glutaminase to form glutamate, thus replenishing the neurotransmitter pool. In a normal spinal cord and brain stem, the level of extracellular glutamate is kept at low micromolar levels in the extracellular fluid because glial cells, which function in part to support neurons, use the excitatory amino acid transporter type 2 (EAAT2) protein to absorb glutamate immediately. A deficiency in the normal EAAT2 protein in patients with ALS, was identified as being important in the pathology of the disease {See e.g., Meyer et al., J. Neurol. Neurosurg. Psychiatry, 65: 594-596 (1998); Aoki et al., Ann. Neurol. 43: 645-653 (1998); Bristol et al, Ann Neurol. 39: 676-679 (1996)). One explanation for the reduced levels of EAAT2 is that, EAAT2 is spliced aberrantly (Lin et al., Neuron, 20: 589-602 (1998)). The aberrant splicing produces a splice variant with a deletion of 45 to 107 amino acids located in the C-terminal region of the EAAT2 protein (Meyer et ah, Neureosci Lett. 241 : 68-70 (1998)). Due to the lack of, or defectiveness of EAAT2, extracellular glutamate accumulates, causing neurons to fire continuously. The accumulation of glutamate has a toxic effect on neuronal cells because continual firing of the neurons leads to early cell death. Accordingly, one aspect of the invention provides methods for selectively transfecting cells to express a chimeric form of EAAT2 under the control of a dimerizer.

(e) Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic disease that is characterized by "attacks," during which areas of white matter of the central nervous system, known as plaques, become inflamed. Inflammation of these areas of plaque is followed by destruction of

myelin, the fatty substance that forms a sheath or covering that insulates nerve cell fibers in the brain and spinal cord. Myelin facilitates the smooth, high-speed transmission of electrochemical messages between the brain, spinal cord, and the rest of the body. Damage to the myelin sheath can slow or completely block the transmission of these electrochemical messages, which can result in diminished or lost bodily function.

The most common course of MS manifests itself as a series of attacks, which are followed by either complete or partial remission, during which the symptoms lessen only to return at some later point in time. This type of MS is commonly referred to as "relapsing-remitting MS." Another form of MS, called "primary-progressive MS," is characterized by a gradual decline into the disease state, with no distinct remissions and only temporary plateaus or minor relief from the symptoms. A third form of MS, known as "secondary-progressive MS," starts as a relapsing-remitting course, but later deteriorates into a primary-progressive course of MS. The symptoms of MS can be mild or severe, acute or of a long duration, and may appear in various combinations. These symptoms can include vision problems such as blurred or double vision, red-green color distortion, or even blindness in one eye, muscle weakness in the extremities, coordination and balance problems, muscle spasticity, muscle fatigue, paresthesias, fleeting abnormal sensory feelings such as numbness, prickling, or "pins and needles" sensations, and in the worst cases, partial or complete paralysis. About half of the people suffering from MS also experience cognitive impairments, such as for example, poor concentration, attention, memory and/or judgment. These cognitive symptoms occur when lesions develop in those areas of the brain that are responsible for information processing.

(f) Huntington 's Disease

Huntington's disease (HD) is a hereditary disorder caused by the degeneration of neurons in certain areas of the brain. This degeneration is genetically programmed to occur in certain areas of the brain, including the cells of the basal ganglia, the structures that are responsible for coordinating movement. Within the basal ganglia, Huntington's disease specifically targets nerve cells in the striatum, as well as cells of the cortex, or outer surface of the brain, which control thought, perception and memory. Neuron degeneration due to HD can result in uncontrolled movements, loss of intellectual

capacity and faculties, and emotional disturbance, such as, for example, mood swings or uncharacteristic irritability or depression.

As discussed above, neuron degeneration due to HD is genetically programmed to occur in certain areas of the brain. Studies have shown that Huntington's disease is caused by a genetic defect on chromosome 4, and in particular, people with HD have an abnormal repetition of the genetic sequence CAG in the HD gene, which has been termed ITl 5. The IT 15 gene is located on the short arm of chromosome 4 and encodes a protein called huntingtin. Exon I of the ITl 5 gene contains a polymorphic stretch of consecutive glutamine residues, known as the polyglutamine tract (D. Rubinsztein,

"Lessons from Animal Models of Huntington's Disease," TRENDS in Genetics, 18(4): 202-9 (April 2002)). Asymptomatic individuals typically contain fewer than 35 CAG repeats in the polyglutamine tract.

The inherited mutation in HD is an expansion of the natural CAG repeats within the sequence of exon 1 of the human HD gene. This leads to an abnormally long stretch of polyglutamines. The length of the polyglutamine repeats correlates with the severity of the disease. One of the pathological hallmarks of HD is a buildup of intracellular protein aggregates composed of these abnormal HD proteins with long polyglutamine repeats. In one aspect the invention provides for a method to selectively transfect cells to express the chimeric form of the anti-apoptotic protein XIAP, known to play a role in blocking neuronal death. This demonstrates that expression of an anti-apoptotic gene can protect from mutant Huntington-induced neuronal death.

(g) Other Degenerative Diseases This invention also relates to compositions and methods of treatment of other degenerative disorders. These include, but are not limited to the following: head and spinal cord trauma; cardiac cell death due to ischemia; tissue and organ death due to transplant rejection; and hearing loss due to autotoxicity.

III. Vectors

The vectors of the invention can be delivered to the cells of the central nervous system by using viral vectors or by using non-viral vectors. In a preferred embodiment, the invention uses adeno-associated viral vectors comprising the a nucleotide sequence

encoding a chimeric receptor for gene delivery. AAV vectors can be constructed using known techniques to provide at least the operatively linked components of control elements including a transcriptional initiation region, a exogenous nucleic acid molecule, a transcriptional termination region and at least one post-transcriptional regulatory sequence. The control elements are selected to be functional in the targeted cell. The resulting construct which contains the operatively linked components is flanked at the 5' and 3' region with functional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. The ITR sequences for AAV-2 are described, for example by Kotin et al. (1994) Human Gene Therapy

5:793-801; Berns "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) The skilled artisan will appreciate that AAV ITR' s can be modified using standard molecular biology techniques. Accordingly, AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to, AAV-I, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAVX7, AAV-8 and the like. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as the ITR' s function as intended, i.e., to allow for excision and replication of the bounded nucleotide sequence of interest when AAV rep gene products are present in the cell.

The skilled artisan can appreciate that regulatory sequences can often be provided from commonly used promoters derived from viruses such as, polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Use of viral regulatory elements to direct expression of the protein can allow for high level constitutive expression of the protein in a variety of host cells. Ubiquitously expressing promoters can also be used include, for example, the early cytomegalovirus promoter Boshart et al. (1985) Cell 41 :521-530, herpes virus thymidine kinase (HSV-TK) promoter (McKnight et al. (1984) Cell 37: 253-262), beta-actin promoters {e.g., the human beta-actin promoter as described by Ng et al. (1985) MoI. Cell Biol. 5: 2720-2732) and colony stimulating factor-1 (CSF-I) promoter (Ladner ef α/. (198I) EMBO J. 6: 2693-2698).

The AAV vector harboring the nucleotide sequence encoding a protein of interest, e.g., chimeric growth factor receptor, and a post-transcriptional regulatory sequence (PRE) flanked by AAV ITRs, can be constructed by directly inserting the nucleotide sequence encoding the protein of interest and the PRE into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, as long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. These constructs can be designed using techniques well known in the art. {See, e.g., Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor

Laboratory Press); Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka (1992) Current Topics in Microbiol, and Immunol. 158:97-129; Kotin (1994) Human Gene Therapy 5:793-801; Shelling et al. (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875). Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al , Supra. Several AAV vectors are available from the American Type Culture Collection ("ATCC") under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

In order to produce recombinant AAV particles, an AAV vector can be introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, N. Y., Davis et al. (1986) Basic Methods in

Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13: 197. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transduction (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73).

Suitable host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a exogenous nucleic acid molecule. Host cells containing the above-described AAV vectors must be rendered capable of providing AAV helper functions in order to replicate and encapsidate the expression cassette flanked by the AAV ITRs to produce recombinant AAV particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV vectors. Thus, AAV helper functions include one, or both of the major AAV open reading frames (ORFs), namely the rep and cap coding regions, or functional homologues thereof.

Alternatively, a vector of the invention can be a virus other than the adeno- associated virus, or portion thereof, which allows for expression of a nucleic acid molecule introduced into the viral nucleic acid. For example, replication defective retroviruses, adenoviruses, herpes simplex virus, and lentivirus can be used. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include Crip, Cre, 2 and Am. The genome of adenovirus can be manipulated such that it encodes and expresses the protein of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See e.g., Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68: 143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus {e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Alternatively, the vector can be delivered using a non-viral delivery system. This includes delivery of the vector to the desired tissues in colloidal dispersion systems that include, for example, macromolecule complexes, nanocapsules, microspheres, beads,

and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genetic material at high efficiency while not compromising the biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al. (1988)

Biotechniques, 6:682). Examples of suitable lipids liposomes production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Additional examples of lipids include, but are not limited to, polylysine, protamine, sulfate and 3b -[N- (N',N' dimethylaminoethane) carbamoyl] cholesterol.

IV. Dimerization

The invention relies on the use of a dimerizer molecule to bring together the chimeric growth factor receptors, and mediate desired biological events. Dimerization refers to the association of like components, e.g., two chimeric proteins, to forms dimers.

Dimerization can be induced using homo-dimerizers, molecules that link two like components or hetero-dimerizers, molecules that link two non-related components. Dimerization, triggers cellular processes normally associated with signal transduction. Exemplary receptor proteins and processes triggered via dimerization events include EPO receptor, G-CSF receptor, GH-receptor, IL-2, IFN-α and IFN-β receptors, Insulin receptors, and Trk receptors to name a few.

These receptors are activated via the extracellular association of various polypeptides, growth hormones, and cytokines and result in intracellular signaling events (Ullrich et. al., Signal transduction by receptors with tyrosine kinase activity, Cell 61: 203-212, 1990). These receptors are composed of three regions: and extracellular ligand-binding domain, a transmembrane domain and an intracellular signal transduction domain. Many signal transducing proteins are tyrosine kinases or receptors complexed to tyrosine kinases, e.g., CD3 zeta, IL-2R, IL-3R etc. (Cantley, et. al., Cell 64: 281,

1991). Tyrosine kinase receptors which are activated by cross-linking include the Trk family of neurotrophic receptors such as the NGF-R, Rorl,2 etc. Receptors that associate with the tyrosine kinase receptors upon cross-linking include the CD3 zeta family of receptors, CD3 eta, CD3 gamma, CD3 delta and CD3 epsilon found in T-cells.

The foregoing is in no way an exhaustive list, but provides exemplary systems for use with the subject invention.

V Dimerizers Generally speaking, the dimerizer is capable of binding to two (or more) protein molecules, in either order or simultaneously, preferably with a Kd value below about 10 ^{" 6} , more preferably below about 10 ^"7 , even more preferably below about 10 ^"8 , and in some embodiments below about 10 ^"9 M. The dimerizer preferably is a non-protein and has a low molecular weight. The proteins so oligomerized may be the same or different. In one exemplary embodiment of this invention, it is preferable that the dimerizer is a molecule capable of traversing the cell membrane in its bioactive form so as to crosslink dimerization domains of two proteins within the cell. In order to achieve this many dimerizers are hydrophobic molecules. However, if this is not the case, the dimerizers can be made more hydrophobic via chemical modification. In another embodiment, the dimerizer acts extracellularly to bring together proteins that act in concert to initiate intracellular physiological events. For such dimerizers, the molecule need not display any cellular permeability.

Applicable and readily observable or measurable criteria for dimerizers include: (a) dimerizer is physiologically acceptable (i.e., lacks undue toxicity towards the cell or animal for which it is to be used), (b) it has a reasonable therapeutic dosage range, (c) desirable (for applications in whole animals), it can be taken orally ( is stable in the gastrointestinal system and absorbed into the vascular system), (d) it can cross the cellular and other membranes, as necessary, and (e) binds to the target protein(s) with reasonable affinity for the desired application. Preferably the dimerizer is relatively inert physiologically, but for its activating capability with the target protein(s).

(a) Design of Dimerizers

One method for preparing a "dimerizers" for use in the instant invention involves the step of identifying a first compound capable of binding one of the protein mediators and a second compound capable of binding to the other protein mediator. The two compounds are then covalently joined to one another to form a dimerizer which is capable of binding to both mediators (at the same time). In a favored embodiment of the current invention, a synthetic analog is used as the dimerizer, to bring together the ligand binding domains of the human protein FKBPl 2. In a more preferred embodiment, a high affinity synthetic ligand (e.g., AP20187) is used to bring together the ligand binding domain of human FKBP 12 genetically engineered into a chimeric growth factor receptor used in the treatment of neurodegenerative disorders. This chimeric protein comprises a membrane targeting domain at the N-terminus, a ligand binding domain, and an intracellular signaling domain. A number of chemical techniques known to one of skill in the art can be used to link the first and second compounds comprising the dimerizer.

VI Examples-Treatment of Neurodegenerative Disorders

The methods of the instant invention can be tested using in-vitro as well as in- vivo assays.

(a) In- Vitro Studies

(i) Plasmid encoding the chimeric receptor protein:

A gene for the chimeric receptor was constructed and has the following regions:

(a) a nucleotide sequence that encodes for a membrane targeting domain, (b) a sequence that codes for a ligand-binding dimerization domain, and (c) an intracellular signaling domain. The gene was cloned into an AAV plasmid, and the plasmid was packaged to generate high titre r-AAV-chimeric receptor viral particles using an optimized protocol.

For in-vitro studies the chimeric receptor protein was expressed fused to green fluorescence protein (GFP) at its C-terminus. Two plasmids designated aspMP-F-TrkB and p-M-F-TrkB were engineered and designed to express the chimeric proteins inside cells. The chimeric proteins contain a myristoylation and/or palmitoylation membrane honing signal at the N-terminus. This domain is followed by a ligand induced dimerization domain, specifically the ligand

binding domain of human FKBP 12 designed to bind AP20187. The C-terminus of the chimeric receptor comprises an intracellular signaling domain of a neurotropin receptor, e.g., the intracellular domain of TrkA, TrkB, or TrkC. In addition, two other plasmids pMP-GFP and pM-GFP containing the membrane targeting sequence operably linked to the sequence for GFPwere constructed as negative controls. In addition, control plasmids p-MP-TrkB and p-M-TrkB contain the membrane targeting sequence operably linked to the sequence for TrkB (i.e., they lack the dimerization domain), and as should exist as inactive monomers. In the absence of dimerizer, the inactive form of the chimeric receptor accumulates at the inner surface of the plasma membrane as a monomer. However, addition of the dimerizer causes two chimeric molecules to come together and activate the signal cascade responsible for the stepwise recruitment and activation of neurotrophic proteins.

(ii) Transfection:

In a preferred embodiment of this invention, the rat pheochromocytoma PC 12 cells commonly used as a cell culture model were transfected using the viral vectors. Following transfection, the expressed protein tends to localize along the plasma membrane as visualized using fluorescent microscopy (Figure 1).

(iii) Analysis:

Figure IA shows rat pheochromocytoma PC 12 cells transiently transfected with pMP-GFP, a negative control using fluorescent microcroscopy. As seen in this figure, all of the cells express GFP, however, in the absence of a ligand-binding domain or the intracellular signaling domain for neurotrophic factor the cells appear as clumps of round masses devoid of any morphological features attributed to neurons (e.g., a cell body and a filamentous axon). In Figure IB, the cells were transfected with pMP-TrkB, a control plasmid comprising the myristoylation/ palitoylation signal domains operably linked to the gene for human TrkB signal domain. This image is similar to that seen in

Figure IA, and lacks any morphological characteristics attributed with neurons. Finally, Figure 1C depicts a fluorescence micrograph of rat pheochromocytoma PC 12 cells transiently transfected with pMP-F-TrkB, plasmid containing the

myristoylation/palitoylation signal domains operably linked to the gene human FKBP 12 dimerization domain and further linked to the gene for human TrkB domain, linked to GFP. This figure clearly shows fluorescent cells morphologically similar to neuronal cells, i.e., having a cell body and filamentous structures.

Figure 2 shows a phase contrast microscopic image of rat pheochromocytoma PC 12 cells transiently transfected with either the two control plasmids or a plasmid encoding the chimeric receptor protein in the absence of dimerizer AP20187 (Figure 2A) or in the presence of a therapeutic concentration of AP20187. Figure 2A, panel (1) shows cells transiently transfected with (1) pMP-GFP, a negative control comprising the myristoylation/palitoylation signal domains operably linked to the gene for green fluorescent protein (GFP). Panel (2), shows cells transfected with pMP-TrkB, a control plasmid lacking the dimerization domain, and Panel (3), shows cells transfected with pMP-F-TrkB, plasmid containing the membrane targeting, dimerization, and TrkB signaling domains. In all three panels of Figure 2 A, neither vector had any significant influence on cellular morphology, with all panels of Figure 2A showing cells as round clumps. However, in the presence of dimerizer AP20187, the cells transfected with plasmid pMP-F-TrkB, showed morphological changes similar to those of neurons, while the cells transfected with pMP-GFP, or pMP- TrkB continued to remain as clumped round masses. This result clearly demonstrated the ability to control cell differentiation and neurite growth using the methods of the instant invention.

Figure 3A-C show a phase contrast image depicting the survival of rat pheochromocytoma PC12 cells 24 hours after exposure to 6-OHDA (lOOμM) and in the presence of AP-20187. Cells expressing the chimeric receptor were first differentiated in culture using AP20187 for three days, followed by treatment with the neurotoxin 6- OHDA at a final concentration of lOOμM. Figures 3 A and 3B shows cells transiently transfected with pMP-GFP and pMP-TrkB, control plasmids, while Figure 3C depicts cells containing pMP-F-TrkB, plasmid. Cell survival was monitored 24 hours post- treatment with 6-OHDA. The untransfected and cells transfected with control plasmid both died and detached from the tissue culture plates after treatment with 6-OHDA (Figures 3A, 3B). However, as seen in Figure 3C, the cells transfected with gene for the chimerical receptor, essentially remain attached to the tissue culture plates and also

maintain their morphology. These results indicate that the method of this invention can be used to promote survival of neuronal cells used in therapy for neurodegenerative disorders.

VII Use of Stem Cells In The Treatment of Neurodegenerative Disorders

Another preferred embodiment of this invention, is the use of chimeric neurotrophic receptors to control the survival and differentiation of stem cells. Stem cells and neuro-progenitor cells hold promise as treatments for many neurodegenerative conditions such as PD, Alzheimer's, Huntington's disease etc. However, significant challenges have to be overcome to use these cells as therapeutics, most importantly, the challenge of increasing the survival of implanted stem or neuro-progenitor cells, and the ability to selectively target and differentiate these cells into neurons rather than glial cells. According to one embodiment of the current invention undifferentiated neural progenitor cells stably transfected with the expression plasmid for the chimerical receptor, are implanted into a diseased region of the brain. Following implantation, the cells are differentiated by exposure to a synthetic dimerizer. This allows for the controlled differentiation of progenitor cells into neurons rather particularly neurons that exhibit cellular characteristics similar to those in the target region of the brain. Thus, the instant invention provides methods for treating neurodegenerative disorders, as well as a method for controlling the differentiation and survival of progenitor cells used in therapy.

VII Pharmaceutical Compositions And Pharmaceutical Administration

The vector or the synthetic dimerizer used in this invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the vector of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and

the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.

The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal). In one embodiment, the vector is administered by intravenous infusion or injection. In another embodiment, the vector is administered by intramuscular or subcutaneous injection. In another embodiment, the vector is administered perorally. In the most preferred embodiment, the vector is delivered to a specific location using stereostaxic delivery. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antigen, antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such

as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

The vector of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g. , Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the vectors of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the vector may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the vector to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the vector are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response {e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage

unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.