AGENTS FOR THE PREVENTION AND TREATMENT OF PARKINSON'S DISEASE

FIELD OF THE INVENTION;

The invention relates to therapeutic agents for the prevention and treatment of Parkinson's disease, especially in humans. More specifically, the invention relates to a gene implicated in causing Parkinson's disease, to antagonists of such genes and gene products. The invention additionally relates to nucleic acid molecules that influence the expression of such genes . The invention also relates to therapeutic methods that employ such agents.

BACKGROUND OF THE INVENTION;

Parkinson's disease is a serious neurodegenerative disorder, which usually affects individuals over 45 years of age (Schoenberg, B.S., Adv. Neurology 4 _. 5:277 (1986) ) , and is a leading cause of neurologic disability in individuals older than sixty years old (Yahr, M.D., In: Merritt's Textbook of Neurology. 658-671, ed. Rowland, .P., Lea and Febiger, Philadelphia, PA (1989) ) . The disease is characterized by neurological symptoms including akinesia, tremor, muscular rigidity, and loss of postural reflexes . In humans, Parkinson's disease can arise as a manifestation of other diseases or conditions of the nervous system (secondary Parkinsonism) including viral disease, stroke, toxic brain lesions, cerebral trauma, but the majority of cases are idiopathic (primary Parkinsonism) (Yahr, M.D., In: Merritt's Textbook of

Neurologγ, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ; German, D.C. et al .. Ann. N.Y. Acad. Science 648:42-62 (1992) ; Parkinson, J., In: An Essay on the Shaking Palsy, London (1817) ) . Although, there is a separation of this disease into two main clinical groups, evidence has shown that all forms of this disease have a common feature: the degeneration of dopaminergic neurons in the suJbstantia nigra that innervate the striatum (Yahr, M.D., In: Merritt' s Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ; German, D.C. et al. , Ann. N.Y. Acad. Sci. 648:42-62 (1992); Hornykiewicz, 0. et al . , Adv. Neurology 45, 19 (1986)) , particularly the neuromelanin-pigmented sub-population (Hirsch, E. et al . , Nature 334:345-348 (1988)) . Loss of neurons and pigment also occur in the locus ceruleus and in the dorsal vagal nucleus of the brain stem (Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) . These areas are further characterized by the presence of Lewy bodies (eosinophilic intraneuronal inclusion bodies) , which are the hallmark of Parkinson's disease.

Dopamine, a neurotransmitter found in high concentration in the neostriatum, is synthesized from tyrosine in a sequential pathway, in which the enzyme tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to form L-DOPA, which is then decarboxylated by the enzyme, aromatic amino acid decarboxylase, to form dopamine (Rawn, J.D., In: Biochemistry, Neil Patterson Publishers, Burlington, NC (1989) ; Yahr, M.D., In:

Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) .

This neurotransmitter interacts with two main types of receptors: D-l which is linked to the enzyme adenylate cyclase, and D-2 which is not associated with this enzyme (Kebabian, J.W. et al . , Nature 277 :93-96 (1979)) . The neurological symptoms of Parkinson's

disease appear to be associated with ineffective transmission at certain D-2 receptors. In addition, molidine a specific antagonist of the D-2 receptor has been shown to induce the syndrome (Ayd, F.J., Pis. Nerv. Syst. 35:447-452 (1974)) ; Kebabian, J. . et al . , Nature 277:93-96 (1979)) . Both receptors have been cloned (Bunzow, J.R., WO 91/18005; Bunzow, J.R. et al . , Nature 33_6:783-787 (1988) ) .

In humans with Parkinson's disease, a correlation exists between the loss of dopamine in selected areas and the degeneration of the sui_.sta.ntia nigra, in that the greater the degeneration of neurons, the lower the concentration of dopamine and the greater the severity of Parkinson's disease (Yahr, M.D., In: Merritt' s Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . However, the mechanism by which this degeneration of neurons occurs has not been previously elucidated. Indeed, it is not known for certain if the loss of dopamine alone is the only defect in Parkinson's disease, or whether it is responsible for all of neurological symptoms of this disorder.

It has been well-established that the dopaminergic system exerts an inhibitory modulation on numerous excitatory cholinergic neurons of the striatum (Yahr,

M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ; Stadler, H. et al . , Brain Res. 55:476-480 (1973) ; Guyenet, P.G. et al . , Brain Res. 84:227-244 (1975)) ; Ladinsky, H. et al .. Brain Res. 84:221-226 (1975)) .

Studies have also suggested that presynaptic cholinergic receptors of the muscarinic and nicotinic types can modulate the spontaneous release of dopamine from striatal dopaminergic terminals (Giorguieff, M.F. et al., J. of Pharm. and Exper. Therap. 200:535-543

(1977)) . Acetylcholine, the neurotransmitter of the cholinergic system, is plentiful in the striatum and its

concentration has been shown to be unaltered in Parkinson's disease. In the normal healthy state, the effects of acetylcholine and dopamine appear to balanced (Yahr, M.D., In: Merritt's Textbook of Neurology, 658- 671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ) . However, the loss of dopamine-containing neurons disrupts the balance between the two systems, and the cholinergic system prevails.

It has been suggested that other neurotransmitters such as noradrenalin, GABA, or serotonin, or other factors such as neuropeptides, free radical processes and infectious agents may play a role in Parkinson's disease (In: Neurodegenerative disorders: The role played by endotoxins and xenobiotics. ed. Nappi, G. et al., Raven Press, New York (1988); Yahr, M.D., In:

Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989); Krogsgaard-Larsen, P., Pharmacology & Toxicology 70:95- 104 (1992) ) . Recently, in monkeys, the neurotoxin MPTP (n- methyl-4- phenyl-1,2,3,6-tetrhydropyridine) has been shown to produce a chronic Parkinson syndrome similar to idiopathic Parkinson's disease (German, D.C. et al. , Ann. N.Y. Acad. Science 648:42-62 (1992); Yahr, M.D., In: Merritt's Textbook of Neurology. 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)). These studies further demonstrated a similar pattern of midbrain dopamine cell loss in both idiopathic Parkinson's disease and MPTP-treated monkeys suggesting that a similar mechanism of destruction of these neurons is involved in both Parkinson's disease and MPTP (German, D.C. et al. , Ann. N.Y. Acad. Science 648:42-62 (1992)) . Midbrain dopamine neurons have been shown to contain the calcium-binding protein calbindin- D _28k (CaBP) which buffers intracellular calcium concentrations (German, D.C. et al.. Ann. N.Y. Acad. Science 648:42-62 (1992); Gerfen, C.R. et al. , Proc.

Natl. Acad. Science (U.S.A.) 82:8780-8784 (1985)) , and protects neurons from toxic levels of calcium (Feldman, S.C. et al . , In: Stimulus Response Coupling: The Role of Intracellular Calcium-Binding Proteins, ed. Smith, V.L. et al. , CRC Press, Boston, MA (1990) ; German, D.C. et al. , Ann. N.Y. Acad. Science 648:42-62 (1992)) . Recent evidence has indicated that MPTP toxicity results in increased levels of intracellular calcium (Sun, C.J. et. al. , J. Neural Transm. 74:75-86 (1988) ; Kass, G.E. et al., Arch. Biochem. Biophys . 260:789-797 (1988) ; German, D.C. et al . , Ann. N.Y. Acad. Science 648:42-62 (1992)) . Interestingly, the location of CaBP-containing cells in the normal brain was found to correspond closely with the location of midbrain dopaminergic neurons that were spared in Parkinson's disease and MPTP-treated animals suggesting that: 1) both Parkinson's disease and MPTP cause neuron degeneration through alteration in the balance of intracellular calcium, and 2) CaBP may protect these neurons from cell loss caused by both Parkinson's disease and MPTP (German, D.C. et al . , Ann. N.Y. Acad. Science 648:42-62 (1992)) .

Previous family and twin studies had concluded that genetic factors play a minimal role in the etiology of the disease (Duvoisin, R.C., Adv. Neurology 45 :307-312 (1986) ) . However, a reevaluation of the limitations of previous studies (Duvoisin, R.C. Adv. Neurology 45 :307- 312 (1986) ; Vieregge, P. et al. , Neurology 41:255 (1991) ; Golbe, L.I., Neurology 40:7-14 (1990)) , recent documentation on numerous pedigrees of histologically confirmed Lewy body Parkinson's disease (Duvoisin, R.C, Adv. Neurology 4j5:305-312 (1986) , and the use of fluoro- dopa position emission tomography (PET-detects subclinical disruption of the dopaminergic nigrostriatal system) in asymptomatic twins and at-risk relatives (Duvoisin, R.C, Adv. Neurology 45:307-312 (1986) ;

Zimmerman, T.R. et al . , Neurology, 41:255 (1991) ; Mark, M.H. et al . , Neurology 41:255 (1991)) have provided

evidence suggesting that Parkinson's disease is an autosomal dominant disorder with decreased penetrance, age-related expression, and a wide range of clinical manifestations (Duvoisin, R.C, Adv. Neurology 60 :306- 314 (1993) ) .

In addition to the classical motor impairments that characterize the clinical diagnosis of Parkinson's disease, studies have established that dementia particularly of the Alzheimer's type frequently occurs in Parkinson's disease. Indeed, various clinicopathological and neurochemical correlations exist between Parkinson's and Alzheimer's disease (Boiler, F. et al . , Ann. Neurology 7:329-335 (1980) ; Lees, A., In: Senile Dementia of the Alzheimer Type, ed. Traber, J. et al. , 60-71, Springer-Verlag, Berlin (1985) ; Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . For example, fibrillary tangles, senile plaques, and cerebral atrophy, all pathological features of Alzheimer's disease, have been observed in cases of Parkinson's disease (Boiler, F. et al . , Ann. Neurology 2:329-335 (1980) ; Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . Lewy bodies, the pathologic hallmark of Parkinson's disease have been reported in patients having Alzheimer's disease (Forno, L.S. et al . , Arch. Neurology 35:818-822 (1978) ; Lees, A.J., In: Senile Dementia of the Alzheimer Type, 60-71, ed. by Traber, J. et al . , Springer-Verlag, Berlin (1985) ; Boiler, F. et al . , Ann. Neurology 7:329-335 (1980)) .

Additionally, loss of neurons in the locus ceruleus and the nucleus of Meynert (Lees, A.J., In: Senile Dementia of the Alzheimer Type, 60-71, ed. Traber, J. et al . , Springer-Verlag, Berlin (1985)) ; Whitehouse, P.J. et al .. Ann. Neurology 13:243-248 (1983)) , hyperinnervation of surviving nucleus of Meynert neurons by galanin (a peptide which controls cholinergic

neurons) (Chan-Palay, V., Adv. Neurol . 51:253-255 (1990) ) , a decrease in both cortical somatostatin (Epelbau , J. et al . , Brain Res. 278:376-379 (1983) ; Lees, A.J., In: Senile Dementia of the Alzheimer Type, ed. Traber, J. et al . , 60-71 (1985)) and choline acetyltransferase activity occur in both Parkinson's and Alzheimer's disease. These correlations suggest a relationship between the degenerative processes characterizing both diseases (Epelbaum, J. et al . , Brain Res. 278:376-379 (1983)) .

The diagnosis of Parkinson's disease can be made by recognition of one of the cardinal clinical symptoms (Yahr, M.D., In: Merritt's Textbook of Neurology, 658- 671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ) . However, the time of onset of the disease may be difficult to ascertain due to the absence of one of the cardinal symptoms, or subtlety of early symptoms. Hoehn et al . (Neurology 12=427-442 (1967)) have devised a system for grading the severity of the disease into five stages based on the level of clinical disability. The routine examination of blood, CSF, and urine is normal in patients with the disease (Yahr, M.D., In: Merritt's Textbook of Neurology. 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . However, the CSF of idiopathic Parkinson's disease shows a deficiency of the dopamine metabolite, homovanillic acid (Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ; Benarroch, E.E. et al . , J. Clin. Neurophysiology 10 _. :39-50 (1993)), and contains an antibody that reacts with midbrain dopaminergic neurons (Carvey, P.M., et al . , Neurology 41:53-58 (1991) ; Benarroch, E.E., et al .. J.Clin. Neurophysiology 10 :39- 50 (1993)) . An EEG reading is usually normal, although a diffuse slowing may occur (Yahr, M.D., In: Merritt' s Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . Computed

tomography (CT) scans are often abnormal in Parkinson's disease patients having organic mental syndromes. Recently, the use of fluorodopa PET has been used to detect subclinical degeneration in nigrostriatal pathways in at-risk individuals (Duvoisin, R.C, Adv.

Neurology 60:306-315 (1993) ; Mark, M. et al . , Neurology 41:255 (1991) ) .

During the initial phase of the disease, dopamine deficiency is minimal, and the remaining nerve cells compensate by synthesizing more dopamine (Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . This phase, termed the compensated phase, is usually treated with agents that will enhance existing dopaminergic activity. As the disease progresses with continued loss of dopaminergic neurons, a decompensated phase of the disease occurs, which is best treated by agents that promote the production of dopamine. One of the established treatments having a beneficial effect on the symptoms of Parkinson's disease involves the oral administration of the dopamine precursor, L-DOPA with a peripheral decarboxylase inhibitor (i.e., carbidopa) (Horellou, P. et al . , J. Physiology, Paris 85:158-170 (1991) ; Yahr, M.D., In: Merritt's Textbook of Neurology, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . L-DOPA is able to cross the blood-brain barrier, and is converted to dopamine by remainder dopamine neurons, leading to significant improvement in motor function. Although this drug can slow the progression of the disease, it loses its effectiveness over months or years, and side effects appear such as the "on-off" effect, abnormal involuntary movements (Marsden, CD. et al., The Lancet 1:292-296 (1976) ; Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) , and psychosis (Scholz, E. et al . , Eur. Arch. Psychiatr.

Neurological Science 235 :60-64 (1985) ; Moskovitz, C e al. , Amer. J. Psychiatr. 135:218-226 (1978)) . Classical neuroleptic drugs which preferentially block D-2 receptors have been used to treat drug-induced psychosis (Klockgether, T. et al . , J. Neurology 237:221-225 (1990) ) .

Antidepressants such as imipramine and amitriptyline, have been used alone or in conjunction with L-DOPA in the early phases of the disease, and improve some of the symptoms such as akinesia, depression, and rigidity (Yahr, M.D., In: Merritt' s Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . These agents exert their action by blocking the storage and uptake of dopamine, thereby making it more available at the synaptic cleft in the striatum. In addition, they also have anticholinergic action.

Bromocriptine, an ergoline compound which activates dopamine receptors is less potent than L-DOPA, and has been used often as an adjunct to L-DOPA when the latter loses its effectiveness (Yahr, M.D., In: Merritt' s Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989) ) . However, various side effects such as toxic psychoses, limb edema, and phlebitis have been noted with long-term usage.

L-Deprenyl, a monamine oxidase inhibitor has been effective as an anti-Parkinson' s agent by inhibiting the enzyme monamine oxidase MAO-B in the striatum (Yahr, M.D., In: Merritt's Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA

(1989)) . Dopamine is a major substrate of this enzyme, therefore, inhibition of MAO-B allows dopamine to accumulate thereby enhancing its action. This agent is usually used as an adjunct with L-DOPA and helps control the "on-off" effect. Some reports indicate that this drug loses its effectiveness after a couple of years,

while other findings indicate that the drug may retard the progression of the disease.

Anticholinergic agents have been shown to moderately improve symptoms of the disease, and elicit their effect by opposing the muscarinic effects of acetylcholine in the CNS (Yahr, M.D., In: Merritt' s

Textbook of Neurology, 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA (1989)) . However, their use has been restricted by side effects. Antihistamineε, which have anticholinergic properties have been shown to have a mild effect on symptoms . Usually these agents are used in combination with more potent compounds such as L-DOPA (Yahr, M.D.,

In: Merritt's Textbook of Neurology. 658-671, ed. Rowland, L.P., Lea and Febiger, Philadelphia, PA

(1989) ) .

Moon, M.W. et al . (U.S. Patent No. 5,157,124) have reported the use of heterocyclic acetylenic amine compounds having cholinergic agonist or antagonist activity, in the treatment of cognitive disorders, extrapyramidal motor disorder, and other central nervous system disorders .

Dahl, B.H. et al . (U.S. Patent No. 5,164,404) disclose the use of hydrazone derivatives for antagonizing the biological effects of excitatory amino acids, useful in the treatment of central nervous system disorders .

Regnier, G. et al . (U.S. 4,782,054) disclosed tetrahydroquinonline morpholine compounds which maintain catecholaminergic neurotransmission, useful for treatment cerebral ageing and ischemic syndromes .

Amantadine, an antiviral agent has some beneficial effects on the symptoms of Parkinson's disease. This agent may act directly on striatal dopaminergic mechanisms, or block cholinergic mechanisms (Yahr, M.D.,

In: Merritt's Textbook of Neurology, 658-671, ed.

Rowland, L.P., Lea and Febiger, Philadelphia, PA

(1989) ) . However, the beneficial effects are not lasting, and side effects similar to those of the anticholinergic agents occur.

Recently, treatment of MPTP-induced Parkinsonian syndrome in monkeys with GMl ganglioside (a modulator of neuronal differentiation and development, protein phosphorylation, and synaptic function) has been reported to result in improvement of neurological function, increased striatal dopamine and enhanced dopaminergic innervation of the striatum (Schneider,

J.S. et al . , Science 256:843-846 (1992)) , suggesting its use as in the treatment of Parkinson's disease.

Neural grafting has also represented a possible therapeutic approach to the disease. Striatal transplantation of polymer encapsulated catecholamine- releasing cells was shown to reduce experimental Parkinsonism in rats (Winn, S.R et al .. Soc. For Neuroscience Abst. 17:1077 (1991)) . Clinical trials of dopamine neural grafting of adrenal medulla to the striatum has achieved variable success in humans with the disease (Backlund, E-0. et al. , J.Neurosurg. 62 :169- 173 (1985) ; Ozand, P.T. et al. , J. Child Neurology 2=S132-S140 (1992) and encouraging results in monkeys (Redmond, D.E. et al .. The Lancet 1:1125-1127 (1986)) . Recently, grafts of fetal dopamine tissue implanted into the putamen of a patient affected with idiopathic Parkinson's disease, were shown to survive, grow and restore striatal dopamine synthesis and storage (Lindvall, 0. et al.. Science 247:574-577 (1990)) . These changes were accompanied by a marked improvement in the patient's motor functions, although this treatment required immunosuppressive therapy.

Somatic gene therapy studies on fibroblaεt and striatal cells that were genetically engineered to produce tyrosine hydroxylase, released DOPA and dopamine, and upon grafting both types of modified cells showed behavioral improvement in animal models of

Parkinson's disease . (Horellou, P. et al .. J. Physiology, Paris 85:158-170 (1991) ; (Freese, A., Soc. For Neuroscience Abst. 17:1077 (1991)) .

In addition, a potent glial-cell line-derived neurotrophic factor (GDNF) was shown to enhance survival of cultured rat midbrain dopaminergic neurons (Lin, L.H. et al .. Science 260 :1130-1132 (1993)), suggesting a future therapeutic approach of transplanting cells genetically engineered to secrete GDNF into the brains of Parkinson's patients (Weiss, R., Science 260 : 1072- 1073 (1993) ) .

Thus, despite the use of dopamine and other therapeutic approaches in the treatment of this disease, the degeneration of dopamine neurons still progresses . Accordingly, a therapy that would prevent the neurological symptoms, but also arrest the neurodegenerative process characterizing this disease would be highly desirable. The present invention provides such agents and methods.

BRIEF DESCRIPTION OF THE FIGURE;

Figure 1A shows the nucleotide sequence of Cadrac cDNA. The sequence is 100% homologous to nucleotideε 553-1224 inclusive on the antisense strand of human APP mRNA. Figure IB shows the amino acid sequence of the protein encoded by the Cadrac cDNA sequence.

Figure 1C shows the nucleotide sequence of Cadrac promoter (Cadracpro) which controls the transcription of Cadrac mRNA. SUMMARY OF THE INVENTION;

The invention concerns agents and methods for the diagnosis and therapy of Parkinson's disease. Such agents include antisense molecules capable of

influencing the transcription of Cadrac. The invention also includes antagonists of the product of this gene. In detail, the invention provides a nucleic acid molecule (termed "Cadrac") , substantially free of natural contaminants . The cDNA sequence of Cadrac is

SEQ ID N0:1, and the sequence of Cadrac promoter (termed Cadracpro) SEQ ID NO:3.

The invention also provides the protein (termed "Cadop") that is encoded by the Cadrac cDNA, substantially free of natural contaminants . The amino acid sequence of this protein is SEQ ID N0:2.

The invention additionally includes a reagent capable of diagnosing the presence of a molecule selected from the group consisting of a Cadrac gene sequence, a Cadrac mRNA transcript, and a Cadrac gene product .

The invention particularly concerns the sub- embodiments wherein the reagent is a nucleic acid molecule (such as a ribozyme, or a nucleic acid molecule obtainable by mutating a nucleic acid molecule having a sequence of SEQ ID NO:l , or a nucleic acid sequence that is complementary to the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3) or to the complement of one of these sequences, or a protein (such as an antibody, or a fragment of an antibody) .

The invention also provides a method of treating Parkinson' s disease which comprises providing to an individual, in need of such treatment, an effective amount of an inhibitor of Cadrac, Cadracpro or of Cadop, such as a protein inhibitor or a nucleic acid molecule.

DESCRIPTION OF THE PREFERRED EMBODIMENTS;

The present invention relates to the discovery of a gene, designated "Cadrac," that encodes a protein ("Cadop") with the potential to be a common domain for interacting with the human D-2 dopamine receptor and the

human brain Ca ²⁺ channel protein. The present invention provides the sequences of these molecules . These molecules may be used in the diagnosis, prognosis and treatment of Parkinson's disease. The Cadrac gene is antisense to regions of the human APP gene, and thus the transcript of this gene cannot be expressed in humans carrying the wild type APP gene. The Cadrac gene thus cannot be expressed under normal conditions. The Cadrac gene can be expressed, however, if mutations or other changes occur in the APP gene which impair or prevent the transcription of the APP gene . Silent mutations which might occur in the Cadrac gene will also influence the expression or non- expression of the antisense protein.

I. The Molecules of the Present Invention

The negative strand of exons and introns in the human APP gene was evaluated in a search for genes responsible for Parkinson's disease. This evaluation led to the recognition that the APP antisense strand encoded a protein.

The Cadrac cDNA sequence is shown in Figure 1A (SEQ ID NO:l) , the deduced sequence of the encoded protein is shown in Figure IB (SEQ ID NO:2) and the sequence of Cadrac promoter(TATA box and correlated cap site) is shown in figure 1C (SEQ ID NO:3) .

SEQ ID NO:l ATG CTG GAT AAC TGC CTT CTT ATC AGC TTT

AGG CAA GTT CTT TGC TTG ACG TTC TGC CTC

TTC CCA TTC TCT CAT GAC CTG GGA CAT TCT

CTC TCG GTG CTT GGC CTC AAG CCT CTC TTT GGC TTT CTG GAA ATG GGC ATG TTC ATT CTC

ATC CCC AGG TGT CTC GAG ATA CTT GTC AAC

GGC ATC AGG GGT ACT GGC TGC TGT TGT AGG

AAC TCG AAC CAC CTC TTC CAC AGA CTC TGT

GGT GGT GGT GGT GGT GGT GGC AAT GCT GGT GGT TCT CTC TGT GGC TTC TTC GTA GGG TTC

CTC AGC CTC TTC CTC TAC CTC ATC ACC ATC

CTC ATC GTC CTC GTC ATC ATC GGC TTC TTC

TTC TTC CAC CTC AGC CAC TTC TTC CTC CTC

TGC TAC TTC TAC TAC TTT GTC TTC ACT CCC ATC TGC ATA GTC TGT GTC TGC TCC GCC CCA CCA GAC ATC CGA GTC ATC CTC CTC CGC ATC AGC AGA ATC CAC ATT GTC ACT TTC TTC AGC CAG TGG GCA ACA CAC AAA CTC TAC CCC TCG

GAA CTT GTC AAT TCC GCA GGG CAG CAA CAT GCC GTA GTC ATG CAA GTT GGT ACT CTT CTC ACT GCA TGT CTC TTT GGC GAC GGT GTG CCA GTG AAG ATG AGT TTC GCA AAC ATC CAT CCT CTC CTG GTG TAA

SEQ ID NO:2 MLDNCLLISFRQVLCLTFCLFPFSHDLGHSLSVLGLKPLFG

FLEMGMFILIPRCLEIL NGIRGTGCCCRNSNHLFHRLCGG

GGGGGNAGGSLCGFFVGFLSLFLYLITILIVLVIIGFFFFH

LSHFFLLCYFYYFVFTPICIVCVCSAPPDIRVILLRISRIH IVTFFSQWATHKLYPSSLVNSAGQQHAWMQVGTLLTACLF

GDGVPVKMSFAΝIHPLLV

SEQ ID NO:3 TCATAAATCACACGGAGGTGTGTCATAAC

The Cadrac gene sequence (Figure 1A; SEQ ID NO:l) is 100% homologous to nucleotides 553-1224 inclusive on the antisense strand of APP mRNA.

The Cadrac promoter sequence (Figure 1C; SEQ ID NO:3) is located -1002 to -1043 upstream of the Cadrac open reading frame.

The 223 amino acid trans-membrane protein encoded by the antisense cDNA is shown in Figure IB (SEQ ID

NO:2) . The protein has four potential trans membrane helices one of which contains a 25 amino acid domain (amino acids 128-152) , which is can interact with both the human D-2 dopamine receptor and the human brain L- type Ca ²⁺ channel protein, and may be an activator of these two the latter proteins. Additionally, Cadop contains domains capable of interacting with the muscarinic acetylcholine receptor (Acr, amino acids 49- 52) and the cystic fibrosis transmembrane regulator (CFTR, amino acids 93-117) as shown in Figure IB. Sequences in Cadop can also interact with the dihydropyridine sensitive channel protein, and with the artriuetic peptide receptor precursor. The latter

receptor is intimately involved in stress response in humans .

II. The Physiological Role of the Molecules of the Present Invention

Under normal physiological conditions, the sense/ antisense relationship between APP and the Cadrac gene will not allow Cadrac expression in healthy humans; however, acquired mutations in regions of the APP gene involved in the antisense repression of these genes will allow Cadrac and Cadop to be expressed, and thus result in Parkinson's disease.

The human APP gene encodes a membrane glycoprotein called amyloid precursor protein, which is the precursor of the peptide B-amyloid (Hardy, J.A. et al .. Science 25 :184-185 (1992) ; Kosik, K.K., Science 256:780-783

(1992) ; Kang, J. et al.. Nature 325:733 (1987)) . This peptide is deposited in plaques in the brain and vasculature (Glenner, G. et al.. Biochem. Biophys. Res. Commun. 120:885 (1984) ; Glenner, G. et al .. Biochem. Biophys. Res. Commun. 122.:1131 (1984)) of Alzheimer's patients and is currently thought to be a causative agent of Alzheimer's pathology, subsequently resulting in neurofibrillary tangles, neuronal cell loss, vascular damage and dementia (Hardy, J.A. et al .. Science 2_5 :184-185 (1992)) .

Mutations in the APP gene have been found to be responsible for hereditary Alzheimer's disease (Hardy, J.A. et al.. Science 256:184-185 (1992) ; Kosik, K.S., Science 256:780-783 (1992) ; Goate, A. et al . , Nature 349:704-706 (1992) ; Murrell, J. et al .. Science 254:97- 99 (1991) ; Chartier-Harlin, M.-C, Nature:353 :844-846 (1991) ; Cai X.-D, et al .. Science 259:514-516 (1993)) .

The APP gene has been mapped to chromosome 21 (St.' George-Hyslop, P.H. et al .. Science 235:885-890 (1987)) and cloning of the gene by several groups has shown the

existence of three precursor forms, 695 to 770 amino acids long (Goldgaber, D. et al . , Science 235 : 877-880 (1987) ; Kang, J. et al . ■ Nature 325:733-736 (1987) ; Tanzi, R. et al . , Science 235:880-884 (1987) ; Robakis, N.K. et al .. Proc. Natl . Acad. Science (U.S.A.) 84:4190- 4194 (1987) ; Kitaguchi, N. et al .. Nature 331:530-532 (1988) ; Ponte, P. et al .. Nature 331:525-527 (1988) ; Cole, G.M. et al . , In: Molecular biology and genetics of Alzheimer's disease. 113-119 (1990) ) . The precise function of the APP protein has yet to be identified, although secreted APP has been shown to have growth- regulating activity (Saitoh, T. et al .. Cell 58:615-622 (1989) ; Cole, G.M. et al .. In: Molecular biology and genetics of Alzheimer's disease. 113-121, ed. Miyatake, T. et al .. Elsevier Science Publishers (1990)) , which was inhibited by using an antisense cDNA construct of APP (Cole, G.M. et al . , In: Molecular biology and genetics of Alzheimer's disease. 113-121, ed. Miyatake, T. et al .. Elsevier Science Publishers (1990)) . As stated previously in the application, pathologically and biochemically established Alzheimer's changes and dementia are prevalent in patients with Parkinson's disease (Boiler, F. et al . , Ann. Neurology 2=329-335 (1980) ; Lee, A.J., In: Senile Dementia of the Alzheimer Type. 60-71, ed. Traber, J. et al .. Springer- Verlag, Berlin (1985) , a finding consistent with the recognition of the present invention that certain aspects of both diseases have a similar etiology, and that the APP gene plays a major role in Parkinson's disease.

The cDNA reconstructed from the antisense strand of human APP gene encodes a 223 amino acid protein Cadop, which contains a 25 amino acid domain capable of interacting with the human D-2 dopamine receptor and the human brain Ca ²⁺ channel protein. Cadop has the potential to activate the D2-dopamine receptor and the human brain Ca ²⁺ channel protein simultaneously, hence it

can be assumed that the protein might function as a dimer.

There are four possible ways in which Cadop can cause the symptoms which are characteristic of Parkinson's disease: 1) by simultaneously inhibiting the dopamine receptor and the Ca ²⁺ channel protein, 2) by inhibiting one or the other protein, 3) by activating one or the other protein, and 4) by simultaneously activating both proteins . The mode of action of both the dopamine receptor and the brain Ca ²⁺ channel protein favor the fourth possibility.

Because of the sense-antisense relationship between Cadrac and the APP gene it is unlikely that Cadop can be produced in healthy humans. The loss of antisense control of Cadrac could be due to mutations occurring in the region of the APP gene corresponding to Cadrac.

The sense - antisense relationship between APP and Cadrac makes the latter protein an ideal candidate for the direction of immunotherapeutic reagents to treat and cure Parkinson's disease. Also, because this protein is not expressed in humans, side effects from any such therapeutics should be nonexistent .

Irrespective of the manner of the involvement of the Cadrac gene in Parkinson's disease, any therapeutic method which blocks the expression of this protein or blocks the protein will significantly contribute towards the control of the disease. The diagnostic and/or therapeutic reagents of the invention additionally include fusion proteins, antipeptide reagents, etc., made from the deduced amino acid sequence described in the embodiment of Figures IB. The invention also includes any cellular protein activated by the protein or domains of the protein described in the embodiment of Figures IB.

III. The Uses of the Molecules of the Present Invention

The elucidation of the significance of Cadrac in the etiology of Parkinson's disease provides an improved means for diagnosing the presence and clinical grade of the disease. Moreover, it provides an improved means for predicting whether an asymptomatic individual is predisposed to the disease. It further provides a means for treating the disease.

In particular, the protein described in Figures IB, or mutants thereof, may be used in the treatment of, or in the development of reagents for the treatment of, these diseases and conditions.

A. Diagnostic Uses

Since Cadrac cDNA is not expressed by normal cells, the detection of this molecule in a tissue or fluid sample -- such as a biopsy sample, spinal fluid or blood or lymph fluid sample -- is indicative of the presence of Parkinson's disease.

The detection of these molecules may be done by any of a variety of methods. In one embodiment, antibodies are employed that are capable of binding to the product of the Cadrac gene, and the presence of such molecules is determined via an immunoassay. A large number of suitable immunoassay formats have been described (Yolken, R.H. , Rev. Infect. Pis. 4:35 (1982) ; Collins, W.P., In: Alternative Immunoassays, John Wiley & Sons, NY (1985) ; Ngo, T.T. et al . , In: Enzyme Mediated Immunoassay. Plenum Press, NY (1985) ; incorporated by reference herein.

Suitable antibodies can be either polyclonal or monoclonal, of either a species homologous to or heterologous to the species from which the sample was derived. In lieu of such antibodies, equivalent binding molecules, such as antibody fragments (F(ab') , F(ab') ₂ , single chain antibodies, etc.) , recombinant antibodies, chimeric antibodies, etc. may be employed. Such

antibodies can be obtained using conventional methods with the gene product, Cadop, as an antigen. The Cadrac gene product is preferably obtained through the expression of the gene sequences described herein. The simplest immunoassay involves merely incubating an anti-Cadop antibody with a sample suspected to contain the target molecule -- the protein product of the Cadrac gene. The presence of the target molecule is determined by the presence, and proportional to the concentration, of any antibody bound to the target molecule. In order to facilitate the separation of target-bound antibody from the unbound antibody initially present, a solid phase is typically employed. Thus, for example the sample can be passively bound to a solid support, and, after incubation with the antibody, the support can be washed to remove any unbound antibody.

In more sophisticated immunoassayε, the concentration of the target molecule is determined by binding the antibody to a support, and then permitting the support to be in contact with a sample suspected to contain the target molecule. Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the preεence of a labelled, second antibody that is capable of binding to a second epitope of the target molecule. Immobilization of the labelled antibody on the support thus requires the presence of the target, and iε proportional to the concentration of the target in the sample. In an alternative asεay, the target is incubated with the sample and with a known amount of labelled target. The presence of any target molecules in the sample competes with the labelled target molecules for antibody binding εiteε. Thus, the amount of labelled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecule in the sample.

Aε indicated above, immunoassay formats may employ labelled antibodies to facilitate detection. Radioisotopic immunoassays ("RIAs") have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kineticε. Such aεεayε suffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require special handling and disposal, and entail the use of complex and expensive analytical equipmen . RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S., et al. , North Holland Publishing Company, NY (1978) , with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related

Techniques" by Chard, T., incorporated by reference herein.

Enzyme-based immunoassay formats (ELlSAs) have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies -- colorimetric, pH, gas evolution, etc. -- can be used to quantitate the assay. In addition, the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use. ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, D.M. et al .. Eds.) , John Wiley & Sons, NY (1988), incorporated by reference herein. For these reasons, enzyme labels are particularly preferred. No εingle enzyme is ideal for use as a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular asεay system. Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time) , purity of the enzyme preparation, sensitivity of detection of its product,

eaεe and εpeed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and itε conjugate, and the like. Exampleε of εuitable enzymeε include peroxidaεe, acetylcholine esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, /3-galactosidaεe, catalase, delta-5-steroid isomerase, glucose oxidase, glucose-6- phosphate dehydrogenase, glucoamylase, glycoamylase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phoεphate isomerase, ureaεe, yeaεt-alcohol dehydrogenase, etc. Peroxidase and urease are among the more preferred enzyme labelε, particularly becauεe of chromogenic pH indicatorε which make its activity readily visible to the naked eye.

In lieu of such enzyme labels, radioiεotopic, chemilumineεcent or fluoreεcent labels may be employed. Examples of εuitable radioisotopic labels include ³ H, ^lxl In, ¹²⁵ I, ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ⁵⁷ To, ^S8 Co, ⁵⁹ Fe, ⁷⁵ Se, ¹⁵² Eu, ⁹⁰ Y, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, etc. Examples of suitable chemiluminescent labelε include a luminal label, an iεoluminal label, an aromatic acridinium eεter label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, an aequorin label, etc. Examples of suitable fluoreεcent labels include a fluoreεcein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, a fluorescamine label, etc.

As an alternative to such immunoaεεay formats, the presence of Cadrac cDNA in a cell can be determined by any means capable of detecting Cadrac mRNA. Thus, molecules comprising nucleic acid probes capable of hybridizing to the Cadrac sequence of Figure 1 may be

uεed in the diagnoεiε of Parkinεon'ε disease. As used herein, a "probe" is a detectably labelled nucleic acid molecule that is capable of hybridizing to a defined εite of a target molecule. Any of the nucleotide εequenceε disclosed herein can be used to define a probe; the general requirement for such use being merely that the nucleic acid molecule be sufficiently long (generally 10 or more nucleotides in length) that it possesses the capacity to form stable hybridization products with the target molecule. Any of a wide variety of labels (see above) may be used to label nucleic acids: enzyme labels (Kourilsky et al. , U.S. Patent 4,581,333) , radioisotopic labels (Falkow et al . , U.S. Patent 4,358,535; Berninger, U.S. Patent 4,446,237), fluorescent labels (Albarella et al . , EP 144914), chemical labels (Sheldon III et al .. U.S. Patent 4,582,789; Albarella et al .. U.S. Patent 4,563,417) , modified baseε (Miyoshi et al .. EP 119448) , etc. Such nucleic acid based assays may use either DNA or RNA to detect the Cadrac mRNA. In one embodiment, the assayε may be performed on RNA that has been extracted from samples of neuronal or other preselected tissue. Alternatively, and more preferably, the asεays may be done in situ on biopsied tissue.

Where the concentration of Cadrac mRNA in a sample is too low to be detected, such mRNA may be specifically amplified through the use of any of a variety of amplification protocols, such as PCR (Mullis, K.B., Cold Spring Harbor Symp. Quant . Biol . 51:263-273 (1986) ;

Saiki, R.K., et al.. Bio/Technology 3:1008-1012 (1985) ; Mullis K. et al .. U.S. Patent 4,683,202; Erlich, H. , U.S. Patent 4,582,788; Saiki, R. et al .. US 4,683,194 and Mullis, K.B., et al.. Met. Enzymol. 155:335-350 (1987) , transcription-based amplification systemε (Kwoh, D. et al . , Proc. Natl . Acad. Science (U.S.A.) 86 :1173 (1989) ; Gingeraε, T.R. et al .. PCT appl . WO 88/10315;

Davey, C et al . ,- European Patent Application Publication no. 329,822), etc.

In yet another embodiment, the diagnosis of Cadrac expresεion iε performed using a ribozyme produced from nucleic acid molecule having a sequence of SEQ ID N0:1. Ribozymes (RNA enzymes) are catalytic RNA sequenceε (containing no protein) that can cleave RNA target moleculeε with which they hybridize (Cech, T. et al .. Cell 27:487 (1981) ; Cech, T., Science 236:1532-1539 (1987) ; Cech, T. et al .. Ann. Rev. Biochem. 55:599-630 (1986) ; James, W., Antiviral Chemistry & Chemotherapy 2:191-214 (1991)) . Often the substrate is part of the ribozyme itεelf.

An artificial ribozyme can be deεigned to εpecifically cleave a target RNA by flanking εequenceε complementary to the target (Haεeloff, J. et al . , Nature 334:585-591 (1988) ; Cameron, F. et al .. Proc. Natl. Acad. Science (U.S.A.) 86:9139-9143 (1989) ; Jameε, W., Antiviral Chemistry S. Chemotherapy 2:191-214 (1991) . The minimum requirement for cleavage within the target RNA is the location of a suitable three base sequence GUC, GUA, or GUU preceding the cleavage site. Artificial ribozymes having a characteriεtic "hammerhead" secondary structure have been designed by Haεeloff, J. et al . (Nature 334:585-591 (1988) ; Jeffrieε, A. et al . , Nucleic Acidε Res. 12=1371-1377 (1989) ; Gerlach et al . WO Patent No. 8905852 (1989) ; Goodchild, J. et al .. Arch. Biochem. Biophyε . 284:386-391 (1991) ; Jameε, W., Antiviral Chemiεtry & Chemotherapy 2:191-214 (1991)) . Variouε modificationε in ribozymeε have also been shown to be neceεεary to obtain in vivo activity (Cameron, F. et al .. Proc. Natl. Acad. Science (U.S.A.) 86:9139-9143 (1989) ; Cotten, M. et al .. EMBO J. 1:3861-3866 (1989) ; Sarver, N. et al .. Science 247:1222-1225 (1990) ; James, W., Antiviral Chemistry & Chemotherapy 2:191-214 (1991) ) .

B. Prognostic Uses

The present invention additionally provides a capacity to predict whether an individual is at riεk for Parkinεon'ε diεease. Thus, any of the above-described assays may be performed on an asymptomatic individual in order to assesε that individual'ε predisposition to Parkinson' ε disease.

C. Therapeutic Uses

Significantly, the present invention provides a means for treating Parkinson's diseaεe. Such treatment may be either "prophylactic" or "therapeutic." A prophylactic treatment iε one that is provided in advance of any symptom of Parkinεon'ε disease in order to prevent or attenuate any subsequent onset of the diεeaεe. A therapeutic treatment iε one that iε provided in responεe to the onset of a symptom of Parkinson's disease, and serves to attenuate an actual symptom of the diseaεe.

In one embodiment, εuch treatment iε provided by adminiεtering to a patient in need of εuch treatment an effective amount of an antibody, or an antibody fragment (F(ab') , F(ab') ₂ , single chain antibodies, etc.) that is capable of binding to the product of the Cadrac gene. As used herein, an effective amount is an amount sufficient to mediate a clinically significant change in the severity of a symptom, or a clinically significant delay in the onset of a symptom.

As will be appreciated, for acute administration, polyclonal or monoclonal antibodies (or fragments of either) may be adminiεtered. More preferably, and eεpecially for chronic administration, the use of non- immunogenic antibodies is preferred. Such moleculeε can be pεeudo-homologous (i.e. produced by a non-human species, but altered to a form that iε immunologically

indistinct from human antibodieε) . Exampleε of εuch pseudo-homologous molecules include "humanized" (i.e. non-immunogenic in a human) prepared by recombinant or other technology. Such antibodieε are the equivalentε of the monoclonal and polyclonal antibodies, but are leεε immunogenic, and are better tolerated by the patient.

Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al . , International Patent Publication PCT/US86/02269; Akira, K. et al .. European Patent Application 184,187; Taniguchi, M. , European Patent Application 171,496; Morrison, S.L. et al.. European Patent Application 173,494; Neuberger, M.S. et al . , PCT Application WO 86/01533; Cabilly, S. et al . , European Patent Application 125,023; Better, M. et al. , Science 240:1041-1043 (1988) ; Liu, A.Y. et al .. Proc. Natl. Acad. Science (U.S.A.) 84:3439-3443 (1987); Liu, A.Y. et al .. J. Immunol. 139:3521-3526 (1987) ; Sun, L.K. et al.. Proc. Natl. Acad. Science (U.S.A.) 84:214-218 (1987) ; Nishimura, Y. et al . , Cane. Reε . 47:999-1005 (1987) ; Wood, C.R. et al . , Nature 314:446-449 (1985)) ; Shaw et al . , J. Natl.Cancer Inεt. 80:1553-1559 (1988) ; all of which references are incorporated herein by reference) . General reviews of "humanized" chimeric antibodies are provided by Morrison, S.L. (Science, 229:1202-1207 (1985)) and by Oi, V.T. et al .. BioTechniques 4:214 (1986) ; which referenceε are incorporated herein by reference) .

Suitable "humanized" antibodies can alternatively be produced by CDR or CEA substitution (Jones, P.T. et al. , Nature 321:552-525 (1986) ; Verhoeyan et al . , Science 239:1534 (1988) ; Beidler, C.B. et al . ■ J\_

Immunol . 141:4053-4060 (1988) ; all of which references are incorporated herein by reference) .

In another embodiment, the nucleic acid molecules of the present invention may be mutated and expressed in order to identify Cadrac mutant gene products that can complex with and εignificantly inactivate normal Cadrac gene products present in Parkinson's disease. In one sub-embodiment, such mutated protein molecules may be administered to a patient. Alternatively, nucleic acid expreεsing such molecules may be administered.

In yet another embodiment, "antisense" or "triplex" nucleic acid molecules may be used to provide the desired therapy. As used herein, an "antisense oligonucleotide" is a nucleic acid (either DNA or RNA) whoεe εequence is complementary to the sequence of at least part of the Cadrac protein-encoding sequences described herein, such that it is capable of binding to, or hybridizing with, an endogenouε Cadrac mRNA molecule, and can thereby impair (i.e. attenuate or prevent) its the translation into the Cadrac gene product. A "triplex" molecule is a nucleic acid molecule that is capable of binding to double-stranded DNA in a manner sufficient to impair its transcription.

To act as a triplex oligonucleotide, the nucleic acid molecule must be capable of binding to the Cadrac gene sequence of the double-stranded DNA genome in a manner sufficient to impair the transcription of the gene. Triplex oligonucleotides are disclosed by Hogan, U.S. Patent 5,176,996 and by Var a et al . , U.S. Patent 5,175,266. To act as an antiεenεe oligonucleotide, the nucleic acid molecule muεt be capable of binding to or hybridizing with that portion of the Cadrac mRNA molecule which mediateε the tranεlation of the target mRNA. Antisense oligonucleotides are disclosed in European Patent Application Publication Nos . 263,740; 335,451; and 329,882, in U.S. Patent 5,097,617 and in PCT Publication No. WO90/00624, all of which referenceε are incorporated herein by reference. Such a molecule can be of any length that is effective for this purpose.

Preferably, the antisense oligonucleotide will be about 10-30 nucleotides in length, most preferably, about 15- 24 nucleotides in length.

Thus, in one embodiment of this invention, an antisense oligonucleotide that is designed to specifically block transcription from Cadrac promoter or translation of a Cadrac mRNA tranεcript can be uεed to impair the expreεεion of the Cadrac gene in a cell, and thereby provide a treatment for Parkinεon's disease. In general, the antisense oligomer is prepared in accordance with the nucleotide sequence of Cadrac or Cadracpro as reported herein.

The εequence of the antisense oligonucleotide may contain one or more insertions, substitutionε, or deletionε of one or more nucleotideε provided that the resulting oligonucleotide is capable of binding to or hybridizing with the above-deεcribed translation locus of the Cadrac mRNA.

Any means known in the art to synthesize the antisense oligonucleotides of the present invention may be used (Zamechik et al .. Proc. Natl. Acad. Science (U.S.A. ) £2=4143 (1986) ; Goodchild et al .. Proc. Natl . Acad. Science (U.S.A.) 5=5507 (1988) ; Wickstrom et al . , Proc. Natl. Acad. Science (U.S.A.) 5:1028; Holt, J.T. et al. , Molec. Cell. Biol . 8:963 (1988) ; Gerwirtz, A.M. et al.. Science 242:1303 (1988) ; Anfossi, G., et al.. Proc. Natl. Acad. Science (U.S.A.) 86:3379 (1989) ; Becker, D., et al .. EMBO J. 8:3679 (1989) ; all of which references are incorporated herein by reference) . Automated nucleic acid synthesizers may be employed for this purpose. In addition, desired nucleotides of any εequence can be obtained from any commercial εupplier of εuch custom molecules.

Most preferably, the antisense oligonucleotides of the present invention may be prepared using solid phaεe "phoεphoramidite εynthesis." The synthesis is performed with the growing nucleotide chain attached to a εolid

εupport derivatized with the nucleotide which will be the 3'-hydroxyl end of the oligonucleotide. The method involves the cyclical synthesis of DNA using monomer units whose 5'-hydroxyl group is blocked (preferably with a 5' -DMT (dimethoxytrityl) group) , and whose amino groups are blocked with either a benzoyl group (for the amino groups of cytoεine and adenosine) or an isobutyryl group (to protect guanosine) . Methods for producing such derivativeε are well known in the art . In yet another embodiment of the invention, a ribozyme that can be designed to cleave a target Cadrac mRNA transcript can be used to block expression of the Cadrac gene in a cell, and provide another treatment for Parkinson's disease. As stated previouεly in thiε application, an artificial ribozyme can be designed to specifically cleave a target RNA by flanking ribozyme sequences complementary to sequences of the target mRNA (Cameron, F. et al . , Proc. Natl. Acad. Science (U.S.A.) £6=9139-9143; James, W., Antiviral Chemistry & Chemotherapy 2=191-214 (1991) . The minimum requirement in designing a ribozyme to cleave a target mRNA is that the sequence around the cleavage εite should be preceded by the triplet GUC, GUA or GUU. Means known in the art to syntheεize ribozymeε may be used as disclosed previously in this application.

Additionally, in yet another embodiment an antisense nucleotide and ribozyme may be used in combination to block expresεion of the Cadrac gene (Cameron, F. et al .. Proc. Natl. Acad. Science (U.S.A.) £6=9139-9143 (1989) ; Jameε, W., Antiviral Chemistry & Chemotherapy 2:191-214 (1991)) .

IV. Administration of the Molecules of the Present Invention

The above-described therapeutic agents of the preεent invention can be formulated according to known

methods used to prepare pharmaceutically useful compositions, whereby these materialε, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington' ε Pharmaceutical Sciences (16th ed., Oεol, A., Ed., Mack, Easton PA (1980)) . In order to form a pharmaceutically acceptable composition suitable for effective adminiεtration, εuch compositions will contain an effective amount of such agents, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparationε may be achieved through the use of polymers to complex or absorb the agentε . The controlled delivery may be exercised by selecting appropriate macromoleculeε (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methyl- celluloεe, carboxymethylcelluloεe, or protamine, sulfate) and the concentration of macromolecules as well aε the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the agents into particles of a polymeric material εuch aε polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, inεtead of incorporating these agents into polymeric particleε, it iε poεεible to entrap these materials in microcapsuleε prepared, for example, by coacervation techniqueε or by interfacial polymerization, for example, hydroxymethylcelluloεe or gelatine-microcapεuleε and poly(methylmethacylate) microcapεules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microsphereε, microemulεionε, nanoparticles, and

nanocapsules or in macroemulsionε . Such techniques are disclosed in Remington's Pharmaceutical Scienceε (1980) . In one embodiment of the preεent invention, nucleic acid molecule (ε) compriεing antisense or triplex molecules, or encoding mutated Cadrac gene product may be administered using viral or retroviral vectors in accordance with the methods of "gene therapy" .

The principles of gene therapy are disclosed by Oldham, R.K. (In: Principles of Biotherapy, Raven Press, NY, 1987), and similar texts. Disclosures of the methods and uses for gene therapy are provided by Boggs, S.S. (Int. J. Cell Clon. 8:80-96 (1990)) ; Karson, E.M. (Biol. Reorod. 42:39-49 (1990)) ; Ledley, F.D., In: Biotechnology. A Comprehensive Treatise, volume 7B, Gene Technology. VCH Publishers, Inc. NY, pp 399-458 (1989)) ; all of which references are incorporated herein by reference.

Although, as indicated above, such gene therapy can be provided to a recipient in order to treat (i.e. εuppress, or attenuate) an existing condition, the principles of the present invention can be used to provide a prophylactic gene therapy to individuals who, due to inherited genetic mutations, or somatic cell mutation, are predisposed to breast cancer. Having now generally deεcribed the invention, the same will be more readily underεtood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unlesε εpecified.

Example 1

Syntheεiε and Expression of the Cadrac Gene

The Cadrac gene waε prepared by oligonucleotide synthesiε using solid phaεe gene aεεembly. The cDNA is constructed in three seperate parts which are linked together to produce the full length cDNA. Successive

oligonucleotides phosphorylated and added at molar exceεs were attached stepwise to a growing chain anchored to a solid phase support. The assembled oligonucleotides were ligated with T4 DNA ligaεe and detached from the εupport by cleaving at an added reεtriction enzyme εite. Full length strands are gel purified, mixed in equimolar amounts and annealed and the double εtranded cDNA is re-purified. The constructed genes were procesεed in the EXPRESS system (Invitrogen Corp. : pTrcHiε Xpress-Prokaryotic Expression and

Purification system) according to the manufacturer's instructionε .

In thiε manner, a DNA molecule, encoding a protein of 223 amino acids was constructed. The nucleic acid sequence of this molecule is 100% homologous to regionε on the antisense strand of human APP gene. The expression of these proteins iε repreεsed by the normal wild type APP gene healthy in humans; however, mutationε, inherited or acquired, in regions of the APP gene which are crucial for maintaining repression could permit the expression of various levelε of this antisenεe protein. The antiεenεe protein is a potential activator of human D-2 dopamine receptor and brain Ca ²⁺ channel protein. The antisense relationship with APP gene and the function of this protein make them ideal candidates for causative factorε of APP protein related to Parkinεon's disease. Since the Cadop protein is not expressed in normal humans it iε an excellent target for developing humanized antibodieε against to prevent and cure Parkinson's disease.

While the invention has been deεcribed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including εuch departures from the present

diεcloεure aε come within known or cuεtomary practice within the art to which the invention pertains and aε may be applied to the eεεential featureε hereinbefore εet forth and as follows in the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Preddie, Rick E.

Bergmann, Johanna E. (ii) TITLE OF INVENTION: Agents For The Prevention And Treatment

Of Parkinson's Disease

(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Weil, Gotshal & Manges

(B) STREET: 1615 L Street, N.W., Suite 700

(C) CITY: Washington

(D) STATE: DC (E) COUNTRY: United States

(F) ZIP: 20036

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Auerbach, Jeffrey I.

(B) REGISTRATION NUMBER: 32,680 (C) REFERENCE/DOCKET NUMBER: 683-105

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 682-7033

(B) TELEFAX: (202) 857-0939

(2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 672 baεe pairε

(B) TYPE: nucleic acid

(C) STRANDEDNESS : εingle

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

( iv) ANT I - SENSE : YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Homo Sapienε

(vii) IMMEDIATE SOURCE: (B) CLONE: APPL6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

ATGCTGGATA ACTGCCTTCT TATCAGCTTT AGGCAAGTTC TTTGCTTGAC

50

GTTCTGCCTC TTCCCATTCT CTCATGACCT GGGACATTCT CTCTCGGTGC 100

TTGGCCTCAA GCCTCTCTTT GGCTTTCTGG AAATGGGCAT GTTCATTCTC

150 ATCCCCAGGT GTCTCGAGAT ACTTGTCAAC GGCATCAGGG GTACTGGCTG

200 CTGTTGTAGG AACTCGAACC ACCTCTTCCA CAGACTCTGT GGTGGTGGTG

250 GTGGTGGTGG CAATGCTGGT GGTTCTCTCT GTGGCTTCTT CGTAGGGTTC

300

CTCAGCCTCT TCCTCTACCT CATCACCATC CTCATCGTCC TCGTCATCAT 350

CGGCTTCTTC TTCTTCCACC TCAGCCACTT CTTCCTCCTC TGCTACTTCT

400 ACTACTTTGT CTTCACTCCC ATCTGCATAG TCTGTGTCTG CTCCGCCCCA

450 CCAGACATCC GAGTCATCCT CCTCCGCATC AGCAGAATCC ACATTGTCAC

500 TTTCTTCAGC CAGTGGGCAA CACACAAACT CTACCCCTCG GAACTTGTCA

550

ATTCCGCAGG GCAGCAACAT GCCGTAGTCA TGCAAGTTGG TACTCTTCTC 600

ACTGCATGTC TCTTTGGCGA CGGTGTGCCA GTGAAGATGA GTTTCGCAAA

650 CATCCATCCT CTCCTGGTGT AA

(2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 223 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Homo Sapiens

(vii) IMMEDIATE SOURCE: (B) CLONE: APPL6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :

Met Leu Asp Asn Cys Leu Leu lie Ser Phe Arg Gin Val

1 5 10

Leu Cys Leu Thr Phe Cys Leu Phe Pro Phe Ser His Asp

15 20 25

Leu Gly His Ser Leu Ser Val Leu Gly Leu Lys Pro Leu

25 30

Phe Gly Phe Leu Glu Met Gly Met Phe lie Leu lie Pro

35 40 45

Arg Cys Leu Glu lie Leu Val Asn Gly lie Arg Gly Thr

50 55 60

Gly Cys Cys Cyε Arg Aεn Ser Asn Hiε Leu Phe His Arg

65 70

(2) INFORMATION FOR SEQ ID NO:3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 baεe pairs

(B) TYPE: nucleic acid (C) STRANDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI_SENSE : YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Homo Sapiens

(vii) IMMEDIATE SOURCE: (B) CLONE: APPL6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

TCATAAATCA CACGGAGGTG TGTCATAACC 30