WO1994024279A2 - Agents for the prevention and treatment of huntington's disease and other neurological disorders - Google Patents

Abstract

Agents and methods for the diagnosis and therapy of Huntington's Disease and related conditions are disclosed. Such agents include antisense molecules of genes implicated in Huntington's Disease and related diseases as well as analogues and derivatives of these molecules, and protein molecules expressed by these genes.

Description

TITLE OF THE INVENTION;

AGENTS FOR THE PREVENTION AND TREATMENT OF HUNTINGTON'S DISEASE AND OTHER NEUROLOGICAL DISORDERS

FIELD OF THE INVENTION: The invention relates to therapeutic agents for the prevention and treatment of Huntington's disease myotonic dystrophy, fragile x syndrome, spino bulbar atrophy and spino cerebellar ataxia type 1 in humans. More specifically, the invention relates to six genes that are implicated in causing Huntington's Disease, myotonic dystrophy, fragile x syndrome, spino bulbar atrophy and spinocerebrellar ataxia type 1, to antagonists of these genes and their gene products. The invention additionally relates to nucleic acid molecules that influence the expression of these genes. The invention also relates to therapeutic methods that employ any such agents.

CROSS-REFERENCE TO RELATED APPLICATIONS;

This application is a continuation-in-part of pending U.S. Patent Application Serial No. 08/050,578, filed April 20, 1993.

BACKGROUND OF THE INVENTION;

I. Huntington's Disease

Huntington's chorea ("Huntington's Disease") is an autosomal dominant progressive and fatal neurodegenerative disease which starts in humans around age 40. The disease is characterised by involuntary movements, cognitive and psychiatric disturbances and intellectual decline. There is no cure for the disease which progresses to death within 15 - 20 years from the date of onset (Bruyn, R.P.M. et al., J. Neurolog. Sci. £5:29-38 (1990)) . Until now nothing was known about the biochemical basis of the disease, but recently a novel gene containing a trinucleotide repeat (CAG) that is expanded in chromosomes of Huntington's victims was determined to be the genetic basis of the disease (Huntington's Disease Collaborative Research Group. Cell 72:971-983 (1993) .

The gene affected in Huntington's Disease has been mapped to the short arm of chromosome 4 (Gusella, J.F. et al.. Nature 306:234-238 (1983); exler, N.S. et al.. Nature 326:194-19 7 (1984)). The mutant gene which produces Huntington's Disease causes the premature loss of certain populations of neurons, and results in certain specific biochemical changes in the brain, particularly in the caudate nucleus, putamen, globus pallidus, substantia nigra and occipital cortex (Perry, T.L. et al.. J. Neurolog. Sci. 78:139-150

(1987) ) . Choline acetyltransferase activity has also been found to be seriously diminished (Bruyn, R.P.M. et al.. J.

Neurolog. Sci. £5:29-38 (1990)). Similarly, the concentrations of the neuropeptides P and enkephalin have been found to be markedly depressed in brain tissue of sufferers of Huntington's Disease.

II. Related Neurological Disorders

Myotonic dystrophy, spinocerebellar ataxia type 1, and spino-bulbar atrophy (Kennedy's disease) exemplify neurological disorders that are related to Huntington's disease; fragile x syndrome is linked to the above diseases by the fact that the genetic basis of this disease is also the expansion of unstable trinucleotide repeats in the FMR1 gene. Myotonic dystrophy is an autosomal dominant disorder that combines dystrophic muscular weakness with myotonia. The disease affects one in 8,000 individuals. It can occur at any age, and varies in severity. Severe cases show marked peripheral muscular weakness associated with a high incidence of cataracts, testicular atrophy, balding, cardiomuscular defficiencies, and endocrine abnormalities. In severe cases, the disease may be fatal. The genetic basis of the disease is an unstable trinucleotide CTG repeat in the 3'end of a gene encoding a cAMP-dependent protein kinase, (Aslandis. C et al.. Nature 355:348-551 (1992) ) .

Spinocerebellar ataxia type 1 is a progressive neuro¬ degenerative disease characterized by ataxia, opthalmoparesis and variable degrees of muscle weakness (Orr, H.T. et al.. Naturegenetics 4:221-225 (1993)) . The genetic basis of the disease is believed to be unstable expansion of a CAG trinucleotide repeat in a gene localised to human chromosome #6.

X-linked spinal and bulbar atrophy (such as Kennedy's disease) is an adult onset form of motor neuron disease (LaSpanda, A.R. et al.. Nature 352:77-79 (1991)) . The genetic basis of the disease is believed to be unstable trinucleotide expansion in a CAG repeat in the coding region of the androgen receptor gene.

Fragile x syndrome is an x linked heritable disease which affects about 1 in 1250 males. The disease is characterized by mental retardation and macroorchidism. The genetic basis of the disease is believed to be unstable trinucleotide expansion in a CGG repeat in the 5'end of the gene (Verkerk et al.. Cell. 65:905-914 (1991) )

BRIEF DESCRIPTION OF THE FIGURES: Figure 1A presents the cDNA and protein sequences of a 162 bp cDNA (huntL) . The sequence of the novel 33 amino acid neuropeptide (hormone) precursor (phuntH) encoded by this cDNA is shown in Figure IB. The amino acid sequence of the active huntH hormone is shown in Figure 1C.

Figures 2A-2C present the cDNA, encoded amino acid sequences of gene sequences implicated in myotonic dystrophy and the organisation of the gene including 5'regulatory sequences (promoter and four GC boxes) . Figure 2A provides the sequence of a 126 bp cDNA molecule that encodes MMD1L. The protein encoded by MMD1L, MMD1H, is shown in Figure 2B. The location of the TATA box, four GC boxes and a transcription termination signal relative to the MMD1L orf is shown in Figure 2C. Figure 3A provides the sequence of a 93 bp cDNA molecule that encodes FMR1L. Figure 3B provides the sequence of the encoded 30 amino acid polypeptide (FMR1H) . Figure 3C provides the sequences of promoter elements and correlated cap sites in the 5'regulatory region of FMR1L gene (FMRlLreg) . Figure 3D provides the sequence of the 159 bp cDNA molecule which encodes FMR1L2. Figure 3E provides the sequence of the encoded 52 amino acid polypeptide (FMR1H2) . Figure 3F provides the sequence of the promoter elements and correlated cap sites in the regulatory region of the FMR1L2 gene (FMRlL2reg) .

Figure 4A presents the 168 bp cDNA sequence of the andr2L gene. Figure 4b provides the sequence of the encoded 56 amino acid polypeptide. Figure 4C provides the sequence of a promoter element ("CAATT box") in the regulatory region of the andr2L gene and the organisation of the gene.

Figure 5A presents the 234 cDNA sequence of the SCA1L gene. Figure 5B presents the sequence of the 78 amino acids polypeptide encoded by SCA1L cDNA. Figure 6A illustrates the structural inter¬ relationships between the predicted antisense neuropeptides of the present invention. SUMMARY OF THE INVENTION;

The invention concerns agents and methods for the diagnosis and therapy of Huntington's disease and related conditions, such as myotonic dystrophy, spinocerebellar ataxia, spino-bulbar atrophy and fragile x syndrome. Such agents include antisense molecules capable of influencing the transcription of genes encoded by the antisense strands of the myotonic protein kinase gene, the androgen receptor gene, the FMR1 gene, the SCA1 gene and the huntingtin gene, The invention also includes antagonists of the products of these genes.

In detail, the invention provides a nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of MMD1LH, FMR1LH, FMR1LH2, phuntH, huntH, SCA1LH and andr2LH. The invention particularly pertains to such nucleic acid molecules whose sequence is selected from the group consisting of SEQ ID N0:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 SEQ NO:10, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:16.

The invention also provides a protein, substantially free of natural contaminants, selected from the group consisting of MMD1LH, FMR1LH, FMR1LH2, phuntH, huntH andr2LH and SCAILH. The invention particularly pertains to such proteins whose sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO:11, SEQ ID NO:14 and SEQ ID NO:17.

The invention also provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of a gene sequence that encodes MMD1LH, a gene sequence that encodes FMR1LH, a gene sequence that encodes FMR1LH2, a gene sequence that encodes phuntH, a gene sequence that encodes huntH a gene sequence that encodes andr2LH and a gene that encodes SCAILH. The invention also provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of an RNA transcript that encodes MMD1LH, an RNA transcript that encodes FMR1LH, an RNA transcript that encodes FMR1LH, an RNA transcript that encodes phuntH, an RNA transcript that encodes huntH, an RNA transcript that encodes andr2H and an RNA transcript that encodes SCAILH.

The invention further provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of MMD1LH, FMR1LH, phuntH, huntH, andr2LH and SCAILH.

The above reagents may be a nucleic acid molecule, especially a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO: 7, or SEQ ID NO:10, SEQ ID: 13 and SEQ ID: 16. Alternatively such reagents may be obtained by mutating a nucleic acid molecule having a sequence of SEQ ID NO:l, SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:7, or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:16. Alternatively, the reagent may comprise a nucleic acid sequence that is complementary to the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO:9 or SEQ ID NO:9 SEQ ID NO:10 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 16. The above reagent may alternatively be a protein, especially an antibody, or a fragment of an antibody.

The invention further pertains to a method of treating Huntington's disease or myotonic dystrophy or spinobulbar atrophy or spinocerebella ataxia or fragile x syndrome which comprises providing to an individual, in need of such treatment, an effective amount of an inhibitor of the MMD1L gene, the FMR1L gene, the huntL gene, the andr2L gene and the SCA1L gene.

The inhibitor may be a protein, especially an antibody, or fragment thereof, or may be a nucleic acid molecule. DESCRIPTION OF THE PREFERRED EMBODIMENTS:

Huntington's disease affects .001% of most European populations. It is a progressive and fatal neurological disease which starts in humans around the age of 40, but sometimes, although rarely, occurs in its most serious form in juveniles. There is no cure for the disease. Nothing is known about the biochemical cause of the disease.

The present invention, however, relates to the discovery of a novel gene located on the antisense strand of the untingtin gene, which encode a tachykinin related neuropeptide phunth, and its amidated form (activated) hunth. Hunth administered to rats causes lesions in the striatum and interferes with oxidative phosphorylation in rat brain mitochrondia. Hunth is expressed in brains of Huntington's disease victims but not in brains of normal humans and appears to act through cognate receptors located on the surface of neurons in the putamen, caudate nucleus and selected regions of the cortex in human brains. Other sites of action of huntH are the key glycolytic enzymes phosphofructokinase, and pyruvate dehydrogenase where it intereferes with the breakdown of pyruvate causing this substrate to be converted eventually into lactic acid. Acting in glucagon-like fashion, huntH iniatiates glycogenolysis in specific neurons causing accumulation of glucose-6-phosphate which, because of the absence of glucose-6-phosphatase in the affected cells, causes activation of glycogen synthase and inhibition of glycogen phosphorylase complex. The result is that nearly all glucose entering the cells is converted to glycogen.

Some glucose in affected neurons is processed through gluconeogenesis, but the activities of hunth mentioned above, steers this process to the formation of lactic acid which cannot be reconverted into glucose in these neurons. Hunth initiates and maintains the neuropathological symptoms of Huntington's disease by promoting glucose starvation in selected neurons in the presense of adequate levels of glucose entering these neurons. The invention provides methods through which huntL gene can be inactivated to thereby provide a cure and treatment for Huntington's disease. The present invention provides the sequence of this molecules, as well as that of the proteins encoded by it. These molecules may be used in the diagnosis, prediction and treatment of Huntington's disease. Similar molecules related to myotonic dystrophy, spinocerebellar ataxia type 1, spino-bulbar atrophy and fragile x syndrome can be used in the prediction and treatment of these diseases.

I. Huntington's Disease and Related Neurological Disorders A. Huntington's Disease

One of the biochemical changes associated with Huntington's Disease is an overproduction of lactic acid coupled with a progressive loss of neurons from specific regions of the brain of Huntington patients. Recent results have led to the conclusion that faulty metabolism in brain cell mitochondria, causes overproduction of lactic acid in specific populations of neurons in Huntington's patients.

The biological activity of huntH causes abnormal utilization of glucose in selected populations of neurons in human brains which leads to all the devastating clinical symptoms of Huntington's disease in humans.

B. Spino-Bulbar Atrophy (Kennedy's Disease)

As indicated, X-linked spinal and bulbar muscular atrophy is an adult onset form of motor neuron disease. The genetic basis of the disease is believed to be unstable trinucleotide (CAG)_n repeat expansion in the 5' coding region of the androgen receptor gene (AR) . Mutation/s in another region of AR appers to be the genetic basis of another disease which results in testicular feminization in affected males, but trinucleotide expansion does not result in the latter condition; futhermore, deletion of AR gene does not lead to spino bulbar atrophy but causes testicular femination. Other mutations in the AR gene predisposes men to breast cancer, but not to spino bulbar atrophy; therefore it is assumed that a gain of an unknown function for the AR gene leads to expression of the disease phenotype.

Andr2L is a secertory, tachykinin related neuropeptide

(Figure 6A) , encoded on the antisense strand of the andr2L gene. The neuropeptide affects a number of systems including, the oxysterol binding protein, potassium channel protein, sodium channel protein, the ryanodine receptor, type 3 sperm binding protein, creatine kinase, nitric oxide synthase and an octopamine related receptor precursor. These systems are all involved in intracellular regulatory activities the disruption of any combination of which, would result it the pathophysiological symptoms of Kennedy's disease. Andr2L is expressed in humans suffering from Kennedy's disease, but has not been detected in normal healthy humans. C. Spinocerebellar Ataxia (SCA 1)

As indicated, spinocerebellar ataxia type 1 (SCA 1) is a progressive neurodegenerative disease characterized by ataxia, ophthalmoparesis and variable degrees of muscle weakness. The genetic cause of SCAl is believed to be unstable expansion of a CAG trinucleotide repeat (CAG)_n in a gene that has been localised to human chromosome 6p. Only the region of the gene in the vicinity of (CAG)n has been sequenced.

The proteins expressed by the gene harbouring the unstable trinucleotide repeat have not been shown to be involved in the expression of the disease phenotype in any of the four other human diseases, involving unstable trinucleotide repeat expansion, characterized to date, but antisense neuropeptides seem to be involved in expression of the phenotype of all four diseases. SCA1L is a ACTH related neuropeptide (Figure 6F) encoded on the antisense strand of the SCA1 gene. The neuropeptide has biochemical properties to be the causative factor of SCA type 1 including a mitochrondial transit sequence, which allows it to enter mitochrondia. It can influence the activity of NTS proto oncogene (a protein involved in neural tissue development) , the GABA receptor (negative neurotransmitter) , the choline transporter (acetylcholine synthesis) and can activate or mimic the intracellular activity of a number of toxins. SCA1L is present in humans suffering from SCA1 but not in normal humans. The biological properties described above, similarity with the other diseases in which the genetic cause is unstable expansion in a trinucleotide repeat, described herein, and the positional relationship on the antisense strand with respect to the trinucleotide repeat in the sense strand strongly indicates that SCA1L and SCAILH are causative factors of SCA1 in humans.

D. Myotonic Dystrophy As indicated, myotonic dystrophy is an autosomal dominant disease affecting about 1:8,000 individuals. The pathophysiological symptoms of the disease includes myotonia, muscle wasting and weakness, heart abnormalities, intellectual impairment, cataracts and testicular atrophy. There is a mild form of the disease which occurs in older people and a very severe congenital form which affect infants.

The genetic basis of the disease is unstable expansion of a CTG trinucleotide repeat in the 3' non coding region of a gene encoding a cAMP dependent protein kinase. MMD1L is encoded on the antisense strand in the 3 ^λnon-coding region of the androgen receptor gene. The novel neuropeptide MMDILH expressed by MMDIL has a glycosylation site, a itochrondial transit sequence and functions as a transcriptional down-regulator. It can influence the activity of muscle glycogen synthase, ATP synthase, β-1 adrenergic receptor, lysine oxidase, the mullerian inhibiting factor precursor, tetrohydrobioterin and ELAN - 1 receptor. MMDILH is expressed in humans suffering from myotonic dystrophy but not in normal, healthy humans.

The combined effect, or combinations of the biological activities of MMDILH can account for the full range of clinical symptoms associated with myotonic dystrophy.

Fragile X syndrome

As indicated fragile x syndrome is the most common cause of inheritable mental retardation. It occurs in about 1:1250 males. The main clinical features of the disease are mental retardation and macroorchidism. The genetic basis for the disease is believed to be unstable expansion of a CGG repeat in the 5'untranslated region of the FMRl gene which promotes methelyation of a CpG island 250 bp upstream of the expanded repeat. This prevents expression of the FMRl protein and causes the fragile X syndrome; however, other factors lead to expression of the fragile x syndrome phenotype in humans: in one example members of a family have a deletion in chromosome x (1.6 kb) which removes both the CpG island and the unstable repeat from the FMRl gene, but all show the full fragile x syndrome phenotype; in another example a single point mutation in FMRl mRNA resulted in the fragile X syndrome in a patient in the absence of CGG expansion; yet in other examples, especially in mosaic patients, FMRl is expressed, perhaps at a lower level than normal, but the fragile x syndrome phenotype appears in these people. FMR1L and FMR1L2 are two genes encoded on the antisense strand upstream of the CpG island on the FMRl gene. The FMRl protein can be amidation at two classical amidation sites located in the c-terminal one third of the protein. Amidation at these sites will lead to the release of two active polypeptides; one comprising 101 amino acids which contains a 20 amino acid active domain (FMRla l) , the other, (FMRlam2) 89 amino acids. FMR1L2 is a thacykinin-related, opiod type neuropeptide (Figure 6E) ; it is probably involved in the early development of neural plasticity. It can interact with (possibly activate) the dopamine D5 receptor in the limbic section of the brain, the acetylcholine receptor and the mylein phospholipoid protein and (possibly inactivate) toxins which interact with brain sodium channel protein. The effects of deletions and mutations in the FMRl gene on the transcription of FMRl mRNA, and the conditions under which the fragile x syndrome phenotype is expressed, suggest that FMRl might transactivate or activate FMR1L2. From the deduced biological activities, it appears that FMR1L2 is necessary for the proper development of some basic mental functions (i.e., functions relating to mental development) within the limbic section of the brain. FMR1L is an ACTH related neuropeptide, by virtue of strong homology to the 20 aa active domain in FMRlaml, it can mimic or block the biological activity of FMRl. Expression of FMR1L is repressed by FMRl mRNA in normal healthy people, but occurs in the absence of FMRl expression or in any circumstance where FMRl transcription is not optimal, e.g., point mutations which may affect RNA conformation or stability. FMR1L can interact with (probably block) the acetylcholine receptor, the steroid sulfatase precursor and modulates collagen biosynthesis. The Fragile X syndrome results from the expression of FMR1LH in the absence of optimal transcription of the FMRl gene. FMR1LH blocks either the expression or activity of FMR1LH2 whose absence leads to the fragile X syndrome phenotype in predisposed humans. The Molecules of the present Invention.

A. Gene Sequences Whose Expression is Repressed by Huntingtin mRNA

Recently a gene of unknown function, designated "huntingtin, " which contains an expanded, unstable trinucleotide repeat, has been implicated as the causative factor in Huntington's disease. Although discovery of the "huntingtin" gene is a major step forward in the search for a cure for the disease, and huntingtin mRNA greatly improves the accuracy and time period of detecting potential Huntington's disease victims, based on all published and communicated results to date, the huntingtin protein, which appears to be widely expressed, is not expected to play any significant role in preventing or treating Huntington's disease.

One aspect of the present invention concerns the recognition that the "huntingtin" gene encodes a neuropeptide (hormone) precursor (phuntH) . PhuntH is converted into an active hormone (huntH) by post translation amidation. HuntL open reading frame begins at the presumed open reading frame ATG of huntingtin, in the opposite orientation, and closes 99 bp 5' to the latter open reading frame, in the upstream 5' region of huntingtin mRNA. Part of the translational-transcription region of huntL overlaps the unstable CAG trinucleotide repeat region of huntingtin. Mutations in the latter trinucleotide repeat region which cause an increase of trinucleotide repeats is the genetic basis of Huntington's disease. The same event leading to expansion of trinucleotide repeats which obviously have a "negative" effect on transcription of huntingtin mRNA appears to have a positive effect on the transcription of huntL mRNA resulting in the differential expression of huntL and phuntH.

The sequence of the 162 bp cDNA that encodes huntL is shown in the embodiment of Figure 1A (SEQ ID N0:1) . The cDNA encodes a novel tachykinin related neuropeptide

(hormone) precursor (phuntH) of 33 amino acids whose sequence is shown in Figure IB (SEQ ID NO:2) . As indicated, phuntH is converted into an active hormone huntH, whose amino acid sequence is shown in Figure IC

(SEQ ID NO:3) .

SEQ ID N0:1 CTGCTGGAAAGGACTTGAGGGACTCGAAGGCCTTCATC AGCTTTTCCAAGGGTCGCC ATG GCG GTC TCC CGC CCG

GCA CGG CAG TCC CCG GAG GCC TCG GGC CGA CTC GCG GCG CCG CTC AGC ACC GGG GCA ATG AAT GGG GCT CTG GGC CGC AGG TAA AAGCAGAA

SEQ ID NO:2 MAVSRPARQSPEASGRLAAPLSTGAMNGALGRR

SEQ ID NO:3 MAVSRPARQSPEASGRLAAPLSTGAMNGAL-NH₂

PhuntH is a basic (pi 12.65), 3.34 kd polypeptide. The structure predicted for phuntH is shown in the embodiment of Figure 1A. It is expected that gly₃₁ will donate an amide to leu₃₀ and the secretion/ activation signal arg₃₂-arg₃₃ will be cleaved between residues 31 and 32 producing a c-terminal amidated active hormone (huntH) . The neuropeptide has a putative protein kinase c phosphorylation site at ser₁₄.

B. Gene Sequences Whose Expression is Repressed by

Myotonic Protein Kinase mRNA

Expanding trinucleotide repeats appear to be the genetic basis of Huntington's Disease, as well as other important human diseases. A DNA sequence containing a variable number of trinucleotide (CTG) repeats in the 3' end of a gene encoding a cAMP-dependent protein kinase appears to be the genetic basis of Myotonic Dystrophy.

One aspect of the present invention concerns the recognition of a 126 bp cDNA (MMDIL) , whose sequence is shown in Figure 2A (SEQ ID NO:4) which encodes a novel 41 amino acid neuropeptide (MMDILH) whose sequence is shown in Figure 2B (SEQ ID NO:5) . The coding region of MMDIL which is 100% homologous to nucleotides 10,273 -10,398 of the antisense strand of the myotonic protein kinase, starts 279 bp upstream from the GTC expansion region. This location places the trinucleotide expansion area within the regulatory regions of MMDIL gene. The organization of the regulatory region of the MMDIL gene (MMDlLreg)is shown in Figure 2C (SEQ ID NO:6A-E) . It contains one promoter element ("TATA box") indicated with a "p", and four "GC boxes" (indicated by gc) . The regulatory region is divided into two sub-regions one from "-142 to - 222" and the other from "-439 - 649". The trinucleotide repeat region is located betwween the two regulatory sub regions. The multiple promoter system indicates that MMDIL gene may be expressed in a tissue specific manner which require the use of different promoters.

SEQ ID NO:4 ATG CAG CCC AGG GCG GCG GCA CGA GAC AGA ACA

ACG GCG AAC AGG AGC AGG GAA AGC GCC TCC GAT AGG CCA GGC CTA GGG ACC TGC GGG GAG AGG GCG AGG TCA ACA CCC GGC ATG GGC CTC TGA

SEQ ID NO:5 MQPRAAARDRTTAMRSSESA

SSRPGLGTCGGRARSTPGMGL

SEQ ID NO:6 AGGTCAATAAATATCCAAA..//...GCGGGCGGAGCCGG..

GATAGGTGGGGTG....// GGGGGCGGGCCCGG

GGTGGGCGCGGCTT

C. Gene Sequences whose Expression is Repressed by FMRl mRNA.

A DNA sequence containing a variable number of trinucleotide CGG repeats in the 5' noncoding region of the FMRl gene appear to be the genetic basis of Fragile X Syndrome. Another aspect of this invention is the discovery of a 93 bp cDNA molecule (FMRIL) , whose sequence is shown in Figure 3A (SEQ ID NO:7) . FMRIL encodes a novel 30 amino acid ACTH related neuropeptide (FMR1LH) whose sequence is shown in Figure 3B and (SEQ ID NO:8) . FMR1LH is very closely related to ACTH, the other antisense polypeptides described herein, and tachykinins as shown in the embodiment of Figure 6 and 6D) . The coding region of FMRIL which is 100% homologous to nucleotides 2356-2448 on the antisense strand of the FMR-1 gene, opens 261 bp upstream from the CGG repeat region. FMRIL regulatory region (FMRlLreg) in the 5' end of the gene, shown in Figure 3C (SEQ ID N0:9), is located within the protein coding region of FMRl gene. FmrlL protein coding region opens a 11 nucleotides from the CpG island. The CGG repeat lies within the regulatory region (but on the opposite strand) of the FMRIL gene. The location of FMRIL with respect to FMRl means that transcription of FMRIL is normally inhibited by FMRl mRNA. FMRlLreg contains four promoter elements, "TATA" boxes, (indicated by "p" in figure) , two TATA boxes contain a pair of tandem promoter elements each, while two have single elements. All the promoter elements are correlated with "cap sites", (indicated by "cp" in figure) . Like in the case of MMDIL, FMRIL may be subjected to tissue specific ecpxession programmed by different promoters. SEQ ID NO:7 ATG CGC GCT GCT GGG AAC CGG CCG GGG TGC CGG GTC GAA AGA CAG ACG CGC GGG CCG GGC GTG CGC GGG CTT GGT GGA GGG CGG GAA GGC TGA

SEQ ID NO:8 MRAAGNRPGCRVERQTRGPGVRGLGGGREG

SEQ ID NO:9 GTGTAACTTTAAAAAGCACAGTAAACCCATGAATGCCTATATAAA

GCCTCAAO^TTC-AGTCCACTGCAACACCACAAATTCAAAGG7AA

TGTGGTO^GTTCTTAGGGGCACΑAAAATATTTTCGCTCAGTTAA

AAAAAAAAGCCCTAGTAATAATATACTCTCAGTTAATTACCTGAA GTTTCATGGCATATATTTAGGTCTTTGGAAACTTAAAC-ACΑCΑCA

CΑCΑATTTTTAAGTAGCΑGCGCIOCTAATGGTTTTAGACGCTGAA GCATGTGCATTCCTGAATTTACCCCGTTT

A second part of this aspect of the invention is the discovery of a 159 bp cDNA molecule, FMR1L2, whose sequence is shown in Figure 3D (SEQ ID NO:10) . FMR1L2 encodes a novel 52 amino acid protein " FMR1L2H" which is shown in Figure 3E (SEQ ID NO:11) . The protein has sequence features of a HC:HC or HC:CC zinc finger, DNA binding protein. The orf which opens 478 down stream from the stop translation codon of FMRIL is 100% homologous to nucleotides 1661 - 1770 on the antisense strand of the

FMRl gene; the coding region, and 5'regulatory region of

FMR1L2 (FMRlL2reg) which includes two tandem TATA box elements (indicated with "p" in the figure) with a correlated cap site (indicated with "cp" in the figure) and an upstream GC box shown in Figure 3F (SEQ ID NO:12) ,

(indicated by "gc" in the figure) are located entirely within the 3'nontranslated region of FMRIL and hence its expression might be effectively blocked by expression of FMRIL.

SEQ ID NO:10 ATG CGT CCT GTC CAC AGG GGC CGA TGC ACC TCC TTG CAA CCC TTT ACA TTC CAC TGT GAA ACA AAC CTC AAC TTT TTC TTA TTC CTG TTT TTA CAC CGT GCT TAT AGC TGC CTT AAT CCA TGT CCC CTT CGG GAT GCT GGT ATC CAA CTG AGA AGT TGA

SEQ ID NO:11 MRPVHRGRCTSLQPFTFHCETNLNFFLFLFLHRAY SCLNPCPLRDAGIQLRS

SEQ ID NO:12 TTTCACTTGACTATATATTTTTTAAAAATTGTGTTAAGCACTT GGAGGTTCATTTCTGCCCCTACTGTATGTGCACCCTGTGCCAG

AGGGTGGGGTGAACACGTGTGTAGCAGTC

D. Gene Sequences Whose Expression is Repressed by the Androgen Receptor Protein (AR) mRNA.

Unstable expansion of a CAG trinucleotide repeat in the 3'untranslated region of the AR gene is thought to be the genetic basis of spino bulbar atrophy. The present invention also involves a gene "andr2L" antisense to AR. The cDNA, Figure 4A (SEQ ID NO:13) is 100 % homologous to a region on exon #1 on the leftward reading strand of AR ^vorf starting 80 bp 3' to the CAG repeat and extending downstream to nucleotide 312. The "andr2LH" protein Figure 4B (SEQ ID NO:14) encoded by andr2L is a tachykinin related neuropeptide (see Figure 6F) . Andr2LH has a potential mitochondrial transit secjuence which will allow entrance into mitochondria, and a potential cleavage site between amino .acid residues T53-Q54 which can split the neuropeptide into two active peptides. Protein kinase c sites are present at residues S2, S7 . A casein kinase site is present at residues S61. The organisation of the andr2L gene is shown in Figure 4C. The 5'regulatory region contains a "ccaat box" promoter element (SEQ ID NO:15) which programs the transcription of andr2L mRNA from several cap sites well placed with respect to the ccaat box..

SEQ ID NO:13 ATG TCT TTA AGG TCA GCG GAG CAG CTG CTT

AAG CCG GGG AAA GTG GGG CCC AGC AGG GAC

AAC GTG GAT GGG GCA GCT GAG TCA TCC TCG TCC GGA GGT GCT GGC AGC TGC TGC GGC AGC

CCC TTG CTG GCG GCC ACG GCG GCT CCA GGC

TCT GGG ACG CAA CCT CTC TCG GGG TGG CAC

TCC AGG GCC GAC TGC GGC TGT GAA GGT TGC

TGT TCC TCA TCC AGG ACC AGG TAG

SEQ ID NO:14 MSLRSAEQLLKPGKVGPSRDNVDGAAES

SSSGGAGSCCGSPLLAATAAPGSGTQPL SGWHSRADCGCEGCCSSSRTR

SEQ ID NO: 15 TTCGGCCAATGG

E. Gene Sequences Whose Expression is Repressed by SCA1 mRNA

Unstable expansion of a trinucleotide CAG repeat also appears to be the genetic cause of spinocerebellar ataxia. The present invention also relates to the reconstruction of a cDNA "SCA1L" Figure 5A (SEQ ID NO:16) which is 100 % homologous to a region of the anti sense strand of the SCA1 gene. The homology which starts 23 bp 3' of nCGA spans 170 bp. The 56 amino acid protein "SCAILp" encoded by SCA1L, Figure 5B (SEQ ID NO:17) appears to be a neuropeptide by similarity to the other antisense polypeptides already described (see Figure 6) . The protein has a casein kinase site at residues 2-3. It also contains a mitochondrial transit sequence which allow it to enter and influence mitochondrial metabolic activity.

SEQ ID NO:16 ATG ACG ACC TGG GAG GGG GGC CCC AGG GTG AGC GTG TGT GGG ATC GTC TGG TGG GGG TGG AGG TGG ACG GGG ATG GCC GGA GGA GAG GCG GTC CGG CCG GTG TTC TGC GGA GAA CTG GAA ATG TGG ACG TAC TGG TTC TGC TGG GCT GGT GGG GGG GAC CCG GGG TGA

SEQ ID NO:17 MTTWEGGPRVSVCGI VW GWRWTGMAG

GEAVRPVFCGELEM TYWFCWAGGGDPG

III. The Physiological Role of the Molecules of the Present Invention A. Role of Gene Sequences whose Expressiopn is

Repressed by the "Huntington" mRNA

It is known that cells are regulated by many hormones and hormone-like factors through reaction of the hormone/factor with cognate receptors on the cell surface. Therefore the cell membrane is, in general, the site of action of polypeptide hormones. Cellular communication through hormones is extremely specific and there is a large number of hormones in the body with new ones constantly being discovered. Many hormones cause the release of other substances, some of which may be other hormones. The property of stimulating the release of other factors explains how a single hormone causes a wide variety of effects in the body and how it becomes involved in cascade effects among many types of cells. Some actions of hormone-receptor complexes in cell membranes increase the flow of ions into cells, especially calcium, others activate or suppress activities of enzymes in contact with the receptor or transducer with which the receptor reacts.

HuntH resembles, but does not have the complete address sequence which is characteristic of tachykinins, a group of active neuropeptides, that include among others, substance P, neurokinin and neuropeptide Y. These are active peptides which excite neurons, evoke behavioral responses, are potent vasodilators and secreatagogues and contact directly or indirectly) many smooth muscles. With respect to the latter activity, huntH stimulates glycogenolysis by interacting with cognate receptors on the plasma membranes of neurons located in the human brain striatum, blocks gluconeogenes by interacting with phosphofructokinase and causes the accumulation of lactic acid in affected neurons by inhibiting the activity of a key component of the pyruvate dehydrogenase enzyme complex. All the neuropathological symptoms of Huntington's disease can be explained by the hormone activities outlined above.

Cell death in Huntington's disease is confined to specific neurons in particular regions of the brain. This is consistent with the specific, hormone-receptor reaction in cells containing a cognate receptor for huntH. The latter interaction can also explain the observation that neurons which die in the brains of Huntington' s disease victims contain a high concentration of lactic acid. The role of faulty glucose metabolism in Huntington's disease is consistient with recent results from investigations using image technology, which show that the first signals of Huntington's disease in predisposed humans, before any clinical symtoms are discernible, is disruption in glucose metobalism.

The antisense relationship between huntL and huntingtin indicates that expression of phuntH is repressed by wild type huntingtin; therefore, the neuropeptide cannot be expressed in brains of normal humans; however, it can be expressed in brains of Huntington's disease victims when trinucleotide expansion in huntingtin gene diminishes/abolishes repressor function of huntinting mRNA. Therefore, huntL and phuntH and huntH, described herein, are ideal targets against which anti- nucleotide, or anti-peptide or immunological reagents which can traverse the blood brain barrier, can be directed against. Such reagents will prevent the onset and/or progression of Huntington's disease in those people who are genetically predisposed to become victims. Since huntL, phuntH and huntH are related only to the neuropathological symptoms in Huntington's disease patients, side effect-free, therapeutic reagents directed against these substances can be readily developed.

C. Role of Gene Sequences Whose Expression is Repressed by the Myotonic Protein Kinase mRNA

The known biochemical activity of the Myotin protein kinase cannot account for the variety of clinical symptoms including, myotonia, muscle wasting and weakness, heart abnormalities, intellectual impairment, testicular atrophy and cataracts, associated with myotonic dystrophy. Like huntH, MMDILH is evolutionary related to tachykinins (figure 6&6B) . The neuropeptide has sites for: phosphorylation (2 casein kinase II and 1 kinase C) , N- glycosylation (1) and mitochondrial membrane transit which can confers versatility of biological activity to the neuropeptide. In addition 5'regulatory region of MMDIL gene harbours five promoter elements which can be used for tissue specific expression of MMDILH. The neuropeptide appears to be a transcriptional inhibitor. The cDNA encoding MMDILH has been cloned from an autopsied diseased heart, but has not been found in tissue from normal humans. The biochemical properties of MMDILH viz.,inhibiting: (i) glycogen synthase which is required to produce glucose from stored glycogen reserves for rapid mobilization in skeletal muscle during periods of stressful activtity; (ii) ATP synthase which is required especially for heart muscle mitochrondial oxidative phosphorylation; (iii) the β-adrenergic receptor which is transduced by the neurotransmitters norepinephrine and epinephrine. The latter hormones are involved in synaptic transmission and affects the physiological function of most organs in the body including cardaic and skeletal muscle, very rapidly. The β-adrenergic receptor is the cognate receptor for epinephrine which stimulates glycogen degradation in skeletal muscle and the cascade of events, including intracellular changes in Ca²⁺ levels. Norepinephrine is also involved in DOPA and melanin formation. DOPA is required for synthesis of dopamine. Melanin is stored in melanocytes which are specifically located in the retina and ciliary body of the eye and the substantia negra of the brain; (iv) mύllerian inhibiting factor which is produced in sertoli cells in the testiε and stimulates regression of the mύllerian tublues, which in the developing female form ovaries and fallopian tubes,

(v) ELAN-1 endothelial cell leukocyte adhesion molecule is involved in inflammation, thrombosis and vasculitis.

Negative regulation by MMDILH of all or a combination of the physiological systems described above, will lead to some or all of the clinical symptoms characteristic of myotonic dystrophy. Since MMDILH is only expressed in the diseased condition and not in healthy humans it is the perfect target against which anti-nucleotide, anti-peptide or immunological reagents can be directed to prevent and cure myotonic dystrophy in humans, without any side effects.

C. The Role of Gene sequences whose Expression is Repressed by FMRl mRNA. The fragile X mental retardation syndrome is believed to be caused by the elongation of a small target DNA fragment, containing (CGG)_n number of repeats in the 5' untranslated region of the FMR-1 gene which results in methylation of a CpG island 250bp upstream from the repeat and the subsequent shut down of transcription of the FMRl mRNA, which it is believed, leads directly to the main clinical characteristics of fragile x syndrome, i.e., mental retardation and testicular atrophy in predisposed humans. This may not be the case, however, since there are several examples of the fragile x syndrome phenotype in humans in the presence of FMRl mRMA transcription and FMRl protein expression.

FMRl protein is related to MER1 type RNA binding proteins. It interacts with the small ribonuclear protein (srnp) whose function is to facilitate the transport of proteins through the endoplasmic reticulum, and also appears to have sperm chromatin decondensing activity. The expressed protein and can undergo post translational amidation at two sites in the c terminal one third of the protein to form three seperate molecules including, a c-terminal polypeptide of 101 amino acids (101 aa) which harbours a 20 aa domain with high homology to FMR1LH (described below) , and an 89 aa polypeptide; these appear to have biological activities unrelated to fragile x syndrome.

FMR1LH is an ACTH type neuropeptide distantly related to tachykinins. It is involved inactivation/activation of the acetycholine receptor protein, collagen alpha 1&2, the steroid sulphatase protein precursor and inhibition of FMR1LH2 which is described below. FMR1LH is expressed in patients with fragile x syndrome but not in normal, healthy humans. Its expression is repressed by FMRl mRNA.

FMR1LH2 is a tachykinin related opiod type neuropeptide.

It appears to activate the dopamine D5 receptor in the limbic section of the human brain, the muscarinic acetylcholine receptor, the myelin proteolipid protein, the anti xenobiotic cytochrome system, and systems which protect humans from the harmful action of endogenous toxins and proteases.

FMR1LH is present in fragile x syndrome patients and FRMl is not, the opposite holds true for normal, healthy humans. Because the transcription regulatory region of FMRIL lies within the coding section of FRMl (on the opposite strand) it cannot be transcribed in the presence of FMRl mRNA in the correct conformational form; however, any event which affects the conformation of FMRl mRNA permits the expression of FMRIL in variable amounts, and in proportion to the severity of the effect of ^xthe event'on FMRl mRNA expression. Also, in some examples FMRl and FMR1LH are missing due to a large deletion in the FMRl gene, but the fragile x phenotype is expressed in affected patients. This latter situation suggest that another factor, a positive regulator, which is required for the preventing the fragile x syndrome in humans is also removed by the deletion. This factor is FRM1LH2, which is probably reςpiired during early development for the establishment of neural plasticity. When it is allowed to be expressed, FMR1LH prevents the expression of FRM1LH2 which lies within it's 3^xnon coding region. Because FMRIL and FMR1LH are only expressed under diseased conditions they are ideal targets for therapeutic intervention to prevent and stop fragile x syndrome in pedisposed humans.

D. Role of Gene Sequences Whose Expression is Repressed by the AR mRNA Although trinucleotide expansion in the 5'region of the androgen receptor gene is probably the genetic cause of spino bulbar atrophy, the androgen receptor protein does not play a significant role, if any, in the pathophysiological symptoms of the disease which include: proximal muscle tremor, cramps and fasiculations, weakness, atrophy, gynecomastia (in males) , impaired spermatogenis and dysphagia,- therefore it is likely that the clinical symptoms must be caused by another factor. The andr2p protein, which is antisense to the region of exon 1 in the AR gene which contains a transcriptional modulatory domain, has the required biological properties to be the causative factor of the disease. It has features of transcriptional inhibitors and by similarity it appears to act on sterols and steroid type receptors; it is closer related to tachykinins than any of the other antisense neuropeptideε described in this invention (Figure 6F) . It can influence the expression and activity of: ERR2 type steroid receptors; potassium channel and sodium channel proteins; nitric oxide synthase which affects neurotransmission and metabolism in brain and skeletal muscle; the ryanodine receptor which is involved in CA²⁺ utilization and control in skeletal muscle; the protein 3-type sperm binding protein which inactivates sperms; the oxysterol binding protein which is involved in regulation of sterol metabolism; an octapine type receptor which affects neuromuscular and neurotransmitter function; creatine kinase which plays a control role in energy transduction in skeletal muscle, heart, brain and spermatozoa; 6- phosphofructosekinase, which is a key control step in glycolysis; glycine dehydrogenase, which plays a unique role in nerve transmission; and the interleukin 4 receptor precursor.

Andr2H is not expressed in healthy humans due to antisense inhibition by wild type androgen receptor mRNA; however, when trinucleotide expansion is 'induced' transcription of the AR mRNA is affected in proportion to the size of the expansion (which is probably dependant on inducer level) and expression of andr2H occurs at a corresponding level. Andr2L is transcribed from the single promoter in the 5'regulatory region. Andr2L is not expressed when AR is deleted since it is localised on the same region of the x-chromosome as is AR and is 'co-deleted'; as can be expected spino bulbar atrophy does not occur in humans when the AR gene is deleted.

Since Andr2L and andr2LH are not expressed in normal, healthy humans they are ideal molecules to target for developing diagnostics and side effect free therapeutics to prevent, treat and cure the symptoms of Kennedy's disease in humans.

E. Role of Gene Sequences whose Expression is Repressed by SCAl mRNA.

Spinocerebellar Ataxia Type 1 (SCAl) is characterised by ataxia, ophthalmoparesis and variable degree of muscle weakness. Although the gene has been sequenced the orf of the SCAl protein is not yet known. Expansion of a CAG trinucleotide repeat which is believed to be the genetic cause of the disease. By comparison with the four other diseases which involve expansion of unstable trinucleotite repeats, the location of the antisense mRNA (SCA1L) with respect to the CAG repeat, the close relationship of the expressed neuropeptide "SCAILH" with the other antisense neuropeptides of this invention and with ACTH, and the biochemical characteristics of the antisense protein, indicates that SCAILH is the putative biochemical causative factor of SCAl. SCAILH contains a mitochrondial transit sequence, can be highly phosphorylated and can mimic or influence the intracellular activity of the following proteins: WNTS proto oncogene, a protein involved in neural tissue development; the GABA receptor, a negative regulator of neurotransmitter activity; the choline transporter, a protein involved in acetylcholine synthesis and certain intracellular toxins, e.g., the cytolytic toxin aerolysin . SCA1L is not expressed in normal humans because of the antisense relationship with wild type SCAl mRNA; however, when unstable trinucleotide expansion disrupts normal transcription of the SCAl mRNA, SCAILH is expressed and can cause the SCAl phenotype in affected humans.

SCA1L and SCAILH are ideal targets against which diagnostics and side effect free therapeutics for detection, prevention and cure of spinocerebellar ataxia type 1, in humans, can be rapidly developed.

IV. The Uses of the Molecules of the Present Invention

The elucidation of the significance of the above- described gene sequences and gene products in the etiology of Huntington'ε avd related diseases provides improved means for diagnosing the presence and clinical grade of Huntington's disease and related disorders. Moreover, it provides an improved means for predicting whether an aεymptomatic individual iε predisposed to such conditions. It further provides a meanε for treating εuch conditionε. In particular, any of the proteins described above, or mutants thereof, may be uεed in the treatment of, or in the development of reagentε for the treatment of, these diseaseε and conditionε.

A. Diagnostic Uses

Since the gene productε of the present invention are not expresεed by normal cellε, the detection of theεe moleculeε in a tiεsue or fluid sample -- εuch aε a biopεy εample, or a blood or lymph fluid sample -- is indicative of the presence of Huntington's diεeaεe and related diεorders in a patient.

The detection of these molecules may be done by any of a variety of methods. In one embodiment, antibodieε are employed that are capable of binding to the productε of MMDILH, FMRlLHp, phuntH and/or huntH, or SCAILH or andr2LH gene(ε), and the preεence of εuch moleculeε iε determined via an immunoaεεay. A large number of εuitable immunoaεεay formatε have been deεcribed (Yolken, R.H., Rev. Infect. Pis. 4:35 (1982); Collins, W.P., In: Alternative Immunoasεayε. John Wiley & Sonε, NY (1985) ; Ngo, T.T. et al. , In: Enzyme Mediated Immunoaεεay, Plenum Press, NY (1985) ; incorporated by reference herein.

Suitable antibodies can be either polyclonal or monoclonal, of either a εpecieε homologouε 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 antibodieε, chimeric antibodies, etc. may be employed. Such antibodies can be obtained using conventional methods with the gene productε of MMDILH, FMR1LH, huntH, SCAILH or andr2LH gene(s) as an antigen. Such gene productε are preferably obtained through the expreεεion of the gene εequenceε deεcribed herein. The εimpleεt immunoaεsay involves merely incubating an antibody capable of binding a molecule of the present invention with a sample suεpected to contain the target molecule -- the protein product of, MMDIL, FMRIL, huntL, SCA1L or andr2L gene(ε) . The preεence of the target molecule iε determined by the preεence, and proportional to the concentration, of any antibody bound to the target molecule. In order to facilitate the εeparation 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 immunoaεεayε, the concentration of the target molecule iε determined by binding the antibody to a εupport, and then permitting the εupport to be in contact with a sample suεpected to contain the target molecule. Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of wayε. For example, the εupport can be incubated in the presence of a labelled, second antibody that is capable of binding to a εecond epitope of the target molecule. Immobilization of the labelled antibody on the εupport thus requires the presence of the target, and is proportional to the concentration of the target in the sample. In an alternative assay, the target is incubated with the sample and with a known amount of labelled target. The preεence of any target moleculeε in the εample competeε with the labelled target moleculeε for antibody binding sites. Thus, the amount of labelled target molecules that are able to bind the antibody iε inversely proportional to the concentration of target molecule in the εample.

Aε indicated above, immunoaεsay formats may employ labelled antibodies to facilitate detection. Radioisotopic immunoaεεayε ("RIAs") have the advantages of simplicity, senεitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kineticε. Such aεεayε εuffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require εpecial handling and diεpoεal, and entail the uεe of complex and expenεive analytical equipment. RIAε are deεcribed in Laboratory Techniqueε and Biochemiεtry in Molecular Biology, by Work, T.S., et al. , North Holland Publiεhing Company, NY (1978) , with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniς∑ues" by Chard, T., incorporated by reference herein.

Enzyme-based immunoasεay formats (ELISAs) have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, εuch that a large number of detection strategieε colorimetric, pH, gaε evolution, etc. -- can be uεed 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 & Sonε, NY (1988), incorporated by reference herein. For theεe reasons, enzyme labels are particularly preferred.

No single enzyme is ideal for use aε a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular assay system. Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of εubεtrate molecules converted to product per enzyme site per unit of time) , purity of the enzyme preparation, εenεitivity of detection of itε product, ease and speed of detection of the enzyme reaction, absence of interfering factorε or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and coεt of the enzyme and its conjugate, and the like. Examples of suitable enzymes include peroxidase, acetylcholine eεteraεe, alpha-glycerol phoεphate dehydrogenaεe, alkaline phoεphataεe, aεparaginaεe, β- galactosidase, catalaεe, delta-5-εteroid iεomerase, glucose oxidase, glucose-6-phosphate dehydrogenaεe, glucoamylaεe, glycoamylaεe, luciferaεe, malate dehydrogenase, peroxidase, ribonuclease, εtaphylococcal nucleaεe, trioεe phoεphate isomerase, urease, yeaεt- alcohol dehydrogenaεe, etc. Peroxidaεe and ureaεe are among the more preferred enzyme labelε, particularly becauεe of chromogenic pH indicatorε which make itε activity readily viεible to the naked eye.

In lieu of εuch enzyme labelε, radioiεotopic, chemilumineεcent or fluoreεcent labelε may be employed. Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, "Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵Eu, ⁹⁰Y, ⁶Cu, ²¹⁷Ci, ¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. Examples of suitable chemiluminescent labelε include a luminal label, an isolu inal label, an aromatic acridinium eεter label, an imidazole label, an acridinium salt label, an oxalate eεter label, a luciferin label, an aequorin label, etc. Examples of suitable fluorescent labels include a fluorescein 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 immunoassay formats, the presence of MMDIL, FMRIL, huntL, SCAIL or andr2L genes, in a cell can be determined by any means capable of detecting these genes or their mRNA. Thus, molecules comprising nucleic acid probeε capable of hybridizing to, MMDIL, FMRIL, huntL, SCAIL or andr2L, εequenceε may be uεed in the diagnoεis of Huntington's Diseaεe or myotonic dyεtrophy. Aε 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 sequences discloεed herein can be uεed to define a probe; the general requirement for εuch use being merely that the nucleic acid molecule be εufficiently long (generally 10 or more nucleotideε in length) that it poεεeεεeε the capacity to form εtable hybridization productε with the target molecule. Any of a wide variety of labels (εee above) may be uεed to label nucleic acidε: enzyme labelε (Kourilεky et al.. U.S. Patent 4,581,333), radioiεotopic labelε (Falkow et al.. U.S. Patent 4,358,535; Berninger, U.S. Patent 4,446,237), fluoreεcent labelε (Albarella et al. , EP 144914) , chemical labelε (Sheldon III et al.. U.S. Patent 4,582,789; Albarella et al.. U.S. Patent 4,563,417), modified bases (Miyoshi et al.. EP 119448), etc.

Such nucleic acid based aεεays may use either DNA or RNA to detect, MMDIL, FMRIL, huntL, SCAIL or andr2L mRNA. In one embodiment, the assays may be performed on RNA that has been extracted from brain, muscle, heart or testicular cellε. Alternatively, and more preferably, the aεsays may be done in situ on biopsied tissue. Where the concentration of MMDIL, FMRIL, huntL, SCAIL or andr2L 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. JLl:263-273 (1986); Saiki, R.K., et al.. Bio/Technology 2:1008-1012 (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 systems (Kwoh, D. et al. , Proc. Natl. Acad. Sci. (U.S.A.)

16 11 3 (1989); Gingeras, T.R. et al. , PCT appl. WO

88/10315 (priority: US Patent applicationε εerial noε. 064,141 and 202,978); Davey, C. et al. (European Patent Application Publication no. 329,822), etc.

In yet another embodiment, the diagnoεis of the expression of MMDIL, FMRIL, huntL, SCAIL or andr2L mRNA is performed using a ribozyme produced from nucleic acid molecules having a sequence of such RNA molecule(s) .

B. Prognostic Uses

The present invention additionally provides a capacity to predict whether an individual is at riεk for Huntington'ε diεease and related disorderε. Thuε, any of the above-deεcribed assays may be performed on an asymptomatic individual in order to assess that individual's predisposition to Huntington's disease and such other conditionε.

C. Therapeutic Uses

Significantly, the present invention provides a means for treating Huntington's Disease and related disorders.

Such treatment may be either "prophylactic" or

"therapeutic." A prophylactic treatment iε one that iε provided in advance of any εymptom of a diεease in order to prevent or attenuate any subsequent onset of that diεeaεe. A therapeutic treatment is one that is provided in response to the onset of a symptom of a disease, and serves to attenuate an actual symptom of that disease. In one embodiment, such treatment is provided by administering to a patient in need of such 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 MMDIL, FMRIL, huntL, SCAIL or andr2L genes. As used herein, an effective amount iε 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 fragmentε of either) may be administered. More preferably, and especially for chronic adminiεtration, the use of non- immunogenic antibodies is preferred. Such molecules can be pseudo-homologouε (i.e. produced by a non-human εpecieε, but altered to a form that iε immunologically indiεtinct from human antibodieε) . Exampleε of εuch pseudo-homologous moleculeε include "humanized" (i.e. non- immunogenic in a human) prepared by recombinant or other technology. Such antibodieε are the ecjuivalentε of the monoclonal and polyclonal antibodies, but are lesε 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. ______________ Science

240:1041-1043 (1988); Liu, A.Y. et al. ■ Proc. Natl. Acad. Sci. USA £4:3439-3443 (1987); Liu, A.Y. et al.. J_^ Immunol. 139:3521-3526 (1987) ; Sun, L.K. et al. , Proc. Natl. Acad. Sci. USA £4:214-218 (1987) ; Nishimura, Y. et al.. Cane. Reε. 47:999-1005 (1987); Wood, CR. et al. , Nature 324:446-449 (1985)); Shaw et al.. J. Natl.Cancer Inεt. .8(1:1553-1559 (1988); all of which referenceε 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.. BioTechniqueε 4_:214 (1986) ; which referenceε are incorporated herein by reference) .

Suitable "humanized" antibodies can alternatively be produced by CDR or CEA εubεtitution (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 mutated MMDILH, FMR1LH,FMR1LH2, phuntH and/or huntH, or the SCAILH or andr2LH mutant gene products that can complex with and significantly inactivate their corresponding normal gene products present in a tumor cell. In one sub-embodiment, such mutated protein molecules may be adminiεtered to a patient. Alternatively, nucleic acid expreεsing such molecules may be adminiεtered.

In yet another embodiment, "antiεenεe" or "triplex" nucleic acid moleculeε may be uεed to provide the deεired therapy. As uεed herein, an "antiεense oligonucleotide" is a nucleic acid (either DNA or RNA) whose sequence is complementary to the sequence of at least part of MMDIL, FMRIL, huntL, SCAIL or andr2L, protein-encoding gene sequenceε described herein, such that it is capable of binding to, or hybridizing with, an endogenouε mRNA molecule, and can thereby impair (i.e. attenuate or prevent) the translation of MMDIL, FMRIL, huntL, SCAIL or andr2L mRNA. A "triplex" molecule iε a nucleic acid molecule that iε capable of binding to double-stranded DNA in a manner sufficient to impair its transcription.

To act aε a triplex oligonucleotide, the nucleic acid molecule must be capable of binding to MMDIL, FMRIL, huntL, SCAIL or andr2L genes of the double-stranded DNA genome in a manner sufficient to impair the tranεcription of that gene. Triplex oligonucleotideε are disclosed by Hogan, U.S. Patent 5,176,996 and by Varma et al.. U.S. Patent 5,175,266. To act as an antisense oligonucleotide, the nucleic acid molecule muεt be capable of binding to or hybridizing with that portion of MMDIL, FMRIL, huntL, SCAIL, andr2L mRNA molecule which mediateε the translation 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, oεt preferably, about 15-24 nucleotides in length.

Thus, in one embodiment of this invention, an antiεenεe oligonucleotide that iε deεigned to εpecifically block tranεcription from MMDlLreg, FMRlLreg and andr2Lreg or tranεlation of MMDIL, FMRIL, huntL, SCAIL or andr2L mRNA tranεcript can be uεed to impair the expression of MMDIL, FMRIL, huntL, SCAIL or andr2L gene(s) in a cell, and thereby provide a treatment for Huntington'ε diεeaεe and related disorders.

In general, the antisense oligomer is prepared in accordance with the nucleotide sequence of MMDIL, FMRIL, huntL, SCAIL or andr2L gene(s) as reported herein.

The sequence of the antisense oligonucleotide may contain one or more insertions, substitutions, or deletions of one or more nucleotides provided that the resulting oligonucleotide is capable of binding to or O I

hybridizing with the above-described translation locus of either MMDIL, FMRIL, huntL, SCAIL or andr2L gene(s), or their RNA or promoter element of MMDlLreg, FMRlLreg or andr2Lreg. Any means known in the art to synthesize the antisense oligonucleotides of the preεent invention may be uεed (Zamechik et al.. Proc. Natl. Acad. Sci. (U.S.A.) 11:4143 (1986); Goodchild et al.. Proc. Natl. Acad. Sci. (U.S.A.) 15_:5507 (1988); Wickεtrom et al.. Proc. Natl. Acad. Sci. (U.S.A.) 15:1028; Holt, J.T. et al.. Molec. Cell. Biol. 8:963 (1988); Gerwirtz, A.M. et al.. Science 241:1303 (1988); Anfossi, G., et al.. Proc. Natl. Acad. Sci. (U.S.A.) 86:3379 (1989); Becker, D., et al.. EMBO J. 1:3679 (1989); all of which references are incorporated herein by reference) . Automated nucleic acid synthesizers may be employed for this purpoεe. In addition, desired nucleotides of any sequence can be obtained from any commercial εupplier of such custom molecules.

Most preferably, the antiεense oligonucleotides of the present invention may be prepared using solid phase "phosphoramidite synthesis. " The synthesis is performed with the growing nucleotide chain attached to a solid support derivatized with the nucleotide which will be the 3'-hydroxyl end of the oligonucleotide. The method involveε the cyclical εyntheεis 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 cytosine and adenosine) or an iεobutyryl group (to protect guanoεine) . Methodε for producing εuch derivatives are well known in the art.

V. Administration of the Molecules of the Present Invention

The above-described therapeutic agents of the present invention can be formulated according to known methods used to prepare pharmaceutically useful compositions, whereby theεe materials, or their functional derivatives, are combined in admixture with a pharmaceutically accep¬ table carrier vehicle. Suitable vehicles and their formulation, incluεive of other human proteins, e.g., human serum albumin, are deεcribed, for example, in Remington'ε Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)) . In orde r to form a pharmaceutically acceptable compoεition εuitable for effective adminiεtration, εuch compoεitions 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 preparations may be achieved through the use of polymers to complex or absorb the agents. The controlled delivery may be exercised by selecting appropriate macromolecules

(for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules aε well as the methods of incorporation in order to control releaεe. Another possible method to control the duration of action by controlled release preparations is to incorporate the agents into particles of a polymeric material such as polyesterε, polyamino acidε, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, inεtead of incorporating theεe agentε into polymeric particleε, it is posεible to entrap these materials in microcapsuleε prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsuleε and poly- (methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systemε, for example, lipoεomes, albumin microsphereε, microemulεions, nanoparticles, and nanocapsules or in macroemulsions. Such techniqueε are discloεed in Remington'ε Pharmaceutical Scienceε (1980) . In one embodiment of the preεent invention, nucleic acid molecule(s) comprising antisense or triplex molecules, or encoding a mutated MMDIL, FMRIL, huntL, SCAIL or andr2L or a wild type FMR1L2 product may be administered using viral or retroviral vectors in accordance with the methods of "gene therapy" .

The principles of gene therapy are discloεed by Oldham, R.K. (In: Principleε of Biotherapy. Raven Preεε, NY, 1987), and similar texts. Disclosures of the methods and useε for gene therapy are provided by Boggs, S.S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E.M. (Biol. Reprod. 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. suppress, or attenuate) an existing condition, the principles of the present invention can be used to provide a prophylactic gene therapy to individualε who, due to inherited genetic mutationε, or somatic cell mutation, are predisposed to Huntington's disease.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Example 1 Synthesis and Expression of cDNA Encoding HuntL

HuntL was prepared by oligonucleotide synthesis using solid phase gene assembly. Succesεive oligonucleotideε phoεphorylated and added at molar excess were attached stepwise to a growing chain anchored to a solid phase support. The assembled nucleotideε were ligated with T4 DNA ligase and detached from the support by cleaving at an added Hind III restriction enzyme site. HuntL was cloned into pBlueBacHis Xpress-baculoviruε Expreεεion εystem in which phuntH is expressed and purified. It is not expected that huntH can be obtained in the latter expresεion εyεtem εince cleaving and amidation doeε not occur.

The predicted epitope, amino acid reεidueε 4-13, iε chemically synthesized and coupled to Keyhole Limpet Hemocyanin (KLH) . The complex iε inoculated into rabbitε to produce antibodieε for diagnostic use.

While the invention has been described 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 principleε of the invention and including εuch departureε from the present disclosure as come within known or cuεtomary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claimε.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Preddie, Rick E. Bergmann, Johanna E. (ii) TITLE OF INVENTION: Agents for the Prevention and Treatment of Huntington 's Disease and Other Neurological Disorders

(iii) NUMBER OF SEQUENCES: 17

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Probevec AG (B) STREET: Mόrikestr. 22

(C) CITY: Hamburg

(D) STATE: Germany

(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: PCT

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT None

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 4940 862553

(B) TELEFAX: 4940 860176

4940 4717 4962

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

(A) LENGTH: 165 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: Antisense to huntingtin gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTGCTGGAAG GACTTGAGGG ACTCGAAGGC CTTCATCAGC TTTTCCAGGG 50 TCGCCATGGC GGTCTCCCGC CCGGCACGGC AGTCCCCGGA GGCCTCGGGC 100 CGACTCGCGG CGCCGCTCAG CACCGGGGCA ATGAATGGGG CTCTGGGCCG 150 CAGGTAAAAG CAGAA 165

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

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

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: phuntH

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

Met Ala Val Ser Arg Pro Ala Arg Gin Ser Pro Glu Ala Ser Gly Arg 1 5 10 15

Leu Ala Ala Pro Leu Ser Thr Gly Ala Met Asn Gly Ala Leu Gly Arg 20 25 30 Arg

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

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

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: huntH (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ala Val Ser Arg Pro Ala Arg Gin Ser Pro Glu Ala Ser Gly Arg 1 5 10 15

Leu Ala Ala Pro Leu Ser Thr Gly Ala Met Asn Gly Ala Leu 20 25 30

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 126 base pairs

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: MMDIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATGCAGCCCA GGGCGGCGGC ACGAGACAGA ACAACGGCGA ACAGGAGCAG 50 GGAAAGCGCC TCCGATAGGC CAGGCCTAGG GACCTGCGGG GAGAGGGCGA 100 GGTCAACACC CGGCATGGGC CTCTGA 126

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

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

(vii) IMMEDIATE SOURCE: (B) CLONE: MMDILH (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Gin Pro Arg Ala Ala Ala Arg Asp Arg Thr Thr Ala Met Arg Ser 1 5 10 15

Ser Glu Ser Ala Ser Ser Arg Pro Gly Leu Gly Thr Cys Gly Gly Arg 20 25 30

Ala Arg Ser Thr Pro Gly Met Gly Leu 35 40

(2) INFORMATION FOR SEQ ID N0:6A:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: MMDIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6A: AGGTCAATAA ATATCCAAA 19

(2) INFORMATION FOR SEQ ID NO:6B:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: MMDIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6B: GCGGGCGGAG CCGG 14 (2) INFORMATION FOR SEQ ID NO:6C:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: MMDIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:C: GATAGGTGGG GTG 13

(2) INFORMATION FOR SEQ ID NO:6D: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: MMDIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6D:

GGGGGCGGGC CCGG 14

(2) INFORMATION FOR SEQ ID NO:6E:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: MMDIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6E:

GGTGGGCGCG GCTT 14

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 93 base pairs

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: FMRIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGCGCGCTG CTGGGAACCG GCCGGGGTGC CGGGTCGAAA GACAGACGCG 50

CGGGCCGGGC GTGCGCGGGC TTGGTGGAGG GCGGGAAGGC TGA 93

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

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

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: FRM1LH

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Arg Ala Ala Gly Asn Arg Pro Gly Cys Arg Val Glu Arg Gin Thr 1 5 10 15

Arg Gly Pro Gly Val Arg Gly Leu Gly Gly Gly Arg Glu Gly 20 25 30

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:299 base pairs

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: FMRlLreg

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GTGTAACTTT AAAAAGCACA GTAAACCCAT GAATGCCTAT ATAAAGCCTC 50 AACAATTCAG TCCACTGCAA CACCACAAAT TCAAAGGAAA TGTGGTCAAG 100 TTCTTAGGGG CACAAAAATA TTTTCGCTCA GTTAAAAAAA AAAGCCCTAG 150 TAATAATATA CTCTCAGTTA ATTACCTGAA GTTTCATGGC ATATATTTAG 200 GTCTTTGGAA ACTTAAACAC ACACACACAA TTTTTAAGTA GCAGCGCTGC 250 TAATGGTTTT AGACGCTGAA GCATGTGCAT TCCTGAATTT ACCCCGTTT 299 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 159 base pairs

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: FMR1L2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATGCGTCCTG TCCACAGGGG CCGATGCACC TCCTTGCAAC CCTTTACATT 50 CCACTGTGAA ACAAACCTCA ACTTTTTCTT ATTCCTGTTT TTACACCGTG 100 CTTATAGCTG CCTTAATCCA TGTCCCCTTC GGGATGCTGG TATCCAACTG 150

AGAAGTTGA 159

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: FMR1LH2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met arg pro val his arg gly arg cys thr ser leu gin pro phe thr 5 10 15 phe his cys glu thr asn leu asn phe phe leu phe leu phe leu his 20 25 30 arg ala tyr ser cys leu asn pro cys pro leu arg asp ala gly ile

35 40 45 gln leu arg ser

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 104 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: FMRlL2reg

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TGACTATATA TTTTTTAAAA ATTGTGTTAA GCACTTGAGG TTCATTTCTG 50 CCCCTACTGT ATGTGCACCC TGTGCCAGAG GGTGGGGTGA ACACGTGTGT 100

AGCAGTC 107

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 234 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS (C) INDIVIDUAL ISOLATE: andr2L (XI)SEQUENCEDESCRIPTION: SEQIDNO:13: ATGTCΓΠAA GGTCAGCGGA GCAGCTGCTT AAGCCGGGGA AAGTGGGGCC SO CAGCAGGGAC AACGTGGATGGGGCAGCTGAGTCATCCTCG TCCGGAGGTG 100 CTGGCAGCTG CTGCGGCAGC CCCTTGCTGG CGGCCACGGC GGCTCCAGGC 150 TCTGGGACGC AACCTCTCTC GGGGTGGCAC TCCAGGGCCG ACTGCGGCTG 200 TGAAGGTTGC TGTTCCTCAT CCAGGACCAG GTAG 234 (2)INFORMATIONFORSEQIDNO:14: (I)SEQUENCECHARACTERISTICS:

(A) LENGTH: 77 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: andr2LH

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Ser Leu Arg Ser Ala Glu Gin Leu Leu Lys Pro Gly Lys Val Gly 1 5 10 15

Pro Ser Arg Asp Asn Val Asp Gly Ala Ala Glu Ser Ser Ser Ser Gly 20 25 30

Gly Ala Gly Ser Cys Cys Gly Ser Pro Leu Leu Ala Ala Thr Ala Ala 35 40 45

Pro Gly Ser Gly Thr Gin Pro Leu Ser Gly Trp His Ser Arg Ala Asp 50 55 60

Cys Gly Cys Glu Gly Cys Cys Ser Ser Ser Arg Thr Arg 65 70 75

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS (C) INDIVIDUAL ISOLATE: andr2Lreg

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

TTCGGCCAAT GG 12

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 168 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: SCAIL

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ATGACGACCT GGGAGGGGGG CCCCAGGGTG AGCGTGTGTG GGATCGTCTG 50

GTGGGGGTGG AGGTGGACGG GGATGGCCGG AGGAGAGGCG GTCCGGCCGG 100 TGTTCTGCGG AGAACTGGAA ATGTGGACGT ACTGGTTCTG CTGGGCTGGT 150 GGGGGGGACC CGGGGTGA 168

(2) INFORMATION FOR SEQ ID NO: 173: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

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

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: SCAILH (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Met Thr Thr Tφ Glu Gly Gly Pro Arg Val Ser Val Cys Gly He 1 5 10 15

Val Tφ Tφ Gly Tφ Arg Tφ Thr Gly Met Ala Gly Gly Glu Ala Val 20 25 30

Arg Pro Val Phe Cys Gly Glu Leu Glu Met Tφ Thr Tyr Tφ Phe Cys 35 40 45

Tφ Ala Gly Gly Gly Asp Pro Gly 50 55

Claims

WHAT IS CLAIMED.

1. A nucleic acid molecule, εubεtantially free of natural contaminants, that encodes a protein selected from the group consiεting of MMDIL, FMRIL, FMRlLreg, FMR1L2, FMRlL2reg, huntL, SCAIL, and andr2L.

2. The nucleic acid molecule of claim l whoεe sequence is selected from the group conεisting of SEQ ID N0:1, SEQ ID N0:4, SEQ ID N0:6A-E, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:16.

3. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID NO:l.

4. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID N0:4.

5. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID NO:6A-E.

6. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID NO:7.

7. The nucleic acid molecule of claim 2 wherein said εequence iε SEQ ID NO:9.

8. The nucleic acid molecule of claim 2 wherein εaid εequence is SEQ ID NO:10.

9. The nucleic acid molecule of claim 2 wherein said sequence iε SEQ ID NO:12.

10. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID NO:13.

11. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID NO:15.

12. The nucleic acid molecule of chain 2 wherein said sequence is SEQ ID NO:16

13. A protein, εubstantially free of natural contaminants, selected from the group consiεting of the group consisting of MMDILH, FMR1LH, FMR1L2H, phuntH, huntH, SCAILH, andr2LH.

14. The protein of claim 13 wherein said protein has a sequence of SEQ ID NO:2.

15. The protein of claim 13 wherein said protein has a sequence of SEQ ID NO:3.

16. The protein of claim 13 wherein said protein has a sequence of SEQ ID NO:5.

17. The protein of claim 13 wherein said protein has a εequence of SEQ ID NO:8.

18. The protein of claim 13 wherein εaid protein haε a sequence of SEQ ID NO:11.

19. The protein of claim 13 wherein said protein has a sequence of SEQ ID NO:14.

20. The protein of claim 13 wherein said protein has a sequence of SEQ ID NO:17.

21. A reagent capable of diagnosing the presence of a molecule selected from the group consiεting of a gene sequence that encodes MMDILH, a gene sequence that encodes FMR1LH, a gene sequence that encodes FMR1L2H, a gene εequence that encodes phuntH, a gene sequence that encodes huntH, a gene sequence that encodes SCAILH, and a gene sequence that encodes andr2LH.

22. A reagent capable of diagnosing the presence of a molecule selected from the group consiεting of an RNA tranεcript that encodeε MMDILH, an RNA tranεcript that encodeε FMRILH, an RNA tranεcript encodeε FMR1L2H, an RNA tranεcript that encodes phuntH, an RNA transcript that encodes huntH, an RNA transcript that encodes SCAILH, and an RNA transcript that encodes andr2LH.

23. A reagent capable of diagnosing the presence of a molecule selected from the group consiεting of MMDILH, FMRILH, FMR1L2H, phuntH, huntH, SCAILH, and andr2LH.

24. The reagent of claim 23, wherein said reagent is a nucleic acid molecule.

25. The reagent of claim 22, wherein said reagent is a ribozyme produced from nucleic acid molecules having a sequence Of SEQ ID N0:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:16.

26. The reagent of claim 24, wherein said reagent is obtainable by mutating a nucleic acid molecule having a sequence of SEQ ID NO:l, SEQ ID N0:4, SEQ ID NO.-6A-E, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:16.

27. The reagent of claim 24, wherein said reagent compriseε a nucleic acid εequence that iε complementary to the nucleotide εequence of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:6A-E, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:16.

28. The reagent of claim 23 wherein εaid reagent is a protein.

29. The reagent of claim 28, wherein said protein is an antibody, or a fragment of an antibody.

30.A method of treating Huntington's Disease, myotonic dystrophy, fragile x syndrome, spino bulbar atrophy or spinocerebellar ataxia which compriseε providing to an individual, in need of εuch treatment, an effective amount of an inhibitor of the MMDIL gene, the FMRIL gene, the huntL gene, the FMRIL gene, the SCAIL gene or the andr2L gene.

31. The method of claim 30, wherein εaid inhibitor iε a protein.

32. The method of claim 31, wherein said inhibitor is an antibody, or fragment thereof.

33. The method of claim 30, wherein said inhibitor is a nucleic acid molecule.