WO1994013798A1 - Agents for the prevention and treatment of human alzheimer's disease - Google Patents

Agents for the prevention and treatment of human alzheimer's disease Download PDF

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Publication number
WO1994013798A1
WO1994013798A1 PCT/EP1993/003581 EP9303581W WO9413798A1 WO 1994013798 A1 WO1994013798 A1 WO 1994013798A1 EP 9303581 W EP9303581 W EP 9303581W WO 9413798 A1 WO9413798 A1 WO 9413798A1
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protein
pac
seq
nucleic acid
tpac
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PCT/EP1993/003581
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French (fr)
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Johanna E. Bergmann
Rick E. Preddie
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Bergmann Johanna E
Preddie Rick E
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Priority claimed from CA 2085924 external-priority patent/CA2085924A1/en
Application filed by Bergmann Johanna E, Preddie Rick E filed Critical Bergmann Johanna E
Priority to AU59689/94A priority Critical patent/AU5968994A/en
Publication of WO1994013798A1 publication Critical patent/WO1994013798A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to therapeutic agents for the prevention and treatment of human Alzheimer's Disease. More specifically, the invention relates to a proteinase/esterase-like protein implicated in Alzheimer's Disease, to analogues and derivatives of this molecule, and to nucleic acid molecules encoding such molecules, or influencing their expression. The invention also relates to therapeutic methods for using such agents.
  • AD Alzheimer's disease
  • a progressive disease of the human central nervous system It is manifested by dementia in the elderly, by disorientation, loss of memory, difficulty with language, calculation, or visual-spacial skills, and by psychiatric manifestations. It is associated with degenerating neurons in several regions of the brain. Alzheimer's disease is reviewed by Price, D.L. et al. (Clin. Neuropharm. 14.S9-S14 (1991)) ; Pollwein, P. et al . (Nucl. Acids Res. 20:63-68 (1992)); Regland, B. et al.
  • amyloid protein AP
  • the principal component of the amyloid protein plaques is a 40 amino acid protein known as the / S/A4 amyloid protein (Price, D.L. et al.. Clin. Neuropharm. 14:S9-S14 (1991)) .
  • This protein forms fibrils, which are concentrated in amyloid deposits in the extracellular space of the brain parenchyma and in the vascular elements of the brain and the pia-arachnoid (Currie, J.R. et al. , J. Neurosci. Res.
  • the j ⁇ /A4 amyloid protein is a 4 kD protein fragment whose sequence is contained within a family of larger proteins, which are alternatively spliced transcripts of a single gene known as Alzheimer 3-amyloid peptide precursor (APP) (Podlisny, M.B. et al. , Science 238 :669- 671 (1987); Currie, J.R. et al.. J. Neurosci. Res.
  • APP Alzheimer 3-amyloid peptide precursor
  • the APP gene is located on chromosome 21 and is preferentially expressed in the neuronal cells of the central nervous system.
  • the different isoforms of APP are generated by proteolytic cleavage of the translational products (Pollwein, P. et al. (Nucl. Acids Res. 2_0:63-68 (1992); Price, D.L. et al.. Clin. Neuropharm. 14:S9-S14 (1991) ) .
  • Type V hyperlipoproteinemia is characterized clinically by hepatosplenomegaly, occasional eruptive xanthomas and an increased incidence of pancreatitis (Ghiselli, G., et al .. Lancet 2:405-407 (1982)) . These patients have striking hypertriglyceridemia due to increased plasma chylomicron and very low density lipoprotein concentrations in the fasting state, without a deficiency of lipoprotein lipase or its activator protein, apolipoprotein (apo) C-II.
  • Apolipoprotein E (apoE) , a protein constituent of triglyceride-rich lipoproteins, has been implicated in the receptor-mediated hepatic uptake of these particles ((Ghiselli, G., et al.. Lancet 2:405-407 (1982)) .
  • ApoE3 is considered the normal isoform.
  • the apoE2 variant possesses a Cys substituted for an Arg at residue 158 in its amino acid sequence.
  • the apoE4 variant possesses an Arg substituted for Cys at residue 112 in its amino acid sequence, and lacks cysteine residues (Weisgraber. K.H., J. Lipid Res. 31:1503-1512 (1990) ; Utermann, G. et al .. J. Lipid Res. 25 :378-382 (1984) ) .
  • Apolipoprotein (apo) E polymorphism has a significant effect on plasma cholesterol and low density lipoprotein cholesterol concentrations (Wardell, M.R. et al . , J. Lipid. Res. 12:521-528 (1991)) .
  • the common variants, apoE2 and apoE4 have a significant impact on interindividual variation of lipid and lipoprotein levels in normal subjects.
  • the common variant apoE2 and more than half a dozen rare variants are defective in binding to the low-density lipoprotein (LDL) receptor, and all are causally associated with the lipid disorder, type III hyperlipoproteinemia (HLP) .
  • LDL low-density lipoprotein
  • the mode of inheritance of the disorder can be either dominant or recessive, depending on the particular mutation(s) in apoE, although the mechanisms involved are not fully understood (Rail, S.C. et al .. J. Intern. Med. 231:653- 659 (1992) ) .
  • the apoE4 isoform is associated both with HPL and type V HPL (Gregg, R.E. et al . , J. Clin. Invest. 21:815-821 (1986) ; Kuusi, T. et al .. J. Lipid Res . 11293-298 (1988) ; Yanagi, H. et al . , Clin. Genet. 18:264-269 (1990)) .
  • Diabetic patients with the ApoE4 allele appear to be more susceptible to HPL than diabetic patients with other alleles (Eto., M. et al .. Diabetes 361301-1306 (1987) ) .
  • diabetic patients with apoE2 were characterized by increased levels of plasma triglyceride, total cholesterol, very low density lipoprotein (VLDL)-chol, and apoE and an increased VLDL-chol/VDLD-triglyceride ratio, i.e. the accumulation of remnants .
  • VLDL very low density lipoprotein
  • VDLD-triglyceride ratio i.e. the accumulation of remnants .
  • fasting plasma glucose and he oglobin- Al levels were significantly higher in hyperlipoproteinemic diabetic patients with apoE2 than in normolipidemic diabetic patients with apoE2 (Eto, M.
  • Alzheimer's disease would probably affect one out of every 10 humans alive today. To date there is no treatment for Alzheimer's disease at any stage of its development. Two therapeutic reagents, Cognex and Menthane, appear to give slight relief to some victims but do not alter the course of the disease. In view of the importance of diagnosing, predicting, and treating Alzheimer's disease and hyperlipoproteinemia and cardiovascular disease, an affective means for achieving these goals would be highly desirable.
  • the present invention supplies such means.
  • Figure IA shows the protein coding sequence of "Pac” cDNA and the deduced sequence of the protein is shown in Figure IB.
  • "Tpac" cDNA is reconstructed from negative strands of exons and from introns in the amyloid precursor related human APRP gene.
  • the protein coding sequence of the cDNA is shown in Figure 1C and the deduced sequence of the protein in the embodiment of Figure ID.
  • Figure 2 shows the sequence of cDNA molecules.
  • the protein coding sequence of "hsaN" cDNA is shown in (A) and the deduced sequence of the protein is shown in (B) .
  • Figure 3A shows the nucleotide sequence of cDNA encoding the apoE4L protein.
  • Figure 3B shows the amino acid sequence of the protein.
  • raised "p” indicates kinase c phosphorylation sites
  • pck identifies casein kinase phosphorylation sites
  • a down arrow indicates the location of a cleavage/secretory site. Double underscoring is used to denote sequences with significant identity and homology to apoE4.
  • Figure 4A shows the nucleotide sequence of cDNA encoding the apoE4Ll protein.
  • Figure 4B shows the amino acid sequence of the protein.
  • the raised "p” indicates kinase c phosphorylation sites,- a down arrow indicates the location of a cleavage/secretory site.
  • Figure 5 shows the upstream (5') regions that are responsible for the transcription of the ApoE4L/Ll/Lx2 proteins.
  • Figure 6A shows the nucleotide sequence of cDNA encoding the apoE4Lx2 protein.
  • Figure 6B shows the amino acid sequence of the protein.
  • the raised "p" indicates kinase c phosphorylation sites,- a down arrow indicates the location of a cleavage/secretory site.
  • the amino acids that join the E4L protein to the E4L1 protein are underlined. Double inderscoring is used to denote sequences with significant identity and homology to apoE4.
  • the invention concerns agents and methods for the diagnosis and therapy of Alzheimer's disease and related conditions, as well as hyperlipoproteinemia and associated cardiovascular disease.
  • agents include a proteinase/esterase-like protein implicated in Alzheimer's Disease, as well as analogues and derivatives of this molecule, and nucleic acid molecules encoding such molecules, or influencing their expression.
  • the invention provides a nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2.
  • the invention provides the above-described nucleic acid molecule wherein the sequence is SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 12.
  • the invention also provides a protein, substantially free of natural contaminants, selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2.
  • the invention provides the above-described protein having a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10 or SEQ ID NO: 13.
  • the invention also provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of Pac, a Pac-encoding nucleic acid molecule, Pac-l, a Pac-l-encoding nucleic acid molecule, Pac-2, a Pac-2-encoding nucleic acid molecule, Pace, a Pace-encoding nucleic acid molecule, Tpac, a Tpac- encoding nucleic acid molecule, Tpac-2, a Tpac-encoding nucleic acid molecule, hsaP, an hsaP-encoding nucleic acid, ApoE4L, an ApoE4L-encoding molecule, ApoE4Ll, an ApoE4Ll-encoding molecule, ApoE4Lx2, and an ApoE4Lx2- encoding molecule.
  • a molecule selected from the group consisting of Pac, a Pac-encoding nucleic acid molecule, Pac-l, a Pac-l-encoding nucleic
  • the reagent is a nucleic acid molecule, (especially a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:12., or a nucleic acid molecule obtainable by mutating a nucleic acid molecule, (especially a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:12., or a nucleic acid molecule obtainable by mutating a
  • nucleic acid molecule having a sequence of SEQ ID NO.-l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:12) or a protein (especially an antibody, or a fragment of an antibody, which is capable of binding to Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2) .
  • the invention also provides a method of treating Alzheimer's disease, Down's Syndrome, Parkinson's
  • Alzheimer's disease reflects the abnormal processing of APP to produce /3/A4 protein
  • the exact mechanism of this processing has not been elucidated.
  • Two of these forms (having 751 and 770 amino acids) contain a domain that has substantial homology to Kunitz-type protease inhibitors.
  • APP is processed to form AP throughout life, however, in Alzheimer's disease, an abnormal AP -- /S/A4 protein -- which is longer at the C-terminal by 2 or 3 amino acids is expressed.
  • APP is a cell surface receptor or a transmembrane protein, in which the ⁇ /A domain is partly embedded in the cell membrane.
  • the secretion of the 3/A4 protein thus reflects the cleavage of the domain from the precursor molecule (see, Roch, J.M. et al.. J. Biol. Chem. 267:2214-2221 (1992)) .
  • the proteolysis of APP into AP is believed to prevent the formation of ⁇ /AA protein.
  • the present invention derives, in part, from the discovery of a protein with proteinase and acetylcholinesterase domains that catalyzes the formation of the ⁇ /AA protein.
  • the protease is encoded by a copy of the antisense strand of the APP gene.
  • the present invention also derives in part from the discovery of a truncated, modified version of the protease that is encoded on the antisense strand of a gene -- the human APRP gene -- that is structurally and functionally related to APP.
  • the present invention provides the sequence of these molecules, as well as that of the cDNA that encodes them. These molecules may be used in the diagnosis, prediction and treatment of Alzheimer's disease.
  • the present invention also derives, in part, from the recognition of the role of the ApoE gene in human Alzheimer's disease, and in cardiovascular disease, such as hyperlipoproteinemia.
  • the invention relates to the discovery two tandem open reading frames ("orfs") on the antisense strand of ApoE4 gene.
  • the first orf designated herein as "ApoE4L” or “E4L, " commences 82 base pairs (bp) upstream from the ApoE4 stop translation signal (nucleotide 4403 on the coding strand) and terminates with a TAG codon at nucleotide 3817.
  • the second orf designated herein as “ApoE4Ll” or “E4L1, " is located within an exon, and encodes a putative 198 amino acid protein. Because the two orfs are in the same reading frame, a mutation that obliterates the termination codon of E4L results in the creation of a fusion protein, designated herein as
  • splicing occurs in many eukaryotic genes, and since different spliced varieties of mRNAs are found in different cell types, it is believed that splicing is tissue specific and may be a method of local environmental control over the function of some gene(s) .
  • the present invention further concerns gene sequences produced via such alternate splicing mechanisms, and the proteins that such gene sequences encode.
  • the molecules of the present invention comprise Pac, Tpac, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2, as well as the analogs of these proteins, and gene sequences that encode them.
  • the molecules of the invention also include the novel set of gene sequences and encoded proteins (Pac-l, Pac-2, Pace, Tpac-1) ithat result from the alternative splicing of the mRNA antisense to the amyloid forming APP gene. These molecules of the present invention are described below in greater detail.
  • the negative strand of exons and introns in the human APP gene was evaluated in a search for the putative Alzheimer's disease protease. This evaluation led to the recognition that the APP antisense strand encoded a protein -- designated herein as "Pac.”
  • the protein-encoding portion of the Pac cDNA sequence is shown in Figure IA (SEQ ID NO:l) and the deduced sequence of the protein in Figure IB (SEQ ID NO:2) .
  • the cDNA encoding "Pac” is 100% homologous to a region of the negative strand of the human APP mRNA, starting at amino acid 7 within the AD protein.
  • the molecular weight of the Pac proteinase/esterase was found to be 12,040 kd, and to comprise 104 amino acid residues.
  • the protein is predicted to be unstable.
  • Tpac amyloid precursor-related gene
  • the Acidic Activator Protein, hsaP A number of patients from families with inherited Alzheimer disease exhibit mutations on chromosome #14 in the region of the major heat shock 70 kb protein (hsp70) .
  • hsp70 major heat shock 70 kb protein
  • the cDNA encodes a 5.45 kD, 51 amino acid, intracellular, acidic activator protein (hsaP) , shown in (B) of Figure 2 (SEQ ID NO:6) .
  • hsaP shares >75% homology with a 45 amino acid domain of hsp70 in which 16 of 18 possible hydrophobic amino acid residues match, strongly indicating that hsaP can interact specifically with hsp70.
  • hsaP Under normal physiological conditions, the sense/ antisense relationship between APP and hsaP will not allow the expression of hsaP in health humans,- however, acquired mutations in regions of the APP gene involved in antisense repression of hsaP will allow hsaP to be expressed. This free, intracellular, hsaP is "neutralized” through interaction with wild type hsp70, as is the case with some other undesirable intracellular proteins.
  • hsp70 Mutations in any of several dozen genes which share the hsp70 region in chromosome #1 can be the cause of inherited Alzheimer's disease.
  • the role of hsp70 in the disease may be caused by inherited mutations that reside in the region of the gene and that are critical for interaction with expressed hsaP.
  • the pathophysio- logical symptoms of Alzheimer's disease in these families may be the result of the inherited mutation on chromosome #14 and an acquired mutation on APP.
  • either hsa or pac may be an Alzheimer's locus on human chromosome #14.
  • humanized antibodies prepared against hsaP, or ribozymes prepared from selected regions of the APP gene against hsaN will safely and efficiently prevent and cure Alzheimer's disease in such families.
  • Tpac The deduced protein encoded by "Tpac” is a truncated form of "Pac.”
  • the protein was found to be truncated at amino acid #23 and four new amino acids --
  • CITS were found to have been added to the truncated c-terminal end of the protein. It comprises only the proteinase domain of "Pac.” This domain has significant identity and homology with domains in, viper Russelli proteinase, human cathepsin A, human complement c3 convertase, RNA 1 polyprotein protease cofactor, and a species of yeast killer toxin with carboxypeptidase-like activity.
  • Tepac has strong similarity and homology to the powerful, 50 amino acid, testes DNA binding "protamine Zl” protein. The latter protein forms highly condensed chromatin packets consisting of membranes and chromosomal DNA in sperm heads.
  • the 12.4 kd native Pac protein may associate with other dissimilar protein subunits to form a polyprotein which can mediate such enzymatic activities.
  • the 12.4 kd protein acting may act as an activator of inactive proteinase and acetylcholinesterase zymogens to form active enzyme molecules which can carry out the enzymatic activity.
  • Tpac may be involved as a causative agent in Alzheimer's disease.
  • the 27 amino acid protein which lacks an acetylcholinesterase domain can function. Tpac may act as a transducer of protease activity in an inactive proteinase. In analogy to the action of its homologue,
  • Tpac may bind to chromosomal DNA and influence the expression of critical genes. Tpac may alternatively compact the relatively insoluble abnormal AD protein, together with neural cell chromatin and dendrites, into the "intracellular neurofibrillary tangles" found in advanced stages of Alzheimer's disease.
  • the diagnostic and/or therapeutic reagents of the invention additionally include fusion proteins, antipeptide reagents, etc., made from the deduced amino acid sequences described in the embodiment of Figures IB and ID.
  • the invention also includes any cellular proteinase or acetylcholinesterase activated by the proteins or domains of the proteins described in the embodiment of Figures IB and ID.
  • the first orf identified in the ApoE antisense sequence encodes the apoE4L protein.
  • the nucleotide sequence of ApoE4L cDNA is shown in Figure 3A (SEQ ID NO: 7) .
  • the apoE4L protein sequence is shown in Figure 3B (SEQ ID NO: 8) .
  • Of particular interest is the sequence 89-104 of SEQ ID NO: 8, which possesses significant identity and homology to apoE4 sequences.
  • Kinase c phosphorylation sites are present at positions 3, 42, 43, 54, 87, 112, 122, and 129 of SEQ ID NO: 8.
  • Casein kinase phosphorylation sites are present at 176- 177 and 193-194 of SEQ ID NO: 8.
  • Cleavage/secretory sites occur at positions 43-44 and 182-183.
  • the second orf identified in the ApoE antisense sequence encodes the apoE4Ll protein.
  • the second orf commences 16 bp downstream from E4L stop codon, nucleotide 3801, in the same reading frame, and terminates within the adjoining intron at nucleotide 3558.
  • This second orf encodes a putative 107 amino acid long protein designated herein as "E4L1". Only 21 bp of exon sequence is included in the E4L1 sequence.
  • the nucleotide sequence of cDNA encoding the apoE4Ll protein is shown in Figure 4A (SEQ ID NO: 9) .
  • the apoE4Ll protein sequence is shown in Figure 4B (SEQ ID NO:10) .
  • a kinase c phosphorylation site is present at position 54 of SEQ ID NO: 10.
  • a cleavage/secretory site occurs at positions 46-47.
  • Both the Apo4L and Apo4Ll orfs can be transcribed from promoter elements and coordinated GC box and cap sites that are located 300-500 bp upstream from the
  • E4L/L1 orf The sequence of this upstream regulatory region is SEQ ID NO: 11 ( Figure 5) .
  • Promoter elements are present at positions 12-26 and 322-335 in SEQ ID NO:11.
  • a GC box is present at position 206-219.
  • Cap sites are present at positions 55 and 338 in SEQ ID NO:
  • a ribosome binding site is present 5 nucleotides from the end of the sequence.
  • the sequence "TATAA" located 100 bp downstream of the E4L1 stop codon might be used as a transcription termination signal, however, there is a perfect consensus termination signal at nucleotide 2495 which may alternatively signal the end of the putative antisense prime transcript.
  • E4Lx2 The nucleotide sequence that encodes E4Lx2 is presented in Figure 6A (SEQ ID NO:12) ; the six new codons are underlined.
  • E4Lx2 The amino acid sequence of E4Lx2 (SEQ ID NO: 13) is ⁇ hown in Figure 6B. Of particular interest is the sequence 89-104 of SEQ ID NO:13, which possesses significant identity and homology to apoE4 sequences.
  • Kinase c phosphorylation sites are present at positions 3, 42, 43, 54, 87, 122, 129, 152 and 303 of SEQ ID NO:13.
  • Casein kinase phosphorylation sites are present at 176-177 and 193-194 of SEQ ID NO:13.
  • Cleavage/secretory sites occur at positions 35-36, 43-44 and 182-183 and 295-296.
  • a glycosylation site is present at position 202.
  • the E4Lx2 protein has the combined primary characteristics of the E4L protein as well as those of the E4L1 protein, plus a potential glycosylation site provided by the codons linking the two proteins.
  • the E4Lx2 protein has several additional potentially functional characteristics including significant identity and homology with the herpes associated "infected cell protein, " and potential protease activity.
  • the primary structure of E4Lx2 appears to make this protein more likely than apo4E to be responsible for the role in growth and repair of the nervous system during development and injury ascribed to apoE4,- and, also, for the tight binding to the 3-amyloid protein -- which, as indicated above is the central player in the pathophysiology of human Alzheimer's disease.
  • E4Lx2 rather than mutations in the ApoE4 protein, is implicated in causing or contributing to late-onset familial Alzheimer's disease.
  • E4L and E4L1 appear to be involved in other diseases associated with ApoE2, ApoE3, and ApoE4 -- such as hyperlipoproteinemia and related cardiovascular conditions.
  • splicing occurs in many eukaryotic genes, and since different spliced varieties of mRNAs are found in different cell types, it is believed that splicing is tissue specific and may be a method of local environmental control over the function of some gene(s) .
  • the present invention further concerns gene sequences produced via such alternate splicing mechanisms, and the proteins that such gene sequences encode.
  • spliced DNA and encoded proteins described here are probably involved in the neuropathology of Alzheimer's disease. Alternate splicing of the mRNA antisense to the amyloid forming APP gene, gives rise to a set of novel gene sequences and encoded proteins (Pac- 1, Pac-2, Pace, Tpac-1) whose sequences are presented below.
  • Tpac-2 Tpac is unchanged and can be independently expressed, a different protein (“Pace") is in the acetylcholinesterase domain, and a neuropeptide-type protein, Tpac-2, is encoded in the central domain.
  • Pace a different protein
  • Tpac-2 a neuropeptide-type protein
  • Pac-2 (SEQ ID NO: 15) : ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA
  • the cDNA sequence of Pace is as follows
  • Tpac-1 The cDNA sequence of Tpac-1 is as follows
  • the deduced amino acid sequences of the proteins expressed by antisense mRNAs, Pac-l and Pac-2, produced by the alternative/differential splicing in the pac antisense gene is as follows:
  • Pac-l (SEQ ID NO:18) :
  • Tpac-2 (SEQ ID NO:19) : MQKGDNVCSSLGPRCGEAQVKRF
  • the elucidation of the significance of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2 in the etiology of Alzheimer's disease provides 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.
  • the molecules and methods of the present invention can be used in the diagnosis and treatment of these diseases and conditions.
  • any of the proteins described herein, or mutants thereof may be used in the treatment of or in the development of reagents for the treatment of these diseases and conditions.
  • antibodies are employed that are capable of binding to the Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 molecules, and the presence of such molecules is determined via and immunoassay.
  • 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.
  • 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 Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 as an antigen.
  • Such molecules are preferably obtained through the expression of the gene sequences described herein.
  • the simplest immunoassay involves merely incubating an anti-Pac, anti-Tpac, anti-hsaP, anti-ApoE4L, anti- ApoE4Ll, anti-ApoE4Lx2, etc. antibody with a sample suspected to contain the target molecule -- Pac, Tpac, hsaP, ApoE4L, ApoE4Ll, ApoE4Lx2, etc.
  • the presence of the target molecule is determined by the presence, and proportional to the concentration, of any antibody bound to the target molecule.
  • a solid phase is typically employed.
  • 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.
  • 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.
  • the support can be incubated in the presence 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 is proportional to the concentration of the target in the sample.
  • 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 sites.
  • 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.
  • 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 kinetics. Such assays 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 equipment.
  • 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 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.
  • 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
  • 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, ease and speed 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 its conjugate, and the like.
  • Suitable enzymes include peroxidase, acetylc oline esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, 3-galactosidase, catalase, delta-5-steroid isomerase, glucose oxidase, glucose-6- phosphate dehydrogenase, glucoamylase, glycoamylase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phosphate isomerase, urease, yeast-alcohol dehydrogenase, etc.
  • Peroxidase and urease are among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.
  • radioisotopic, chemiluminescent or fluorescent labels may be employed.
  • suitable radioisotopic labels include 3 H, lll ln, 15 I, 131 I, 32 P, 3S S, 14 C, 51 Cr, 57 To, 5 ⁇ C ⁇ , 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 21 Ci, 11 At, 12 Pb, 47 Sc, 109 Pd, etc.
  • suitable chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, an aequorin label, etc.
  • fluorescent labels examples 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.
  • the presence of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 in a cell can be determined by any means capable of detecting mRNA encoding these proteins.
  • molecules comprising nucleic acid probes capable of hybridizing to such molecules may be used in the diagnosis of Alzheimer's disease.
  • a "probe" is a detectably labelled nucleic acid molecule that is capable of hybridizing to a defined site of a target molecule.
  • nucleic acid sequences disclosed herein can be used as 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 may be used to label nucleic acids: enzyme labels
  • Such nucleic acid based assays may use either DNA or RNA to detect the Pac, Pac-l, Pac-2, Pace, Tpac,
  • the assays may be performed on RNA that has been extracted from neuronal cells. Alternatively, and more preferably, the assays may be done in situ on biopsied tissue.
  • 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 Svmp. Quant. Biol. 51:263-273 (1986);
  • the diagnosis of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 expression is performed using a ribozyme produced from nucleic acid molecules having a sequence of such molecules.
  • the present invention additionally provides a capacity to predict whether an individual is at risk for Alzheimer's disease.
  • any of the above-described assays may be performed on an asymptomatic individual in order to assess that individual's predisposition to Alzheimer's disease.
  • the present invention provides a means for treating Alzheimer's disease.
  • Such treatment may be either “prophylactic” or "therapeutic.”
  • a prophylactic treatment is one that is provided in advance of any symptom of Alzheimer's disease in order to prevent or attenuate any subsequent onset of the disease.
  • a therapeutic treatment is one that is provided in response to the onset of a symptom of Alzheimer's disease, and serves to attenuate an actual symptom of the disease.
  • 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'),, single chain antibodies, etc.) that is capable of binding to Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 .
  • 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.
  • polyclonal or monoclonal antibodies may be administered. More preferably, and especially for chronic administration, the use of non- immunogenic antibodies is preferred.
  • Such molecules can be pseudo-homologous (i.e. produced by a non-human species, but altered to a form that is immunologically indistinct from human antibodies) .
  • pseudo-homologous molecules include "humanized” (i.e. non-immunogenic in a human) prepared by recombinant or other technology.
  • Such antibodies are the equivalents of the monoclonal and polyclonal antibodies, but are less 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.
  • chimeric antibodies i.e. chimeric antibodies
  • nucleic acid molecules of the present invention may be mutated and expressed in order to identify Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mutant molecules that can complex with and significantly inactivate Pac, Pac- l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Li or ApoE4Lx2 molecules present in a cell.
  • such mutated protein molecules may be administered to a patient.
  • nucleic acid expressing such molecules may be administered.
  • an “antisense” or “triplex” nucleic acid molecules may be used to provide the desired therapy.
  • an “antisense oligonucleotide” is a nucleic acid (either DNA or RNA) whose sequence is complementary to the sequence of at least part of the Pac-, Pac-l-, Pac-2-, Pace-, Tpac-, Tpac-2-, hsaP-, ApoE4L-, ApoE4Ll- or ApoE4Lx2-encoding sequences described herein, such that it is capable of binding to, or hybridizing with, an endogenous Pac, Pac- l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA molecule, and thereby impair (i.e.
  • 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.
  • the nucleic acid molecule To act as a triplex oligonucleotide, the nucleic acid molecule must be capable of binding to the region of the double-stranded DNA genome that encodes Pac, Pac- l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 in a manner sufficient to impair the transcription of either gene.
  • Triplex oligonucleotides are disclosed by Hogan, U.S. Patent 5,176,996 and by Varma et al . , U.S. Patent 5,175,266.
  • the nucleic acid molecule must be capable of binding to or hybridizing with that portion of the Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA molecule which mediates the translation of the target mRNA.
  • Antisense oligonucleotides are disclosed in European Patent Application Publication Nos. 263,740; 335,451; and
  • the antisense oligonucleotide will be about 10-30 nucleotides in length, most preferably, about 15-24 nucleotides in length.
  • an antisense oligonucleotide that is designed to specifically block translation of a Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA transcript can be used to impair the expression of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 in a cell, and thereby provide a treatment for Alzheimer's disease.
  • the antisense oligomer is prepared in accordance with the nucleotide sequence of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 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 hybridizing with the above-described translation locus of either Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 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. Sci. (U.S.A.
  • the antisense 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 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 cytosine and adenosine) or an isobutyryl group (to protect guanosine) .
  • Methods for producing such derivatives are well known in the art.
  • compositions can be formulated according to known methods used to prepare pharmaceutically useful compositions, whereby these materials, 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's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)) .
  • 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 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, methyl- cellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • appropriate macromolecules for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methyl- cellulose, carboxymethylcellulose, or protamine, sulfate
  • concentration of macromolecules as well as 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 such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • coacervation techniques for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • nucleic acid molecule(s) comprising antisense or triplex molecules, or encoding mutated Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 molecules may be administered using viral or retroviral vectors in accordance with the methods of "gene therapy" .
  • 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 individuals who, due to inherited genetic mutations, or somatic cell mutation, are predisposed to Alzheimer's disease.
  • Oligonucleotides corresponding to "Pac” and “Tpac” cDNA were prepared by oligonucleotide synthesis on an Applied Biosystems synthesizer using the column method according to the recommendations of the manufacturer, and gel purified.
  • Pac the positive and negative strands were synthesized as three fragments, which were ligated to form a continuous strand, and gel purified.
  • Tpac full-length strands were synthesized and gel purified. Appropriate strands were mixed together in equimolar amounts and processed in the EXPRESS system (Invitrogen corporation pTrcHis Xpress-Prokaryotic Expression and Purification system) according to the recommendations of the manufacturer.
  • ADDRESSEE WEIL, GOTSHAL & MANGES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • Trp Ala Arg Ser Cys Ser Gly Trp Pro Ala Arg Glu Pro Thr Val Ala 65 70 75 80
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomicl (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • Trp Ala Arg Ser Cys Ser Gly Trp Pro Ala Arg Glu Pro Thr Val Ala 65 70 75 80
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES

Abstract

Agents and methods for the diagnosis and therapy of Alzheimer's disease and related conditions are disclosed. Such agents include a proteinase/esterase-like protein implicated in Alzheimer's disease, as well as analogues and derivatives of this molecule, and nucleic acid molecules encoding such molecules, or influencing their expression.

Description

TITLE OF THE INVENTION:
AGENTS FOR THE PREVENTION AND TREATMENT OF HUMAN ALZHEIMER'S DISEASE
FIELD OF THE INVENTION:
The invention relates to therapeutic agents for the prevention and treatment of human Alzheimer's Disease. More specifically, the invention relates to a proteinase/esterase-like protein implicated in Alzheimer's Disease, to analogues and derivatives of this molecule, and to nucleic acid molecules encoding such molecules, or influencing their expression. The invention also relates to therapeutic methods for using such agents.
CROSS-REFERENCE TO RELATED APPLICATIONS:
This application is a continuation-in-part of U.S. patent applications serial nos. 08/029,401 (filed on March 4, 1993) and 08/115,013 (filed on September 1, 1993) .
BACKGROUND OF THE INVENTION:
Alzheimer's disease ("AD") is a progressive disease of the human central nervous system. It is manifested by dementia in the elderly, by disorientation, loss of memory, difficulty with language, calculation, or visual-spacial skills, and by psychiatric manifestations. It is associated with degenerating neurons in several regions of the brain. Alzheimer's disease is reviewed by Price, D.L. et al. (Clin. Neuropharm. 14.S9-S14 (1991)) ; Pollwein, P. et al . (Nucl. Acids Res. 20:63-68 (1992)); Regland, B. et al.
(Med. H poth. 38:11-19 (1992)) and Johnson, S.A. (In: Review of Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.), iley-Liss, NY, 163-170 (1990)) .
I. Amyloid Protein
Pathologically, the disease is recognized by the presence of intracellular tangles, and extracellular deposits or "plaques" of amyloid protein ("AP") in the neuropil and in blood vessels. The principal component of the amyloid protein plaques is a 40 amino acid protein known as the /S/A4 amyloid protein (Price, D.L. et al.. Clin. Neuropharm. 14:S9-S14 (1991)) . This protein forms fibrils, which are concentrated in amyloid deposits in the extracellular space of the brain parenchyma and in the vascular elements of the brain and the pia-arachnoid (Currie, J.R. et al. , J. Neurosci. Res. .10:687-689 (1991)) . All cases of Alzheimer's disease show such deposition of amyloid in brain parenchyma. The jβ/A4 amyloid protein is a 4 kD protein fragment whose sequence is contained within a family of larger proteins, which are alternatively spliced transcripts of a single gene known as Alzheimer 3-amyloid peptide precursor (APP) (Podlisny, M.B. et al. , Science 238 :669- 671 (1987); Currie, J.R. et al.. J. Neurosci. Res.
10:687-689 (1991)) . This precursor is normally cleaved to form the /3-amyloid protein ("AP") . The isolation of cDNA encoding the S/A4 protein has led to the recent recognition that the protein is, in fact, synthesized as part of APP, and that 3/A4 protein comprises an abnormal form of AP (Zain, S.B. et al. , Proc. Natl. Acad. Sci. (U.S.A.) 85:929-933 (1988); Vitek, M.P. et al.. Molec. Brain Res. 4:121-131 (1988); Johnson, S.A. (In: Review of Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.), Wiley-Liss, NY, 163-170 (1990)) . The APP gene is located on chromosome 21 and is preferentially expressed in the neuronal cells of the central nervous system. In addition to alternate splicing programs, the different isoforms of APP are generated by proteolytic cleavage of the translational products (Pollwein, P. et al. (Nucl. Acids Res. 2_0:63-68 (1992); Price, D.L. et al.. Clin. Neuropharm. 14:S9-S14 (1991) ) .
Although the accumulation of 8/A4 protein in Alzheimer's disease is believed to result from the faulty processing of one or more of the APP isoforms (Currie, J.R. et al .. J. Neurosci. Res. 30:687-689 (1991) ) , the exact mechanism of β/A4 protein formation is not yet known (see, Johnson, S.A. (In: Review of Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.), Wiley-Liss, NY, 163-170 (1990); Roch, J.M. et al., J. Biol. Chem. 267:2214-2221 (1992)) .
II. Apolipoprotein E
Type V hyperlipoproteinemia (HLP) is characterized clinically by hepatosplenomegaly, occasional eruptive xanthomas and an increased incidence of pancreatitis (Ghiselli, G., et al .. Lancet 2:405-407 (1982)) . These patients have striking hypertriglyceridemia due to increased plasma chylomicron and very low density lipoprotein concentrations in the fasting state, without a deficiency of lipoprotein lipase or its activator protein, apolipoprotein (apo) C-II.
Apolipoprotein E (apoE) , a protein constituent of triglyceride-rich lipoproteins, has been implicated in the receptor-mediated hepatic uptake of these particles ((Ghiselli, G., et al.. Lancet 2:405-407 (1982)) . The protein is important in modulating the catabolism of remnants of triglyceride-rich lipoprotein particles (Gregg, R.E. et al.. J. Clin. Invest. 78 =815-821 (1986)), in particular, by facilitating the plasma transport of cholesterol (Lalazar, A. et al .. J. Biol. Chem. 263:3542-3545 (1988)) . It is a polymorphic protein with the three common alleles coding for apoE2, apoE3 , and apoE4 (Utermann, G. et al . , J. Lipid Res . 5:378-382 (1984) ; Utermann, G., J. Inner. Metab. Pis. 11:74-86 (1988) ; Ghiselli, G., et al .. Lancet 2:405-407 (1982) ) . In addition to these major isoforms of apoE, minor variant isoforms (apoEl, apoE5 , and apoE7) have been detected by isoelectric focusing (Maeda, H. et al .. J. Biochem. (Tokyo) 105:51-54 (1989)) .
ApoE3 is considered the normal isoform. The apoE2 variant possesses a Cys substituted for an Arg at residue 158 in its amino acid sequence. The apoE4 variant possesses an Arg substituted for Cys at residue 112 in its amino acid sequence, and lacks cysteine residues (Weisgraber. K.H., J. Lipid Res. 31:1503-1512 (1990) ; Utermann, G. et al .. J. Lipid Res. 25 :378-382 (1984) ) .
Apolipoprotein (apo) E polymorphism has a significant effect on plasma cholesterol and low density lipoprotein cholesterol concentrations (Wardell, M.R. et al . , J. Lipid. Res. 12:521-528 (1991)) . The common variants, apoE2 and apoE4 have a significant impact on interindividual variation of lipid and lipoprotein levels in normal subjects. The common variant apoE2 and more than half a dozen rare variants are defective in binding to the low-density lipoprotein (LDL) receptor, and all are causally associated with the lipid disorder, type III hyperlipoproteinemia (HLP) . The mode of inheritance of the disorder can be either dominant or recessive, depending on the particular mutation(s) in apoE, although the mechanisms involved are not fully understood (Rail, S.C. et al .. J. Intern. Med. 231:653- 659 (1992) ) . The apoE4 isoform is associated both with HPL and type V HPL (Gregg, R.E. et al . , J. Clin. Invest. 21:815-821 (1986) ; Kuusi, T. et al .. J. Lipid Res . 11293-298 (1988) ; Yanagi, H. et al . , Clin. Genet. 18:264-269 (1990)) . Diabetic patients with the ApoE4 allele appear to be more susceptible to HPL than diabetic patients with other alleles (Eto., M. et al .. Diabetes 361301-1306 (1987) ) . In contrast, diabetic patients with apoE2 were characterized by increased levels of plasma triglyceride, total cholesterol, very low density lipoprotein (VLDL)-chol, and apoE and an increased VLDL-chol/VDLD-triglyceride ratio, i.e. the accumulation of remnants . In addition, fasting plasma glucose and he oglobin- Al levels were significantly higher in hyperlipoproteinemic diabetic patients with apoE2 than in normolipidemic diabetic patients with apoE2 (Eto, M. et al .. J. Clin. Endocrinol . Metab. 69:1207-1212 (1989) ) . Indeed, homozygosity for ApoE2 invariably gives rise to dysbetalipoproteinemia, and when associated with obesity or a gene for hyperlipidemia, results in type III hyperlipoproteinemia (Wardell, M.R. et al . , J. Lipid Res. 31:535-544 (1990) ) . Thus, diabetes appears to predispose individuals having the ApoE2 allele to hyperlipoproteinemia (particularly type III) and may be a factor linking diabetes with hyperlipoproteinemia and cardiovascular disease (Eto, M. et al .. J. Clin. Endocrinol. Metab. 69:1207-1212 (1989)) . Gene sequences that encodes several of the ApoE isoforms has been cloned (Gill, L.L. et al .. Biochem. Biophys. Res. Commun. 130:1261-1266 (1985) ; Maeda, H. et al.. J. Biochem. (Tokvo) 105:491-493 (1989) ; Horie, Y. et al.. J. Biol. Chem. 267:1962-1968 (1992)) .
Without some type of effective treatment,
Alzheimer's disease would probably affect one out of every 10 humans alive today. To date there is no treatment for Alzheimer's disease at any stage of its development. Two therapeutic reagents, Cognex and Menthane, appear to give slight relief to some victims but do not alter the course of the disease. In view of the importance of diagnosing, predicting, and treating Alzheimer's disease and hyperlipoproteinemia and cardiovascular disease, an affective means for achieving these goals would be highly desirable. The present invention supplies such means.
BRIEF DESCRIPTION OF THE FIGURES:
Figure IA shows the protein coding sequence of "Pac" cDNA and the deduced sequence of the protein is shown in Figure IB. "Tpac" cDNA is reconstructed from negative strands of exons and from introns in the amyloid precursor related human APRP gene. The protein coding sequence of the cDNA is shown in Figure 1C and the deduced sequence of the protein in the embodiment of Figure ID.
Figure 2 shows the sequence of cDNA molecules. The protein coding sequence of "hsaN" cDNA is shown in (A) and the deduced sequence of the protein is shown in (B) . Figure 3A shows the nucleotide sequence of cDNA encoding the apoE4L protein. Figure 3B shows the amino acid sequence of the protein. In Figure 3B, raised "p" indicates kinase c phosphorylation sites,- "pck" identifies casein kinase phosphorylation sites; a down arrow indicates the location of a cleavage/secretory site. Double underscoring is used to denote sequences with significant identity and homology to apoE4.
Figure 4A shows the nucleotide sequence of cDNA encoding the apoE4Ll protein. Figure 4B shows the amino acid sequence of the protein. In Figure 4B, the raised "p" indicates kinase c phosphorylation sites,- a down arrow indicates the location of a cleavage/secretory site.
Figure 5 shows the upstream (5') regions that are responsible for the transcription of the ApoE4L/Ll/Lx2 proteins. Figure 6A shows the nucleotide sequence of cDNA encoding the apoE4Lx2 protein. Figure 6B shows the amino acid sequence of the protein. In Figure 6B, the raised "p" indicates kinase c phosphorylation sites,- a down arrow indicates the location of a cleavage/secretory site. The amino acids that join the E4L protein to the E4L1 protein are underlined. Double inderscoring is used to denote sequences with significant identity and homology to apoE4.
SUMMARY OF THE INVENTION:
The invention concerns agents and methods for the diagnosis and therapy of Alzheimer's disease and related conditions, as well as hyperlipoproteinemia and associated cardiovascular disease. Such agents include a proteinase/esterase-like protein implicated in Alzheimer's Disease, as well as analogues and derivatives of this molecule, and nucleic acid molecules encoding such molecules, or influencing their expression. In detail, the invention provides a nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2. In particular, the invention provides the above-described nucleic acid molecule wherein the sequence is SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 12.
The invention also provides a protein, substantially free of natural contaminants, selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2. In particular, the invention provides the above-described protein having a sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10 or SEQ ID NO: 13. The invention also provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of Pac, a Pac-encoding nucleic acid molecule, Pac-l, a Pac-l-encoding nucleic acid molecule, Pac-2, a Pac-2-encoding nucleic acid molecule, Pace, a Pace-encoding nucleic acid molecule, Tpac, a Tpac- encoding nucleic acid molecule, Tpac-2, a Tpac-encoding nucleic acid molecule, hsaP, an hsaP-encoding nucleic acid, ApoE4L, an ApoE4L-encoding molecule, ApoE4Ll, an ApoE4Ll-encoding molecule, ApoE4Lx2, and an ApoE4Lx2- encoding molecule.
The invention particularly concerns the embodiments wherein the reagent is a nucleic acid molecule, (especially a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:12., or a nucleic acid molecule obtainable by mutating a
•$ nucleic acid molecule having a sequence of SEQ ID NO.-l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:12) or a protein (especially an antibody, or a fragment of an antibody, which is capable of binding to Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2) .
The invention also provides a method of treating Alzheimer's disease, Down's Syndrome, Parkinson's
Disease Schizophrenia, hyperlipoproteinemia, or other cardiovascular disease which comprises providing to an individual, in need of such treatment, an effective amount of an inhibitor of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2.
DESCRIPTION OF THE PREFERRED EMBODIMENTS:
Although it has been recognized that Alzheimer's disease reflects the abnormal processing of APP to produce /3/A4 protein, the exact mechanism of this processing has not been elucidated. Studies have demonstrated that at least 3 different forms of APP are produced in neuronal cells (Roch, J.M. et al . , J. Biol. Chem. 267:2214-2221 (1992)) . Two of these forms (having 751 and 770 amino acids) contain a domain that has substantial homology to Kunitz-type protease inhibitors. Normally, APP is processed to form AP throughout life, however, in Alzheimer's disease, an abnormal AP -- /S/A4 protein -- which is longer at the C-terminal by 2 or 3 amino acids is expressed. The deposition of this abnormal protein in plaques coupled with a constant destruction of acetylcholine in neurons cause the dementia and other degenerative symptoms that characterize the advanced stages of the disease. Researchers have proposed that APP is a cell surface receptor or a transmembrane protein, in which the β/A domain is partly embedded in the cell membrane. The secretion of the 3/A4 protein thus reflects the cleavage of the domain from the precursor molecule (see, Roch, J.M. et al.. J. Biol. Chem. 267:2214-2221 (1992)) . Under normal physiological conditions, the proteolysis of APP into AP is believed to prevent the formation of β/AA protein. In sum, the consensus of all experimental evidence suggests that the β/AA variant of AP protein plays a major role in the expression of Alzheimer's disease, and that the β/AA protein is produced via proteolysis. The identity of the protease responsible for β/AA formation has hitherto been unknown.
The present invention derives, in part, from the discovery of a protein with proteinase and acetylcholinesterase domains that catalyzes the formation of the β/AA protein. The protease is encoded by a copy of the antisense strand of the APP gene. The present invention also derives in part from the discovery of a truncated, modified version of the protease that is encoded on the antisense strand of a gene -- the human APRP gene -- that is structurally and functionally related to APP. The present invention provides the sequence of these molecules, as well as that of the cDNA that encodes them. These molecules may be used in the diagnosis, prediction and treatment of Alzheimer's disease. The present invention also derives, in part, from the recognition of the role of the ApoE gene in human Alzheimer's disease, and in cardiovascular disease, such as hyperlipoproteinemia. In particular, the invention relates to the discovery two tandem open reading frames ("orfs") on the antisense strand of ApoE4 gene. The first orf, designated herein as "ApoE4L" or "E4L, " commences 82 base pairs (bp) upstream from the ApoE4 stop translation signal (nucleotide 4403 on the coding strand) and terminates with a TAG codon at nucleotide 3817. The second orf, designated herein as "ApoE4Ll" or "E4L1, " is located within an exon, and encodes a putative 198 amino acid protein. Because the two orfs are in the same reading frame, a mutation that obliterates the termination codon of E4L results in the creation of a fusion protein, designated herein as
"ApoE4Lx2" or "E4Lx2."
Alternate splicing occurs in many eukaryotic genes, and since different spliced varieties of mRNAs are found in different cell types, it is believed that splicing is tissue specific and may be a method of local environmental control over the function of some gene(s) . The present invention further concerns gene sequences produced via such alternate splicing mechanisms, and the proteins that such gene sequences encode.
I. The Molecules of the Present Invention
The molecules of the present invention comprise Pac, Tpac, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2, as well as the analogs of these proteins, and gene sequences that encode them. The molecules of the invention also include the novel set of gene sequences and encoded proteins (Pac-l, Pac-2, Pace, Tpac-1) ithat result from the alternative splicing of the mRNA antisense to the amyloid forming APP gene. These molecules of the present invention are described below in greater detail.
A. The Pac Protease
The negative strand of exons and introns in the human APP gene was evaluated in a search for the putative Alzheimer's disease protease. This evaluation led to the recognition that the APP antisense strand encoded a protein -- designated herein as "Pac." The protein-encoding portion of the Pac cDNA sequence is shown in Figure IA (SEQ ID NO:l) and the deduced sequence of the protein in Figure IB (SEQ ID NO:2) . The cDNA encoding "Pac" is 100% homologous to a region of the negative strand of the human APP mRNA, starting at amino acid 7 within the AD protein. The molecular weight of the Pac proteinase/esterase was found to be 12,040 kd, and to comprise 104 amino acid residues. The protein is predicted to be unstable.
B. The Tpac Protease
In a similar manner to that described above, the introns and exons of the antisense strand of the amyloid precursor-related gene ("APRP") was evaluated, and found to encode a protein designated herein as "Tpac." The protein-encoding portion of the Tpac cDNA sequence is shown in Figure 1C (SEQ ID NO:3) and the deduced amino acid sequence of the protein is shown in Figure ID (SEQ ID NO:4) . The mRNA encoding "Tpac" is 100% homologous with a region of the negative strand of APRP mRNA which traverses a portion of the AD protein, the protease inhibitor domain and an intron of APRP protein.
C. The Acidic Activator Protein, hsaP A number of patients from families with inherited Alzheimer disease exhibit mutations on chromosome #14 in the region of the major heat shock 70 kb protein (hsp70) . We have constructed a cDNA (hsaN) shown in (A) of Figure 2 which is 100% homologous to nucleotides 313 to 471 inclusive on the antisense strand of the human APP gene (SEQ ID NO:5) . The cDNA encodes a 5.45 kD, 51 amino acid, intracellular, acidic activator protein (hsaP) , shown in (B) of Figure 2 (SEQ ID NO:6) . hsaP shares >75% homology with a 45 amino acid domain of hsp70 in which 16 of 18 possible hydrophobic amino acid residues match, strongly indicating that hsaP can interact specifically with hsp70.
Under normal physiological conditions, the sense/ antisense relationship between APP and hsaP will not allow the expression of hsaP in health humans,- however, acquired mutations in regions of the APP gene involved in antisense repression of hsaP will allow hsaP to be expressed. This free, intracellular, hsaP is "neutralized" through interaction with wild type hsp70, as is the case with some other undesirable intracellular proteins.
Mutations in any of several dozen genes which share the hsp70 region in chromosome #1 can be the cause of inherited Alzheimer's disease. The role of hsp70 in the disease may be caused by inherited mutations that reside in the region of the gene and that are critical for interaction with expressed hsaP. Thus, the pathophysio- logical symptoms of Alzheimer's disease in these families may be the result of the inherited mutation on chromosome #14 and an acquired mutation on APP. Alternatively, either hsa or pac may be an Alzheimer's locus on human chromosome #14.
Nevertheless, regardless of the mechanisms of intracellular interaction between hsaP and hsp70, humanized antibodies prepared against hsaP, or ribozymes prepared from selected regions of the APP gene against hsaN, will safely and efficiently prevent and cure Alzheimer's disease in such families.
D. Comparison between Pac and Tpac
It was observed, from similarities, that the deduced protein encoded by Pac and Tpac are highly related, and that both are related to lysosomal membrane proteins and have distinct domains. Three domains "Pase" (endo-proteinase) ,- "Acase" (acetylcholinesterase) and "Ease" (esterase) are indicated in Figure IB. A domain "Btox" for botulism toxin (a zinc metalloprotease which blocks neurotransmitter release form neurons) was found to partially overlap the "Ease" domain.
The deduced protein encoded by "Tpac" is a truncated form of "Pac." The protein was found to be truncated at amino acid #23 and four new amino acids --
CITS were found to have been added to the truncated c-terminal end of the protein. It comprises only the proteinase domain of "Pac." This domain has significant identity and homology with domains in, viper Russelli proteinase, human cathepsin A, human complement c3 convertase, RNA 1 polyprotein protease cofactor, and a species of yeast killer toxin with carboxypeptidase-like activity. In addition "Tpac" has strong similarity and homology to the powerful, 50 amino acid, testes DNA binding "protamine Zl" protein. The latter protein forms highly condensed chromatin packets consisting of membranes and chromosomal DNA in sperm heads.
E. Mechanisms of Pac and Tpac Action
The factors responsible for inducing the expression of "Pac" and "Tpac" have not been fully identified, but such information is unnecessary for the purposes of the present invention. Presumably, these proteins are not normally expressed in healthy humans because the mRNA encoding them is in an antisense relationship with APP/APRP mRNA. Without limitation, there are several ways in which "Pac" may be involved as the causative factor in Alzheimer's disease. The 12.4 kd native Pac protein or two or more similar subunits, acting together, may actively cut out the abnormal β/AA protein from APP and actively destroy acetylcholine. Alternatively, the 12.4 kd native Pac protein may associate with other dissimilar protein subunits to form a polyprotein which can mediate such enzymatic activities. In another alternative, the 12.4 kd protein acting may act as an activator of inactive proteinase and acetylcholinesterase zymogens to form active enzyme molecules which can carry out the enzymatic activity. There are several ways in which "Tpac" may be involved as a causative agent in Alzheimer's disease. The 27 amino acid protein which lacks an acetylcholinesterase domain, can function. Tpac may act as a transducer of protease activity in an inactive proteinase. In analogy to the action of its homologue,
"protamine Zl, " Tpac may bind to chromosomal DNA and influence the expression of critical genes. Tpac may alternatively compact the relatively insoluble abnormal AD protein, together with neural cell chromatin and dendrites, into the "intracellular neurofibrillary tangles" found in advanced stages of Alzheimer's disease.
Irrespective of the manner of the involvement of the "Pac" and "Tpac" in Alzheimer's disease any therapeutic method which blocks the expression of these proteins or blocks the proteins 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 sequences described in the embodiment of Figures IB and ID. The invention also includes any cellular proteinase or acetylcholinesterase activated by the proteins or domains of the proteins described in the embodiment of Figures IB and ID.
F. The ApoE4L Protein
The first orf identified in the ApoE antisense sequence encodes the apoE4L protein. The nucleotide sequence of ApoE4L cDNA is shown in Figure 3A (SEQ ID NO: 7) . The apoE4L protein sequence is shown in Figure 3B (SEQ ID NO: 8) . Of particular interest is the sequence 89-104 of SEQ ID NO: 8, which possesses significant identity and homology to apoE4 sequences. Kinase c phosphorylation sites are present at positions 3, 42, 43, 54, 87, 112, 122, and 129 of SEQ ID NO: 8. Casein kinase phosphorylation sites are present at 176- 177 and 193-194 of SEQ ID NO: 8. Cleavage/secretory sites occur at positions 43-44 and 182-183.
G. The ApoE4Ll Protein
The second orf identified in the ApoE antisense sequence encodes the apoE4Ll protein. The second orf commences 16 bp downstream from E4L stop codon, nucleotide 3801, in the same reading frame, and terminates within the adjoining intron at nucleotide 3558. This second orf encodes a putative 107 amino acid long protein designated herein as "E4L1". Only 21 bp of exon sequence is included in the E4L1 sequence. The nucleotide sequence of cDNA encoding the apoE4Ll protein is shown in Figure 4A (SEQ ID NO: 9) .
The apoE4Ll protein sequence is shown in Figure 4B (SEQ ID NO:10) . A kinase c phosphorylation site is present at position 54 of SEQ ID NO: 10. A cleavage/secretory site occurs at positions 46-47. H. Transcriptional Regulation of the ApoE4L and ApoE4Ll Gene Sequences
Both the Apo4L and Apo4Ll orfs can be transcribed from promoter elements and coordinated GC box and cap sites that are located 300-500 bp upstream from the
E4L/L1 orf. The sequence of this upstream regulatory region is SEQ ID NO: 11 (Figure 5) . Promoter elements are present at positions 12-26 and 322-335 in SEQ ID NO:11. A GC box is present at position 206-219. Cap sites are present at positions 55 and 338 in SEQ ID
NO:11. A ribosome binding site is present 5 nucleotides from the end of the sequence. The sequence "TATAA" located 100 bp downstream of the E4L1 stop codon might be used as a transcription termination signal, however, there is a perfect consensus termination signal at nucleotide 2495 which may alternatively signal the end of the putative antisense prime transcript.
I. The ApoE4Lx2 Protein
Despite the allelic differences of ApoE2, ApoE3 and ApoE4, the antisense molecules encoded by each of these alleles still encodes the E4L and E4L1 proteins,- however ApoE4 comprises two alleles. In one form the stop codon (TAG) which closes E4L is replaced by GCT. As a result of this mutation, the E4L and E4L1 combine with the addition of six new codons to form a hybrid orf, designated herein as "E4Lx2" which has coding information for 311 amino acids. The nucleotide sequence that encodes E4Lx2 is presented in Figure 6A (SEQ ID NO:12) ; the six new codons are underlined. The amino acid sequence of E4Lx2 (SEQ ID NO: 13) is εhown in Figure 6B. Of particular interest is the sequence 89-104 of SEQ ID NO:13, which possesses significant identity and homology to apoE4 sequences. Kinase c phosphorylation sites are present at positions 3, 42, 43, 54, 87, 122, 129, 152 and 303 of SEQ ID NO:13. Casein kinase phosphorylation sites are present at 176-177 and 193-194 of SEQ ID NO:13.
Cleavage/secretory sites occur at positions 35-36, 43-44 and 182-183 and 295-296. A glycosylation site is present at position 202.
The E4L protein is extremely basic (pi = 12.57, charge at pH 7.0 = 38.34, MW= 22 kD, 9 potential phosphorylation sites) . It appears to be a DNA binding, trans-activating transcriptional activator-type protein with the structural characteristics of powerful protamines. There are three secretory sites which could provide four different biologically "active" polypeptides. The E4L1 protein is a basic protein (pi 11.09, MW = 11.8 kD, one potential phosphorylation site) ,- it appears to be a DNA binding protein and may also act as a coupled G-receptor signal transducer. The E4Lx2 protein has the combined primary characteristics of the E4L protein as well as those of the E4L1 protein, plus a potential glycosylation site provided by the codons linking the two proteins.
The E4Lx2 protein has several additional potentially functional characteristics including significant identity and homology with the herpes associated "infected cell protein, " and potential protease activity. The primary structure of E4Lx2 appears to make this protein more likely than apo4E to be responsible for the role in growth and repair of the nervous system during development and injury ascribed to apoE4,- and, also, for the tight binding to the 3-amyloid protein -- which, as indicated above is the central player in the pathophysiology of human Alzheimer's disease.
In view of the involvement of apolipoprotein in Alzheimer's disease, it is therefore likely that E4Lx2, rather than mutations in the ApoE4 protein, is implicated in causing or contributing to late-onset familial Alzheimer's disease. Moreover, E4L and E4L1 appear to be involved in other diseases associated with ApoE2, ApoE3, and ApoE4 -- such as hyperlipoproteinemia and related cardiovascular conditions.
Molecules resulting from Alternative/Differential Splicing
As indicated above, alternate splicing occurs in many eukaryotic genes, and since different spliced varieties of mRNAs are found in different cell types, it is believed that splicing is tissue specific and may be a method of local environmental control over the function of some gene(s) . The present invention further concerns gene sequences produced via such alternate splicing mechanisms, and the proteins that such gene sequences encode.
The spliced DNA and encoded proteins described here are probably involved in the neuropathology of Alzheimer's disease. Alternate splicing of the mRNA antisense to the amyloid forming APP gene, gives rise to a set of novel gene sequences and encoded proteins (Pac- 1, Pac-2, Pace, Tpac-1) whose sequences are presented below.
The above-described Tpac molecule plays a central role in the formation of the amyloid plaques that are characteristic of Alzheimer's disease. In Pac-l the separate domains for protease/protamine and for acetylcholinesterase associated activity is unchanged and the protein's changes in amino acid composition occur only in the "center" domain of the protein which has not been associated with any known activity in pac. In pac-2, Tpac is unchanged and can be independently expressed, a different protein ("Pace") is in the acetylcholinesterase domain, and a neuropeptide-type protein, Tpac-2, is encoded in the central domain. Both Pac-l and Pac-2 have potential mitochondrial transit sequences .
The sequences of cDNA molecules Pac-l and Pac-2 are as follows:
Pac-l (SEQ ID NO: 14) :
ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA TATTTGTCAACCCAGAACCTGGTCGAGTGGTCAGTCCTCGGTCGGCAGCA GGGCGGGCATCAACAGGCTCAACTGGGCACAGGAAGCAAGGGACACACAA AGCAAACAAGACAAATCAAGATGGAGAACGCCCTTGCTGGCTCAGGGGAC TCTTACCTTCGTTTTCTGTGTTGGCTGGCACAGAGTCAGCCCCAAAAGAA
TGCCACGGCTGGAGATCGTCCAGGCTGAACTCTCCATTCACGGGAAGGAG CTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGCATGAGAGCATCGTTTC CGTAACTGATGGTTGGTTCACTAATCATGTTGGCCAAGACGTCATCTGA
Pac-2 (SEQ ID NO: 15) : ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA
TATTTGTCAACCCAGAACCTGTATTACATCATAATTAAAGGGTCACTTCA AATTCTACTCTGCAGTAAGATCLAATTGAGAGAGGCTTAAAATGCAGAAAG GAGACAACGTCTGCTCGAGCTTAGGCCCAAGATGCGGAGAGGCAGAAGTC AAGCGGTTGTGATACCTGGTCGAGTGGTCAGTCCTCGGTCGGCAGCAGGG CGGGCATCAAGATGGAGAACGCCCTTGCTGGCTCAGGGGACTCTTACCTT
CGTTTTCTGTGTTGGCTGGCACGAGTCAGCCCCAAAAGAATGCCACGAGT CAGCCCCAAAAGAATGCCACGGCTGGAGATCGTCCAGGCTGAACTCTCCA TTCACGGGAAGGAGCTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGCAT GAGAGCATCGTTTCCGTAACTGATGGTTGGTTCACTAATCATGTTGGCCA AGACGTCATCTGA
The cDNA sequence of Pace is as follows
(SEQ ID NO: 16) :
ATGCGGAGAGGCACAAGTCAAGCGGTTCTGATACCTGGTCGAGTGGTCAG TCCTCGGTCGGCAGCAGGGCGGGCATCAACAGGCTCAAGTGGGCACAGGA AGCAAGGGACACAGAAAGCAAACAAGACAAATCAAGATGGAGAACGCCCT
TGCTGGCTCAGGGGACTCTTACCTTCGAAATCTGTGTTGGCTGGCACAGA GTCAGCCCCAAAAGAATGCCACGGCTGGAGATCGTCCAGGCTGAACTCTC CATTCACGGGAAGGAGCTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGC ATGAGAGCATCGTTTCCGTAA
The cDNA sequence of Tpac-1 is as follows
(SEQ ID NO: 17) :
ATGCAGAAAGCAGACAACGTCTGCTCGAGCTTAGGCCCAAGATGCGGAGA GGCACAAGTCAAGCGGTTCTGA
The deduced amino acid sequences of the proteins expressed by antisense mRNAs, Pac-l and Pac-2, produced by the alternative/differential splicing in the pac antisense gene is as follows:
Pac-l (SEQ ID NO:18) :
MSEFCIHLHFRDLLRLDICQPRTWSSGQSSVGSRAGINRLKWAQEARDTE SKQDKSRWRTPLLAQGTLTFEICVGWHRVSPKRMPRLEIVQAELSIHGKE
PKRMPRLEIVQAELSIHGKELHGGFRFGQRWHESIVSVTDGWFTNHVGQDVI
Tpac-2 (SEQ ID NO:19) : MQKGDNVCSSLGPRCGEAQVKRF
Pace (SEQ ID NO:20) :
MRRGTSQAVLIPGRWSPRSAAGRASTGSSGHRKQGTQKANKTNQDGERP CWLRGLLPSKSVLAGTESAPKECHGWRSSRLNSPFTGRSSTWFVSVKDG
MRASFP
II. The Uses of the Molecules of the Present Invention
The elucidation of the significance of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2 in the etiology of Alzheimer's disease provides 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 addition, because of the pathological similarity between Alzheimer's disease and Down's Syndrome, Parkinson's Disease and Schizophrenia, the molecules and methods of the present invention can be used in the diagnosis and treatment of these diseases and conditions. In particular, any of the proteins described herein, 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 neither Pac, Tpac, hsaP nor the other antisense sequences described herein are expressed by normal cells, the detection of these molecules in a tissue or fluid sample -- such as a biopsy sample, or a blood or spinal fluid sample -- is indicative of the presence of Alzheimer's disease in a patient. Similarly, detection of ApoE4L, ApoE4Ll, and, in particular, ApoE4Lx2 in such a sample is indicative of Alzheimer's disease, as well as cardiovascular disorders (such as hyperlipoproteinemia, etc.) .
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 Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 molecules, and the presence of such molecules is determined via and 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 Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 as an antigen. Such molecules are preferably obtained through the expression of the gene sequences described herein. The simplest immunoassay involves merely incubating an anti-Pac, anti-Tpac, anti-hsaP, anti-ApoE4L, anti- ApoE4Ll, anti-ApoE4Lx2, etc. antibody with a sample suspected to contain the target molecule -- Pac, Tpac, hsaP, ApoE4L, ApoE4Ll, ApoE4Lx2, etc. 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 immunoassays, 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 presence 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 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 presence of any target molecules in the sample competes with the labelled target molecules for antibody binding sites. 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.
As 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 kinetics. Such assays 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 equipment. 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 (ELISAs) 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 single enzyme is ideal for use as 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 substrate molecules converted to product per enzyme site per unit of time) , purity of the enzyme preparation, sensitivity of detection of its product, ease and speed 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 its conjugate, and the like. Examples of suitable enzymes include peroxidase, acetylc oline esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, 3-galactosidase, catalase, delta-5-steroid isomerase, glucose oxidase, glucose-6- phosphate dehydrogenase, glucoamylase, glycoamylase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phosphate isomerase, urease, yeast-alcohol dehydrogenase, etc. Peroxidase and urease are among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.
In lieu of such enzyme labels, radioisotopic, chemiluminescent or fluorescent labels may be employed. Examples of suitable radioisotopic labels include 3H, lllln, 15I, 131I, 32P, 3SS, 14C, 51Cr, 57To, 5βCθ, 59Fe, 75Se, 152Eu, 90Y, 67Cu, 21Ci, 11At, 12Pb, 47Sc, 109Pd, etc. Examples of suitable chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester 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 Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 in a cell can be determined by any means capable of detecting mRNA encoding these proteins. Thus, molecules comprising nucleic acid probes capable of hybridizing to such molecules may be used in the diagnosis of Alzheimer's disease. As used herein, a "probe" is a detectably labelled nucleic acid molecule that is capable of hybridizing to a defined site of a target molecule. Any of the nucleotide sequences disclosed herein can be used as 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 bases (Miyoshi et al .. EP 119448), etc.
Such nucleic acid based assays may use either DNA or RNA to detect the Pac, Pac-l, Pac-2, Pace, Tpac,
Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA. In one embodiment, the assays may be performed on RNA that has been extracted from neuronal cells. Alternatively, and more preferably, the assays may be done in situ on biopsied tissue.
Where the concentration of such 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 Svmp. 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 systems (Kwoh D et al .. Proc. Natl . Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras TR et al.. PCT appl. WO 88/10315 (priority: US Patent applications serial nos. 064,141 and 202,978); Davey, C. et al. (European Patent Application Publication no. 329,822), etc.
In yet another embodiment, the diagnosis of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 expression is performed using a ribozyme produced from nucleic acid molecules having a sequence of such molecules.
B. Prognostic Uses
The present invention additionally provides a capacity to predict whether an individual is at risk for Alzheimer's disease. Thus, any of the above-described assays may be performed on an asymptomatic individual in order to assess that individual's predisposition to Alzheimer's disease.
C. Therapeutic Uses
Significantly, the present invention provides a means for treating Alzheimer's disease. Such treatment may be either "prophylactic" or "therapeutic." A prophylactic treatment is one that is provided in advance of any symptom of Alzheimer's disease in order to prevent or attenuate any subsequent onset of the disease. A therapeutic treatment is one that is provided in response to the onset of a symptom of Alzheimer's disease, and serves to attenuate an actual symptom of the 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'),, single chain antibodies, etc.) that is capable of binding to Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 . 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 administered. More preferably, and especially for chronic administration, the use of non- immunogenic antibodies is preferred. Such molecules can be pseudo-homologous (i.e. produced by a non-human species, but altered to a form that is immunologically indistinct from human antibodies) . Examples of such pseudo-homologous molecules include "humanized" (i.e. non-immunogenic in a human) prepared by recombinant or other technology. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies, but are less 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 14^:1041-1043 (1988); Liu, A.Y. et al .. Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987) ; Liu, A.Y. et al . , _X_ Immunol. 139:3521-3526 (1987); Sun, L.K. et al.. Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. et al.. Cane. Res. 47:999-1005 (1987); Wood, CR. et al .. Nature 314:446-449 (1985)); Shaw et al .. J^ Natl.Cancer Inst. 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.. BioTechniσues 4:214 (1986) ,- which references 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 .. X_ 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 Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mutant molecules that can complex with and significantly inactivate Pac, Pac- l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Li or ApoE4Lx2 molecules present in a cell. In one sub- embodiment, such mutated protein molecules may be administered to a patient. Alternatively, nucleic acid expressing 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) whose sequence is complementary to the sequence of at least part of the Pac-, Pac-l-, Pac-2-, Pace-, Tpac-, Tpac-2-, hsaP-, ApoE4L-, ApoE4Ll- or ApoE4Lx2-encoding sequences described herein, such that it is capable of binding to, or hybridizing with, an endogenous Pac, Pac- l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA molecule, and thereby impair (i.e. attenuate or prevent) its the translation into Pac, Pac- 1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2. 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 region of the double-stranded DNA genome that encodes Pac, Pac- l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 in a manner sufficient to impair the transcription of either gene. Triplex oligonucleotides 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 must be capable of binding to or hybridizing with that portion of the Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA molecule which mediates the translation of the target mRNA. Antisense oligonucleotides are disclosed in European Patent Application Publication Nos. 263,740; 335,451; and
329,882, and in PCT Publication No. WO90/00624, all of which references 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 translation of a Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 mRNA transcript can be used to impair the expression of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 in a cell, and thereby provide a treatment for Alzheimer's disease.
In general, the antisense oligomer is prepared in accordance with the nucleotide sequence of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 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 hybridizing with the above-described translation locus of either Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 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. Sci. (U.S.A. ) 81:4143 (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.. Mol. Cell. Biol. 8:963 (1988); Gerwirtz, A.M. et al . , Science 242:1303 (1988); Anfossi, G., et al. , Proc. Natl. Acad. Sci. (U.S.A.) 86:3379 (1989); Becker, D., e_t 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 sequence can be obtained from any commercial supplier of such custom molecules.
Most preferably, the antisense 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 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 cytosine and adenosine) or an isobutyryl group (to protect guanosine) . Methods for producing such derivatives are well known in the art.
III. 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 these materials, 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's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)) . In order to form a pharmaceutically acceptable composition suitable for effective administration, such 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 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, methyl- cellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as 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 such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) . In one embodiment of the present invention, nucleic acid molecule(s) comprising antisense or triplex molecules, or encoding mutated Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2 molecules 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 Boggε, 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 individuals who, due to inherited genetic mutations, or somatic cell mutation, are predisposed to Alzheimer'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 Cloning and Expression of "Pac" and "Tpac" cDNA
Oligonucleotides corresponding to "Pac" and "Tpac" cDNA were prepared by oligonucleotide synthesis on an Applied Biosystems synthesizer using the column method according to the recommendations of the manufacturer, and gel purified. For "Pac" the positive and negative strands were synthesized as three fragments, which were ligated to form a continuous strand, and gel purified. For "Tpac" full-length strands were synthesized and gel purified. Appropriate strands were mixed together in equimolar amounts and processed in the EXPRESS system (Invitrogen corporation pTrcHis Xpress-Prokaryotic Expression and Purification system) according to the recommendations of the manufacturer. The sense-antisense relationship between the cDNAs encoding these proteins and the cDNA encoding the amyloid forming APP gene, and the similarities between the amino acid sequences deduced for the protein, and amino acid sequences in proteinases and esterases which are involved in pathogenic processes, make the proteins perfect candidates for factors that cause and maintain human Alzheimer's disease. Since the mRNAs encoding these proteins in healthy humans are repressed by mRNA encoding the normal APP gene and the gene encoding the amyloid related protein APRP363, they are potentially excellent targets for a safe, effective, immunological approach aimed at preventing, perhaps even stopping and reversing, the course of this, and related diseases. 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 principles of the invention and including such departures from the present disclosure as come within known or customary 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 claims. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: PREDDIE, RICK
BERGMANN, JOHANNA
(ii) TITLE OF INVENTION: AGENTS FOR THE PREVENTION AND TREATMENT
OF HUMAN ALZHEIMER DISEASE
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: WEIL, GOTSHAL & MANGES
(B) STREET: 1615 L STREET, N.W., SUITE 700
(C) CITY: WASHINGTON
(D) STATE: D.C.
(E) COUNTRY: US
(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: 683-001-CIP2
(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: 315 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: PAC
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :
ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGCTCTCC TCCGTCTTGA 50
TATTTGTCAA CCCAGAACCT GGTCGAGTGG TCAGTCCTCG GTCGGCAGCA 100
GGGCGGGCAT CAACAGGCTC AACTTCGTTT TCTGTGTTGG CTGGCACAGA 150
GTCAGCCCCA AAAGAATGCC ACGGCTGGAG ATCGTCCAGG CTGAACTCTC 200
CATTCACGGG AAGGAGCTCC ACGGTGGTTT TCGTTTCGGT CAAAGATGGC 250
ATGAGAGCAT CGTTTCCGTA ACTGATCCTT GGTTCACTAA TCAAGTTGGC 300
CAAGACGTCA TGTGA 364
(2) INFORMATION FOR SEQ ID NO:2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 104 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: PAC
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :
Met Ser Glu Phe Cys lie His Leu His Phe Arg Asp Leu Leu Arg Leu 1 5 10 15
Asp lie Cys Gin Pro Arg Thr Trp Ser Ser Gly Gin Ser Ser Val Gly 20 25 30
Ser Arg Ala Gly lie Asn Arg Leu Asn Phe Val Phe Cys Val Gly Trp 35 40 45 His Arg Val Ser Pro Lys Arg Met Pro Arg Leu Glu He Val Gin Ala 50 55 60
Glu Leu Ser He His Gly Lys Glu Leu His Gly Gly Phe Arg Phe Gly 65 70 75 80
Gin Arg Trp His Glu Ser He Val Ser Val Thr Asp Asp Trp Phe Thr
85 90 95
Asn His Val Gly Gin Asp Val He 100
(2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: TPAC
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC TCCGTCTTGA 50 TATTTGTCAA CCCAGAACCT GTATTACATC ATAA 84
(2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: TPAC
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 :
Met Ser Glu Phe Cys He His Leu His Phe Arg Asp Leu Leu Arg Leu 1 5 10 15
Asp He Cys Gin Pro Arg Thr Cys He Thr Ser 20 25
(2) INFORMATION FOR SEQ ID NO: 5 :
(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: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(B) STRAIN: hsaP
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : ATGGGTCTTG CACTGCTTGC GGCCCCGCTT GCACCAGTTC TGGATGGTCA 50 CTGGTTGGTT GGCTTCTACC TCATTGGTGA TCTGCAGTTC AGGGTAGACT 100 TCTTGGCAAT ACTGCAGGAT GCCTTCCTTG GTATCAATGC AGGTTTTGGT 150 CCCTGATGG 159
(2) INFORMATION FOR SEQ ID NO: 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(B) STRAIN: hsaP
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 :
Met Gly Leu Ala Leu Leu Ala Ala Pro Leu Ala Pro Val Leu Asp Gly 1 5 10 15
His Trp Leu Val Gly Phe Tyr His He Gly Asp Leu Gin Phe Arg Val 20 25 30
Asp Phe Leu Ala He Leu Gin Asp Ala Phe Leu Gly He Asn Ala Gly 35 40 45
Phe Gly Pro 50
(2) INFORMATION FOR SEQ ID NO: 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 597 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
(vii) IMMEDIATE SOURCE:
(B) CLONE: APOE4L
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 : ATGTCTTCCA CCAGGGGCTC GAACCAGCTC TTGAGGCGGG CCTGGAAGGC 50 CTCGGCCTGC AGGCGTATCT GCTGGGCCTG CTCCTCCAGC TTGGCGCGCA 100 CCTCCGCCAC CTGCTCCTTC ACCTCGTCCA GGCGGTCGCG GGTCCGGCTG 150 CCCATCTCCT CCATCCGCGC GCGCAGCCGC TCGCCCCAGG CCTGGGCCCG 200 CTCCTGTAGC GGCTGGCCGG CCAGGGAGCC CACAGTGGCG GCCCGCACGC 250 GGCCCTGTTC CACCAGGGGC CCCAGGCGCT CGCGGATGGC GCTGAGGCCG 300 CGCTCGGCGC CCTCGCGGGC CCCGGCCTGG TACACTGCCA GGCGCTTCTG 350
CAGGTCATCG GCATCGCGGA GGAGCCGCTT ACGCAGCTTG CGCAGGTGGG 400
AGGCGAGGCG CACCCGCAGC TCCTCGGTGC TCTGGCCGAG CATGGCCTGC 450
ACCTCGCCGC GGTACTGCAC CAGGGCCCCG CGCACGTCCT CCATGTCCGC 500
GCCCAGCCGG GCCTGCGCCG CCTGCAGCTC CTTGGACAGC CGTGCCCGCG 550
TCGCCTCCGC CACCGGGGTC AGTTGTTCCT CCAGTTCCGA TTTGTAG 597
(2) INFORMATION FOR SEQ ID NO: 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: APOE4L
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :
Met Ser Ser Thr Arg Gly Ser Asn Gin Leu Leu Arg Arg Ala Trp Lys
1 5 10 15
Ala Ser Ala Cys Arg Arg He Cys Trp Ala Cys Ser Ser Ser Leu Ala 20 25 30
Arg Thr Ser Ala Thr Cys Ser Phe Thr Ser Ser Arg Arg Ser Arg Val 35 - 40 45
Arg Leu Pro He Ser Ser He Arg Ala Arg Ser Arg Ser Pro Gin Ala 50 55 60
Trp Ala Arg Ser Cys Ser Gly Trp Pro Ala Arg Glu Pro Thr Val Ala 65 70 75 80
Ala Arg Thr Arg Pro Cys Ser Thr Arg Gly Pro Arg Arg Ser Arg Met
85 90 95
Ala Leu Arg Pro Arg Ser Ala Pro Ser Arg Ala Pro Ala Trp Tyr Thr 100 105 110
Ala Arg Arg Phe Cys Arg Ser Ser Ala Ser Arg Arg Ser Arg Leu Arg 115 120 125
Ser Leu Arg Arg Trp Glu Ala Arg Arg Thr Arg Ser Ser Ser Val Leu 130 135 140
Trp Pro Ser Met Ala Cys Thr Ser Pro Arg Tyr Cys Thr Arg Ala Pro 145 150 155 160
Arg Thr Ser Ser Met Ser Ala Pro Ala Arg Ala Cys Ala Ala Cys Ser
165 170 175
Ser Leu Asp Ser Arg Ala Arg Val Ser Ser Ala Thr Gly Val Ser Cys 180 185 190
Ser Ser Ser Ser Asp Leu 195
(2) INFORMATION FOR SEQ ID NO:9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 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
(vii) IMMEDIATE SOURCE:
(B) CLONE: AP0E4L1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :
ATGGTCTCGT CCATCTGCGC CGTCCGGCCG AGAGGGCGGG AGGGTGTCGC 50
ACAGTGGGGA GGCGGGGGAG AAGGGCTGGG ATGGGGCGGG CGAGATGGGG 100
ATGAGCCAGA GAGACCCAAG AGGGAGAGAA GGAACGGGGC AGAGGCCGAG 150
AGAAGGAGAC AGAGACAGAT GCAGAGAGCA GATGCAGAGG GCAGAGGCAG 200
AGACGAAGAA GGAGCTAGGA GGCCGGGCAA GGTGCTCATG CCTCTAATCC 250
CAGCACTTTG GGAGGCCGAG GCGGGAGGAT CGCTTGAGCC CAGAAGTTCA 300
AGACCAGCCT GGGCAACACA GTGA 324 \ 2 ) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: APOE4L1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Val Ser Ser He Cys Ala Val Arg Pro Arg Gly Arg Glu Gly Val 1 5 10 15
Ala Gin Trp Gly Gly Gly Gly Glu Gly Leu Gly Trp Gly Gly Arg Asp 20 25 30
Gly Asp Glu Pro Glu Arg Pro Lys Arg Glu Arg Arg Asn Gly Ala Glu 35 40 45
Ala Glu Arg Arg Arg Gin Arg Gin Met Gin Arg Ala Asp Ala Glu Gly 50 55 60
Arg Gly Arg Asp Glu Glu Gly Ala Arg Arg Pro Gly Lys Val Leu Met 65 70 75 80
Pro Leu He Pro Ala Leu Trp Glu Ala Glu Ala Gly Gly Ser Leu Glu
85 90 95
Pro Arg Ser Ser Arg Pro Ala Trp Ala Thr Gin 100 105
[ 2 ) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomicl (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTCTACTAAA AATACAAAAA TTAGCCGGGT GTGGTGGTGT GAGCCTGTAA 50 TCCCAGCTAC TGAGGCAGCA GAATCGCTTG AACCCAAGAG GCAGAGGTTG 100 CAGTGAGCCA AGATCGTGCC ACTGCACTCT AGCCTGGGTG ACAGAGCCAG 150 ACTCCGTCTA AAAAAAAAAA AAGGGCAGAG AGAAAGATAC ACACAGACAC 200 AGATGGAGAG GAGGGGGCTG AGAATTGTGT GGCAGTATGT GGGCAGAAAG 250 AGAAACTGAG GCTGGGGCTT AGAGGAAATC ACAGGGGGAG GCCAGCAGAT 300 GCGTGAAACT TGGTGAATCT TTATTAAACT AGGGTCCACC CCAGGAGGAC 350 GGCTGGGGCG GGGACAGGGT CTCCCGCTGC AGGCTGCGCG GAGGCAGGAG 400 GCACGGGGTG GCGTGGGGTC GCATGGCTGC AGGCTTCGGC GTTCAGTGAT 450 TGTCGCTGGG CACAGGGGCG GCGCTGGTGC CCACGGCAGC CTGCACCTTC 500 TCCACCAGCC CGGCCCACTG GCGCTGCATG TC 532
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 936 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
(vii) IMMEDIATE SOURCE:
(B) CLONE: APOE4LX2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATGTCTTCCA CCAGGGGCTC GAACCAGCTC TTGAGGCGGG CCTGGAAGGC 50 CTCGGCCTGC AGGCGTATCT GCTGGGCCTG CTCCTCCAGC TTGGCGCGCA 100 CCTCCGCCAC CTGCTCCTTC ACCTCGTCCA GGCGGTCGCG GGTCCGGCTG 150 CCCATCTCCT CCATCCGCGC GCGCAGCCGC TCGCCCCAGG CCTGGGCCCG 200 CTCCTGTAGC GGCTGGCCGG CCAGGGAGCC CACAGTGGCG GCCCGCACGC 250 GGCCCTGTTC CACCAGGGGC CCCAGGCGCT CGCGGATGGC GCTGAGGCCG 300 CGCTCGGCGC CCTCGCGGGC CCCGGCCTGG TACACTGCCA GGCGCTTCTG 350 CAGGTCATCG GCATCGCGGA GGAGCCGCTT ACGCAGCTTG CGCAGGTGGG 400 AGGCGAGGCG CACCCGCAGC TCCTCGGTGC TCTGGCCGAG CATGGCCTGC 450 ACCTCGCCGC GGTACTGCAC CAGGGCCCCG CGCACGTCCT CCATGTCCGC 500 GCCCAGCCGG GCCTGCGCCG CCTGCAGCTC CTTGGACAGC CGTGCCCGCG 550 TCGCCTCCGC CACCGGGGTC AGTTGTTCCT CCAGTTCCGA TTTGGCTGCC 600 TTCAACTCCT CCATGGTCTC GTCCATCTGC GCCGTCCGGC CGAGAGGGCG 650 GGAGGGTGTC GCACAGTGGG GAGGCGGGGG AGAAGGGCTG GGATGGGGCG 700 GGCGAGATGG GGATGAGCCA GAGAGACCCA AGAGGGAGAG AAGGAACGGG 750 GCAGAGGCCG AGAGAAGGAG ACAGAGACAG ATGCAGAGAG CAGATGCAGA 800 GGGCAGAGGC AGAGACGAAG AAGGAGCTAG GAGGCCGGGC AAGGTGCTCA 850 TGCCTCTAAT CCCAGCACTT TGGGAGGCCG AGGCGGGAGG ATCGCTTGAG 900 CCCAGAAGTT CAAGACCAGC CTGGGCAACA CAGTGA 936
(2) INFORMATION FOR SEQ ID NO : 13 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM : HOMO SAPIENS
(vii) IMMEDIATE SOURCE: (B) CLONE: APOE4LX2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ser Ser Thr Arg Gly Ser Asn Gin Leu Leu Arg Arg Ala Trp Lys 1 5 10 15
Ala Ser Ala Cys Arg Arg He Cys Trp Ala Cys Ser Ser Ser Leu Ala 20 25 30
Arg Thr Ser Ala Thr Cys Ser Phe Thr Ser Ser Arg Arg Ser Arg Val 35 40 45
Arg Leu Pro He Ser Ser He Arg Ala Arg Ser Arg Ser Pro Gin Ala 50 55 60
Trp Ala Arg Ser Cys Ser Gly Trp Pro Ala Arg Glu Pro Thr Val Ala 65 70 75 80
Ala Arg Thr Arg Pro Cys Ser Thr Arg Gly Pro Arg Arg Ser Arg Met
85 90 95
Ala Leu Arg Pro Arg Ser Ala Pro Ser Arg Ala Pro Ala Trp Tyr Thr 100 105 110
Ala Arg Arg Phe Cys Arg Ser Ser Ala Ser Arg Arg Ser Arg Leu Arg 115 120 125
Ser Leu Arg Arg Trp Glu Ala Arg Arg Thr Arg Ser Ser Ser Val Leu 130 135 140
Trp Pro Ser Met Ala Cys Thr Ser Pro Arg Tyr Cys Thr Arg Ala Pro 145 150 155 160
Arg Thr Ser Ser Met Ser Ala Pro Ala Arg Ala Cys Ala Ala Cys Ser
165 170 175
Ser Leu Asp Ser Arg Ala Arg Val Ser Ser Ala Thr Gly Val Ser Cys 180 185 " 190
Ser Ser Ser Ser Asp Leu Ala Ala Phe Asn Ser Ser Met Val Ser Ser 195 200 205
He Cys Ala Val Arg Pro Arg Gly Arg Glu Gly Val Ala Gin Trp Gly 210 215 220
Gly Gly Gly Glu Gly Leu Gly Trp Gly Gly Arg Asp Gly Asp Glu Pro 225 230 235 240
Glu Arg Pro Lys Arg Glu Arg Arg Asn Gly Ala Glu Ala Glu Arg Arg
245 250 255
Arg Gin Arg Gin Met Gin Arg Ala Asp Ala Glu Gly Arg Gly Arg Asp 260 265 270 Glu Glu Gly Ala Arg Arg Pro Gly Lys Val Leu Met Pro Leu He Pro 275 280 285
Ala Leu Trp Glu Ala Glu Ala Gly Gly Ser Leu Glu Pro Arg Ser Ser 290 295 300
Arg Pro Ala Trp Ala Thr Gin 305 310
, 2 ) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 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
(vii) IMMEDIATE SOURCE:
(B) CLONE: Pac-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC TCCGTCTTGA 50 TATTTGTCAA CCCAGAACCT GGTCGAGTGG TCAGTCCTCG GTCGGCAGCA 100 GGGCGGGCAT CAACAGGCTC AACTGGGCAC AGGAAGCAAG GGACACACAA 150 AGCAAACAAG ACAAATCAAG ATGGAGAACG CCCTTGCTGG CTCAGGGGAC 200 TCTTACCTTC GTTTTCTGTG TTGGCTGGCA CAGAGTCAGC CCCAAAAGAA 250 TGCCACGGCT GGAGATCGTC CAGGCTGAAC TCTCCATTCA CGGGAAGGAG 300 CTCCACGGTG GTTTTCGTTT CGGTCAAAGA TGGCATGAGA GCATCGTTTC 350 CGTAACTGAT GGTTGGTTCA CTAATCATGT TGGCCAAGAC GTCATCTGA 399
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 513 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
(vii) IMMEDIATE SOURCE:
(B) CLONE: Pac-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC TCCGTCTTGA 50 TATTTGTCAA CCCAGAACCT GTATTACATC ATAATTAAAG GGTCACTTCA 100 ' AATTCTACTC TGCAGTAAGA TCAATTGAGA GAGGCTTAAA ATGCAGAAAG 150 GAGACAACGT CTGCTCGAGC TTAGGCCCAA GATGCGGAGA GGCAGAAGTC 200 AAGCGGTTGT GATACCTGGT CGAGTGGTCA GTCCTCGGTC GGCAGCAGGG 250 CGGGCATCAA GATGGAGAAC GCCCTTGCTG GCTCAGGGGA CTCTTACCTT 300 CGTTTTCTGT GTTGGCTGGC ACGAGTCAGC CCCAAAAGAA TGCCACGAGT 350 CAGCCCCAAA AGAATGCCAC GGCTGGAGAT CGTCCAGGCT GAACTCTCCA 400 TTCACGGGAA GGAGCTCCAC GGTGGTTTTC GTTTCGGTCA AAGATGGCAT 450 GAGAGCATCG TTTCCGTAAC TGATGGTTGG TTCACTAATC ATGTTGGCCA 500 AGACGTCATC TGA 523
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 321 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
(vii) IMMEDIATE SOURCE: ( B ) CLONE : Pace
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
ATGCGGAGAG GCACAAGTCA AGCGGTTCTG ATACCTGGTC GAGTGGTCAG 50
TCCTCGGTCG GCAGCAGGGC GGGCATCAAC AGGCTCAAGT GGGCACAGGA 100
AGCAAGGGAC ACAGAAAGCA AACAAGACAA ATCAAGATGG AGAACGCCCT 150
TGCTGGCTCA GGGGACTCTT ACCTTCGAAA TCTGTGTTGG CTGGCACAGA 200
GTCAGCCCCA AAAGAATGCC ACGGCTGGAG ATCGTCCAGG CTGAACTCTC 250
CATTCACGGG AAGGAGCTCC ACGGTGGTTT TCGTTTCGGT CAAAGATGGC 300
ATGAGAGCAT CGTTTCCGTA A 321
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 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
(vii) IMMEDIATE SOURCE:
(B) CLONE: Tpac-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ATGCAGAAAG CAGACAACGT CTGCTCGAGC TTAGGCCCAA GATGCGGAGA 50 GGCACAAGTC AAGCGGTTCT GA 72
2 ) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 152 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: Pac-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Ser Glu Phe Cys He His Leu His Phe Arg Asp Leu Leu Arg Leu 1 5 10 15
Asp He Cys Gin Pro Arg Thr Trp Ser Ser Gly Gin Ser Ser Val Gly 20 25 30
Ser Arg Ala Gly He Asn Arg Leu Lys Trp Ala Gin Glu Ala Arg Asp 35 40 45
Thr Glu Ser Lys Gin Asp Lys Ser Arg Trp Arg Thr Pro Leu Leu Ala 50 55 60
Gin Gly Thr Leu Thr Phe Glu He Cys Val Gly Trp His Arg Val Ser 65 70 75 80
Pro Lys Arg Met Pro Arg Leu Glu He Val Gin Ala Glu Leu Ser He
85 90 95
His Gly Lys Glu Pro Lys Arg Met Pro Arg Leu Glu He Val Gin Ala 100 105 110
Glu Leu Ser He His Gly Lys Glu Leu His Gly Gly Phe Arg Phe Gly 115 120 125
Gin Arg Trp His Glu Ser He Val Ser Val Thr Asp Gly Trp Phe Thr 130 135 140
Asn His Val Gly Gin Asp Val He 145 150
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: Tpac-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Gin Lys Gly Asp Asn Val Cys Ser Ser Leu Gly Pro Arg Cys Gly
1 5 10 15
Glu Ala Gin Val Lys Arg Phe 20
) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 106 amino acids
(B) TYPE-, amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: Pace
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 20 :
Met Arg Arg Gly Thr Ser Gin Ala Val Leu He Pro Gly Arg Val Val
1 5 10 15
Ser Pro Arg Ser Ala Ala Gly Arg Ala Ser Thr Gly Ser Ser Gly His 20 25 30
Arg Lys Gin Gly Thr Gin Lys Ala Asn Lys Thr Asn Gin Asp Gly Glu 35 40 45
Arg Pro Cys Trp Leu Arg Gly Leu Leu Pro Ser Lys Ser Val Leu Ala 50 55 60
Gly Thr Glu Ser Ala Pro Lys Glu Cys His Gly Trp Arg Ser Ser Arg 65 70 75 80
Leu Asn Ser Pro Phe Thr Gly Arg Ser Ser Thr Val Val Phe Val Ser
85 90 95
Val Lys Asp Gly Met Arg Ala Ser Phe Pro 100 105

Claims

WHAT IS CLAIMED IS:
1. A nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2.
2. The nucleic acid molecule of claim 1 that encodes Pac.
3. The nucleic acid molecule of claim 2 wherein said sequence is SEQ ID NO:l.
4. The nucleic acid molecule of claim 1 that encodes Tpac.
5. The nucleic acid molecule of claim 4 wherein said sequence is SEQ ID NO:3.
6. The nucleic acid molecule of claim 1 that encodes hsaP.
7. The nucleic acid molecule of claim 6 wherein said sequence is SEQ ID NO:5.
8. The nucleic acid molecule of claim 1 that encodes ApoE4L.
9. The nucleic acid molecule of claim 8 wherein said sequence is SEQ ID NO:7.
10. The nucleic acid molecule of claim 1 that encodes ApoE4Ll.
11. The nucleic acid molecule of claim 10 wherein said sequence is SEQ ID NO:9.
12. The nucleic acid molecule of claim 1 that encodes ApoE4Lx2.
13. The nucleic acid molecule of claim 12 wherein said sequence is SEQ ID NO: 12.
14. A protein, substantially free of natural contaminants, selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2.
15. The protein of claim 14, wherein said protein is Pac.
16. The protein of claim 15 wherein said protein has a sequence of SEQ ID NO:2.
17. The protein of claim 14, wherein said protein is Tpac .
18. The protein of claim 17, wherein said protein has a sequence of SEQ ID NO:4.
19. The protein of claim 14, wherein said protein is hsaP.
20. The protein of claim 19, wherein said protein has a sequence of SEQ ID NO: 6.
21. The protein of claim 14, wherein said protein is apoE4L.
22. The protein of claim 21 wherein said protein has a sequence of SEQ ID NO: 8.
23. The protein of claim 14, wherein said protein is apoE4Ll.
24. The protein of claim 23, wherein said protein has a sequence of SEQ ID NO: 10.
25. The protein of claim 14, wherein said protein is apoE4Lx2.
26. The protein of claim 25, wherein said protein has a sequence of SEQ ID NO:13.
27. A reagent capable of diagnosing the presence of a molecule selected from the group consisting of Pac, a Pac-encoding nucleic acid molecule, Pac-l, a Pac-1- encoding nucleic acid molecule, Pac-2, a Pac-2-encoding nucleic acid molecule, Pace, a Pace-encoding nucleic acid molecule, Tpac, a Tpac-encoding nucleic acid molecule, Tpac-2, a Tpac-encoding nucleic acid molecule, hsaP, an hsaP-encoding nucleic acid, ApoE4L, an ApoE4L- encoding molecule, ApoE4Ll, an ApoE4Ll-encoding molecule, ApoE4Lx2, and an ApoE4Lx2-encoding molecule.
28. The reagent of claim 27, wherein said reagent is a nucleic acid molecule.
29. The reagent of claim 28, wherein said reagent is a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:12.
30. The reagent of claim 28, wherein said reagent is obtainable by mutating a nucleic acid molecule having a sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5 , SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:12.
31. The reagent of claim 27, wherein said reagent is a protein.
32. The reagent of claim 31, wherein said protein is an antibody, or a fragment of an antibody.
33. The antibody or fragment of an antibody of claim 32, which is capable of binding to a protein selected from the group consisting of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll and ApoE4Lx2.
34. A method of treating Alzheimer's disease, Down's Syndrome, Parkinson's Disease or Schizophrenia, which comprises providing to an individual, in need of such treatment, an effective amount of an inhibitor of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll or ApoE4Lx2.
35. The method of claim 34, wherein said inhibitor is a protein.
36. The method of claim 35, wherein said inhibitor is an antibody, or fragment thereof.
37. The method of claim 34, wherein said inhibitor is a nucleic acid molecule.
38. A method of treating Alzheimer's disease, Down's Syndrome, Parkinson's Disease, Schizophrenia or hyperlipoproteinemia which comprises providing to an individual, in need of such treatment, an effective amount of an inhibitor of ApoE4L, ApoE4Ll or ApoE4Lx2.
39. The method of claim 38, wherein said inhibitor is a protein.
40. The method of claim 39, wherein said inhibitor is an antibody, or fragment thereof.
41. The method of claim 38, wherein said inhibitor nucleic acid molecule.
PCT/EP1993/003581 1992-12-16 1993-12-16 Agents for the prevention and treatment of human alzheimer's disease WO1994013798A1 (en)

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CA2,085,924 1992-12-16
CA 2085924 CA2085924A1 (en) 1992-12-16 1992-12-16 Putative therapeutic targets for the prevention and stoppage of human alzheimer's disease: cdna and amino acid sequence of a proteinase/esterase-like protein and a proteinase/protamine, truncated, modified form of the protein, anti ense to the alzheimer elated human app gene
US29140193A 1993-03-04 1993-03-04
US08/291,401 1993-03-04

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US5969100A (en) * 1995-01-06 1999-10-19 Sibia Neurosciences, Inc. Peptide, peptide analog and amino acid analog protease inhibitors
US5863902A (en) * 1995-01-06 1999-01-26 Sibia Neurosciences, Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US5714471A (en) * 1995-01-06 1998-02-03 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
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US5872101A (en) * 1995-01-06 1999-02-16 Sibia Neurosciences, Inc. Methods of treating neurodegenerative disorders using protease inhibitors
US5962419A (en) * 1995-01-06 1999-10-05 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US6017887A (en) * 1995-01-06 2000-01-25 Sibia Neurosciences, Inc. Peptide, peptide analog and amino acid analog protease inhibitors
US5804560A (en) * 1995-01-06 1998-09-08 Sibia Neurosciences, Inc. Peptide and peptide analog protease inhibitors
US7964598B2 (en) 1995-10-17 2011-06-21 The J. David Gladstone Institutes ApoE4 domain interaction inhibitors and methods of use thereof
US5955472A (en) * 1995-11-02 1999-09-21 Warner-Lambert Company Naphthylazo inhibition of amyloidosis
WO1998007850A2 (en) * 1996-08-22 1998-02-26 Bergmann Johanna E Agents for pre-symptomatic detection and therapeutic targeting of alzheimer's disease and down syndrome in humans
WO1998007850A3 (en) * 1996-08-22 1998-09-17 Johanna E Bergmann Agents for pre-symptomatic detection and therapeutic targeting of alzheimer's disease and down syndrome in humans
US6046381A (en) * 1998-04-30 2000-04-04 The Regents Of The University Of California Apolipoprotein E transgenic mice and assay methods
US6017913A (en) * 1999-05-03 2000-01-25 Warner-Lambert Company Naphthylazo inhibition of amyloidosis
US8101578B2 (en) 2006-04-28 2012-01-24 Kagoshima University Amyloid [β] Beta fibrillogenesis-inhibiting peptide
US8697651B2 (en) 2006-04-28 2014-04-15 Kagoshima University Amyloid β fibrillogenesis-inhibiting peptide

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