Methods are disclosed for delivering a heterologous gene to a cell body of a neuron by contacting a muscle tissue innervated by the neuron with a viral vector comprising a heterologous gene, wherein the viral vector enters said neuron and is retrogradely moved to the cell body. Additionally, methods for expressing secreted proteins from a nerve cell body as well as methods for treating neurodegenerative disorders such as amyotrophic lateral sclerosis are described.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention described herein relate to methods for delivering heterologous genes to a cell body of a neuron by contacting muscle tissue innervated by the neuron with a vector having the heterologous gene. It was discovered that contacting muscle tissue with a heterologous gene resulted in retrograde transportation of the heterologous gene to the cell body of the neuron. In one embodiment, the heterologous gene is contained within a viral vector so that administering the viral vector via direct injection into the innervated muscle tissue results in stable transduction of the heterologous gene into the neuron cell body. Moreover, although some embodiments of the invention include expression of the heterologous gene once it reaches the cell body, the invention is not limited to expressing the heterologous gene. For example embodiments of the invention include retrogradely transporting the gene to the cell body without resultant expression. As discussed in detail below, preferred heterologous genes include genes encoding trophic factors and anti-apoptotic factors.

It should be realized that contacting the muscle includes any method for providing the heterologous gene to the muscle tissue. It is not required to provide to the heterologous gene directly into the muscle cell for it to be retrogradely transported to the cell body of the neuron. It was discovered that simply contacting the muscle bundle, or muscle tissue, with the heterologous gene was sufficient to induce retrograde delivery of the gene to the cell body. Accordingly, the invention should not be construed to require direct injection of the genetic material into a muscle cell.

The vectors which are introduced into the muscle tissues are taken up by the synaptic regions of these muscle-associated neurons and then transported along the axon of the neuron in a direction opposite the action potential (retrograde transport) and into the body (cellular portion) of the neuron. As used herein, "taken up" is meant to imply either a passive or an active mechanism for moving the vectors into the synaptic end of the neuron. Examples of such mechanisms are receptor mediated processes, endocytosis and vesicle mediated processes. Once present in the cell body of the neuron, the heterologous genes delivered by the vector are transported into the nucleus where they were found to be expressed by the neuron.

Other embodiments of the invention relate to methods for treating neurodegenerative diseases in a patient, wherein the disease affects a particular target neuron. In this embodiment muscle tissue innervated by the target neuron is contacted with a vector having a heterologous gene. The vector enters the neuron and is then retrogradely moved to the cell body of the target neuron. Once the heterologous gene has entered the cell body, it is transported to the nucleus and then expressed. The expression of particular heterologous genes was found to result in a reduction in said neurodegenerative disease. These experiments are described in detail below.

In one embodiment, the invention provides methods of treating Amyotrophic Lateral Sclerosis (ALS) by administering to a patient an viral vector carrying a therapeutically effective amount of a gene encoding insulin-like growth factor I (IGF-1). In this embodiment, the vector, termed herein "AAV-IGF-1" is preferably administered to a patient's diaphram muscles so that it is retrogradely transported to the cell bodies of the neurons that control the diaphram muscles. As is known, one of the leading causes of death among ALS patients is their inability to breath due to degeneration of the nerves and muscles that control breathing. This treatment can prevent, or reduce the instance, of such clinical indications by directly administering the trophic factor IGF-1 to the nerve cells controlling breathing.

In some embodiments of the present invention, the heterologous gene that is administered to a patient can be associated with signal sequences that directed the expressed protein to be secreted following expression. Expression of the secreted protein can be, for example, constitutive or regulatable. Thus, administration of a gene delivery vector into a muscle facilitates the secretion of a protein at a site that is distant from the site of injection. This is especially useful for delivering trophic factors to the tissue surrounding the cell body.

The term amino acid or amino acid residue, as used herein, refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants. The commonly used on- and three-letter abbreviations for amino acids are used herein (Bruce Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994)).

The term "disease state" refers to a physiological state of a cell or of a whole mammal in which an interruption, cessation, or disorder of cellular or body functions, systems, or organs has occurred.

The term "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

Retrograde Transport

Embodiments of the invention relate to methods for stably transfecting neuronal cells through retrograde transport of viral particles through the axon to the nucleus. By administering the proper dose of viral vectors carrying a gene of interest to a particular site, it was discovered that these vectors were capable of retrograde transport and stable transduction of the neuron. Herein, what is meant by "retrograde transport" is uptake of the vector at the axon terminal, and transport through the axon in a direction opposite to the direction of propagation of action potentials (and thus "retrograde") and into the body of the neuron in which the viral particles enter the nucleus, underwent single strand synthesis, and became transcriptionally and translationally active.

Such delivery is advantageous in many cases in which the projection neurons themselves are inaccessible, but their terminal projection fields, which define the neurons, are available for delivery of the genetic vector. Successful delivery to such a terminal projection field of a genetic vector capable of retrograde transport would thus result in retrograde transport and infection of the vulnerable projection neurons. In addition to delivering therapeutic transgenes, the identification of such viral transport mechanisms may advance study of CNS circuitry by combining neural tracing with functional modulation of targeted populations resulting from expression of experimental transgenes to effect a gain or loss of function.

Treatment of Neurodegenerative Diseases

Embodiments of the invention involve delivery of a substantially nontoxic, recombinant adeno-associated virus vector having a heterologous gene of interest in order to provide retrograde gene delivery with stable gene expression. Such a vector can be employed in retrograde gene mapping if a marker gene is packaged in the vector. Alternatively, such a vector can be used for the retrograde delivery of a therapeutic gene, such as a growth factor, an anti-apoptotic gene, or an antisense gene. Such therapeutic use would be especially advantageous where the target neuron population is distributed or difficult to reliably access, such as in the central nervous system. For example, therapeutic gene-bearing vectors can be delivered to the hippocampus or striatum, which results in the infection of projection neurons in the entorhinal cortex and the substantia nigra. This demonstrates a targeted delivery strategy of potential use for gene therapy of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Furthermore, an anti-apoptotic gene such as Bcl-xL can be delivered in vivo to a pathway-specific projection neuron population and the retrograde transport, infection, and expression of this gene product can protect these targeted neurons from subsequent injury. Neuroprotective (antiapoptotic) signaling pathways involving neurotrophic factors, cytokines and "conditioning responses" can counteract the effects of aging and genetic predisposition in neurodegenerative disorders. Thus, targeted delivery of anti-apoptotic genes to vulnerable projection neurons may be a useful neuroprotective strategy for early stages of neurodegenerative disease.

By greatly increasing the viral titer at the point of delivery, it was possible to effect retrograde stable transduction of neurons projecting to the delivery field of the AAV vector. This retrograde transport is thought to be mediated by the microtubules of the axon after uptake of the AAV vector at the axon terminal.

It should be noted here that the way in which viral titers are measured in the literature is not standardized. One method involves simply assessing the number of virions containing the viral genome, regardless of infectivity or functionality, using DNA dot blot, Southern blot, or semiquantitative PCR. These numbers are generally reported as "particles/ml." An assessment of the viral titer using an infectious center assay, in which the rAAV is infected into cells with sufficient helper virus (wild-type AAV and adenovirus) to allow rAAV amplification, provides the number of infectious and replication-competent rAAV particles. This number is generally reported as "infectious units (or infectious particles)/ml." Lastly, an assessment of the viral titer using a rAAV transgene functional assay, which assesses specific transgene expression, provides the number of "transducing units/ml."

Previous experimental use of recombinant AAV vectors have involved relatively low viral titers and have assessed infection of local neurons or anterograde neuronal tracing only. In contrast, embodiments of the invention include methods of raising the virus titer at the point of delivery to preferably 1.times.10.sup.7 infectious particles, or more preferably 1.times.10.sup.8 infectious particles or more, and most preferably 1.times.10.sup.9 infectious particles or more. By using these titer levels, it was possible to detect retrograde transduction of neurons projecting to the delivery field of the AAV vector. Thus, by using a marker gene we were able to identify the nucleus, cell body, and projections for each nerve cell that projected into a predetermined location.

Embodiments of the invention, however, are not necessarily limited to the use of AAV vectors. Any genetic vector may be used to practice the methods disclosed in this application. Of course, the vector should be substantially nontoxic to the contacted cells and enable stable, long-term gene expression. Such vectors may include, for example, lentivirus vectors, liposomal vectors, and the like (see, e.g., Latchman & Coffin, Rev Neurosci. (2001) 12(1):69 78, incorporated by reference herein).

In addition, it is possible to improve the qualities of the rAAV vector by methods well-known in the art, such as chemical modification of the AAV virion structure or capsid gene shuffling. Such methods may be employed to develop AAV strains with new tropism, such as tropism towards axon terminal receptors, as well as strains resistant to naturally occurring neutralizing antibody. Such methods are well within the capabilities of those of ordinary skill in virology.

In accordance with yet another embodiment of the present invention, there are provided methods of treating a neurological disease (including injuries, dysfunctions and disorders) in a mammal comprising administering a therapeutically effective amount or an effective amount of vectors as described herein. The present invention concerns the therapeutic application of vectors as described herein to enhance survival of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system. The ability of vectors as described herein to regulate neuronal differentiation and survival during development of the nervous system and also in the adult state indicates that vectors as described herein can be reasonably expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and prevention of degeneration and premature death which result from loss of differentiation in certain pathological conditions.

In light of this understanding, embodiments of the present invention specifically contemplate applications of vectors containing heterologous genes to the treatment of (prevention and/or reduction of the severity of) neurological conditions deriving from injuries, diseases or disorders, including: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), together with infectious/inflammatory and tumor-induced injury; (ii) aging of the nervous system, including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis, and the like, as well as spinocerebellar degenerations; (iv) chronic immunological diseases of the nervous system or affecting the nervous system, including multiple sclerosis; (v) disorders of sensory neurons as well as degenerative diseases of the retina; and the like.

CNS disorders encompass numerous afflictions such as neurodegenerative diseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g. stroke, head injury, cerebral palsy) and a large number of CNS dysfunctions (e.g. depression, epilepsy, and schizophrenia). In recent years neurodegenerative disease has become an important concern due to the expanding elderly population which is at greatest risk for these disorders. These diseases, which include Alzheimer's Disease, Multiple Sclerosis (MS), Huntington's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease, have been linked to the degeneration of neural cells in particular locations of the CNS, leading to the inability of these cells or the brain region to carry out their intended function.

Further disease conditions contemplated for treatment in accordance with the invention include cerebral ischemia, chronic neurodegeneration, psychiatric disorders, schizophrenia, mood disorders, emotion disorders, disorders of extrapyramidal motor function, obesity, disorders of respiration, motor control and function, attention deficit disorders, concentration disorders, pain disorders, neurodegenerative disorders, epilepsy, convulsive disorders, eating disorders, sleep disorders, sexual disorders, circadian disorders, drug withdrawal, drug addiction, compulsive disorders, anxiety, panic disorders, depressive disorders, skin disorders, retinal ischemia, retinal degeneration, glaucoma, disorders associated with organ transplantation, asthma, ischemia, astrocytomas, and the like. Further examples of disorders include Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS) and Parkinson's disease.

Many neurological disorders are associated with degeneration of discrete populations of neuronal elements and may be treatable with a therapeutic regimen which includes vectors as described herein. For example, Alzheimer's disease is associated with deficits in several neurotransmitter systems, both those that project to the neocortex and those that reside with the cortex. For instance, the nucleus basalis in patients with Alzheimer's disease were observed to have a profound (75%) loss of neurons compared to age-matched controls. Although Alzheimer's disease is by far the most common form of dementia, several other disorders can produce dementia. Several of these are degenerative diseases characterized by the death of neurons in various parts of the central nervous system, especially the cerebral cortex. However, some forms of dementia are associated with degeneration of the thalmus or the white matter underlying the cerebral cortex. Here, the cognitive dysfunction results from the isolation of cortical areas by the degeneration of efferents and afferents. For example, Huntington's disease involves the degeneration of intrastriatal and cortical cholinergic neurons and GABAergic neurons (see, e.g., Hefti et al., Ciba Found Symp. (1996)196:54 69; Koliatsos V. E., Crit Rev Neurobiol (1996) 10(2):205 38). Pick's disease is a severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes accompanied by death of neurons in the striatum. Treatment of patients suffering from such degenerative conditions can include the application of vectors as described herein, in order to manipulate, for example, the de-differentiation and apoptosis of neurons which give rise to loss of neurons. In preferred embodiments, the vectors as described herein are stereotactically provided within or proximate the area of degeneration.

In addition to degenerative-induced dementias, a preparation of invention vectors can be applied opportunely in the treatment of neurodegenerative disorders which have manifestations of tremors and involuntary movements. Parkinson's disease, for example, primarily affects subcortical structures and is characterized by degeneration of the nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism is typically associated with damage to the subthalmic nucleus, often due to acute vascular accident. Also included are neurogenic and myopathic diseases which ultimately affect the somatic division of the peripheral nervous system and are manifest as neuromuscular disorders. Examples include chronic atrophies such as amyotrophic lateral sclerosis, Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which can be manifest as progressive bulbar palsies or spinal muscular atrophies. The present method is amenable to the treatment of disorders of the cerebellum which result in hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the limbs ipsilateral to the lesion. For instance, a preparation of invention vectors can be used to treat a restricted form of cerebellar cortical degeneration involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients.

Other forms of neurological impairment can occur as a result of neural degeneration, such as amyotrophic lateral sclerosis and cerebral palsy, or as a result of CNS trauma, such as stroke and epilepsy. ALS is a name given to a complex of disorders that comprise upper and lower motor neurons. Patients may present with progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, or a combination of these conditions. The major pathological abnormality is characterized by a selective and progressive degeneration of the lower motor neurons in the spinal cord and the upper motor neurons in the cerebral cortex. The therapeutic application of invention vectors prevents and/or reverses motor neuron degeneration in ALS patients.

Other Treatments

In addition to neurodegenerative diseases, acute brain injuries often result in the loss of neural cells, the inappropriate functioning of the affected brain region, and subsequent behavior abnormalities. Probably the largest area of CNS dysfunction (with respect to the number of affected people) is not characterized by a loss of neural cells but rather by abnormal functioning of existing neural cells. This may be due to inappropriate firing of neurons, or the abnormal synthesis, release, and processing of neurotransmitters. These dysfunctions may be the result of well studied and characterized disorders such as depression and epilepsy, or less understood disorders such as neurosis and psychosis.

The vectors of the present invention can also be used in the treatment of autonomic disorders of the peripheral nervous system, which include disorders affecting the innervation of smooth muscle and endocrine tissue (such as glandular tissue). For instance, invention vectors may be useful to treat tachycardia or atrial cardiac arrythmias which may arise from a degenerative condition of the nerves innervating the striated muscle of the heart.

In addition, invention vectors may be employed to support, or alternatively, antagonize the survival and reprojection of several types of central and peripheral ganglionic neurons, sympathetic and sensory neurons, as well as motor neurons (See, e.g., Terenghi G., J Anat (1999) 194 (Pt 1):1 14). To illustrate, such therapeutic vectors may be useful in treatments designed to rescue, for example, retinal ganglia, inner ear and accoustical nerves, and motorneurons, from lesion-induced death as well as guiding reprojection of these neurons after such damage. Such diseases and conditions include CNS trauma, infarction, infection (such as viral infection with varicella-zoster), metabolic disease, nutritional deficiency, toxic agents (such as cisplatin treatment), and the like. Moreover, certain of the vectors described herein (probably antagonistic forms) may be useful in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes.

Accordingly, there are provided methods of treating neuronal trauma in a mammal comprising administering a therapeutically effective amount of invention vectors as described herein. As used herein, the term "Neuronal trauma" refers to any injury to neuronal tissue produced by an exogenous event such as, for example, blunt force or other sudden physical impact that results in neuronal injury or death, either directly or through the abnormal release by dying neurons of toxic levels of endogenous neurotransmitters or metabolites thereof, e.g., glutamate. Neuronal trauma also refers to decreased neurotransmitter production, or a compromise in neuronal function (See, e.g., Fawcett J. W., Spinal Cord (1998) 36(12):811 7).

The vectors of the present invention can also be used in nerve prostheses for the repair of central and peripheral nerve damage. In particular, where a crushed or severed axon is entubulated by use of a prosthetic device, invention vectors can be added to the prosthetic device to increase the rate of growth and regeneration of the dendritic processes. Exemplary nerve guidance channels are described in U.S. Pat. Nos. 5,092,871 and 4,955,892. Accordingly, a severed axonal process can be directed toward the nerve ending from which it was severed by a prosthesis nerve guide which contains invention vectors.

In yet another embodiment, invention vectors can be used in the treatment of neoplastic or hyperplastic transformations, particularly of the central nervous system and lymphatic system. For instance, certain trophic factors are known to have mitotic or apoptotic activity. Thus, certain invention vectors are capable of inducing differentiation of transformed neuronal cells to become post-mitotic or possibly apoptotic. Treatment with certain invention vectors may involve disruption of autocrine loops, such as TGF-beta or PDGF autostimulatory loops, believed to be involved in the neoplastic transformation of several neuronal tumors. Invention vectors may, therefore, be of use in the treatment of, for example, malignant gliomas, medulloblastomas, neuroectodermal tumors, and ependymonas.

Yet another aspect of the present invention concerns the application of the discovery that invention vectors are likely induction signals involved in other vertebrate organogenic pathways in addition to neuronal differentiation as described above, having potential roles in other ectodermal patterning, as well as both mesodermal and endodermal differentiation processes. Thus, it is contemplated that invention vectors can also be utilized for both cell culture and therapeutic methods involving generation and maintenance of non-neuronal tissue, such as in controlling the development and maintenance of tissue from the digestive tract, liver, lungs, and other organs which derive from the primitive gut, as well as dorsal mesoderm-derived structures including muscular-skeletal tissues and connective tissue of the skin; intermediate mesoderm-derived structures, such as the kidney and other renal and urogenital tissues; and head mesenchymal and neural crest-derived tissue, such as cephalic connective tissue and skull and branchial cartilage, occular tissue, muscle and cardiac tissue (see, e.g., Carver and Barness, Clin Perinatol (1996) 23(2):265 85). This should not be construed as a comprehensive list, and other tissues and diseases that may be affected by the invention vectors are envisaged. For example, memory loss or memory enhancement is encompassed as a potential target for invention vectors (see, e.g., Calamandrei and Alleva Behav Brain Res Jan. 23, 1995;66(1 2):129 32). Those of skill in the art will readily recognize additional applications of invention vectors based on the components of the invention vectors, e.g., the activities and, thus, the applications of trophic factors (which have been well characterized and are known to those of skill in the art (Yuen et al., Ann Neurol. (1996) 40(3):346 54)).

Treatment of Muscle-Related Neurodegenerative Diseases

Many neurodegenerative diseases are associated with a progressive loss of muscle function. This loss of muscle function results from the degeneration of neurons which innervate the affected muscle tissue. In some cases, muscle function can be restored and in many cases further loss of function can be prevented by introduction and expression of an appropriate gene within neurons that innervate the affected muscle tissue.

Neurons have shown much potential as gene delivery targets, however, introduction of genes into these cells is often challenging because of their cellular architecture and their location with the body. In many cases, the "cell body" or "cellular portion" (portion containing the nucleus) of the neuron is totally inaccessible to contact with the gene or gene delivery vector. Synaptic regions of motor neurons are associated with muscle tissues which they innervate. Muscle tissues are generally accessible for the administration of agents, such as gene delivery vectors, via intermuscular injection. Accordingly, intermuscular injection provides a route by which the synaptic regions of target neurons can be contacted which a gene delivery vector. As used herein, intermuscular injection includes injection between groups of muscle fibers and also includes injection adjacent a muscle group.

Selection of muscle tissue that is innervated by neurons that are affected by a neurodegenerative disease is an intial step in the treatment of the disease. Relationships between specific motor neurons and the muscle tissues which they innervate are known. For example, there are several known classes of motor neurons, such as the Spinal Cord motor neurons, cranial motor neurons and the brain stem motor neurons. Each of these classes of motor neurons are known to innervate particular muscle groups. Thus, in order to treat a particular set of motor neurons that are degenerating, a physician would only need to determine which muscles are innervated by the neurons and then contact those muscles with a vector that will retrogradely move to the cell body. In one embodiment, the vector includes an anti-apoptotic gene that prevents the cell from dying. In another embodiment, the vector includes a heterologous gene that encodes a wildtype version of a protein in order to replace a defective allele of the same gene in the cell body. In still another embodiment, the heterologous gene is a neurotrophic factor that promotes cell growth.

Examples of trophic factors include, but are not limited to, Agrin, Amphiregulin, Aria, Artemin, BDNF, Cardiotrophin-1, Ciliary neurotrophic factor, c-kit, c-ret, CSF-1, EGF, FGFs: 1, 2, 5, FLT3L, GDNF G-CSF, GM-CSF, Hedgehog, Heregulin (Neuregulin), IGF 1, 2, Interleukin: 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, 15, Leptin, LIF, Midkine, MuSK, Myostatin (GDF 8) NGF, Netrins, Neurturin, NT3, NT4/5, p75, Pleiotrophin, PDGF, Persephin, Saposin C, Stem cell factor, trk A; B; C, and TGF .alpha. and .beta..

Treatment of ALS in Mammals

Amyotrophic lateral sclerosis (ALS) is a prevalent, adult-onset neurodegenerative disease affecting nearly 5 of 100,000 individuals. The disease, first characterized by Charcot in 1869, is a neurodegenerative process selective to motor neurons connecting the brain to the spinal cord and spinal cord to muscles. The neurons typically affected are located in the lower motor neurons of the brainstem and spinal cord and upper motor neurons in the cerebral cortex.

Within 2 to 5 years after clinical onset, the loss of motor neurons leads to progressive atrophy of skeletal muscles, which results in loss of muscular function resulting in paralysis, speech deficits, and death due to respiratory failure. The genetic defects that cause or predispose ALS onset are unknown, although missense mutations in the SOD-1 gene occurs in approximately 10% of familial ALS cases, of which up to 20% have mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), located on chromosome 21. SOD-1 normally functions in the regulation of oxidative stress by conversion of free radical superoxide anions to hydrogen peroxide and molecular oxygen. To date, over 90 mutations have been identified spanning all exons of the SOD-1 gene. Some of these mutations have been used to generate lines of transgenic mice expressing mutant human SOD-1 to model the progressive motor neuron disease and pathogenesis of ALS.

One high expressing mutant SOD-1 mouse (13-fold above endogenous SOD-1) contains an amino acid substitiution of glycine at position 93 by alanine (G93A) in the SOD-1 protein. This mouse model is paralyzed in multiple limbs due to motor neuron cell death in the spinal cord and contains membrane-bound vacuoles in cell bodies and dendrites, which most likely result from degenerating mitochondria (Gurney, et al. (1994) Science 264:1772 5). Leg muscles from end-stage rats have atrophic myofibers and display obvious hindlimb paralysis or paresis in which compound motor action potentials by EMG recordings show markedly reduced amplitudes as well as continuous fibrillation potentials and positive sharp waves compared to wildtype animals. Hematoxylin and Eosin stained sections of end-stage animals reveals dense gliosis with a complete loss of ventral large motor neurons as well as atrophied ventral roots. Degeneration of most axons consists of macrophage infiltration and aggregates of SOD-1 co-localized with ubiquitin as well as accumulation of neurofilaments. Onset of disease progression typically appears as hindlimb abnormal gait which progresses quickly (1 2 days) to overt hindlimb paralysis, typically affecting one limb first. Within 1 2 days, the second hindlimb is involved although the animals can use their forepaws normally. Affected animals show signs of weight loss, poor grooming, and porphyrin staining around the eyes in which the animals progress to end-stage of the disease within 11 days after onset of symptoms. These animals die by 4 to 5 months of age.
 

Claim 1 of 13 Claims

1. A method for delivering a heterologous gene to a cell body of a neuron, comprising: contacting a muscle tissue innervated by said neuron with an AAV viral vector comprising a heterologous gene, wherein said AAV viral vector enters said neuron and is retrogradely moved to the cell body.

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