WO1999036560A2 - Viral vectors encoding neurofilament light proteins and their use - Google Patents

Abstract

Novel viral vectors encoding neurofilament light proteins are described. The invention also provides methods of using these vectors in gene therapy to deliver neurofilament light proteins to subjects having neurodegenerative diseases, neural injuries, and neural degeneration due to aging.

Description

VIRAL VECTORS ENCODING NEUROFILAMENT LIGHT PROTEINS

AND THEIR USE

FIELD OF THE INVENTION

The present invention relates to recombinant viral vectors and their use.

BACKGROUND OF THE INVENTION

Neurons are important cells in the nervous system, being involved in receiving, organizing, and transmitting information. Each neuron contains a cell body, an axon (a thin, tube-like process that arises from the cell body and travels some distance before terminating), and dendrites (neuronal processes of the cell body that are shorter and thicker than axons). The cytoskeleton of the neuron provides mechanical strength to the axons and dendrites and a track for transport of materials between the cell body and the nerve terminal. The cytoskeleton is a system of interconnected macromolecular filaments. Three polymeric structures form the basis of this cytoskeleton: actin filaments (microfilaments), microtubules, and intermediate filaments.

Intermediate Filaments: Intermediate filaments (IFs) are 10 nm filaments found in most eukaryotic cells. There are six classes of IFs recognized according to sequence homology and gene structure: type I and II IFs include the acidic, neutral, and basic keratins; type DI IFs include vimentin, desmin, the glial fibrillary acidic protein (GFAP), peripherin, and plasticin; type IV IFs include neurofilament proteins and α-internexin; type V IFs include the nuclear lamins; and type VI IFs include nestin expressed in neuroepithelial cells.

Neuronal Intermediate Filaments:

Neuronal intermediate filaments (NIFs) include neurofilaments, peripherin, α-internexin, vimentin, and nestin. TheNIF proteins are encoded by a large multigene family displaying cell and tissue-specific expression patterns throughout development. There is a sequential appearance of the NIFs in developing neurons. Nestin is expressed during embryonic development of neuroectodermal cells (Lendahl etα/., (1990) Cell 60:585-595). This is followed by the co-expression of vimentin and α- internexin (Cochard and Paulin ( 1984) J. Neurosci. 4 : 2080-2094; Tapscott et al. , ( 1981 ) Dev. Biol 86:40-45). These NIFs are gradually replaced by NF proteins in maturing CNS neurons (Carden et al, (1987) J. Neurosci. 7:3489-3504; Kaplan et al, (1990) J. Neurosci. 10:2735-2748; Pachter and Liem ( 1984) Dev. Biol. 103 :200-210).

The NIF proteins are made up of an assembly of protein subunits. The current model ofNIF assembly involves 1 ) the bonding of two subunits to form a dimer; 2) the aggregation of two antiparallel dimers to form atetramer, called a protofilament (Steinert and Roop (1988)-4rø.w. Rev. Biochem. 57:593-

625); 3) the joining of about eight protofilaments end on end; and 4) the association of thesejoined protofilaments to other joined protofilaments by staggered overlaps to form a 10 ran filament. The cytoplasmic NIF proteins share a homologous central region of similar size (approximately 310 amino acids) flanked by amino- and carboxy-terminal domains varying greatly in sequence and in length. The central region of NIF proteins forms an extended α-helical rod domain that plays a critical role in protein assembly into 10 ran filaments.

Neurofilaments:

Of all the NIF proteins that participate in the formation of the neuronal cytoskeleton, the neurofilament triplet proteins are the most abundant. These neurofilaments (NFs) are expressed exclusively in neurons. NFs are found predominantly in axons, where they run longitudinally and parallel to each other. While NFs are present in most populations of neurons in the nervous system, they are particularly abundant in large myelinated axons of peripheral nerves that originate from motor and sensory neurons.

NFs provide mechanical support to the neuron and also play a role in modulating the caliber oflarge myelinated axons. Axonal caliber is a determinant of conduction velocity.

NFs are formed by the copolymerization of three NF protein subunits: light (61 kDa) (NF-L), medium (90 kDa) (NF-M), and heavy ( 110 kDa) (NF-H) (Hoffman and Lasek ( 1975) J. Cell. Biol. 66 : 351 - 366). NF-L subunits form the core of the NF and are essential for NF assembly. NF-M and NF-H subunits form side-arm projections in the NF structure, cross-linking NFs and other neuronal structures into a three-dimensional IF matrix. The NF-M and NF-H proj ections appear to modulate the spacing between NFs, thus regulating the caliber of axons.

The three different NF subunits are encoded by three different genes, NF-L, NF-M, and NF-H, each of which is under separate developmental control. During neurogenesis, there is differential expression of the three subunits: the NF-L and NF-M proteins are coexpressed during early embryonic development, while the activation ofNF-H expression is delayed to the postnatal period (Shaw and Weber(1982)Nαtttre298:277-279; Julienet /., (1986) Mol. Brain Res. 1:243-250; Carden etal,

(1987) J. Neurosci. 7:3489-3504).

ΝFs are obligate heteropolymers, requiring ΝF-L with either ΝF-M or ΝF-H for polymer formation (Ching and Lien (1993) J. Cell. Biol. 122: 1323-1335; Lee etal, (1993) J. Cell Biol. 122:1337- 1350). ΝF-H and ΝF-M subunits cannot form polymers by themselves; there is an absolute requirementforΝF-LsubunitsinordertoformIFs(Gardneretα/., (1984)J Neurosci. Res. 11:145-

155; Hisanaga and Hirokawa (1990) J. Mol. Biol. 211 :871-882; Hirokawa (1991) "Molecular architecture and dynamics of the neuronal cytoskeleton" In Burgoyne (ed.) The Neuronal Cytoskeleton (New York: Wiley-Liss) 5-74).

Neurofilaments and Neurodegenerative Diseases: Neurofilaments have been linked to a number of neurodegenerative diseases. Large motor neurons are particularly vulnerable to NF abnormalities because of their high NF content and their long axons. Abnormal depositions ofNF (often called spheroids or Lewy bodies) is a phenomenon observed in many neurodegenerative diseases (Table 1).

Table 1. Human Diseases with Abnormal NF Accumulations Disease Abnormalities Prevalence

ALS NF depositions in motor neurons 70% of cases

• Decline of 60% in NF-L mRNA

Parkinson's disease Lewy bodies in substantia nigra and locus coreuleus 100% of cases

• Declines of 30% NF-L mRNA and 70% NF-H mRNA

Alzheimer's disease Cortical Lewy bodies 20% of cases

• Decline of 70% in NF-L mRNA Lewy body Dementia Cortical Lewy bodies

Guam-Parkinsonism NF depositions in motor neurons 100% of cases

Giant Axonal Neuropathy NF accumulations in peripheral axons

Peripheral Neuropathies NF accumulations in peripheral axons that can be induced by various toxic agents, such as IDPN, hexanedione, acrylamide

As an example, there is evidence that NFs play a central role in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). ALS is an adult-onset and heterogeneous neurological disorder that affects primarily motor neurons in the brain and spinal cord . The degeneration of motor neurons in the brain and spinal cord leads to denervation atrophy of skeletal muscles and, ultimately, to paralysis and death. Although multiple genetic and environmental factors may be implicated in ALS, the striking similarities in the clinical and pathological features of sporadic ALS and familial ALS suggest that similar mechanisms of disease may occur.

A characteristic pathological finding in ALS patients is the presence of abnormal NF accumulations in the cell body and proximal axon of surviving motor neurons. These NF accumulations have been viewed as a marker of neuronal dysfunction, perhaps reflecting defects in axonal transport. Recent evidence suggests that NFs may also play a causative role in ALS and other motor neuron diseases.

Aberrant neuronal swellings that are highly reminiscent of those found in ALS have beenreported in transgenic mice overexpressing the mouse NF-L. High-level expression ofthe wild-type mouse NF-L gene in mice induced an early-onset motor neuron disease accompanied by massive accumulation of NFs in spinal motor neurons and muscle atrophy (Xu et al, (1993) Cell 73:23-33). Similarly, expression of a mutant assembly-disrupting NF-L gene provoked massive accumulation ofNFs in spinal motor neurons, selective death of motor neurons, neuronophagia, and severe denervation atrophy of skeletal muscle (Lee et al, (1994) Neuron 13:975-988).

As additional evidence for NF-L involvement in ALS, a recent report has revealed that there is a 60% decrease in levels ofNF-L mRNA in the motor neurons of patients with ALS (Bergeron etal. , ( 1994)

Brain Res. 659:272-276).

Amutantform of the human copper-zinc superoxide dismutase (SOD) gene is responsible for 2% of ALS cases (Gurney etal. , ( 1994) Science 264 : 1772- 1775). Transgenic mice expressing the human SOD 1 mutation develop a motor neuron disease similar to ALS, in which neuronal swellings occur. These swellings are rich in NFs (Tu et al, (1996) Proc. Natl. Acad. Sci. USA 93(7):3155-3160).

NF-L is also implicated in Parkinson' s disease. The pathological hallmark of idiopathic Parkinson' s disease is the presence ofLewy bodies (LB s), cytoplasmic inclusions made up of altered NF proteins. These LB s are located in neurons of the substantia nigra. A subset of demented elderly patients also exhibit LB-like inclusions in their cortical neurons. The mechanisms involved in the abnormal aggregation of NF proteins to form LBs are still unknown. It has been found that levels of NF-L mRNAs in substantia nigra neurons are reduced in Parkinsonian patients as compared to age-matched controls. There is also reduced NF synthesis in LB-containing neurons.

In patients with Alzheimer's disease, cortical LBs are present in approximately 20% of cases. It has also been discovered that there is a 70% decrease in NF-L mRNA expression in these patients (Crapper McLachlan et α/., (1988) ø/ec. Brain Res. 3:255-262).

Abnormal accumulations of NFs in distinct regions of the neuron also occur in a variety of other neurological disorders, including an inherited giant axonal neuropathy (Carpenter etal, (1974)). Giant axonal neuropathy (GAN) is a comparatively rare neurologic disorder affecting humans and dogs. Neurofilaments collect in focal accumulations at the distal ends of nerves in the peripheral and central nervous systems. These accumulations, which are found multifocally along a single axon, consist of massive collections of abnormally oriented and whorled NFs.

Abnormal accumulations of NFs are also present in toxic neuropathies induced by β,β'- iminodipropionitrile (IDPN), 2,5-hexanedione, acrylamide, and aluminum.

The mechanisms underlying the abnormal aggregation ofNF proteins in neurodegenerative diseases are still unknown. It is very interesting, however, that a decreased level ofNF-L mRNA is associated with degenerative neurons in ALS, Parkinson's disease, Alzheimer's disease, and other neurodegenerative diseases.

NF-L is also a chelator of calcium ions; thus, it can be used as a chelator to restore calcium homeostasis which is affected in neurodegeneration.

Neurofilaments and Injury:

Following injury in mammals, peripheral nervous system (PNS) axons have the capacity to regenerate, whereas central nervous system (CNS) neurons have limited axonal outgrowth. It is widely believed that NFs are required for axonal regeneration following injury. This notion is based on the observation that neurofilament mRNAs decrease two to threefold following axotomy. Although NFs are present in the CNS, their numbers, which are much lower than in the PNS, may not be sufficient to sustain axonal outgrowth.

Neurofilaments and Aging:

Aging is a factor that may contribute to axonal atrophy. There is a normal decline (50-60%) in NF mRNA expression with aging (Parhade/ /., (1995)J Neurosci. Res. 41:355-366). The resulting decrease in NFs may be linked to axonal atrophy and a reduced capacity for compensatory axonal outgrowth during aging. Methods of enhancing neuronal regeneration could attenuate the aging process.

Decreased levels ofNF-L and abnormal NF subunit stoichiometry are associated with degenerative neurons in ALS, Parkinson' s disease, Alzheimer' s disease, and other neurodegenerative diseases, as well as with aging and injury; thus, there is a need for a means of increasing NF-L concentrations in neurons in order to eliminate abnormal accumulations ofNFs and restore the normal stoichiometry of NF subunits. At present, there is no efficient approach to upregulate the levels of NF-L protein in either cultured cells or in vivo.

Oxidative Stress and Neurodegeneration:

Reactive oxygen species (ROS), including hydroxy radicals, superoxide anions, and hydrogen peroxide, are highly reactive substances that can cause tissue injury. ROS are produced in cells by enzymatic, spontaneous, and photochemical oxidation reactions caused by oxidative stress. ROS are produced as by-products of oxidative damage to a wide variety of macromolecules and cellular components (Fridovich in Eichhorn, and Marzilli, ed., Advances in Inorganic Biochemistry (New York:

Elsevier/North Holland, 1979) 67-90; Freeman and Crapo (1982) Laboratory Investigation 47: 412-26). For example, ROS can be generated by the cytotoxic effects of ionizing radiation (Petkau (1980) Ada. Physiol Scand. Suppl.492:81-90;Biaglowetα/., (1983) Radiat. Res. 95:437-455), by various chemotherapeutic agents (Tomasz ( 1976) Chem. Biol. Interact. 13 : 89-97; Lown and Sim (1977) Biochem. Biophys. Res. Commun. 77: 1150- 1157; Borek and Troll (1983) Proc. Natl. Acad.

Sci. USA 80:1304-1307), and by a variety ofbiological processes, including aging (DiGuiseppi and Fridovich (1984) CRC Crit. Rev. Toxicol 12:315-342; Slater (1984) Biochem. J. 222: 1-15).

ROS are highly reactive and can damage biological molecules. Examples of disorders associated with the generation ofROS include synovial inflammation induced by bacterial lipopolysaccharide endotoxin (LPS), inflammation caused by adjuvant-induced arthritis, bleomycin-induced lung fibrosis, reperfusion injury, transplantation rejection, hyperoxia, and diseases caused by oxygen and light. It has been suggested that ROS may be involved in hyperthermic cell injury as well (Omar et al. , ( 1987) Cancer Res. 47:3473).

ROS havebeen implicated in neurodegeneration (Bowling and Beal (1995) Life Sci. 56: 1151- 1171), particularly in Alzheimer's Disease (Snύthetal, (1995) Trends Neurosci. 18:172-176; Smithetα/.,

(\996)Nature 382: 120-121; Good etal, (1996) Am. J. Pathol 149:21-28; Sayre etal, (1997)

J. Neurochem. 68(5):2092-2097). These studies have identified markers of oxidative stress in neurofibrillary tangles and senile plaques of Alzheimer' s patients, including advanced gly cation end products, nitrotyrosine, free carbonyls, heme oxygenase-1, and advanced lipid peroxidation end products. Other evidence implicating oxidative stress in neurodegerative diseases and aging is listed in Table 2.

The exact mechanism by which ROS cause damage to neurons is yet unknown. During aging and in neurodegenerative disease, such as ALS, Alzheimer' s Disease, and Parkinson' s Disease, there is a dramatic reduction in the levels of neurofilament mRNAs. This could contribute to increased vulnerability of neurons to oxidative stress.

Table 2. Evidence of Oxidative Stress in Neurodegenerative Diseases

Disease Evidence References

ALS Mutations in SOD1

-Tyrosine nitration via peroxynitrite Bruijn et al. (1997) PNAS 94:7606-7611 Wiedau-

-Increased peroxidase activity Pazos et al, (1996) Science 271:515- 518

Alzheimer' s Mutations coding for cytochrome c ^{Davis et al}> (¹⁹⁹⁷) ^PNAS 94:4526-4531 oxidases in mitochondrial DNA

Increased lipid peroxidation Sayre et al. (1997) J. Neurochem. 8:2092-2097

Advanced glycosylation end products Smith _e. «/., (1995) TINS 18:172-176

Increase of ROS by amyloid-β Behl et al., (1994) Cell 77:817-827

Carbonyl modification of NF-H Smith et al, (1997) J. Neurochem. 64:2660- 2666

Parkinson' s Increased iron in substantia nigra Olanow (l 993) Trends Neurosci. 16:439-444

Glycoxidation Castellani et al, (1996) Brain Res. 737:195-200

One study suggests that the ROS peroxynitrite causes tyrosine nitration in NFs of ALS patients, which interruptsNF phosphorylation and assembly. It is suggested that this impaired phosphorylation ofNF subunits may affect axonal transport, causing NF accumulation and motoneuron death (Chou etal, (1996) J. Neurological Sciences 139(Suppl.)16-26).

Protective cellular mechanisms against ROS damage are provided by anti-oxidants and radical scavengers such as β-carotene, glutathione, cysteine, and ascorbic acid, as well as by enzymes such as superoxide dismutase and catalase. For example, when cells are exposed to oxygen-generating compounds or other oxidative stresses, glutathione (GSH) and related cellular sulfhydryl compounds become oxidized (Adams et al, (1983) J. Pharmacol Exp. Ther. 227:749-754).

Superoxide dismutases (SODs) are a group of metalloproteins that provide a defense mechanism against oxygen toxicity: SODs catalyze the conversion ofthe superoxide anionto hydrogen peroxide, which can then be detoxified to water and oxygen by catalase and glutathione peroxidase. There are several known forms of SOD containing different metals and different proteins. Eukaryotic cells contain copper-zinc SOD and manganese SOD.

Mutations in the human copper-zinc SOD (SOD 1 ) gene located on chromosome 21 have been found in approximately 20% offamilial ALS cases. To date, morethan40 different SOD 1 mutationshave been identified (Brown ( 1995) Cell 80 : 687-692). The presence of abnormal NF accumulations in motor neurons of some familial ALS cases caused by SOD 1 mutations (Rouleau etal, ( 1996) Ann. Neurol. 39: 128-131) and the finding of similar NF inclusions in transgenic mice expressing SOD1 mutants (Tu et al. , ( 1996) Proc. Natl. Acad. Sci. USA 93 (7) : 3155 -3160) suggest that NF proteins, which are abundant proteins with long half-lives, are favored targets of oxidative damage by S OD 1 mutants.

Many lines of evidence suggest that S OD 1 mutations cause ALS through mechanisms involving a gain of adverse function rather than a loss of SOD activity (Borchelt et al, (1995) J. Biol. Chem. 270:3234-3238). For example, transgenic mice overexpressing SOD 1 mutations developed motor neuron disease even though the SOD activities in mice were not reduced (Gurney et al, (\ 994) Sci.

264: 1772-1775; Wong etal, (1995)Neuron 14: 1105-1116;Rippsetα/., (1995) Proc. Natl Acad Sci. USA 92:689-693). Moreover, mice homozygous for the targeted disruption ofthe SOD 1 gene do not develop motor neuron disease (Reaume etα/., (1996) N t. Genet. 13:43-47). Anumber of mechanisms have been proposed. One mechanism suggests that SOD 1 mutations render the copper in the active site of SOD 1 more accessible to peroxynitrite to form a nitronium-like intermediate that can nitrate proteins at tyrosine residues (Beckman etα/., (1994) Prog. BrainRes. 103:372-380; Beckman et al, (1993) Nature 364:584-585). The nitration of tyrosine residues in ΝF-L to nitrotyrosine inhibits normal NF-L phosphorylation, which is required for assembly ofNF subunits (Chou et al, (1996) J. Neurological Sciences 139(Suppl.)16-26).

Reactive nitrogen species such as peroxynitrite might also create crosslinks by the formation of dityrosine and thereby induceNF aggregation (Julien (1997) Trends Cell Biol 7:243-249). Oxidative modification ofNF proteins by altered SOD 1 activity could result in the formation of protein crosslinks, for example, through a copper-mediated oxidation of sulfhydryl groups or a production of carbonyls on lysine residues. Carbonyl-related modifications ofNF-H have been reported in the neurofibrillary pathology of Alzheimer's disease (Smith et al, (1996) Nature 382:120-121).

Another proposed mechanism suggests that SOD 1 mutants increase peroxidase activity, producing morehydroxylradicalsfromhydrogenperoxidethanthewild-type SOD (Wideau-Pazosetα/., (1996)

Science 271 : 515-518). This increase in ROS levels then causes increased cellular damage.

Recently, it has been discovered that normal SOD1 protects calcineurin, a phosphatase, from inactivation by preventing oxidation ofthe iron at its catalytic site, whereas SOD 1 mutations can alter the activity of calcineurin (Wang et al, (1996) Nature 383 :434-437). These results suggest that SOD 1 and oxidation could play a role in signal-transduction phosphorylation cascades, with SOD 1 mutations leading to anomalous phosphorylation ofNF proteins.

This review ofNFs, neurodegeneration, and oxidative stress indicates that oxidative stress, decreased levels ofNF-L, and abnormal NF subunit stoichiometry are associated with degenerative neurons in neurodegenerative diseases, as well as with aging and injury; thus, there is a need for a means of protecting neurons against neurodegeneration involving oxidative stresses, and for a means of increasing

NF-L mRNA concentrations in neurons in order to eliminate abnormal accumulations ofNFs and restore the normal stoichiometry ofNF subunits. At present, there is no efficient approach to upregulate the levels of NF-L protein in either cultured cells or in vivo.

Gene Therapy Gene transfer techniques can be used to modify cells, such as those ofthe nervous system, in culture and in vivo. Several techniques have been developed to insert DNA into desired host cells, including the use of viruses, microinjection, physical and chemical treatments, and membrane fusion. For example, DNA can be introduced into a host cell by protoplast fusion (Yoakum (1984) Biotechniques 2:24-26, 28-30), or by micro-injection (Spandidos etα/., (1985) Eur. J. Cell. Biol. 37:234-239; Folger etal, (19S2)Molec. Cell Biol 2: 1372-1387; Gordon etal, (\9S0)Proc. Natl. Acad. Sci.

USA 77:7380-7384). Unfortunately, the above-described techniques are relatively inefficient and unsuitable for use in situations that require that the recombinant molecule be introduced into all or most ofthe cells present in culture or in an animal.

Niral vectors have been employed in order to increase the efficiency of introducing DΝA into host cells. A viral vector, as that term is used herein, is a nucleic acid molecule (preferably of DΝA) in which a gene sequence (which is to be transferred) is fused to a subset of viral sequences. Niral expression vectors have been developed using DΝA viruses, such as papovaviruses (ie. SV40), adenoviruses, herpes viruses, and poxviruses (ie. vaccinia virus,), and RΝA viruses, such as retroviruses. The viral sequences and the total genome size are selected such that the vector is capable ofbeing encapsulated in a virus particle and thus is capable of binding to, and introducing its gene sequences into a virus- sensitive host cell. The infective properties of such a virion are, thus, the same as those containing the wild type viral genome.

Most ofthe approved gene transfer trials in humans rely on retro viral vectors for gene transduction. Retroviral vectors in this context are retroviruses from which all viral genes have been removed or altered so that no viral proteins are made in cells infected with the vector. Viral replication functions are provided by the use of retrovirus packaging cells, which produce all ofthe viral proteins but do not produce infectious virus. Introduction ofthe retroviral vector DΝA into packaging cells results in production of virions that carry vector RΝA and can infect target cells, but no further virus spread occurs after infection. To distinguish this process from a natural virus infection where the virus continues to replicate and spread, the term transduction rather than infection is often used.

The major advantages of retroviral vectors for gene therapy are the high efficiency of gene transfer into replicating cells, the precise integration ofthe transferred genes into cellular DΝA, and the lack of further spread ofthe sequences after gene transduction (Miller ( 1992) Nature, 357:455-460). The use of retroviral vectors is limited, however, since both cell division and DNA synthesis are required in order for the provirus to integrate into the host genome; thus, retroviral vectors can only be used in dividing cells, not in neurons. (Methods for introducing gene sequences into neuronal cells are reviewed byBreakefieldetα/., (\9$7)Molec. Neurobiol 1:339-371, which is herein incorporated by reference in its entirety.)

Non-retroviral vectors are being studied for use in gene therapy. One such alternative is the herpes simplex virus (HSV) (Wolfe et al, (1992) Nature Genetics 1 :379-384). HSV-1 has a wide host range, and infects many cell types in mammals and birds, including chickens, rats, mice, monkeys, and humans (Spear et al, DNA Tumor Viruses, J. Tooze, Ed. (Cold Spring Harbor Laboratory: Cold

Spring Harbor, NY, 1981) 615-746). HSV-1 infects post-mitotic neurons in adult animals and can be maintained indefinitely in a latent state (Stevens (1975) Current Topics in Microbiology and Immunology 70:31). It has been suggested that HSV-1 be used as a vector for transferring the HGPRTgeneinto neuronal cells (Palellaetα/., (1988)M./ec. Cell. Biol. 8:457-460). USPatentNo. 5501979 provides a recombinant specific HSV-1 vector capable of infecting neuronal cells.

Adeno-associated virus (AAV) is a defective member ofthe parvovirus family. The AAV genome is encapsidated as a single-stranded DNA molecule of plus or minus polarity (Berns and Rose ( 1970) J. Virol. 5:693-699; Blackow etal, (1967) J. Exp. Med. 115:755-763). Strands of both polarities are packaged, but in separate virus particles (Berns and Adler (1972) Virology 9:394-396); both strands are infectious (Samulski et al , ( 1987) J. Virol. 61 :3096-3101). Efficient replication of AAV requires coinfection with a helper virus such as adenovirus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, or vaccinia virus; hence, the classification of AAV as a defective virus. AAV vector systems are described in US Patent Nos. 4,797,368, 5,436,146, 5,436,146, and 5,478,745.

The adenovirus is also being studied as a vector for gene transfer (Rosenfeld et al, (1992) Cell 68:143-155;Jaffeetα/., (1992)Nαtwre Ge«et/c5 l :372-378;Lemarchandetα ., (1992) Proc. Natl.

Acad. Sci. USA 89:6482-6486). Major advantages of adenovirus vectors are their potential to carry large segments ofDΝA (36 Kb genome), their ability to produce very high titres, and their ability to infect non-replicating cells. Adenovirus vectors for use in gene therapy have been claimed in US Patent Nos. 5,585,362, 5,559,099, and 5,543,328.

Adenovirus vectors have been used to deliver genes to the central nervous system (Betz etal. , ( 1995) J. Cerebral Blood Flow and Metabolism 15(4): 547-551; Akli et al, (1993) Nature Genetics 3(3):224-228), and to neurons (Levallois et al, (1994) Comptes Rendus de I Academie des

Sciences - Serie III, Sciences de la Vie 317(6):495-8; Doran et al, (1995) Neurosurgery 36:965- 970; Hermens etal, (1997) J. Neurosci. Meth. 71:85-98).

Recently, a recombinant adenoviral vector was constructed encoding the rat NF-M gene (Terada et al, (1996) Science 273:784-788). This vector was used to transfect the fourth lumbar (L4) dorsal root ganglion neurons ofboth normal and transgenic mice. The resulting NF-M proteins were observed to copolymerize into the endogenous intermediate filament network. As well, the NF-M proteins were transported into sciatic nerve axons.

This review ofNFs, neurodegenerative disorders, and viral vectors indicates that a need remains for a means of delivering high level expression of NF-L to cells.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any ofthe preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incorporated by reference in their entireties in this application.

SUMMARY OF THE INVENTION

It is therefore an object ofthe present invention to provide recombinant viral vectors encoding neurofilament light (NF-L) proteins for the introduction ofNF-L proteins into cells. These vectors can be used in vivo and in vitro. In particular, these vectors can be used in gene therapy to deliver NF-L to subjects with neurodegenerative diseases, neural injuries, and neural degeneration due to aging. These vectors can be used alone or in conjunction with other viral vectors encoding NIF proteins, including the NF-H protein.

One embodiment ofthe present invention provides for recombinant viral vectors for infecting target cells comprising:

a) the DNA of or corresponding to at least a portion ofthe genome of a virus, which portion is capable of infecting the target cells; and

b) a normal NF-L gene, or portion thereof, operatively linked to the viral genome and capable of expression in the target cell in vivo or in vitro.

The virus may be an adenovirus, a herpes simplex virus, an adeno-associated virus, an AIDS virus, a retrovirus, or any other suitable virus. Preferably, the viral genome is replication-defective. In a preferred embodiment, the virus is the human adenovirus serotype 5 mutant dl309.

The NF-L gene may be any mammalian NF-L gene. Preferably, the NF-L gene is the human NF-L gene.

The target cells may be any animal cell, including human and mammalian cells. In a specific embodiment ofthe present invention, the target cell is a non-mitotic cell, such as a neuron.

In a preferred specific embodiment ofthe present invention, the recombinant viral vector is Ad5-hNF- L.

Another embodiment ofthe present invention provides for the above recombinant viral vectors further comprising an appropriate promoter sequence . Any suitable promoter or any portion thereof may be employed to mediate expression, including an NF-L gene's own promoter, other neuron-specific promoters such as α-tubulin, NF-H, NSE, Thy- 1 , or prion, or a viral promoter such as the CMV or SV40 promoter.

Preferably, the promoter sequence is neuron-specific. In a specific preferred embodiment, the promoter sequence is the human NF-L gene minimal promoter.

In a further embodiment, the present invention provides for cells that express a normal NF-L gene introduced therein through viral transfection.

In yet another embodiment, the present invention provides a use ofthe recombinant viral vectors to deliver an NF-L gene to a subject, comprising administering to the subject an effective amount ofthe recombinant viral vector. The subject can be any animal, including mammals. Preferably, the subject is human.

In a particular embodiment, the recombinant viral vectors are used to deliver an NF-L gene to a subj ect having a neurodegenerative disorder, such as Amyotrophic lateral sclerosis (ALS), Alzheimer' s disease, and Parkinson's disease.

In another particular embodiment, the recombinant viral vectors are used to deliver an NF-L gene to a subject having a neural injury.

In yet another particular embodiment, the recombinant viral vectors are used to deliver an NF-L gene to a subject requiring axonal regeneration as a result of disease, injury, or aging.

In yet a further embodiment, the recombinant viral vectors are used to deliver an NF-L gene to a subject requiring the restoration of calcium ion homeostasis. The calcium ion homeostasis may be a result of neurodegeneration.

In a further embodiment, the recombinant viral vectors ofthe present invention are employed in conjunction with an effective amount of a recombinant viral vector encoding another neuronal intermediate filament protein.

Alternatively, the recombinant viral vectors ofthe present invention may be employed to infect a desired cell line in vitro, whereby the infected cells produce a desired NF-L protein in vitro.

Various other objects and advantages ofthe present invention will become apparent from the detailed description ofthe invention.

BRIEF DESCRD7TION OF THE DRAWINGS

Figure 1. The genomic structure of the Ad5-hNF-L viral vector.

In a), the genome ofthe vector is displayed in map units (m.u.) with 100 m.u. corresponding to the complete genome. The region containing the human NF-L expression cassette covers m.u. 1.25 to 9.25, and is shown by hatched lines. TR means terminal repeat. In b), the detailed structure ofthe expression cassette is shown. From right to left, it includes: "Promoter" being the human NF-L gene minimal promoter (hatched); "Exon 1 , Exon 2, Exon 3 , Exon 4" being the four coding exons ofthe hNF-L gene (flecked) separated by introns (in black); and "pA" being the hNF-L polyadenylation signal. The direction of transcription is given by an arrow above the cassette.

Figure 2. Adenoviruses to direct β-gal and hNF-L expression to spinal motor neurons.

Adenoviral recombinants containing a CMV-lacZ expression cassette were injected into the right tibialis muscle of 2 month old hNF-H+/+ mice. The lacZ expression in spinal motor neurons sending their axons into the L5 ventral roots was detected in β-gal stained /neutral red counterstained sections at (a) 7 days and (b) 14 days post-injection. The neurofilamentous swellings in hNF-H+/+ mice were not affected by the viral-mediated β-gal expression, (c), hNF-L expression 48h post-infection by Ad5- hNFL in a dissociated culture ofE 1311NF-H+/+ spinal cord. The hNF-L promoter provides neuronal specific expression in this mixed culture, (d), Detection of hNF-L expression in hNF-H+/+ mice spinal cord ipsilateral to the inj ection, 21 day s after inj ection of Ad5 -hNFL into the right tibialis muscle, (e), No hNF-L expression could be detected in the spinal cord contralateral to the injection side, (f), Toluidine blue stained Epon sections show a reduction in the number of perikaryal swellings in the ipsilateral spinal cord (arrows) when compared to (g) the non-injected contralateral side. The hNF-L proteins in spinal motor neurons could be detected 9 months after muscular viruses injection. At this time point, the motor neurons expressing hNF-L had no neurofilamentous swelling (d, insert), unlike motor neurons on the contralateral side (e, insert).

Figure 3. Northern and western blot analysis of double transgenic mice.

(A) Detection ofNF-L and hNF-H mRNAs in spinal cord of transgenic mice. The probe used for the detection ofNF-L hybridizes with both human and mouse NF-L transcripts. An actin cDNA probe was used for standardization. Lane 1 , hNF-H+/+; lane 2, hNF-H+/-; lane 3, hNF-L+/+; lane 4, hNF- L+/-; lane 5, hNF-L+/+;hNF-H+/+; lane 6, hNF-L+/-;hNF-H+/-; lane 7, normal; lane 8, hNF-L+/- ;hNF-H+/+ and lane 9, hNF-L+/+;hNF-H+/- mice. (B) Coomassie stained SDS-PAGE and western blot analysis of Triton-insoluble spinal cord protein extracts from 6 month old mice. Western blots were carried out on replica filters. Lanes identification is identical to figure 1 A.

Figure 4. Reduction of perikaryal swellings by extra hNF-L.

Light micrographs of spinal motor neurons in the lumbar region, (a), normal; (b), hNF-L+/+; (c), hNF- H+/+; (d), hNF-L+/+;hNF-H+/+; (e), hNF-H+/-; (f), hNF-L+/-;hNF-H+/-; (g), hNF-L+/-;hNF-

H+/+; (h), hNF-L+/+;hNF-H+/-. NF swellings appear as lightly stain areas. Thin arrows point to normal appearing motor neurons, large arrows in (h) point to a giant axon found in hNF-L+/+;hNF- H+/- mice.

Figure 5. Restoration of axon calibers by hNF-L co-expression. Light micrographs show transverse sections of L5 ventral roots ofthe various transgenic mice at 6 months of age. (a), normal; (b), hNF-L+/+; (c), hNF-H+/+; (d), hNF-L+/+;hNF-H+/+; (e), hNF- H+/-; (f), hNF-L+/-;hNF-H+/-; (g), hNF-L+/-;hNF-H+/+; (h), hNF-L+/+;hNF-H+/-. Mice overexpressing the hNF-H transgene alone show dramatic decrease in axonal caliber and some degeneration profile c and e). Co-expression of hNF-L restores axonal diameters (d and f).

Figure 6. Caliber distribution of ventral root axons in 6 month old mice.

Cross-section areas of L5 ventral root axons were expressed into diameters of circles with corresponding surface areas. The histograms compare (a), normal versus hNF-L+/+; (b), hNF-H+/+ versushNF-L+/+;hNF-H+/+; (c), hNF-H+/- versus hNF-L+/-;hNF-H+/-; (d), hNF-L+/+;hNF-H+/- versus hNF-L+/-;hNF-H+/+.

Figure 7. Restoration of NF network in axons of doubly transgenic mice. Electron micrographs ofL5 ventral root ultrathin sections of 6 month old mice: (a), normal; (b), hNF-

L+/+; (c), hNF-H+/+; (d), hNF-L+/+;hNF-H+/+; (e), hNF-H+/-; (f), hNF-L+/-;hNF-H+/-; (g), hNF-L+/-;hNF-H+/+; (h), hNF-L+/+;hNF-H+/-. Note the difference of inter-NF spacing between mice overexpressing hNF-H alone © and e) and those overexpressing both hNF-L and hNF-H at different levels (d, f, g and h).

Figure 8. Restoration of slow axonal transport by hNF-L co-expression.

³⁵S-methionine was injected into the spinal cord of 3 month old animals at the entry point ofthe L5 ventral root. After 28 days, the L5 ventral roots, L5 DRGs and 8 successive 3 mm segments of sciatic nerves were isolated. The pooled ventral roots and DRGs, lane vr, represent 12 mm of axonal length. As previously reported, the rate of axonal transport is diminished in hNF-H+/+ mice. The axonal transport of cytoskeletal components in doubly transgenic mice is significantly enhanced as compared to hNF-H+/+ mice. Note the transport rates for mouse NF-M (arrow) and tubulin (asterisk) which are restored in the doubly transgenic mice.

Figure 9. Rescue of abnormal limb contraction.

(a), Normal mice extend their legs when lifted by their tail whereas hNF-H overexpressing mice contract their hindlimbs. (b), In contrast, mice overexpressing both human NF transgenes extend their limbs like normal mice.

Figure 10. Increased lifespan of SODl^G37R transgenic mice by NF-L overexpression.

Survival curves of transgenic mice expressing S OD 1 ^G37R alone or together with the NF-L transgene. The survival probability of transgenic mice is plotted as a function of their ages in weeks. It is remarkable that NF-L overexpression increased the mean longevity of SOD 1 ^G37R transgenic mice by approximately eight weeks. DETAILED DESCRIPTION OF THE INVENTION

The following terms and abbreviations are used throughout the specification and in the claims:

"infection" is generally meant the process by which a virus transfers genetic material to its host or target cell.

"normal gene" means any nucleic acid sequence which codes for a functional gene protein or fragment thereof; thus, variations in the actual sequence ofthe gene can be tolerated provided that functional protein can be expressed. An NF-L gene used in the practice ofthe present invention can be obtained through conventional methods such as DNA cloning, artificial construction or other means.

"mutation" means any alteration ofthe DNA including, but not limited to, deletions, insertions, and missense and nonsense mutations.

"promoter" means a sequence ofDNA involved in binding RNA polymerase to initiate transcription.

The present invention provides viral vectors encoding neurofilament light (NF-L) proteins or fragments thereof.

A recombinant viral vector ofthe present invention comprises:

a) the DNA of or corresponding to at least a portion ofthe genome of a virus, which portion is capable of infecting the target cells; and

b) a normal neurofilament light protein (NF-L) gene, or portion thereof, operatively linked to the viral genome and capable of expression in the target cell in vivo or in vitro.

Viruses: Generally any virus capable of infection and gene transfer can be employed in the present invention. Suitable viruses for this invention include adenoviruses, adeno-associated virus, herpes simplex viruses, the AIDS virus, and retroviruses well known to those skilled in the art.

The viral vector employed may, in one embodiment, be an adenoviral vector that includes essentially the complete adenoviral genome (Shenketα/., (1984) Cwrr. Topics Microbiol. Immun. 111(3):1-

39). Alternatively, the viral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted. Preferably, the viruses used in the construction of viral vectors are rendered replication-defective to remove the effects of viral replication on the target cells.

Neurofilament Light Protein: Generally, any mammalian NF-L can be employed in the present invention. Preferably, the NF-L is human NF-L. The DNA sequences can be either cDNA or genomic DNA. DNA encoding the entire NF-L protein, or any portion thereof, may be used . Due to the degeneracy ofthe genetic code, other DNA sequences that encode substantially the same NF-L protein or a functional equivalent can also be used. Multiple gene copies may also be used.

The sequence ofthe human NF-L gene has been published (Julien et al. , ( 1987) Biochim. Biophys.

Ada 909 : 10-20) . Other NF-L sequences are also published, including the mouse sequence (Lewis and Cowam ( 19S6)Mol. Cell Biol. 6 : 1529- 1534), and the rat sequence (Chin and Lien (1989) Europ. J. CellBiol. 50:475-490). Any other NF-L sequences published in the future are also covered bythis invention.

Promoters:

The DNA sequences encoding the NF-L protein are under the control of a suitable promoter. Any suitable promoter or any portion thereof may be employed to mediate expression, including an NF-L gene's own promoter, other neuron-specific promoters such as α-tubulin, NF-H, NSE, Thy-1, or prion, or a viral promoter such as the CMV or SV40 promoter.

5 The NF-L gene's own promoter is preferred when neuron-specific expression of the NF-L is desired. Construction:

In order to produce the gene constructs used in the invention, recombinant DNA and cloning methods, which are well known to those skilled in the art, may be utilized (see Sambrook et al, Molecular Cloning, A Laboratory Manual, 2d ed. (New York: Cold SpringHarbor Laboratory Press, 1989), including the use of restriction enzymes, site directed mutagenesis, ligation, homologous recombination, and transfection techniques. Appropriate NF-L coding sequences may be generated from cDNA or genomic clones.

Example:

In one embodiment ofthe present invention, the recombinant vector is an adenoviral vector comprising a human NF-L expression cassette. The vector is free ofthe adenoviral El DNA sequences. The expression cassette comprises the human NF-L gene nrinimal promoter, genomic DNA sequences of the human NF-L gene, and the hNF-L polyadenylation signal.

Such vectors may be constructed by removing the El region from the adenovirus using standard techniques, which renders the virus replication defective. The E 1 region in then replaced by DNA encoding the human NF-L gene with its promoter and polyadenylation signal. This may be achieved by first inserting the human NF-L expression cassette into a shuttle plasmid using standard ligation techniques. The shuttle plasmid will contain DNA sequences homologous to the adenovirus genome, which serve as a substrate for homologous recombination with the modified adenovirus. Such sequences may encompass, for example, a segment ofthe adenovirus 5 genome from base 3329 to base 6246 ofthe genome. The shuttle plasmid may also include a selectable marker and an origin of replication. The origin of replication may be a bacterial origin of replication. Representative examples of such shuttle plasmids include pXCJL.1. Homologous recombination is then effected with the modified adenovirus in which the maj ority ofthe E 1 adenoviral DNA sequences have been deleted . Upon homologous recombination, a recombinant adenovirus vector is formed which includes DNA sequences derived from both the shuttle plasmid and the modified adenovirus. Such homologous recombination may be effected through co-transfection ofthe shuttle plasmid and the modified adenovirus into ahelper cell line by calcium phosphate precipitation. The helper cell line may be 293 cells, which are permissive for adenoviruses deleted in the El region. Use:

The viral vectors ofthe present invention may be used to transfect any cells in which the delivery and expression of a NF-L gene is desired.

The viral vectors of the present invention can be used for gene therapy to treat various neurodegenerative disorders such as Amyotrophic lateral sclerosis (ALS), Alzheimer's disease,

Parkinson's disease, Giant Axonal Neuropathy, toxic neuropathies such as those induced by β,β'- iminodipropionitrile (IDPN), 2,5-hexanedione, acrylamide, and aluminum, Lewy body Dementia, and Guam-Parkinsonism.

As discussed earlier, a decreased level of NF-L mRNA is associated with degenerative neurons in ALS, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. It has been suggested that alterations in the stoichiometry ofthe NF subunits may lead to disorganization ofNFs and other cytoskeletal elements. The recombinant viral vectors ofthe present invention can be used to introduce the human NF-L into neurons, thereby restoring the normal stoichiometry ofthe NF subunits and eliminating the NFs accumulations. As shown in Example π, when the Ad5-hNF-L vector was injected into the hNF-H transgenic mouse, a model of ALS, a normal NF ratio was generated, and the neuronal swellings were eliminated.

The recombinant viral vectors ofthe present invention can also be used for gene therapy to treat subjects requiring axonal regeneration because of neural injury or aging. NF-L plays a role in axonal growth. The recombinant viral vectors ofthe present invention can be used to introduce NF-Ls into neurons, thereby enhancing neuronal regeneration.

The recombinant viral vectors ofthe present invention can also be used in conjunction with other viral vectors encoding neuronal intermediate filaments, including the neurofilament heavy protein, in order to slow down disease with oxidative stress involvement or calcium ion involvement.

In order to transfect cells, the cells are put in contact with a suspension containing a recombinant viral vector ofthe present invention. Upon cell contact, the vector enters the cell, the vector genome is transported into the cell nucleus, the NF-L gene is transcribed, and the NF-L mRNA is translated into protein in the cell cytoplasm.

When used for gene therapy to treat subjects with neurodegenerative disorders or subjects requiring axonal regeneration because of neural injury or aging, the cells targeted are preferably neurons. Subjects may be animals, including mammals and humans.

Administration:

It will be appreciated that administration ofthe viral vectors ofthe present invention will be by procedures well established in the pharmaceutical arts, e.g. by direct delivery to the target organ, tissue or site, intranasally, intravenously, intramuscularly, subcutaneously, intradermally and through oral administration, either alone or in combination.

Preferably, the viral vectors are administered by injecting vector suspension into various locations of the nervous system, or by injection into nerves, or injection into peripheral tissues such as skin or muscles, which are innervated by neurons. In the latter case, the vector enters the neurons via the axons or axon teirninals, and the vector genome is transported retrogradely in the axon to the nucleus .

Formulations:

It will be appreciated that formulations suitable for administration ofthe viral vectors ofthe present invention include aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non- aqueous sterile suspensions.

Pharmaceutically acceptable excipients are also well-known to those who are skilled in the art, and are readily available. The choice of excipient will be determined in part by the particular method used to administer the recombinant viral vector. Accordingly, there is a wide variety of suitable formulations for use in the context ofthe present invention. The following methods and excipients are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount ofthe compound dissolved in diluents, such as water, saline, or orange juice;, (b) capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more oflactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

The recombinant viral vector ofthe present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.

Additionally, the recombinant viral vectors ofthe present invention may be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood ofthe intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets ofthe kind previously described. Dosages:

The dosages administered will vary from subject to subject and will be determined by the level of enhancement ofNF-L function balanced against any risk or deleterious side effects. Monitoring levels of transduction, NF-L expression and/or the presence or levels of normal NF-L will assist in selecting and adjusting the dosages administered.

When used as atherapeutic, atherapeutically effective dosage ofthe vectors ofthe present invention will be administered for atherapeutically effective duration. By "therapeutically effective amount" and "therapeutically effective duration" is meant an amount and duration sufficient to achieve a selected desired result in accordance with the present invention without undue adverse or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts.

The dose administered to an animal, particularly a human, in the context ofthe present invention will vary with the gene of interest, the composition employed, the method of administration, and the particular site and organism being treated; however, the dose should be sufficient to effect a therapeutic response.

In vitro use:

In addition to use in vivo, the viral vectors ofthe present invention also have utility in vitro. Cell populations with defective NF-L genes can be removed from a subject or otherwise provided, transduced with a normal NF-L gene in accordance with the principles of the invention, then (re)introduced into the subject.

The viral vectors ofthe present invention are also useful as a research model. The vectors may be employed to infect desired cell lines in vitro, whereby the infected cells produce NF-L in vitro. Such cell lines are useful to study therapeutic approaches to treating neurodegenerative diseases or neuronal injury and to research neuronal physiology in both normal and disease states.

Improvement over Current Tools: The recombinant viral vectors ofthe present invention provide an efficient way to deliver high-level expression ofNF-L to cells in vivo and in vitro. Specifically, they provide a means of efficient gene transfer to neurons. Furthermore, if the gene is under the transcriptional control ofthe NF-L minimal promoter, expression ofthe gene is neuron specific.

The present invention is described in further detail in the following non-limiting examples. It is to be understood that the examples described below are not meant to limit the scope ofthe present invention.

It is expected that numerous variants will be obvious to the person skilled in the art to which the present invention pertains, without any departure from the spirit ofthe present invention. The appended claims, properly construed, form the only limitation upon the scope ofthe present invention.

EXAMPLE I CONSTRUCTION OF THE Ad5-hNF-L VECTOR

A defective adenovirus vector was created containing an expression cassette for the human neurofilament light gene (Ad5-hNF-L). The adenovirus vector was derived from the human adenovirus serotype 5 (Ad5) mutantώ./309 (Jones and Shenk (1979) Cell 17:683-689), by creating a deletion in the early region 1 (El) from nucleotides 452 to 3328 (based on the wild-type Ad5 sequence); this deletion rendered the virus replication defective. A stretch ofDNA containing the coding sequence of the human NF-L gene with its promoter and polyadenylation signal (hNF-L) was first inserted into a pXCJL.1 shuttle plasmid, having sequences homologous to the Ad5 genome. This generated the plasmid pXCJLhNF-L (LW), which was used to insert the hNF-L fragment into the adenovirus by homologous recombination. The resulting Adv-hNF-L plasmid is shown in Figure 1.

Materials:

All restriction enzymes and T4 DNA ligase were obtained from Bethesda Research Laboratories (BRL) (Burlington, ON, Canada). The pXCJL.1 shuttle plasmid, pJM 17 plasmid, and 293N3 S cells were obtained from Dr. Frank Graham (McMaster University, Hamilton, ON, Canada). 293 Cells were obtained fromDr. Philip Branton (McGill University, Montreal, PQ, Canada). Molecular weight markers, agarose, and low-melting agarose were purchased fromBRL. Glassmilk kits were obtained from Bio 101 (Vista, CA). Dulbecco's modified Eagle Medium (DMEM), fetal calf serum, and horse serum were purchased from Gibco. Jocklik-modified DMEM was obtained from Gibco. Yeast extract was obtained fromDifco. The Qiagen maxiprep kit was obtained from Qiagen (Chatsworth, CA). Geneclean was obtained from Bio-Can.

Preparation ofthe hNF-L gene fragment: A 2959 bp DNA fragment containing a genomic clone for the human NF-L gene with its minimal promoter and a polyadenylation signal was excised from plasmid pSKBAM-XB A-hNF-L (J. P. Julien) by digestion with Xbal and BamHl . 10 μl of pSKBA-XBA-hNF-L (approximately 2-3 μg) was digested with lOunitsofXbal and 10 units ofBamHl inMedium SaltBuffer(MSB)inavolumeof 20 μl at 37 ° C for at least one hour. Following digestion, the sample was run in a horizontal agarose gel according to standard techniques (Sambrook etal, (1989) Molecular Cloning: a laboratory manual (Cold SpringHarbor Laboratory: Cold Spring Harbor, NY)). Molecular weight markers and undigested DNA were run in the same gel as controls. Following ethidium-bromide staining, the gel was photographed under UV illumination using a polaroid camera. The 2959 bp Xbal-BamHl fragment of pSKBAM-XBA-hNF-L was purified using the Glassmilk kit according to the manufacturer' s instructions.

Preparation of Shuttle Plasmid:

5 μlpXCJL.1 (approximately 2.5 μg) was digested with lOunitsofXbal in React 2 buffer in a volume of20 μlat37°C. After2hours, 10 units ofBamHl, 3 μlNaC10.5M, 1 μl ofReact2, and 6 μl of water were added. The digestion pursued for 2 hours more. Following digestion, the sample was run in a horizontal agarose gel according to standard techniques (Sambrook etal, (1989) supra). Molecular weight markers and undigested DNA were run in the same gel as controls. Following ethidium- bromide staining, the gel was photographed under UV illumination using a polaroid camera. The 6.64 kb Xbal-BamHl fragment of pXCJL. l was purified using the Glassmilk kit according to the manufacturer's instructions.

The 2959 bp DNA fragment from plasmid pSKBAM-XBA-hNF-L was ligated with the 6.64 kb

Xbal - BamHl fragment ofthe pXCJLl plasmid. 1 μl (approximately 1 ng) ofthe 2959 bp hNF-L fragment was ligated with 1 μl (approximately 1 ng) ofthe 6.64 kbpXCJL.l fragment in a reaction mix containing 1 unit of T4 DNA ligase and 2 μl 5X ligation buffer in a final volume of 10 μl; the reaction was performed at 4 ° C overnight. 5 μl ofthe ligation product was transformed into E. coli strain DH5 bacteria and plated . Ten ampicillin-resistant colonies were selected and grown in Luria-Broth medium according to standard techniques (Sambrook et al, (1989) supra).

Small scale extractions of plasmid DNA were performed using an alkaline lysis protocol (Sambrook etal, ( 1989) sM/?rø). Plasmid DNA was analyzed by restriction endonuclease digestion using 10.5 μl of plasmid DNA with each of the folio wing: A) 10 units ofBamHl and lOunitsofXbal inMSB;B) lOunits ofEcoRl and lOunitsofXbal in High S alt Buffer; or C) lOunits ofEcoRl and 10 units of Sphl in High Salt Buffer. Each digestion was performed in a final volume of 15 μl at 37°C for a minimum of 1 hour. The digested samples were analyzed by electrophoresis in an agarose gel, ethidium-bromide staining, and photographing under UV illumination using a polaroid camera. Desired recombinants generated the following fragments: digestion A: two fragments of 11.4 kb and 6.6 kb; digestion B: three fragments of 500bp, lkb, and 16.5 kb; and digestion C: six fragments of 500 bp, 900 bp, 1.3 kb, 1.7 kb, 2 kb, and 11.4 kb.

Three plasmid preparations showed the desired fragments. One of them, designated pXCJLhNF-L (LW) was selected for subsequent use. Large scale extraction of pXCJLhNF-L (LW) was performed using the Qiagen maxiprep kit according to the manufacturer's instructions.

Construction ofthe Ad5 -hNF-L vector: The pJM 17 vector allows insertion ofDNAinto the adenovirus genome by homologous recombination, since it contains the genome of Ad5 dl309 along with the pBRX plasmid at map unit 3.7 ofthe genome (McGrory et al, (1988) Virology 163 :614-617). The methods for homologous recombination- mediated insertion ofDN A into the adenovirus genome using the pJM 17 plasmid have been described (McGrory et al, (1988) supra).

pJM17 was grown in Terrific Broth medium (Sambrook et al, (1989) supra), and a large-scale extraction was performed using the Qiagen maxiprep kit according to the manufacturer's directions. Ten petri dishes (60mm) were plated with the 293 cells in DMEM plus 10% fetal calf serum.

The pXCJLhNF-L (LW) plasmid and the pJM 17 plasmid were cotransfected into 293 cells using the calcium-phosphate precipitation technique. 4 μg of pJMl 7 DNA was mixed with 6 μg of pXCJLhNF- L(LW)DNAinHepes-bufferedsaline(140mMNaCl, 5mMKCl, 1 mMNa₂HPO₄, 0.1% dextrose, 20 mMHepes pH 7.05) and 125 mM CaCl₂ in a final volume of 1 ml. After 20 minutes, the 0.5 ml ofthe precipitate was added slowly to each of two 293 plated petri dishes. The dishes were then incubated at 37 ° C for 4 hours. The medium containing the precipitate was then removed and the cells in each dish were covered with 5 ml of DMEM containing 5% fetal calf serum, 1% low-melting agarose, and 2% yeast extract. The cells were then returned to the incubator until viral plaques appeared. A total of 10 petri dishes of 293 cells were transfected.

Homologous recombination between pXCJLhNF-L and pJMl 7 led to the appearance of viral plaques in transfected cells. One viral plaque, termed Ad5-hNF-L, was isolated from one ofthe petri dishes. The plaque was collected with a pipette and resuspended in 400 μlofPBS. 200 μl of the suspension was used to infect a 100 mm petri dish containing 293 cells; the remaining 200 μl was frozen. After cytopathic effects had developed (3 days), the contents ofthe petri dish was centrifuged at 2000 rpm for 10 minutes, and the supernatant collection and frozen. The cells were then incubated overnight in 0.5 ml of 0.5 mg/ml pronase in 0.01M Tris, 0.01M EDTA, 0.5% SDS at 37°C. The DNA was extracted with phenol-chloroform and resuspended in 50 μl of TE buffer.

The structure ofthe virus was confirmed by Southern hybridization. For this purpose, 5 μl ofthe isolated DNA was digested with 2 μl ofBam H 1 and 2 μl of Xba 1 in B SB buffer in a final volume of

40 μl at 37° C for at least one hour. As a positive control, 1.5 μg ofthe pXCJL-hNF-L was also digested with Xbal in BSB buffer at 37°C for at least one hour. As a positive control, 1.2 μg of pXC JL-BZRG6 (containing no NF sequence) was digested with S al 1 in OP A buffer at 37 ° C for at least one hour. After digestion, the DNA fragments and molecular weight markers were separated in an agarose gel and blotted onto a nitrocellulose membrane (Sambrook et al, (1989) supra).

A hNF-L DNA probe was prepared by digesting 1 μg of pXCJL-hNF-L DNA with Bglll in React3 buffer at 37 ° C for at least one hour. Following digestion, the sample was run in a horizontal agarose gel according to standard techniques (Sambrook et al. , ( 1989) supra). Molecular weight markers and undigested DNA were run in the same gel as controls. Following ethidium-bromide staining, the gel was photographed under UV illumination using a polaroid camera. A 1 kb fragment was extracted from the agarose gel and purified using Geneclean according to the manufacturer' s instructions. This fragment was nick-translated with ³²P. Probe synthesis was done as described in Sambrook et al, (1989) supra.

The nitrocellulose membrane was hybridized with the hNF-L probe, washed, and exposed to X-ray film according to Sambrook etal, (1989) supra. A positive band of about 4.5 kb was seen in both the DNA extracted from cells infected with the recombinant plaque and the pXCJL-hNF-L plasmid, but not in the pXCJL-BZRGό DNA, confirming insertion ofthe hNF-L expression cassette into the recombinant virus.

A purified stock ofthe Ad5-hNF-L vector was prepared as follows: the Ad5-hNF-L supernatant collected from the 100 mm petri dish was used to infect three 150 mm petri dishes containing a confluent layer of 293 cells, which were incubated at 37°C. Upon development of complete cytopathic effects, the cells were harvested and freeze-thawed three times in a dry ice-ethanol bath. Large debris was removed by centrifugation at 2000g for 10 minutes. The supernatant was combined with the supernatant saved from the petri dish. This solution was then used to infect 3 x 10⁸293N3 S cells, a subclone of 293 cells that has been selected for its ability to grow in suspension cultures. The cells were grown in spinner culture in Jocklik-modified DMEM containing 5% horse serum. Once complete cytopathic effect was obtained, the cells were harvested by centrifugation at 2000g for 10 minutes, freeze-thawed three times, and cleared by low-speed centrifugation. The viral vector was then purified by two rounds of cesium chloride gradient ultracentrifugation, followed by dialysis in a solution of 10% glycerol in 0.01M Tris pH 7.6, and titered by plaque assay on 293 cells (Graham and Prevec ( 1991 ) Manipulation of Adenovirus Vectors.

EXAMPLE II EXPRESSION OF hNF-L IN NEURONS IN VITRO Mixed Spinal Cord Cultures

Cultures of dissociated spinal cord-DRG were prepared from E 13 hNF-H overexpressors or normal mouse embryos as described by Durham et al, (1996) Exp. Neur. 140: 14-29. Briefly, the spinal cords and DRGs were removed from El 3 embryos, finely minced, and dissociated by trituration following 30 minutes incubation in 0.25% trypsin (Gibco BRL). Cells were plated at a density of

200,000 cells/well on round glass coverslips (13 mm) and incubated for 24 hours in a glucose-enriched minimal essential medium (EMEM, 10% FCS and horse serum). Thereafter, cultures were maintained in modified N3 medium with 2% horse serum. On day 5, cultures were treated for 48 hours with 1.4 μg/ml cytosine-b-arabinoside (Calbiochem) to reduce proliferation of non-neuronal cells. Spinal cord- DRG cultures were used in infection experiments with adenoviral vectors 3 weeks after dissociation.

In Vitro Infection of Cultures Motor Neurons

In vitro infection studies were carried out using dissociated spinal cord-DRG cultures from hNF-H transgenic or normal mice. Replication-incompetent adenoviruses were added to the cultures at a final titer of 5 x 10⁶ PFU/ml of Ad5-hNF-L virus (equivalent to 10 viral particles/cell; m.o.L). Cells were exposed to adenoviral vectors for 5 hours at 37 ° C and were then washed in culture medium and further cultured in modified N3 medium.

Immunohistochemistry

Twenty-four, 48, or 72 hours following exposure to adenoviral vectors, cells were fixed in freshly prepared phosphate buffered 4% paraformaldehyde solution and incubated with 0.1% Triton X- 100 in Tris-buffered saline for 10 minutes at room temperature. The cells were blocked for 3 hours at room temperature in IF buffer, 20 mM Hepes pH 7.9, 250 mM KC1, 1% BSA, 0.2% fish skin gelatin (Sigma), 0.1% Triton X- 100, then treated with the primary antibody overnight at 4 ° C in a humid chamber. The primary antibodies were diluted in gelatin-buffer (20mM Tris-HCl pH 7.3 , 150 mM NaCl, 1% fish skin gelatine (Sigma), and 0.1% Tween20) at the following titers: monoclonal mouse anti-humanNF-LDP5-l 12 1:2000 (N.T.L. France); monoclonal mouse anti-NF-LRPN.1105 1 :1000

(Amersham); monoclonal rat anti-human NF-H (OC95) 1 : 200 (kindly provided by V M. - Y. Lee); polyclonal rabbit anti-NF-H 1 : 1000 (Sigma); monoclonal mouse anti-NF-M NN18 1 : 1000 (Boehringer Mannheim); monoclonal mouse anti-β-tubulinKMX- 1 - :200 (Boehringer Mannheim); and monoclonal mouse anti-actin C4 1 : 1000 (Boehringer Mannheim). Following several washes with gelatin buffer, the cells were incubated with secondary antibodies. For indirect fluorescence detection, a fluorescein-conjugated secondary antibody was used (Jackson ImmunoResearch Laboratories, Inc.). Cells were mounted using Slow Fade (Molecular Probes Inc.). Alternatively, we used secondary biotin-conjugated antibodies and streptavidin-peroxidase system (Vectastain Vector Laboratories) to obtain a brown precipitate from DAB (3,3'-diaminobenzidine, Sigma).

Results

Cultures of dissociated spinal cord from hNF-H+/+ embryos were used to demonstrate the cell- specificity ofthe Ad5-hNFL virus. In this in vitro assay, expression ofthe hNF-L gene was observed only in neuronal cells (Fig.2c). In contrast, another viral vector encoding for the β-galactosidase (β-gal) under the control ofthe cytomegalovirus promoter ( Ad5-CMV-LacZ) was expressed in the various cell types present (data not shown).

EXAMPLE III TRANSFECTION OF Ad5-hNF-L INTO MOUSE MODEL OF MOTOR NEURONOPATHY

The Ad5-hNF-L construct was injected into transgenic mice expressing the human NF-H gene, a mouse model of motor neuronopathy.

Generation of Human NF-H Transgenic Mouse

The mouse model of motor neuronopathy was generated as described in Cote et al, (1993) Cell 73 : 35 -46. Briefly, a 39 kb fragment ofthe genomic human NF-H gene, including the complete NF-H transcriptional unit flanked with 9.6 kb of 5' sequences and 13.4 kb of 3 ' sequences, was microinj ected into fertilized mouse eggs. Integration ofthe human transgene into the mouse genome was assessed by Southern blot analysis of genomic DNA isolated from the mouse tail. Transgene copy number was estimated by densitometric analysis. Human NF-H transgene expression was assessed by Northern blot analysis; human NF-H mRNA was detected only in the brain, cerebellum, and spinal cord of transgenic mice, not in liver, kidney, lung, spleen, muscle, or heart. The expression ofthe transgene was limited to nervous tissue. Production of human NF-H protein, determined by SDS gels and immunoblotting, was increased up to 2-fold as compared to the levels of endogenous mouse NF-H protein.

The hNF-H transgenic mice appeared normal during the first few weeks of postnatal development. Then, progressively, the mice began to manifest signs of neurological abnormality : they developed fine tremors, had abnormal limb flexions, and developed signs of weakness. By three to four months of age, the NF-H transgenics showed striking abnormalities in motor neurons ofthe anterior horn and dorsal root ganglia. Many neurons showed prominent swellings ofthe perikarya and proximal axons, consisting of densely packed 10 ran neurofilaments. These filaments are composed by heteropolymerization of multiple NF subunits. Abnormalities indicative of axonal atrophy are also detected. The abnormal accumulation ofNFs plays a central role in motor neuron degeneration by disrupting the intracellular supply of components required for axonal integrity (Collard etal. , ( 1995) Nature 375:61-64). This disruption of axonal transport by NF disorganization is a pathological mechanism consistent with several aspects of ALS. The progressive axonopathy in the NF-H transgenics is accompanied by secondary atrophy of skeletal muscle fibers. In summary, the modest overexpression ofhNF-H proteins in these transgenic mice provokes a progressive neuronopathy with pathological features that resemble those observed in ALS.

Delivery of Recombinant Adenoviruses to Transgenic Mice

The Ad5-hNF-L construct was injected into two month old hNF-H +/+ transgenic and normal mice. All surgical procedures were carried out under general anaesthesia and in accordance with The Guide to the Care and Use of Experimental Animals ofthe Canadian Council on Animal Care. The recombinant adenoviruses were in a 10 mM Tris-HCl buffer solution pH 7.6 at a concentration of 3 x 10⁹ PFU/mL. Ten injections of 2μL each were performed in the right tibialis muscle. Control injection were done using a solution of 5 mg/mL bovine serum albumin (Sigma) in 10 mM Tris-HCl pH 7.6. The mice were killed at 7, 14, or 21 days post-injection and analyzed for β-galactosidase activity or for human NF-L immunodetection.

Detection of β-galactosidase Activity Anaesthetized mice were perfused with 50 mL of PBS pH 7.4 followed by 50 mL of phosphate buffered 2% paraformaldehyde pH 7.4. Tissues were dissected and further fixed for 45 minutes at room temperature in fresh fixative. After 2 washes in PBS pH 7.4, the samples were incubated overnight in a staining solution (lmg/mL X-Gal (Sigma), 5 mMK₃Fe(CH)₆, 5 mMK₄Fe(CH)₆-3H₂O, 2 mMMgCl₂, 0.01% sodiumdeoxycholate, 0.02%NP40inPBS) at37°C. After 3 brief washes with

3% DMSO in PBS, the samples were immersed in 15 sucrose in PBS for cryosectioning. Cryosections of 15 μm were mounted onto gelatin-coated slides, dehydrated, and counterstained with neutral red.

Morphometric Analysis In order to measure axonal calibers, thin sections ( 1 μm) ofEpon-embedded (Marivac) L5 ventral roots from transgenic and normal mice were stained with 2% Toluidine blue (JBS). The images of sections were then digitalized using aHamamatsuCCD camera mounted on aLeitzDiaplan microscope with a 100X objective. The digitalized images were subsequently analyzed using a morphometric software (Image 1, Universal Imaging Corp., USA).

Results

Injections of Ad5-CMV-LacZ viruses into the right tibialis muscle of hNF-H+/+ mice, as shown in figure 2, resulted in a robust and specific expression of β-gal in motor neurons enervating the injected muscle. No obvious cytotoxic effect due to viral infection could be detected. These experiments also revealed that neither the viral infection nor the CMV-driven expression of lacZ reduced the neurofilamentous swellings in hNF-H+/+ mice (Fig.2aandb). Seven days post-injection, itwas noted that normal mice showed prominent β-gal staining of motor neuron perikaiya, whereas the hNF-H+/+ mice had a limited number of positive cells with staining restricted to a spotty pattern. The poor β-gal staining in the hNF-H+/+ mice observed at a relatively short time interval after viral injection is probably reflecting an impairment of retrograde axonal transport, as a consequence ofNF accumulations.

The Ad5-hNFL viral vector was inj ected into the right tibialis muscle ofNF-H+/+ mice, and the spinal cord of these animals was examined 21 days post-infection. As shown in figure 2, the immunodetection ofhNF-L proteins using a specific anti-human NF-L antibody (DP5 - 112) occurred only in spinal motor neurons ipsilaterally to the injected side (Fig.2d and e). Moreover, no perikaryal swellings occurred in the hNF-L-positive motor neurons. Thin sections of Epon-embedded spinal cord further demonstrated that the number of neurofilamentous swellings in the perikarya of motor neurons were reduced in the spinal cord ipsilateral to the Ad5-hNFL injected side, as compared to the non-inj ected contralateral side (Fig.2f and g). Moreover, it was demonstrated that the Ad5-hNFL vector is suitable to direct a long term expression ofhNF-L protein. The insert in figure 2d shows detection of hNF-L proteins in a motor neuron of hNF-H+/+ mice 9 months after muscular injection ofthe viruses.

These results demonstrate that extra hNF-L proteins can suppress motor neuron disease caused by the overexpression of hNF-H proteins.

EXAMPLE IV

MATING TRANSGENIC MICE

To confirm that the results obtained by transfecting the NF-H transgenic mouse with the Ad5-hNF-L construct were not artefacts, transgenic mice overexpressing the hNF-L gene were mated with theNF- H transgenic mice.

The NF-H transgenic mice were generated as described above. The transgenic mice overexpressing hNF-L were generated as described in Julien et al, (1987) Genes & Development 1.1085-1097. Briefly, a 21.5 kb DNA fragment containing the human NF-L gene, including all exon sequences, 5 ' - flanking sequences, and sequences downstream ofthe first polyadenylation site, was microinjected into the male pronucleus of fertilized mouse eggs. Injected eggs were transferred to the oviduct of pseudopregnant females. The presence of hNF-L DNA in offspring of founder mice was determined by Southern blot analysis ofDN A extracted from the tails. Transgenic mice were then examined for the presence of hNF-L transcripts in their brain RNA. The hNF-L protein was identified using a monoclonal antibody raised against bovine NF-L, which recognizes the human but not the mouse NF-L protein. Human NF-L protein was identified in both rain homogenates and in assembled neurofilaments prepared from myelinated axons of transgenic mice. The relative proportion of human NF-L protein detected was equivalent to the relative human NF-L mRNA concentrations observed. Homozygous mice of each parental line were crossbred to obtain mice heterozygous for both transgenes. Further crossbreeding ofthe first generation yielded normal mice and mice heterozygous or homozygous for each transgene. The animals used in this study were not pure inbred mice, but were dominantly of C57BL/6 genetic background. The genotypes of transgenic mice were identified by

Southern blotting of tail genomic DNA. Briefly, approximately 1 cm of mouse tail was digested in 10 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.5% SDS, 2 mM EDTA with 0.6 mg/mL of proteinase K (Boehringer Mannheim) at 55 ° C for 4 hours. The digested tissue was then extracted with phenol- chloroform and the aqueous phase precipitated in ethanol. The resulting genomic DNA pellet was resuspended in TE buffer ( 10 mM Tris-HCl pH 8.0, 1 mM EDTA), and 10 μg ofDNA was digested overnight with a selected restriction enzyme. The digestion product was run on an agarose gel, transferred to a charged nylon membrane (GeneScreen Plus, NEN Life Science Products), and hybridized as described in Sambrook etal, (l9S9)supra. The probe used to detect specifically the hNF-H gene was a PCR product spanning to the fourth exon ofthe hNF-H gene. For detection ofthe hNF-L transgene, the probe corresponded to aPst I fragment from the first exon ofthe mouse NF-L gene that hybridizes with the gene of both species. Filters were exposed on BioMax MR films (Kodak), using Cronex intensifying screens (Dupont).

Northern Blotting

Total RNA was prepared from freshly isolated or flash-frozen spinal cords from transgenic and normal mice. Homogenization was carried out in 5 mL of Trizol (Gibco-BRL) per gram of tissue and total

RNA isolation performed according to manufacturer' s guidelines. Five or 10 μg of total RNA was loaded onto a 1% agarose-formaldehyde gel and processed for northern blotting as described by Sambrook et al, ( 1989) supra. The radiolabeled probes used for the detection of hNF-L and hNF-H transgenes were the same as those used for genomic screening. The loading was standardized using a mouse actin cDNA as a probe. Filters were exposed to BioMax MR films (Kodak), using Cronex intensifying screens (Dupont).

Protein analysis

Mice were sacrificed and the relevant tissues were dissected to be processed immediately or flash- frozen in liquid nitrogen . To obtain Triton-insoluble cytoskeletal fractions, tissues were homogenized in 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF, 10 mg/mL aprotinin, 2 mg/mL leupetin, 2 mg/mL pepstatin and 1 % Triton X- 100. Homogenates were centrifuged for 20 min at 4 ° C at 13 , OOOx g in a microfuge. The Triton-insoluble pellet was re-homogenized in SUB (0.5% SDS, 8 M urea, 2% β-mercaptoethanol). The resuspended material was centrifuged at room temperature for 15 min in a microcentrifiige. Protein concentrations ofthe resulting supematants were measured using Bio-Rad Protein Assay (Bio-Rad), a Bradford-based protein assay. For immunoblotting, protein samples in 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol and 0.7 M β- mercaptoethanol were loaded on 7.5% SDS-PAGE and subsequently transferred onto nitrocellulose. Filters were blocked for 3 hours in gelatin-buffer (20 mM Tris-HCl pH 7.3, 150 mMNaCl, l%fish skin gelatin (Sigma) and 0.1 % Tween 20) and then incubated with primary antibodies for 4 hours at room temperature or overnight at 4 ° C. The primary antibodies were diluted in the gelatin-buffer at the following titers: monoclonal mouse anti-human NF-L DP5-112 1 :2000(N.T.L. France); monoclonal mouse anti-NF-L RPN.1105 1 : 1000 (Amersham); monoclonal rat anti-humanNF-H (OC95) 1 :200 (kindly provided by V. M. - Y. Lee); polyclonal rabbit anti-NF-H 1 : 1000 (Sigma); monoclonal mouse anti-NF-M NN 18 1 : 1000 (Roche Diagnostics); monoclonal mouse anti-β-tubulin KMX- 1 1 :200 (Roche Diagnostics); and monoclonal mouse anti-actin C4 1 : 1000 (Roche Diagnostics). After several washes in gelatin-buffer, membranes were incubated for one hour with peroxidase-conjugated secondary antibodies (anti-mouse, rat or rabbit, Jackson ImmunoResearch Laboratories Inc.) diluted 1 : 1000 in gelatin-buffer. The membranes were washed once in gelatin-buffer and 3 times in 20 mM

Tris-HCl pH 7.3, 150 mM NaCl. Detection of the immune complex was performed with the chemoluminescent ECL detection kit (Amersham).

Morphometric Analysis

In order to measure axonal calibers, thin sections (1 μm) ofEpon-embedded (Marivac) L5 ventral roots from transgenic and normal mice were stained with 2% Toluidine blue ( JB S) . The images of sections were then digitalized using a Hamamatsu CCD camera mounted on a Leitz Diaplan microscope with a 100X objective. The digitalized images were subsequently analyzed using a morphometric software (Image 1, Universal Imaging Corp., USA). Immunohistochemistry

Anaesthetized mice were perfused with 50 mL of PBS pH 7.4 followed by 50 mL of phosphate buffered 4% paraformaldehyde pH 7.4. Tissues were dissected and further fixed for 2 hours to overnight at 4 ° C in fresh fixative. Samples were sectioned using a vibratome and 25 μm sections were mounted on gelatin-coated slides and permeabilized with 0.3% Triton X- 100 in PB S for 5 minutes at room temperature. Sections were then blocked for 2 hours at room temperature in IF buffer (20 mM Hepes pH 7.9, 250 mMKCl, 1% BSA, 0.2% fish skin gelatin (Sigma), 0.1% Triton X- 100). Sections were then incubated with primary and secondary antibodies as described in the immunohistochemistry section in Example II.

Electron Microscopy

Anaesthetized mice were perfused with 50 mL ofPBS pH 7.4 followed by 50 mL of Jone's fixative pH 7.4 (65 mMNaCl, 2.68 mMKCl, 3.26 mMNaH₂PO₄, and 14.42 mMNa₂HPO₄). The tissues were dissected and further fixed for 2 hours to overnight in fresh fixative at 4 ° C . The samples were postfixed in 2% osmium tetraoxide for 2 hours and dehydrated in a graded series of ethanol solutions and Epon (Marivac) embedded according to standard protocols. Ultrathin sections were stained with uranyl acetate and lead citrate prior to observation on a Philips 10 electron microscope.

Axonal Transport Study

Two month old hNF-H+/+, hNF-L+/+;hNF-H+/+, and normal mice were anaesthetized using sodium pentobarbital, following which 2μL ofPBS containing 300μCi of ³⁵S-methionine (Amersham) was injected into the ventral horn ofthe spinal cord at the level ofthe first lumbar segments. Twenty-eight days after injection, the injected region ofthe spinal cord, the L5 ventral roots, L5 DRGs, and both sciatic nerves were removed. The nerves were then cut into 8 segments of 3mm each, and corresponding segments ofthe two nerves were pooled. Each fraction was homogenized in 10 mM Tris-HCl pH7.5, 150mMNaCl, l mMEDTA,2mMPMSF, 10 mg/mL aprotinin, 2 mg/mL leupetin, 2 mg/mL pepstatin, and 1 % Triton X- 100. Triton-insoluble preparations were obtained as described previously in the protein isolation section. Cytoskeleton-enriched preparations and supematants were separated on 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue. After destaining in 30% methanol, 10% acetic acid, the gels were incubated 30 min at room temperature in Amplify (Amersham). Dried gels were exposed to BioMax MR films (Eastman-Kodak, Rochester, NY).

Results

It was previously reported that an approximate 3 -fold increase in levels of mRNAs existed for the parental hNF-L and hNF-H lines (Beaudet et al , (1993 )Brain Research. Molecular Brain Research 18:23-3 l; Coteeta/., (1993) Ce// 73:35-46). As showninFig.3a, the levels ofmRNA in the spinal cord of doubly hNF-L;hNF-H transgenic mice (3 month old) correspond to those found in singly transgenic mice bearing the hNF-L or hNF-H transgenes alone. The levels of hNF-L or hNF-H transcripts were doubled in mice homozygous for the transgenes, as compared to heterozygous mice.

The increases in hNF-L and hNF-H mRNA levels did not result in comparable increases in protein levels, as showninFigure 3b and in Table2. Densitometric analyzes were performed on the Coomassie stained SDS-PAGE of cytoskeletal protein-enriched preparations from the spinal cord. A 3-fold increase of exogenous mRNAs in mice homozygous for hNF-H (hNF-H+/+) or hNF-L (hNF-L+/+) resulted in increased protein levels of 208 ± 12% for NF-H and 115 ±3 % for NF-L, respectively. It was reported previously that in hNF-L transgenic mice the human NF-L protein species constitutes nearly 80% ofthe total NF-L protein content in the spinal cord (Beaudet et al, (1993) Brain

Research. Molecular Brain Research 18:23-31). It is noteworthy that in doubly transgenic hNF-

L+/+;hNF-H+/+ mice, the levels of foreign proteins were further enhanced with a content in NF-L and

NF-H proteins corresponding to 130 ±5% and 251 ±9%, respectively, the levels found in normal mice

(see Table 2). This additional increase is due to a reciprocal stabilization of additional NF-L and NF-H proteins that are able to form heterodimers (Giasson and Mushynski, ( 1998) J. Neurochem. 70 : 1869-

1875).

Table 2. Levels of NF-subunits in the Spinal Cord of 6 Month Old Transgenic Mice normal hNF-H+/+ hNF-L+/+; hNF-L+/+; hNF-H+/+ total NF-H 100 % 208 % (±12) 251 % (±9) 97 % (±9) total NF-M 100 % 61 % (±5) 64 % (±4) 110 % (±10) total NF-L 100 % 87 % (±9) 130 % (±5) 115 % (±3) The expression ofhNF-L and hNF-H species was further confirmed by western blotting, using specific antibodies directed against the human NF-H protein and the human NF-L proteins (Fig.3b). Whereas the levels ofNF-M were down-regulated in transgenic mice expressing the hNF-H proteins, the levels of tubulin and actin remained similar to those of normal mice.

Reduction ofperikaryal NF accumulations in doubly transgenic mice

The spinal cord from 6 month old transgenic mice was examined by light microscopy (Fig.4). Mice homozygous or heterozygous for the hNF-H transgene developed abnormal accumulations ofNFs in the perikarya and proximal axons of spinal motor neurons (Fig.4c and e) (previously reported in Cote etal, (1993) Cell 73 : 35 -46). In contrast, the 3 -fold increase ofNF-L mRNAs in the hNF-L+/+ mice did not lead to abnormal neurofilamentous accumulations in motor neurons (Fig.4b). Remarkably, the co-expression ofhNF-L proteins in the doubly hNF-L;hNF-H transgenic mice reduced dramatically the number and size ofperikaryal swellings (Fig. 4d, f, h). These beneficial effects of extra hNF-L proteins are particularly striking when hNF-H+/- mice (Fig.4c) are compared to hNF-L+/-;hNF-H+/- mice (Fig.4f) whose spinal cord is virtually devoid ofperikaryal swellings, documenting a gene dosage effect. Mice heterozygous for hNF-L and homozygous for hNF-H (hNF-L+/-;hNF-H+/+) developed large perikaryal swellings (Fig.4g), reminiscent of mice expressing hNF-H alone (Fig.4c and e). In contrast, no NF inclusions were detected in perikarya of motor neurons from mice homozygous for hNF-L and heterozygous for hNF-H (hNF-L+/+;hNF-H+/-) (Fig.4h, small arrows); however, hNF- L+/+;hNF-H+/- mice exhibited some giant proximal axons (large arrows). Similar results were obtained with one year old mice ofthe various genotypes (data not shown).

Axonal atrophy in hNF-H mice is alleviated by extra hNF-L

The L5 ventral roots from 6 month old mice were examined by light microscopy. A dramatic atrophy of motor axons in hNF-H+/+ and hNF-H+/- transgenic mice could be observed (Fig.5c and e). The axonal atrophy was more pronounced in the hNF-H+/+ animals (Fig.5 c) than in the hNF-H+/- animals (Fig. 5e), emphasizing again the gene dosage effect of transgenes. m doubly hNF-L;hNF-H transgenic mice, co-expression of hNF-L restored the radial growth of axons (Fig. 5d, f and h). Remarkably, in the doubly heterozygous hNF-L+/-;hNF-H+/- mice (Fig. 5f) and in the hNF-L+/+;hNF-H+/- mice (Fig. 5h), some ventral root axons oflarger caliber than normal were observed. To quantify the changes of axonal calibers, cross-sectional areas ofL5 ventral root axons were analyzed using a morphometric software (Image 1, Universal Imaging Corp., USA). Normal mice and hNF-L mice showed a bimodal distribution of axonal calibers with peaks at 2-3 μm and 7-8 μm (Fig. 6a). In contrast, no bimodal distribution and a significant increase in the percentage of small axons were observed in mice expressing hNF-H alone (Fig. 6b) . The co-expression of hNF-L together with hNF-H restored the radial growth of axons. Thus, the bimodal distribution of axonal calibers was completely reestablished in the doubly heterozygous hNF-L+/-;hNF-H+/- mice. In addition, a rescue of axonal atrophy in the hNF- L+/+;hNF-L+/+ mice (Fig. 6b) and the hNF-L+/-;hNF-H+/+ mice (Fig. 6d) could be detected, although the bimodal di stribution was not fully recovered (Fig. 6b) . Note the presence of axons with diameters exceeding 13 μm in doubly transgenic mice.

Integrity of axonal cytoskeleton recovered in doubly transgenic mice

The L5 ventral roots of 6 months old animals were analyzed by electron microscopy (EM) . Transverse sections of large motor axons from normal mice revealed an abundance ofNF profiles (Fig. 7a). In motor axons of hNF-L+/+ mice, an increased density ofNFs as compared to normal could be observed (Fig.7b). In contrast, in homozygous or heterozygous hNF-H transgenic mice, (Fig. 7c and e), the cytoskeleton was markedly perturbed and the number of intact NF structures was reduced dramatically. In hNF-H+/+ mice at 12 and 24 months of age, EM revealed in these shrunken axons a general disruption ofthe NF network and fewer microtubules, as compared to younger animals (Collard et al, (1995) Nature 375:61-4). Consistent with the above morphometric data, the co- expression of hNF-L with hNF-H led to the reestablishment of a normal cytoskeleton in axons from young and old mice. The expression of hNF-L in hNF-L+/-;hNF-H+/- mice (Fig. 7f) and in hNF- L+/+;hNF-H+/- mice (Fig. 7h) restored a normal NF density and distribution across the axoplasm (Fig. 7d and f). Analysis of 12 month old animals yielded similar results: fewer degenerative axonal profiles could be detected in doubly transgenic mice as compared to mice expressing the hNF-H transgene alone (data not shown). It is remarkable that a relatively low protein ratio ofNF-L to NF-H in hNF- L+/-;hNF-H+/+ transgenic mice was sufficient to dramatically improve the cytoskeletal integrity in motor axons (Fig. 7g). Improved axonal transport in doubly transgenic mice

Defects in axonal transport has been proposed to underlie the pathogenic mechanism in hNF-H transgenic mice (Collard et al, (1995) Nature 375:61-4); therefore, the rate of transport of cytoskeletal proteins into axons of 2 month old doubly transgenic mice was studied by monitoring radiolabeled, slowly transported polypeptides, 28 days after the injection of ³³S-methionine into the spinal cord. In each segment along the length ofthe sciatic nerve, the transported radiolabeled polypeptides present in the Triton-insoluble fraction were loaded onto SDS -PAGE and analyzed by fluorography . Whereas normal mice showed a leading peak corresponding to a transport rate of -0.75 mm/day for the three NF subunits (Fig. 8), axonal transport was impaired in hNF-H+/+ mice with a leading peak for NF-L and NF-M corresponding to an axonal transport rate of -0.64 mm/day (see arrows). The transport of tubulin was also altered in hNF-H+/+ mice with leading edge at -0.96 mm/day instead of -1.18 mm day in normal mice (Fig. 8, asterisk). The co-expression of hNF-L enhanced the anterograde axonal transport rate, not only for NF proteins, but also for tubulin, with transport rate of -0.86 mm/day and -1.18 mm/day, respectively (Fig. 8, bottom panel).

No overt phenotypes in mice co-expressing human NF-L and NF-H

Whereas hNF-H+/+ and hNF-H+/- mice acquired progressive motor dysfunction and weaknesses, mice co-expressing hNF-L and hNF-H did not develop overt clinical symptoms. Moreover, they rarely exhibited the hind limb contraction reflex, characteristic of motoneuronal disorders, observed in mice expressing the hNF-H transgene alone (Fig. 9). It is noted that the hNF-H+/+ mice lost body weight during aging. At one year of age, the hNF-H+/+ mice have a weight of 31.5 ±3.6 g (n=12) instead of

48.5 ±5.2 g (n=10) for normal mice. At the same age, the hNF-L+/+;hNF-H+/+ mice had an average body weight of 39.0 ±4.6 g (n=9), which is closer to normal.

The above results demonstrate that NF-L proteins can suppress motor neuron disease. A gene delivery approach based on the use ofthe recombinant viral vectors encoding NF-L proteins ofthe present invention offer a means of up-regulating NF-L levels in a sustained manner. These vectors can be used for gene therapy to treat neurodegenerative diseases, neural injuries, and neural degeneration due to aging. EXAMPLE V PROTECTIVE EFFECT OF ELEVATING LEVELS OF NF-L PROTEINS IN ALS MICE

To investigate the effect of elevating the levels ofNF-L proteins in neurodegenerative diseases, excess levels of human NF-L protein were expressed in a transgenic mouse model of ALS. To this end, transgenic mice expressing a human SODl mutation (G37R) were bred with transgenic mice overexpressing normal forms ofthe human gene encoding NF-L protein.

Materials and Methods:

The transgenic mice overexpressing hNF-L were generated as described in Juli en etal. , (1987) Genes & Development 1 : 1085-1097. The transgenic mice heterozygous for an SODl mutation (G37R) referred to as line 29 are described in Wong etal, (1995) Neuron 14: 1105-1116. The G37Rmice were mated with mice heterozygous for the human NF-L transgene to produce the following offspring : SODl^G37R transgenics, NF-L transgenics, doubly SODl^G37R;NF-L transgenics, and normal mice.

Physiological Characteristics and Life Expectancy:

The NF-L mRNA was overexpressed by approximately 1.5 fold in the S OD 1 ^G37R;NF-L transgenic mice. Figure 10 shows the survival curves ofSODl^G37R;NF-Ltransgenicmice as comparedto the

SOD l^G37R transgenic mice. The increased levels ofthe human NF-L protein extended the life spans ofthe SODl^G37R transgenic mice by approximately 8 weeks.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications to the invention to adapt it to various usages and conditions. Such changes and modifications are properly, equitably, and intended to be within the full range of equivalence ofthe following claims.

Claims

WE CLAIM:

1. A recombinant viral vector for infecting target cells comprising: a) the DNA of, or corresponding to, at least a portion ofthe genome of a virus, which portion is capable of infecting the target cells; and b) a normal NF-L gene or portion thereof operatively linked to the DNA and capable of expression in the target cell in vivo or in vitro.

2. The recombinant viral vector of claim 1 , wherein the virus is selected from the group consisting of adenovirus, herpes simplex virus, adeno-associated virus, AIDS virus, retrovirus, or any other suitable virus.

3. The recombinant viral vector of claim 1 , wherein the virus is human adenovirus serotype 5 mutant dl309.

4. The recombinant viral vectors of any of claims 1 to 3 , wherein the viral genome is replication- defective.

5. The recombinant viral vector of claim 1 , further comprising an appropriate promoter sequence.

6. The recombinant viral vector of claim 5, wherein the promoter sequence is neuron-specific.

7. The recombinant viral vector of claim 5 , wherein the promoter sequence is selected from the group consisting ofthe human NF-L gene minimal promoter, the human NF-H gene minimal promoter, other neuron-specific promoters such as α-tubulin, NSE, Thy- 1 , or prion, or viral promoters such as CMV or SV40.

8. The recombinant viral vector of claim 1, whereintheNF-LgeneisamammalianNF-Lgene.

9. The recombinant viral vector of claim 1, wherein the NF-L gene is the human NF-L gene.

10. The recombinant viral vector of claim 1, wherein the target cell is an animal cell.

11. The recombinant viral vector of claim 10, wherein the animal cell is mammalian.

12. The recombinant viral vector of claim 11, wherein the mammalian cell is human.

13. The recombinant viral vector of claim 1, wherein the target cell is a non-mitotic cell.

14. The recombinant viral vector of claim 1, wherein the target cell is a neuron.

15. A recombinant adenovirus vector Ad5-hNF-L.

16. A cell transfected with any ofthe recombinant viral vectors of claims 1 through 15.

17. A use of a recombinant viral vector for infecting target cells comprising: a) the DNA of, or corresponding to, at least a portion ofthe genome of a virus, which portion is capable of infecting the target cells; and b) a normal NF-L gene or portion thereof operatively linked to the DNA and capable of expression in the target cell in vivo or in vitro to deliver an NF-L gene to a subject, comprising administering to the subject an effective amount ofthe recombinant viral vector.

18. The use of claim 17, wherein the subject is an animal.

19. The use of claim 18, wherein the animal is a mammal.

20. The use of claim 19, wherein the mammal is a human.

21. The use of claim 17, wherein the subject has a neurodegenerative disorder.

22. The use of claim 21, wherein the neurodegenerative disorder is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease.

23. The use of claim 17, wherein the subject has a neural injury.

24. The use of claim 17, wherein the subj ect requires axonal regeneration as a result of disease, injury, or aging.

25. The use of claim 17, wherein the subject requires the restoration of calcium ion homeostasis.

26. The use of claim 25, wherein the lack of calcium ion homeostasis is a result of neurodegeneration.

27. The use of claim 17, wherein the vector is administered in conjunction with another recombinant viral vector.

28. A use of a recombinant viral vector for infecting target cells comprising: a) the DNA of, or corresponding to, at least a portion ofthe genome of a virus, which portion is capable of infecting the target cells; and b) a normal NF-L gene or portion thereof operatively linked to the DNA and capable of expression in the target cell in vivo or in vitro to deliver an NF-L gene to a cell, comprising in vitro administration of an effective amount of the recombinant viral vector to the cell.