CA2126787A1 - Agents for the prevention and treatment of human alzheimer's disease - Google Patents

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

21267~7 ~ --1--TITLE OF THE lN VL-~ 1 lON:

AGENTS FOR THE PR~vL-.llON AND TREATMENT OF
~UMAN ALZHEIMER'S DISEASE

FIELD OF THE lN V ~ lON:

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).

RA~KG~OUND OF THE lNvL~ ON:

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. Hypoth. 38:11-19 (1992)) and Johnson, S.A. (In:
Review of Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.), Wiley-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 ~/A4 amyloid protein (Price, D.L.
et al., Clin. Neuropharm. I4: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. 30:687-689 (1991)). All cases of Alzheimer's disease show such deposition of amyloid in brain parenchyma.
The ~/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 ~-amyloid peptide precursor (APP) (Podlisny, M.B. et al., Science 238:669-671 (1987); Currie, J.R. et al., J. Neurosci. Res.
30:687-689 (1991)). This precursor is normally cleaved to form the ~-amyloid protein ("AP"). The isolation of cDNA encoding the ~/A4 protein has led to the recent recognition that the protein is, in fact, synthesized as part of APP, and that ~/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)).

21267~7 -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. 20:63-68 (1992); Price, D.L. et al., Clin. Neuropharm. 14:S9-S14 ( 19 9 1 ) ) .
Although the accumulation of ~/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 ~/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

The E4 allele of apoliprotein apoE4 has been associated with late onset familial and sporadic Alzheimer's disease (Saunders, A.M. et al., Neurology 43: 1467-1472 (1993)), (Namba, Y., et al. Brain Res. 514 163-166 (1991)). The apoE gene is located on chromosome 19 and is widely experessed, but is highest concentrated in brain tissue. Apolipoproteins are implicated in other serious human diseases involving faulty lipid metabolism, e.g., cardiovascular diseases.
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 212~7~7 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.
25:378-382 (1984); Utermann, G., J. Inher. Metab. Dis.
11 :74_86 (1988); Ghiselli, G., et al., Lancet 2:405-407 (1982)). In addition to these major isoforms of apoE, minor variant isoforms (apoE1, 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. 32:521-528 (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, 2125~87 -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 (Rall, 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.
78:815-821 (1986); Kuusi, T. et al., J. Lipid Res.
29293-298 (1988); Yanagi, H. et al., Clin. Genet.
38:264-269 (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 hemoglobin-A1 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 212S~87 al., J. Biochem. (Tokyo) 105:491-493 (1989); Horie, Y.
et al., J. Biol. Chem. 267:1962-1968 (1992)).

III c-fos The majority of early onset Alzheimer cases, are related to a genetic lesion (mutation) in the 14q.24 region of chromosome # 14, (St. George-Hyslop, P, et al. Nature Genet. 2: 330-334 (1992)). The gene encoding C-Fos protooncogene is located in the "center" of this region, and abnormal expression of C-Fos has been documented in the hippocampus of Alzheimer's victims, (Zangh. P., et al., Neuroscience 46: 9-21 (1992)). The C-Fos protein has been shown to activate the APP gene promoter;
however, but C-Fos does not display evidence of allelic association with familial Alzheiner's disease,(FAD) and, although it has been concluded that the protein coding region of the C-Fos gene is not the site of FAD mutation on chromosome #14 the possibility exist that mutations in non coding regions of the of the C-Fos gene, which might effect tissue specific expression of C-Fos, could be the FAD mutation site, (Rogarve E.I., et al., Neurology 43: 2275-2279 (1993)). This invention additionally relates to the discovery of a gene "C-Fos5L" which is 100~ homologous to the anti sense strandof a region of the 5' untranslated region of C-Fos protooncogene from nl35-n792 inclusive. The protein encoded by C-Fos5L, "C-Fos5Lp" is a neuropeptide hormone-like molecule made up of 121 amino acids which has all the required biological characteristic for combining with pac and apoE4Lx2 to cause the full range of neuropathological symptoms of human Alzheimer's disease. The neuropeptide harbours two amidation sites and a secretory signal which can be used to produce four distinct, activated, unique neuropepides. In addition C-Fos5Lp, by similarity, is a trans-acting transcriptional activator which can mimic or modulate the activity of a 212~7~7 number of transcription factors including; the SRF
factor, REV protein, ERB-1, nuclear factor NF-1, DNA
binding protein UL42, transforming proteig INF 3, and the androgen receptor transcriptional transactviting region; it can also modulate activity or mimic, the growth arrest specific protein, sperm histone protamine, the acetylcholine receptor protein, fibrillanin, tenacsin, a cysteine protease, the serotonin receptor, the paired amphipathic helix protein, and two serine/threonine protein kinases. In addition amino acids (aa) 91 - 118 in the c terminal region of C-Fos5Lp has significant, unique homology to aa 152 - 166 and 231 - 243 in apoE4; aa 40-59 and aa 102-121 shares homology with regions of apoeLx2.

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 lA shows the protein coding sequence of "Pac" cDNA and the deduced sequence of the protein is shown in Figure lB. "Tpac" cDNA is reconstructed from negative strands of exons and from introns in the amyloid precursor related human APRP gene. The protein 212S7~7 coding sequence of the cDNA is shown in Figure lC and the deduced sequence of the protein in the embodiment of Figure lD.
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 apoE4L1 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 underscoring is used to denote sequences with significant identity and homology to apoE4.
Figure 7 shows the regulatory region (Pac/reg) in the 5 'upstream region of pac. This sequence contain five promoter elements (three individual and two tandem) correlated with "cap" sites. These promoters program the g transcription of the Pac family of mRNA's, probably in a tissue specific manner.
Figure 8A shows the nucleotide sequence of the cDNA
encoding C-Fos5Lp; figure 5B shows the sequence of the C-Fos5Lp neuropeptide, glycine residues (G) at which amidation/activation can occur have a NH2 above, the downwards pointing arrow indicates the secretory signal.
Figure 8C shows the 5' upstream regulatory region of C-Fos5L, "C-Fos5Lreg" the "TATA box" promoter element is underlined, the correlated "cap site" is overlined.

SUMMARY OF THE lNvL-.llON:

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 cont~m;n~nts, that encodes a protein selected from the group consisting of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-Fos5L. In particular, the invention provides the above-described nucleic acid molecule wherein the sequence is SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:12 or SEQ ID NO:22 The invention also provides a protein, substantially free of natural cont~m;n~nts, selected from the group consisting of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp. 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, SEQ ID NO:13 os SEQ ID NO:23.
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-1, 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, ApoE4L1, an ApoE4L1-encoding molecule, ApoE4Lx2, an ApoE4Lx2-encoding molecule and an C-Foc5Lp 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:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12.or SEQ ID NO:22, or a nucleic acid molecule obtainable by mutating a nucleic acid molecule having a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO: 22 or SEQ ID NO:24) or a protein (especially an antibody, or a fragment of an antibody, which is capable of binding to Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-Fos5Lp).
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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp.

212S~87 , DESCRIPTION OF THE PREFERRED EMBODIMENTS:

Although it has been recognized that Alzheimer's disease reflects the abnormal processing of APP to produce ~/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 --~/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 ~/A4 domain is partly embedded in the cell membrane.
The secretion of the ~/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 ~/A4 protein. In sum, the consensus of all experimental evidence suggests that the ~/A4 variant of AP protein plays a major role in the expression of Alzheimer's disease, and that the ~/A4 protein is produced via proteolysis. The identity of the protease responsible for ~/A4 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 ~/A4 protein. The protease is encoded 2126 7~7 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 "E4Ll," 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.
The common factor in all human Alzheimer's disease is the formation of ~ amyloid containing plaques which 212~ 787 indicated that all mechanisms causing Alzheimer's must involv the abnormal formation of ~ amyloid protein and hence the APP gene. The family of pac proteins have all the biological properties required for the full phenotype of Alzheimer's. Hence it is logical to conclude that the disease is caused by the overt expression of these proteins which are expressed in Alzhemier's victims but not at significant or detectable levels in healthy humans. ApoE4Lx2 has the potential to influence the expression of pac and pac-isoforms and C-Fos5Lp can influence the expression of apoE4Lx2 and also mimic the protamine, acetylcholine inhibition (pac by destruction of acetylcholine, C-Fos5Lp by blocking interaction with acetylcholine receptor), and the lysosomal protease activity of pac. Silent mutations like the T-C mutation in exon 2 of C-Fos gene, although insignificant to C-Fos expression can activate expression of the anti-sense gene which, from what we know of other systems we have discovered, is sometimes under subtile repression by the sense gene. Therefore, although mutations have not been found, or looked for, in region of C-Fos gene which encodes C-Fos5L, it appears that interaction between C-Fos5Lp, apoE4Lx2 gene and APP gene, as well as specific mutation in the APP
gene can account for all aspects of human Alzheimer's disease.

I. The Molecules of the Present Invention The molecules of the present invention comprise Pac, Tpac, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-FosL5p, 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-1, Pac-2, Pace, Tpac-1) that result from the alternative splicing of the mRNA
antisense to the amyloid forming APP gene. These 2126 7~7 -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 lA (SEQ ID NO:l) and the deduced sequence of the protein in Figure lB (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 lC (SEQ ID NO:3) and the deduced amino acid sequence of the protein is shown in Figure lD (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 healthy 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 - ~126 ~ 87 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 lB. 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 Z1" 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 ~/A4 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 Z1," 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 lB and lD. The invention also includes any cellular proteinase ~12G 787 or acetylcholinesterase activated by the proteins or domains of the proteins described in the embodiment of Figures lB and lD.

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 apoE4L1 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 apoE4L1 protein is shown in Figure 4A (SEQ ID NO:9).
The apoE4L1 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 ApoE4L1 Gene Sequences Both the Apo4L and Apo4L1 orf's can be transcribed from promoter elements and coordinated GC box and cap sites that are located 300-500 bp upstream from the E4L/Ll 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 "TATAAA"
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 shown 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 ~-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 21~6787 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.

II. The C-Fos5Lp protein The C-Fos5Lp protein is a trans acting transcriptional activator which can function as a family of activated neuro peptides with a very wide range of potential biological activites, many of which are highly relevant to the neuropathological symptoms of Alzheimer's disease. In addition, like pac, C-Fos5Lp can function in a tissue specific manner depending on wheather none, one or both of the potential amidation sites are amidated, and on wheather the secretory signal is used or not used in a specific tissue type. Most significant, however is the unique structural relationship in the c-terminal domain of C-Fos5Lp with domains in apoE4Lx2, and apoE4, which suggest a very specific interaction between these proteins which is highly relevant to the role they play in the etiology of human Alzheimer's disease.

J. 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.

212~787 Pac/reg contains five promoter systems each consisting of a "TATA box" (underlined in figure #7) and correlated "cap site"(overlined in figure #7). These systems are located between -455/-418, -420/-369, -389/-343(two tandem promoters sharing a single correlated cap site) , and -329/-286 and program the transcription of Pac mRNA. Each promoter might power transcription of an alternate spliced mRNA in a tissue specific manner. Pac mRNA also harbours three potential transcription termination sites in the 3' downstream untranslated region located at + 337, + 372 and + 637 relative to the Pac "orf" stop translation codon. Differentially mRNA's might use different transcription termination signals.
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-1 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-1 and Pac-2 have potential mitochondrial transit sequences.
The sequences of cDNA molecules Pac-1 and Pac-2 are as follows:

Pac-1 (SEQ ID NO:14):
ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA
TATTTGTCAACCCAGAACCTGGTCGAGTGGTCAGTCCTCGGTCGGCAGCA
GGGCGGGCATCAACAGGCTCAACTGGGCACAGGAAGCAAGGGACACACAA
AGCAAACAAGACAAATCAAGATGGAGAACGCCCTTGCTGGCTCAGGGGAC
TCTTACCTTCGTTTTCTGTGTTGGCTGGCACAGAGTCAGCCCCAAAAGAA
TGCCACGGCTGGAGATCGTCCAGGCTGAACTCTCCATTCACGGGAAGGAG
CTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGCATGAGAGCATCGTTTC
CGTAACTGATGGTTGGTTCACTAATCATGTTGGCCAAGACGTCATCTGA

Pac-2 (SEQ ID NO:15):
ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA
TATTTGTCAACCCAGAACCTGTATTACATCATAATTAAAGGGTCACTTCA
AATTCTACTCTGCAGTAAGATCAATTGAGAGAGGCTTAAAATGCAGAAAG
GAGACAACGTCTGCTCGAGCTTAGGCCCAAGATGCGGAGAGGCAGAAGTC
AAGCGGTTGTGATACCTGGTCGAGTGGTCAGTCCTCGGTCGGCAGCAGGG
CGGGCATCAAGATGGAGAACGCCCTTGCTGGCTCAGGGGACTCTTACCTT
CGTTTTCTGTGTTGGCTGGCACGAGTCAGCCCCAAAAGAATGCCACGAGT
CAGCCCCAAAAGAATGCCACGGCTGGAGATCGTCCAGGCTGAACTCTCCA
TTCACGGGAAGGAGCTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGCAT
GAGAGCATCGTTTCCGTAACTGATGGTTGGTTCACTAATCATGTTGGCCA
AGACGTCATCTGA

The cDNA sequence of Pace is as follows (SEQ ID NO:16):
ATGCGGAGAGGCACAAGTCAAGCGGTTCTGATACCTGGTCGAGTGGTCAG
TCCTCGGTCGGCAGCAGGGCGGGCATCAACAGGCTCAAGTGGGCACAGGA
AGCAAGGGACACAGAAAGCAAACAAGACAAATCAAGATGGAGAACGCCCT
TGCTGGCTCAGGGGACTCTTACCTTCGAAAT~l~l~llGGCTGGCACAGA
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-1 and Pac-2, produced by the alternative/differential splicing in the pac antisense gene is as follows:

_ 2126787 Pac-1 (SEQ ID NO:18):
MSEFCIHLHFRDLLRLDICQPRTWSSGQSSVGSRAGINRLKWAQEARDTE
SKQDKSRWRTPLLAQGTLTFEICVGWHRVSPKRMPRLEIVQAELSIHGKE
PKRMPRLEIVQAELSIHGKELHGGFRFGQRWHESIVSVTDGWFTNHVGQD
VI

Tpac-2 (SEQ ID NO:19): MQKGDNVCSSLGPRCGEAQVKRF

Pace (SEQ ID NO:20):
MRRGTSQAVLIPGRWSPRSAAGRASTGSSGHRKQGTQKANKTNQDGERP
CWLRGLLPSKSVLAGTESAPKECHGWRSSRLNSPFTGRSST W FVSVKDG
MRASFP
Pac/reg (SEQ ID NO: 21) CTAAATGCAATATAATTTACAATTTATAAACGCAATTAGAAGAATTTCAT
TTCTTAAATGCAGGGGACATTTGGATGAGGTTATATAAAAAGTTTCAGTA
TATTCTCTGCCCAACTGGTTGGTCAAATATTTGATGGTTACTTTAAAAAA
AAAAAAAAAATTTAGTAGAGATGGGGTTTCACCTAATTGGCCAGGCTGGT
CTCGAATTTCTGACCTCGTGATCCTCCCGCCTTGGCCTCCCAAAGTGCTG
C-Fos5L (SEQ ID NO: 22) ATGAGGGGTTTCGGGGATGGCTCCCCCCAGGGCTACAGGGAAAGGCCGTG
GAAACCTGCTGACGCAGATGTCCTAATATGGACATCCTGTGTAAGGGGGG
AGGGATTGACGGGAACTGCTCGCGGGCTGCAGCCAACACCGAGGGTGCAG
TGCGGGGGGAGGCGGGGGCCGCGGCTGGGGGAGGGGAGGCGGGAACGGCG
CAGAATGAGAGAGAACATTCGCACCTGGTTCAATGCGGACCCTTGTTCCC
GAGGTCGGGGGGGATGGGGCAGAGAGCGC~ ACCCTTGTACGGAAA
CTGAAGACAGTTCTGAGGCTCAGAGATAGGAGAAACGGCATCGAGTACAG
GACCCCGAGGACTTAA
C-Fos5Lp MRGFGDGSPQGYRERPWKPADADVLIWTSCVRGEGLTGTARGLQPTPRVQ
CGGRRGPRLGEGRRERRRMRENIRTWFNADPCSRGRGGWGRERLFTLVRK
LKTVLRLRDRRNGIEYRTPRT
C-Fos5Lreg TTCGCTCGCCGCGGCCGCCGGCTCAGTCTTGGCTTCTCAGTTGCTCGCTG
CAGATGCGGTTGGAGTACGAGGCGCCGCAGCCACTG~'l"l"l"l'ATAACAAGC
(:111lATGAATGAGTGTAAAcGTcAcGGGcAcAAccAcGGTGGcGccAGA
GGGGTGGCGCGCGGGCCTGGGCGCTTCTGCGGCCGCCCGGCTGCGTCCCC
AGCGCTCGCAGCTCCCTGCCCCCGCCTTGGCGCGTGTCCTAATCTCGTGA
GCAATTCGCAGTTCCTGTCTCAGAGGTCTCGTGGGCCCCCCAAG

II. The Uses of the Molecules of the Present Invention.

2126 ~ 87 The elucidation of the significance of Pac, Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll, ApoE4Lx2 and C-Fos5Lp 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 or C-Fos5Lp 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 ApoE4Lx2 or C-Fos5Lp molecules, and the presence of such molecules is determined via and immunoassay. A large 2126~87 number of suitable immunoassay formats have been described (Yolken, R.H., Rev. Infect. Dis. 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') 21 single chain antibodies, etc.), recombinant antibodies, chimeric antibodies, etc. may be employed. Such antibodies can be obtained using conventional methods with Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp 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-ApoE4L1, anti-ApoE4Lx2, anti-C-Fos5Lp etc. antibody with a sample suspected to contain the target molecule --Pac, Tpac, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2, C-Fos5Lp 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, acetylcholine esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, ~-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, In, 2sI, l3lI, 32p, 35S, 14C slCr 57To 58Co 59F 75S
Eu, 90Y, 67CU, 2l7ci, 2llAt 2l2Pb 47Sc 109Pd et Examples of suitable chemiluminescent labels include a l-lmi n~l 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L 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 Symp. Ouant. 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L 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.

21267~7 -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' )21 single chain antibodies, etc.) that is capable of binding to Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp. 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 240:1041-1043 (1988); Liu, A.Y. et al., Proc. Natl.
Acad. Sci. USA 84:3439-3443 (1987); Liu, A.Y. et al., J.
Immunol. 139:3521_3526 (1987); Sun, L.K. et al., Proc.
Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y.
et al., Canc. Res. 47:999-1005 (1987); Wood, C.R.
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., BioTechniques 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., J.
Immunol. 141:4053-4060 (1988); all of which references are incorporated herein by reference).
In another embodiment, the nucleic acid molecules of the present invention may be mutated and expressed in order to identify Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos mutant molecules that can complex with and significantly inactivate Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2 or C-FosL5 molecules ~ 2126787 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-1-, Pac-2-, Pace-, Tpac-, Tpac-2-, hsaP-, Pac/reg, ApoE4L-, ApoE4L1-, ApoE4Lx2 or-C-Fos5L -encoding sequences described herein, such that it is capable of binding to, or hybridizing with, an endogenous Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or fosC5L 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, ApoE4L1, ApoE4Lx2 or C-Fos5L. 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2, fosC5L or C-Fos5Lreg 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L 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 transcription or translation of a Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L mRNA transcript can be used to impair the expression of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L
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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2, C-Fos5L or Fos5Lreg 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or Fos5L 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.) 83:4143 (1986); Goodchild et al., Proc. Natl.
Acad. Sci. (U.S.A.) 85:5507 (1988); Wickstrom et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:1028; 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., et -al., EMBO J. 8:3679 (1989); all of which references are incorporated herein by reference). Automated nucleic acid synthesizers may be employed for this purpose. In addition, desired nucleotides of any 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L 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 Boggs, 21~7~7 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 recommen~tions 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.

2~26787 (1) GBNBRAL INPORMATION:
(i) APPLICANT: PRBDDIB, RICK
BBRGMANN, ~OHANNA
(ii) TITLB OF INVBNTION: AGBNTS FOR THB PRBVBNTION AND TRRATMR~lT
OF HUMAN AT.~RTMRu DISBASB
(iii) NUMBBR OF I _ : 24 (iv) ~u~.~k~ ADDRBSS:
1 0 IAI ~nRR.csRR: RICK PRBDDIB
:B.I STRBBT: 4855 COTB ST LUC RD, Suite# 201 C CITY: MONTRBAL
D STATB: QUBBBC
l~BI COUNTRY: CANADA
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ID:I SOFTWARB: PatentIn Release #1.0, Version #1.25 (vi) CURRBNT APPLICATION DATA:
(A) APPLICATION NUMBBR:CAN#
(B) FILING DATB:
(C) CLASSIFICATION:
2 5 (viii) ATTORNBY/AGBNT INFORMATION:
(A) NAMB: NONB
(B) RBGISTRATION NUMBBR:
(C) RBFBRBNCB/DOCKBT NUMBBR:
(ix) TRn- CATION INFORMATION:

3 0 (A) TBLBPHONB: (0014940) 86 25 53 (B) T LBFAX: (0014940) 86 01 76 (2) INFORMATION FOR SBQ ID NO:1:
(i) SBQUBNCB CHARACTBRISTICS:
~A) LBNGTH: 315 hase pairs I:B) TYPB: nucleic acid ~:C) STRA : single l:D) TOPOLOGY: linear (ii) MOLBCULB TYPB: cDNA
(iii) ~Y~u~ CAL: NO
(iv) ANTI-SBNSB: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: PAC

(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:1:
ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGCTCTCC l~l-ll~A l~Ill~l~AA 60 ~rrA~AA~T GGTCGAGTGG TCAGTCCTCG GTCGGCAGCA wG~T CAACAGGCTC 120 AACTTCGTTT I~l~l~ll~G CTGGCACAGA GTCAGCCCCA AAAGAATGCC ACGGCTGGAG 180 ATCGTCCAGG CTGAACTCTC CATTCACGGG AAGGAGCTCC A~l~lll l~lll~l 240 CAAAGATGGC ATGAGAGCAT ~lll~ A ACTGATCCTT GGTTCACTAA TCAAGTTGGC 300 (2) INFORMATION FOR SBQ ID NO:2:
(i) SBQUBNCB CHARACTLRISTICS:
(A) LBNGTH: 104 amino acids (B) TYPB: amino acid (D) TOPOLOGY: linear (ii) MOLBCULB TYPB: protein (iii) ~Y~ul~llCAL: NO
(vi) ORIGINAL SOURCB:
6 0 (A) ORGANISM: HOMO SAPIBNS

~26 7~7 (vii) TMMRnTATR SOURCE:
(B) CLONB: PAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:2:
Met Ser Glu Phe Cys Ile His Leu His Phe Arg Asp Leu Leu Arg Leu 1 s lo lS
A3p Ile Cys Gln Pro Arg Thr Trp Ser Ser Gly Gln Ser Ser Val Gly Ser Arg Ala Gly Ile Asn Arg Leu Asn Phe Val Phe Cy8 Val Gly Trp 1 0 His Arg Val Ser Pro Lys Arg Met Pro Arg Leu Glu Ile Val Gln Ala Glu Leu Ser Ile His Gly Lys Glu Leu His Gly Gly Phe Arg Phe Gly Gln Arg Trp His Glu Ser Ile Val Ser Val Thr Asp Asp Trp Phe Thr Asn His Val Gly Gln Asp Val Ile (2) INFORMATION POR SBQ ID NO:3:
(i) SBQUBNCB rHARArTRRT5TIcs 2 0 (A LBNGTH: 84 baoe pairs (Bl TYPB: nucleic acid (Cl sTRAt~RnNRq~s: single (Dl TOPOLOGY: linear (ii) MOLBCULB TYPB: cDNA
(iii) H~Vl~llCAL: NO
(iv) ANTI-SBNSB: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) TMMRnTATB SOURCB:
3 0 (B) CLONB: TPAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:3:
ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC l`~ ~A TATTTGTCAA 60 rrrAr.AArrT GTATTACATC ATAA 84 (2) INPORMATION POR SBQ ID NO:4:
3 5 (i) SBQUBNCB rRARArTRRT.5TICS:
~A LBNGTH: 27 amino acids B I TYPB: amino acid C:I sTRpt~RnNRe~s: single Dl TOPOLOGY: linear 4 0 ( i i ) MOLBCULB TYPB: cDNA

(iii) HY~Old~llCAL: NO
(iv) ANTI-SBNSB: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: TPAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:4:
Met Ser Glu Phe Cys Ile His Leu His Phe Arg Asp Leu Leu Arg Leu 5 0 Asp Ile Cys Gln Pro Arg Thr Cys Ile Thr Ser (2) INPORMATION POR SEQ ID NO:S:
(i) SBQUBNCB rRARArTRRT~eTIcs (A~ LBNGTH: 159 base pairs 5 5 (B:l TYPB: nucleic acid (C, STRP ~~: single (Dl TOPOLOGY: linear 2~26 737 -(ii) MOLBCULB TYPB cDNA
(iii) ~Y~uln~ CAL NO
(iv) ANTI-SENSB NO
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(B) STRAIN hsaP
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 5 CACTGCTTGC ~ ,l GCACCAGTTC TGGATGGTCA ~ll~l- 60 GGCTTCTACC TCATTGGTGA TCTGCAGTTC ArrrTArArT TCTTGGCAAT ACTGCAGGAT 120 10 ~iu~.~C~.. `~ GTATCAATGC A~.. ~. CCCTGATGG 159 (2) INFORMATION FOR SBQ ID NO 6 (i) SBQUBNCB CHARACTBRISTICS
(A) LBNGTH 51 amino acid~3 (B) TYPB amino acid (D) TOPOLOGY linear (ii) MOLBCULB TYPB protein (iii) IIY~O~ llCAL NO
(iv) ANTI-SBNSB NO
(vi) ORIGINAL SOURCB
2 0 (A) ORGANISM HOMO SAPIBNS
(B) STRAIN huaP
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 6 Met Gly Leu Ala Leu Leu Ala Ala Pro Leu Ala Pro Val Leu AE~p Gly 2 5 Hi3 Trp Leu Val Gly Phe Tyr Hi~ Ile Gly Asp Leu Gln Phe Arg Val Aop Phe Leu Ala Ile Leu Gln A~p Ala Phe Leu Gly Ile A~n Ala Gly 3 o Phe Gly Pro (2) INFORMATION FOR SBQ ID NO 7 (i) SEQUBNCB CHARACTBRISTICS
~A) LBNGTH 597 ba3e pair~
IB) TYPB nucleic acid 3 5 , c) STRANDBDNBSS E~ingle lD~ TOPOLOGY linear (ii) MOLBCULB TYPB cDNA
(iii) tlY~ CAL NO
(iv) ANTI-SBNSB YBS
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4L
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 7 4 5 A~ ll. A rrAr-r~r-GrTc rAArrArrTc TTGAGGCGGG CCTGGAAGGC ~lC~C~l~,C 60 A~,.~T~,l G~.~l~, CTCCTCCAGC ll~ --ACCTCCGCCAC Cl~lC~llc 120 AC~il~ lC A W~ li ~lCl~ i C~IClC~l CCATCCGCGC GCGCAGCCGC 180 TCGCCCCAGG ~ CTCCTGTAGC GG~ ~ rr~ CC CACAGTGGCG 240 GCCCGCACGC b~ rArr~r-r~r~r-c CCCAGGCGCT CGCGGATGGC GCTGAGGCCG 300 5 0 ~/~.c~,c -~ ~c cc~ w~ TACACTGCCA ~,~ l~ CAGGTCATCG 360 i.~l.~l~C I.l~iGC~iAG CATwC~l,c A - ~;--~-' GGTACTGCAC rArr,r,rrrrr 480 -- ~12~ ~ ~7 CGCACGTCCT CCATGTCCGC GCCCAGCCGG GC~17c~b CCTGCAGCTC CTTGGACAGC 540 ~71~7~ 7~Ll~7~ rAc~ Tc A~711,11~1 CCAGTTCCGA TTTGTAG 597 (2) INPORMATION POR SBQ ID NO 8 (i) SBQUBNCB ChABACTBRISTICS
(A) LBNGTH 198 amino acid~
(B) TYPB amino acid (D) TOPOLOGY linear (ii) MOLBCULB TYPB protein (iii) nr~ulnhllCAL NO
1 0 (vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4L
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 8 Met Ser Ser Thr Arg Gly Ser Ann Gln Leu Leu Arg Arg Ala Trp Ly~

Ala Ser Ala Cy3 Arg Arg Ile Cy~ Trp Ala cy8 Ser Ser Ser Leu Ala Arg Thr Ser Ala Thr Cyo Ser Phe Thr Ser Ser Arg Arg Ser Arg Val Arg Leu Pro Ile Ser Ser Ile Arg Ala Arg Ser Arg Ser Pro Gln Ala Trp Ala Arg Ser Cy~ Ser Gly Trp Pro Ala Arg Glu Pro Thr Val Ala 2 5 Ala Arg Thr Arg Pro Cy3 Ser Thr Arg Gly Pro Arg Arg Ser Arg Met Ala Leu Arg Pro Arg Ser Ala Pro Ser Arg Ala Pro Ala Trp Tyr Thr Ala Arg Arg Phe Cy~ Arg Ser Ser Ala Ser Arg Arg Ser Arg Leu Arg Ser Leu Arg Arg Trp Glu Ala Arg Arg Thr Arg Ser Ser Ser Val Leu Trp Pro Ser Met Ala Cy~ Thr Ser Pro Arg Tyr Cy~ Thr Arg Ala Pro 3 5 Arg Thr Ser Ser Met Ser Ala Pro Ala Arg Ala Cy9 Ala Ala Cys Ser Ser Leu A~p Ser Arg Ala Arg Val Ser Ser Ala Thr Gly Val Ser Cy~

(2) INFORMATION POR SBQ ID NO 9 (i) SEQUBNCB ~ARArTRRTqTIcs lAI LBNGTH 324 ba~e pairo IBI TYPB nucleic acid ~c sTRANnRr ~.c ~ingle Dl TOPOLOGY linear (ii) MOLBCULB TYPB cDNA
(iii) ~r~ln~llCAL NO
(iv) ANTI-SBNSB YBS
5 0 (vi ) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4Ll (xi) SBQUBNCB DBSCRIPTION SBQ ID NO 9 A'~71~ ,1 CCATCTGCGC ~ ~GC~ ArAr~,cr,r~, A~,l~,lCG~ ACAGTGGGGA 60 ~- AAGGGCTGGG A~ ~7 CGAGATG~3G ATGAGCCAGA ~rrAA~ 120 A~A~ArAA ~AAr~C AGAGGCCGAG A~AA~A~A~ A~A~A~A~AT G~ ~A 180 2i2~787 -GATGCAGAGG r.rAr.Arr.rA Ar.Arr.AArAA GGAGCTAGGA G~ w AA GGTGCTCATG 240 CCTCTAATCC CAGCACTTTG rrArGrrrAr Grr-r-r-Ar~r-AT CGCTTGAGCC CAGAAGTTCA 300 (2) INFORMATION POR SBQ ID NO 10 (i) SBQUBNCu CHARACTERISTICS
(A) LENGTH 107 amino acids (B) TYPB amino acid (D) TOPOLOGY linear (ii) MOLBCULE TYPB protein 1 0 (iii) dY~u-dhllCAL NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONE APOB4Ll (xi) SBQUBNCE DBSCRIPTION SBQ ID NO 10 Met Val Ser Ser Ile Cys Ala Val Arg Pro Arg Gly Arg Glu Gly Val Ala Gln Trp Gly Gly Gly Gly Glu Gly Leu Gly Trp Gly Gly Arg Asp 2 0 Gly Asp Glu Pro Glu Arg Pro Lyo Arg Glu Arg Arg Asn Gly Ala Glu Ala Glu Arg Arg Arg Gln Arg Gln Met Gln Arg Ala Asp Ala Glu Gly 2 5 Arg Gly Arg Asp Glu Glu Gly Ala Arg Arg Pro Gly Lys Val Leu Met Pro Leu Ile Pro Ala Leu Trp Glu Ala Glu Ala Gly Gly Ser Leu Glu Pro Arg Ser Ser Arg Pro Ala Trp Ala Thr Gln 3 0 (2) INFORMATION POR SBQ ID NO ll (i) SEQUBNCB CHARACTBRISTICS
l,A,I LBNGTH 532 base pairs I B I TYPB nucleic acid 'Cl sTRr ~ single 3 5 ~D TOPOLOGY linear (ii) MOLBCULB TYPB DNA (genomic) (iii) dY~uldhLlcAL NO
(iv) ANTI-suNsB NO
(vi) ORIGINAL SOURCB
4 0 (A) ORGANISM HOMO SAPIBNS
(xi) SBQUENCB DBSCRIPTION SBQ ID NO ll CTCTACTAAA AATArAAAAA TTAGCCGGGT ~l~ GAGCCTGTAA TCCCAGCTAC 60 TGAGGCAGCA GAATCGCTTG AArrrAArAr GCAGAGGTTG CAGTGAGCCA AGATCGTGCC 120 ACTGCACTCT A~C~ ~l~ ACAGAGCCAG A~ lA AAAAr~rr~r~r~r~ AArr.~rrArAr. 180 4 5 ArAAArATAr ArA~ArArAr AGATGGAGAG rDrr,r,rrrTG AGAATTGTGT GGCAGTATGT 240 GGGCAGAAAG AGAAACTGAG G~1~GW~11 ArArrAAATc ArArrrrrAr GCCAGCAGAT 300 GCGTGAAACT TGGTGAATCT TTATTA-DACT AGGGTCCACC rrArr.ArrDr ~l~GW~ 360 r,rrArArGr~T ~ VC AWL-1G~L~ rArr.rArr.A~. GCACGGwTG G~l~l~ 420 Gu~l~G~l~C AGGCTTCGGC GTTCAGTGAT I~l.~l~ w rArDr~r~r~r~rG G~l~l~ 480 5 0 rrArGGrAr~c CTGCACCTTC TcrArrAr~rr rr~rrrArTG GCGCTGCATG TC 532 (2) INPORMATION POR SBQ ID NO 12:
(i) SBQUBNCB r~ARArTRRT~qTICS
(A LPNGTH 936 base pairs (B I TYPB nucleic acid 5 5 (c sTRANnun~uq~q~ single (D TOPOLOGY linear (ii) MOLBCULB TYPB cDNA
(iii) ~Y~uld~llCAL NO
(iv) ANTI-SBNSE YBS
(vi) ûRIGINAL SOURCB:
5(A) ORGANISM HOMû SAPIENS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4LX2 (xi) SBQUBNCB DBSCRIPTION SBQ ID Nû 12 Arl~l.ll~A CCA wGGCTC GAACCAGCTC TTGAwCwG CCTGGAAGGC ~l~W~l~C 60 1 0 AGGCGTATCT G~l~ WC~l~ CTCCTCCAGC ll~G~ a C~l~C~AC ~l~lC~llc 120 A~lC~l~a W~b~l~C~ ~blC~W~l~ CCCATCTCCT CCATCCGCGC GCGCAGCCGC 180 TCGCCCCAGG ~l~ WC~ CTCCTGTAGC W~ W rrArrrArrr CACAGTGGCG 240 GCCCGCACGC W~ ArrAr-r~r-r rrrAr~r,rCrT CGCwATGGC GCTGAGGCCG 300 ~ W~C ~l~W~C CCCwCCT w TACACTGCCA W~llCl~ CA wTCATCG 360 GCATCGCwA wAGCCGCTT ACGCAGCTTG CGCAwT ww ArrrrArrrr CACCCGCAGC 420 TC~l~bbl~C TCT wCCGAG CA'l~b~.~C A~ ~C wTACTGCAC rArrr.rrrrr 480 CGCACGTCCT CCATGTCCGC GrrrAr~crw GC~ ~C-~ CCTGCAGCTC CTT wACAGC 540 ~ ~ T.~C~.C~C rArrr~GcrTc A~l.~..~. CCAGTTCCGA lll~G~l~'C 600 TTCAACTCCT CCAT wTCTC ~l.~I.l~ GC~l~wC CGAGAwwCG wAwwTGTC 660 2 0 GCACAGT ww r~Ar~rr~r~r~r~r- ArAArrrrTG wAT w wCG wCGAGAT w wATGAGCCA 720 aAr-Ar-ArrrA A~Arr~ArAr AAr~rAArGrr GCAGAGGCCG ArArAArrAr ArArArArAr 780 ATGCAGAGAG CAGATGCAGA r~r-rAr~Ar~r-c ArArArrAAr AArrArrTAr rArrrrr,r~C 840 AAGGTGCTCA TGCCTCTAAT rrrAr-rArTT TGc~r-Ar~r/rrr- Arr,rrrrArr. AT~ll~AG 900 CCCAGAAGTT rAArArrArC CTGwCAACA CAGTGA 936 (2) INFORMATION POR SEQ ID NO 13 (i) SBQUENCB ruARArTR~TcTIcs (A) LBNGTH 311 amino acids (B) TYPB amino acid (D) TOPOLOGY linear 3 0 (ii) MOLBCULB TYPB protein (iii) ~Y~Ol~hllCAL NO
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS

(vii) TMMRnTATR SOURCB
3 5 (B) CLONB APOB4LX2 (xi) SBQUBNCB DBSCRIPTION SBQ ID NO 13 Met Ser Ser Thr Arg Gly Ser A3n Gln Leu Leu Arg Arg Ala Trp Ly~

Ala Ser Ala Cy~ Arg Arg Ile Cy9 Trp Ala Cyo Ser Ser Ser Leu Ala Arg Thr Ser Ala Thr C,v8 Ser Phe Thr Ser Ser Arg Arg Ser Arg Val Arg Leu Pro Ile Ser Ser Ile Arg Ala Arg Ser Arg Ser Pro Gln Ala 4 5 Trp Ala Arg 5er Cys Ser Gly Trp Pro Ala Arg Glu Pro Thr val Ala Ala Arg Thr Arg Pro Cy~ Ser Thr Arg Gly Pro Arg Arg Ser Arg Met Ala Leu Arg Pro Arg Ser Ala Pro Ser arg Ala Pro Ala Trp Tyr Thr 5 0 loo 105 110 Ala Arg Arg Phe Cy~ Arg Ser Ser Ala Ser Arg Arg Ser Arg Leu Arg Ser Leu Arg Arg Trp Glu Ala Arg Arg Thr Arg Ser Ser Ser Val Leu 212~ 787 Trp Pro Ser Met Ala Cyo Thr Ser Pro Arg Tyr Cys Thr Arg Ala Pro Arg Thr Ser Ser Met Ser Ala Pro Ala Arg Ala CYB Ala Ala Cyn Ser Ser Leu Asp Ser Arg Ala Arg Val Ser Ser Ala Thr Gly Val Ser Cys Ser Ser Ser Ser Asp Leu Ala Ala Phe Asn Ser Ser Met Val Ser Ser 1 0 Ile Cy9 Ala Val Arg Pro Arg Gly Arg Glu Gly Val Ala Gln Trp Gly Gly Gly Gly Glu Gly Leu Gly Trp Gly Gly Arg Asp Gly Asp Glu Pro 22s 230 235 240 Glu Arg Pro Ly~ Arg Glu Arg Arg Asn Gly Ala Glu Ala Glu Arg Arg Arg Gln Arg Gln Met Gln Arg Ala Asp Ala GlU Gly Arg Gly Arg Asp Glu Glu Gly Ala Arg Arg Pro Gly Lys Val Leu Met Pro Leu Ile Pro 2 0 Ala Leu Trp Glu Ala Glu Ala Gly Gly Ser Leu Glu Pro Arg Ser Ser Arg Pro Ala Trp Ala Thr Gln (2) INFORMATION FOR SBQ ID NO:14:
(i) SBQUBNCB r~A~ArTR~T~TIcs:
(A,l L8NGTH: 399 base pairs (Bl TYPB: nucleic acid (Cl STRANDBDNESS: single (D:l TOPOLOGY: linear (ii) MOLBCULB TYPB: cDNA
(iii) ~Y~l~llCAL: NO
(iv) ANTI-SBNSB: YBS
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: Pac-1 (xi) SEQU8NCB DBSCRIPTION: SBQ ID NO:14:
ATGTCGGAAT TCTGCATCCA TCTT QCTTC AGAGATCTCC T~ iA TATTTGTCAA 60 rrrArAArrT GGTCGAGTGG TCAGTCCTCG GTCGGCAGCA ~ T CAACAGGCTC 120 4 0 AACTGGGCAC AGGAAGCAAG cc~ArArAA ArrAAArAA~ ACAAATCAAG ATGGAGAACG 180 ~-llG~l~ CTCAGGGGAC TCTTACCTTC ~llll~l~li ll~i~l~i~A CAGAGTCAGC 240 rrrAAAArAA TGCCACGGCT GGAGATCGTC CAGGCTGAAC TCTCCATTCA rr,r~ARr~r 300 CTCCACGGTG ~llll~illl CGGTCAAAGA TGGCATGAGA GCATCGTTTC CGTAACTGAT 360 ~ill~ll~A CTAATCATGT TGGCCAAGAC GTCATCTGA 399 (2) INFORMATION FOR SBQ ID NO:15:
(i) SBQUBNCB r~A~ArTR~T.CTICS:
(A) LBNGTH: 513 base pairs (B) TYPB: nucleic acid (C) STR~.J~uN~SS: single (D) TOPOLOGY: linear (ii) MOLBCULB TYPB: cDNA
(iii) ~Y~ul~llCAL: NO
(iv) ANTI-SBNSB: YBS
(vi) ORIGINAL SOURCB:
5 5 (A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: Pac-2 2125~7 (xi) SEQUBNCB DBSCRIPTION SBQ ID NO 15 ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC TC~--ll~A TATTTGTCAA 60 rrrArAArrT GTATTACATC ATAATTAAAG GGTCACTTCA AATTCTACTC Tr-rAr-TAAr-A 120 TCAATTGAGA GAGGCTTAAA ATGCAGAAAG rArArAArrT CTGCTCGAGC TTArrrrrAA 180 GATGCGGAGA rGrArAArTc AAGCGGTTGT GATACCTGGT CGAGTGGTCA ~l~ ~lC 240 rrrArrArrr CGGGCATCAA GATGGAGAAC GCC~ ~ GCTCAGGGGA CTCTTACCTT 300 ~llll~l~l ~ll~lvG~ ACGAGTCAGC rrrAAAArAA TGCCACGAGT CAGCCCCAAA 360 ~l~llllC ~lll~l A AAGATGGCAT GAGAGCATCG TTTCCGTAAC TGATGGTTGG 480 1 0 TTCACTAATC Ai~ll~A AGACGTCATC TGA513 (2) INPORMATION FOR SBQ ID NO 16 (i) SBQUBNCB CHARACTBRISTICS
~A) LBNGTH 321 base pairs B) TYPB nucleic acid c) STRr-~RT)NRqq: Eiingle l~D) TOPOLOGY linear (ii) MOLBCULB TYPB cDNA
(iii) dY~ld~llCAL NO
(iv) ANTI-SENSB YES
2 0 (vi ) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB Pace (xi) SBQUBNCB DBSCRIPTION SBQ ID NO 16 ATGCGGAGAG G QCAAGTCA AG~ll.l~ ATACCTGGTC GAGTGGTCAG l~l-~l~ 60 rr~rrArrrC GGGCATCAAC AGGCTCAAGT Grr,rArArrA Arrr~ ArA~AAAr~rA 120 AArAArArAA ATCAAGATGG AGAACGCCCT l~ A GGGGACTCTT ACCTTCGAAA 180 I.l~l~ll~ CTGwCACAGA GTCAGCCCCA AAAGAATGCC ACwCTGwAG ATCGTCCAGG 240 CTGAACTCTC CATTCACGw AAGGAGCTCC A~wl~ll~ 1~111 - Wl CAAAGATGwC 300 3 0 ATGAGAGCAT ~lll.~lA A 321 (2) INFORMATION FOR SBQ ID NO 17 (i) SBQUBNCB rHARArTRRTqTICS
(A) LBNGTH 72 base pairo (B) TYPB nucleic acid (c) STR~'~)Rr'--"9: oingle (D) TOPOLOGY linear (ii) MOLBCULB TYPB cDNA
(iii) HY~VlHhllCAL NO
(iv) ANTI-SBNSB YBS
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB Tpac-1 (xi) SBQUBNCB DBSCRIPTION SBQ ID NO 17 ATGCAGAAAG rArArAA~rT CTGCTCGAGC TTAr-GrrrAA GATGCGwAGA wCACAAGTC 60 (2) INFORMATION FOR SBQ ID NO 18 (i) SBQUBNCB r~ARArTRRT~qTIcs (A) LBNGTH 152 amino acido 5 0 (B) TYPB amino acid (D) TOPOLOGY linear (ii) MOLBCULB TYPB protein 212 ~ r~ ~ r~

(iii) ~IYt~ bLlCAL: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMBDIATE SOURCE:
(B) CLONE: Pac-1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Ser Glu Phe Cys Ile His Leu His Phe Arg Asp Leu Leu Arg Leu Asp Ile Cys Gln Pro Arg Thr Trp Ser Ser Gly Gln Ser Ser Val Gly Ser Arg Ala Gly Ile Asn Arg Leu Lys Trp Ala Gln Glu Ala Arg Asp Thr Glu Ser Lys Gln Asp Lys Ser Arg Trp Arg Thr Pro Leu Leu Ala Gln Gly Thr Leu Thr Phe Glu Ile Cys Val Gly Trp His Arg Val Ser Pro Lys Arg Met Pro Arg Leu Glu Ile Val Gln Ala Glu Leu Ser Ile 2 o His Gly Lys Glu Lys His Gly Gly Phe Arg Phe Gly Gln Arg Trp His Glu Ser Ile Val Ser Val Thr Asp Gly Trp Phe Thr Asn His Val Gly Gln Asp Val Ile (2) INFORMATION FOR SEQ ID NO:19:
(i) S--QUENCE rH~R Z~rTRR T .qTICS:
(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 3 0 (ii) MOLECULE TYPE: protein (iii) ~IY~UldbLlCAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: Tpac-2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Gln Lys Gly Asp Asn Val Cys Ser Ser Leu Gly Pro Arg Cys Gly Glu Ala Gln Val Ly~3 Arg Phe (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE rul~R~rTRRT~qTIcs:
(A) LENGTH: 106 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) ~IY~ULrlbLlcAL: 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 Gln Ala Val Leu Ile Pro Gly Arg Val Val 5 5 Ser Pro Arg Ser Ala Ala Gly Arg Ala Ser Thr Gly Ser Ser Gly His Arg Lys Gln Gly Thr Gln Lys Ala Asn Lys Thr Asn Gln Asp Gly Glu Arg Pro Cys Trp Leu Arg Gly Leu Leu Pro Ser Lys Ser Val Leu Ala Gly Thr Glu Ser Ala Pro Lys Glu Cys His Gly Trp Arg Ser Ser Arg 6s 70 75 80 Leu Asn Ser Pro Phe Thr Gly Arg Ser Ser Thr Val Val Phe Val Ser Val Lys Asp Gly Met Arg Ala Ser Phe Pro (2) INFORMATION POR SEQ ID NO 21 (i) SEQUENC_ CHARACTERISTICS
(A) LENGTH 250 base pairs (B) TYPE nucleic acid (c) STR~ ~q single (D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA
(iii) ~Y~ulrl~llCAL NO
(iv) ANTI-SBNSE YES
(vi) ORIGINAL SOURCE
(A) ORGANISM HOMO SAPIENS
(vii) IMMEDIATE SOURCR
(B) CLONE Pac/reg (xi) SEQUENCE DESCRIPTION SEQ ID NO 21 CTAAATr-r3ATATAATTTAcAATTTATAAACGc~AATTArAAr-AA~llcAlll.l.~AATG 60 rArrrrArATTTGGATGAGGTTATATAAAAArTTTCAGTATA~ l~CC AACTGGTT 120 GGTcAAATATTTGATGcTTAcTTTA7~AAAAAAAAAAAA7\7\TTTAGTAGAGA~ 180 ACCATATTGGCCA~G~l~l.l.iAATTTCTGACCTCGTGA~C~l.~iC~ ~l.C 240 3 0 (2) INFORMATION FOR SEQ ID NO 22 (i) SEQUENCE CHARACT8RISTICS
(A) LENGTH 366 base pairs (B) TYPE nucleic acid (C) STRANnRnNRcc single 3 5 (D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA
(iii) 11Y~Ul ~ CAL NO
(iv) ANTI-SENSE YES

(vi) ORIGINAL SOURC_ 4 0 (A) ORGANISM HOMO SAPIENS
(vii) IMMEDIATE SûURCE
(B) CLONE C-FosSL

212~787 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
ATGA~lll.~-''-'''''Arrr.rTA~rrr~AA~"~l~AAACCTGCT 60 GAcGcAGATGTccTAATATGGAcA~lccl~~ Ar~r,r~rArcrATTr-Arrrr.AArTGcT 120 ~ r~rrAArArrr-Ar~r~r~TGcA~l~-l~x:~xx/~ l~G~ l~bb~ 180 r~rr~r~rrrrraAArGr~rr~cAGAATr~r~r~r~ArATTcGcAc~-l~bll~AATGcGGAc 240 ~A~lll~ ~X-~l~rArArAl-~ IllllACCCTTGTACGGAAA 300 cTr~AAr~ArAr~TTcTGAGGcTrAr~Ar~ATAr~r~Ar~AAArr-r~rATcGAGTArAr~r~Arrrr~r~Ar~r~ 360ACTTAA 420 (2) INFORMATION FOR SEQ ID NO:23:
1 0 (i) SBQUENCE rHARArTR~T~cTIcs:
(A) LENGTH: 121 amino acido (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) ~Y~ul~llCAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: C-Fos5Lp (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Arg Gly Phe Gly Asp Gly Ser Pro Gln Gly Tyr Arg Glu Arg Pro Trp Lys Pro Ala Asp Ala A~p Val Leu Ile Trp Thr Ser Cy5 Val Arg 2 5 Gly Glu Gly Leu Thr Gly Thr Ala Arg Gly Leu Gln Pro Thr Pro Arg Val Gln Cy~ Gly Gly Arg Arg Gly Pro Arg Leu Gly Glu Gly Arg Arg Glu Arg Arg Arg Met Arg Glu Asn Ile Arg Thr Trp Phe Asn Ala Asp Pro Cys Ser Arg Gly Arg Gly Gly Trp Gly Arg Glu Arg Leu Phe Thr Leu Val Arg Lyo Leu Ly~ Thr Val Leu Arg Leu Arg Asp Arg Arg A~n 3 5 Gly Ile Glu Tyr Arg Thr Pro Arg Thr (2) INFORMATION EOR SEQ ID NO:24:
(i) SEQUENCE ChARACTERISTICS:
(A) LENGTH: 294 ba~e pairs (B) TYPE: nucleic acid (C) STRP ~: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic DNA

(iii) ~Y~ul~llCAL: NO

(iv) ANTI-SBNSB YBS
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIENS
(vii) TMMRnTATR SOURCB
(B) CLONB Fo~SLreg (xi) SBQUBNCB DBSCRIPTION SBQ ID NO 24 TTcG~l~(ic~w~ ~l~AGTcTT wcTTcTcA~ ~AGATGcwT 60 TGr-Ar-TArr-Ar~r-cGccGcAGccA~ TAArAAr-cGTTTTATGAATGAGTGTAAA 120 cGTcAcwwrArAArrA~ l~r-Ar~-l~i((-((-((~-(-(l(iW~i~ll.l~C i80 0 l~AG-l~ lvG~ 240 AAI~l~l~AGcATTTcGcA~ll~ AGAh~l~ l~ wl~ Ar~ 300

Claims (46)

1. A nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2, C-Fos5L
and C-Fos5Lreg.

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:1.

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 ApoE4L1.

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. The nucleic acid of claim 1 that encodes fosC5Lp

15. The nucleic acid of claim 14 wherein said sequence is SEQ ID NO:22.

16. The nucleic acid of claim 1 wherein said sequence is SEQ ID NO:24.

17. A protein, substantially free of natural contaminants, selected from the group consisting of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-Fos5Lp.

18. The protein of claim 17, wherein said protein is Pac.

19. The protein of claim 18 wherein said protein has a sequence of SEQ ID NO:2.

20. The protein of claim 17, wherein said protein is Tpac.

21. The protein of claim 20, wherein said protein has a sequence of SEQ ID NO:4.

22. The protein of claim 17, wherein said protein is hsaP.

23. The protein of claim 22, wherein said protein has a sequence of SEQ ID NO:6.

24. The protein of claim 17, wherein said protein is apoE4L.

25. The protein of claim 24 wherein said protein has a sequence of SEQ ID NO:8.

26. The protein of claim 17, wherein said protein is apoE4L1.

27. The protein of claim 26, wherein said protein has a sequence of SEQ ID NO:10.

28. The protein of claim 17, wherein said protein is apoE4Lx2.

29. The protein of claim 28, wherein said protein has a sequence of SEQ ID NO:13.

30. The protein of claim 17, wherein said protein is fosC5Lp

31. The protein of claim 28, wherein said protein has a sequence of SEQ ID NO:23.

32. A reagent capable of diagnosing the presence of a molecule selected from the group consisting of Pac, a Pac-encoding nucleic acid molecule, Pac-1, 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, ApoE4L1, an ApoE4L1-encoding molecule, ApoE4Lx2, an ApoE4Lx2-encoding molecule, C-Fos5Lp and an C-Fos5Lp encoding molecule.

33. The reagent of claim 32, wherein said reagent is a nucleic acid molecule.

34. The reagent of claim 33, wherein said reagent is a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12 and SEQ ID
NO:22.

35. The reagent of claim 32, wherein said reagent is obtainable by mutating a nucleic acid molecule having a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:21, SEQ ID
NO:22 and SEQ ID NO:24.

36. The reagent of claim 32, wherein said reagent is a protein.

37. The reagent of claim 36, wherein said protein is an antibody, or a fragment of an antibody.

38. The antibody or fragment of an antibody of claim 37 which is capable of binding to a protein selected from the group consisting of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-Fos5Lp.

39. 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-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2, C-Fos5L or C-Fos5Lreg.

40. The method of claim 39, wherein said inhibitor is a protein.

41. The method of claim 41, wherein said inhibitor is an antibody, or fragment thereof.

42. The method of claim 39, wherein said inhibitor is a nucleic acid molecule.

43. 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, ApoE4L1 or ApoE4Lx2.

44. The method of claim 43, wherein said inhibitor is a protein.

45. The method of claim 44, wherein said inhibitor is an antibody, or fragment thereof.

46. The method of claim 43, wherein said inhibitor is a nucleic acid molecule.