CA2221986A1 - Method for identifying alzheimer's disease therapeutics using transgenic animal models - Google Patents

Abstract

The construction of transgenic animal models for testing potential treatments for Alzheimer's disease are described. The models are characterized by pathology similar to that observed in Alzheimer's disease, based on expression of all three forms of the .beta.-amyloid precursor protein (APP), APP695, APP751, and APP770, as well as various point mutations based on naturally occurring mutations, such as the London and Indiana familial Alzheimer's disease (FAD) mutations at amino acid 717, predicted mutations in the APP gene, and truncated forms of APP that contain the A.beta. region. The APP gene constructs are prepared using the human platelet derived growth factor B(PDGF-B) chain gene promoter, or other promoters able to express A$(b) or mutant forms APP at a high level in transgenic animal brain tissue. Animal cells can be isolated from the transgenic animals or prepared using the same constructs with standard techniques such as lipofection or electroporation. The transgenic animals, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer's disease as measured by their effect on the amount of APP and .beta.-amyloid peptide, neuropathology, and behavioral alterations.

Description

-W O 96/40895 PCTAUS96~0967g METHOD FOR IDEl~ Yl~G ~T~ /IER~s DISEASE
T~RAPEVTICS USING TRANSGENIC ANIMAL MODELS
Back~l ou"d of the Invention Transgenic animal models of Alzheimer's disease are described along with a nnethod of using the transgenic animal models to screen for therapeutics useful for the treatment of Alzheimer's disease.
Alzheimer's disease (AD) is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after ex~mining one of his patients who suf~ered drastic reduction in cognitive abilities and had generalized dementia (The early story of Alzheimer's Disease, edited by Bick et al.
(Raven ]?ress, New York 1987)). It is the leading cause of dementia in elderly persons. AD patients have increased problems with memory loss and intellechl~l functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibitpersonality changes, socially i~ lo~liate actions and schizophrenia (A
Guide to the Understanding of Alzheimer's Disease and Related Disorders, edited by Jorm (New York University Press, New York 1987). AD is dev~t~ting for both victims and their families, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration.
The impact of AD on society and on the national economy is enormous. It is expected that the demented elderly population in the United States will increase by 41% by the year 2000. It is expensive for the health care systems that must provide institutional and ancillary care for the AD
patients at an estim~tec~ annual cost of $40 billion (Jorm (1987); Fisher, "Alzheimer's Disease", New York Times, August 23, 1989, page D1, edited by Reisberg (The Free Press, New York & London 1983)). These factors imply action must be taken to generate effective treatments for AD.
~ A~ a macroscopic level, the brains of AD patients are usually smaller, sometimes weighing less than 1~000 grams. At a microscopic level, the histopathological h~llm~rks of AD include neurofibrillary tangles (NFT), neuritic plaques, and degeneration of neurons. AD patients exhibit degeneration of nerve cells in the frontal and temporal cortex of the cerebral W O 96/40895 PCT~US96/09679 cortex, pyramidal neurons of hippocampus, neurons in the medial, medial central, and cortical nuclei of the amygdala, noradrenergic neurons in the locus coeruleus, and the neurons in the basal forebrain cholinergic system.
Loss of neurons in the cholinergic system leads to a consistent deficit in cholinergic presynaptic markers in AD (Fisher (1983); Alzheimer's Disease and Related Disorders, Research and Development edited by Kelly (Charles C. Thomas, Springfield, IL. 1984)). In fact, AD is defined by the neuropathology of the brain.
AD is associated with neuritic plaques measuring up to 200 ~rlm in ~ m~ter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the p~ ci~al constituents of neuritic plaques is amyloid, which is stained by Congo Red (Fisher (1983); Kelly (1984)). Amyloid plaques stained by Congo Red are extracellular, pinK or rust-colored in bright field, and birefringent in polarized light. The plaques are composed of polypeptide ~lbrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mech~ni~m~ of AD neuropathy. However, available evidence strongly inrlic~Ps that there are distinct types of genetic predisposition for AD. First,molecular analysis has provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate et al.
Nature 349:704-706 (1991); Murrell et al. Science 254:97-99 (1991);
Chartier-Harlin et al. Nature 353:844-846 (1991); Mullan et al., Nature Genet. 1:345-347 (1992)). Additional genes for dominant forms of early onset AD reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature 376:775-778 (1995); Levy-Lahad et al., Science 269:973-977 (1995);
Sherrington et al., Nature 375:754-760 (1995)). Another loci associated with AD resides on chromosome l9 and encodes a variant form of apolipoprotein E (Corder, Science 261: 921 -923 (1993) .
Arnyloid plaques are abundantly present in AD patients and in Down's Syndrome individuals surviving to tne age of 40. The overexpression of APP

. CA 02221986 1997-11-24 W O 96/40895 PCT~US96/09679 in Down's Syndrome is recognized as a possible cause of the development of AD in Down's patients over thirty years of age (Rumble et al., New England J. Med. 320: 1446-1452 (1989); Mann et al., Neurobiol. Aging 10:397-399 (1989)). The plaques are also present in the normal aging brain, although at 5 a lower number. These plaques are made up primarily of the amyloid ~B
peptide l'~A,~; som~timPs also referred to in the liLel~lule as ~-amyloid peptide or ,~ pep~tide) (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is also the primary protein constituent in cerebrovascular amyloid deposits. The amyloid is a filamentous material that is arranged in 10 beta-pleated sheets. A,B is a hydrophobic peptide C~ liSillg up to 43 amino acids. The determination of its amino acid sequence led to the cloning of the APP cDNA (Kang et al., Nature 325:733-735 (1987); Goldgaber et al., Science 235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci. 84:4190-4194 (1987); T~n7i et al., Nature 331:528-530 (1988)) and genomic APP
DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989); Yoshikai et al., Gene 87, 257-263 (1990)). A number of forms of APP cDNA have been identifiedl, including the three most abundant forms, APP695, APP751, and APP770. These forms arise from a single precursor RNA by alternate splicing. The gene spans more than 175 kb with 18 exons (Yoshikai et al.
20 (1990)). APP contains an extracellular domain, a tran~m~nhrane region and a cytopla~smic domain. A,B consists of up to 28 amino acids just outside the hydrophobic Lldlls",el,lbrane ~lom~in and up to 15 residues of this tr~n~memhrane domain. Thus, A~B is a cleavage product derived from APP
which is normally found in brain and other tissues such as heart, kidney and 25 spleen. However, A~ deposits are usually found in abl-n~l~n~e only in the brain.
The larger alternate forms of APP (APP751, APP770) consist of APP695 plus one or two additional domains. APP751 consists of all 695 amino acids of APP695 plus an additional 56 amino acids which has 30 homology to the Kunitz family of serine protease inhibitors (KPI) (Tanzi et al. (1988); Weidemann et al., Cell 57:115-126 (1989); Kitaguchi et al., Nature 331:530-532 (1988); Tanzi et al., Nature 329:156 (1987)). APP770 W O 9G/1rB95 PCT~US96/09679 contains all 751 amino acids of APP751 and an additional 19 amino acid domain homologous to the neuron cell surface antigen OX-2 (Weidem~nn et al. (1989); Kitaguchi et al. (1988)). Unless otherwise noted, the amino acid positions referred to herein are the positions as they appear in APP770. The amino acid number of equivalent positions in APP695 and APP751 differ in some cases due to the absence of the OX-2 and KPI domains. By convention, the amino acid positions of all forms of APP are ler~renced by the equivalent positions in the APP770 form. Unless otherwise noted, this convention is followed herein. Unless otnerwise noted, all forms of APP and fr~Ement~ of APP, including all forms of A,~, referred to herein are based on the human APP amino acid sequence. APP is post-translationally modified by the removal of the leader sequence and by the addition of sulfate and sugar groups.
Van Broeckhaven et al., Science 248:1120-1122 (1990), have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This was confirmed by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al., Science 248:1124-1128 (1990)). The mutation substituted a gl(~ t? for ~hlt~mic acid at position 22 of the A~ (position 618 of APP695, or position 693 of APP770). In addition, certain families are genetically predisposed to Alzheimer's disease, a condition referred to as famili~l Alzheimer's disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full length protein (Goate et al.
(1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the families and are absent in families with late-onset AD. This mutation at amino acid 717 increases the production of the A~,~ form of A,B from APP (Suzuki et al., Science 264:1336-1340 (1994)). Another mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al.
(1992)). This mutation to amino acids 670 and 671 increases the production of total A~ from APP (Citron et al., Nature 360:622-674 (1992)).

W O ~GI~E9~ PCT~US96/09679 There are no robust animal models to study AD, although aging nonhllm~n primates seem to develop amyloid plaques of A~6' in brain parenchyma and in the walls of some meningeal and cortical vessels.
Although aged primates and canines can serve as animal models, they are S expensive to m~inf~in, need lengthy study periods, and are quite variable in the extent of pathology that develops.
There are no spontaneous animal mutations with sufficient similarities to AD to be useful as experimental models. Various models have been proposed in which some AD-like symptoms may be in(lllced by electrolysis, transpla31tation of AD brain samples, ~Illmimlm chloride, kainic acid or choline ,analogs (Kisner et al., Neurobiol. Aging 7:287-292 (1986); Mistry et al., J Med Chem 29:337-343 (1986)). Flood et al., Proc. Natl. Acad. Sci.
88:3363-3366 (1986), reported ~mn~stir effects in mice of four synthetic peptides homologous to the A~. Rec~llce none of these share with AD either common symptoms, biochemistry or pathogenesis, they are not likely to yield much useful information on etiology or treatment.
Several transgenic rodent lines have been produced that express either the human APP gene or human APP complementary DNA regulated by a variety of promoters. Transgenic mice with the human APP promoter linked to E. coli ~-galactosidase (Wirak et al., The EMBO J 10:289-296 (1991)) as well as transgenic mice expressing the human APP751 cDNA (Quon et al.
Nature 352:239-241 (1991)) or subfr~gm~ntc of the cDNA including the A,~
(Wirak et al., Science 253:323-325 (1991); Sandhu et al., J. Biol. Chem.
266:21331-21334 (1991); Kawabata et al., Nature 354:476-478 (1991)) have been produced. Results obtained in the different studies appear to depend upon the source of promoter and the protein coding sequence used. For example, Wirak et al., Science 253:323-325 (1991), found that in transgenic mice exp~ressing a form of the A,l~, intracellular ~c~lm~ tion of "amyloid-like" material, reactive with antibodies prepared against A~B were observed but did not find other histopathological disease symptoms. The intracellular nature of the antibody-reactive material and the lack of other symptoms suggest that this particular transgenic animal is not a faithful model system CA 0222l986 l997-ll-24 W O ~G/~Cg95 PCT~US96/09679 for Alzheimer's disease. Later studies have shown that similar st:~inin~ iS
seen in non-transgenic control mice and Wirak et al., Science 253:323-325 (1991) was partially retracted in a comment in Science 255:143-145 (1992).
Thus, the st~ining seen by Wirak et al. appears to be artif~
Kawabata et al. (1991) report the production of amyloid plaques, neurofibrillary tangles, and neuronal cell death in their transgenic anim~l.c.
In each of these studies, A,~ or a fragment cont~ining A,~ was expressed.
Wirak et al. (1991), used the human APP promoter while Kawabata et al.
(1991) used the human thy-1 promoter. However, Kawabata et al. (1991) 10 was later retracted by Kawabata et al., Nature 356:23 (1992) and Kawabata et al., Nature 356:265 (1992). In transgenic mice expressing the APP751 cDNA from the neuron-specific enolase promoter of Quon et al. (1991), rare, small extracellular deposits of material reactive with antibody prepared against synthetic A,~ were observed. A review of the papers describing these 15 early transgenic mice in-lir~te that do not produce characteristic Al~heillle pathologies (see Marx, Science 255:1200-1202 (1992)).
Transgenic mice e~ s~hlg APP751 from a neuron-specific enolase (NSE) promoter were recently described by McConlogue et al., Neurobiol.
Aging 15:S12 (1994), Higgins et al., Ann Neurol. 35:598-607 (1995), Mucke 20 et al., Brain Res. 666:151-167 (1994), Higgins et al., Proc. Natl. Acad. Sci.USA 92:4402-4406 (1995), and U.S. Patent 5,387,742 to Cordell. Higgins et al., Ann Neurol. 35:598-607 (1995) describe results with the sarne mice as described by Quon et al. (1991). Such mice have only sparse A,B deposits which are more typical of very early AD and young Down's syndrome cases.
25 The deposits seen in this transgenic mouse were also seen, although at a lower abnn~l~nre7 in non-transgenic control ~nim~ls. Mature lesions such as frequent compacted plaques, neuritic dystrophy and extensive gliosis are not seen in these mice (Higgins et al.~ Ann Neurol. 35:598-607 (1995)).
McConlogue et al. (1994) reported finding no A,~ deposits in these mice.
Transgenic mice in which APP is expressed from the neuronal specific synaptophysin promoter express APP at low levels equivalent to that in brain PCT/US96/0967g W O 3~ 3J

tissue from the NSE APP mice described above. These mice were also reportedl not to display any brain lesions (Higgins et al.).
Tlallsgenic mice cont~inin~ yeast artificial chromosome (YAC) APP
constructs have also been made (Pearson and Choi, Proc. Natl. Acad. Sci.
S USA 90 10578-10582 (1993); Lamb et al., Nature Genetics 5:22-30 (1993);
Buxbaum et al., Biochem. Biophys. Res. Comm. 197:639-645 (1993)). These mice contain the entire human APP genomic gene and express human APP
protein at levels similar to endogenous APP; higher levels of e,~ e;,sion than that obtained in mice using the NSE promoter. None of these mice, however, show evidence of pathology similar to AD.
Alzheimer's disease animal models, including transgenic models, have been recently reviewed by Lannfelt et al., Behavioural Brain Res. 57:207-213 (1993), and Fukuchi et al., Ann. N. Y. Acad. Sci. 695:217-223 (1993).
Lannfelt et al. points out that none of the prior transgenic ~nim~lc that show ap~al~ plaques demonstrate neuropathological changes characteristic of AD.
Lannfelt et al. also ~liccllcces possible reasons for the "failure" of previous transgenic animal models. Similarly, Fukuchi et al. tliccllc~es the failure of prior transgenic animal models to display most of the characteristics known to be associated with AD. For example, the transgenic mouse reported by Quon et al. is reported to produce A~B immnnoreactive deposits that stain only infrequently with thioflavin S and not at all with Congo Red, in contrast to the staining pattern of AD A~B deposits.
It is therefore an object of the present invention to provide an animal model for Alzheimer's disease that is constructed using transgenic technology.
It is a further object of the present invention to provide transgenic ~nim~lc characterized by certain genetic abnorm~liti~os in the expression of theamyloid precursor protein.
It is a further object of the present invention to provide transgenic ~nim~lc exhibiting one or more histopathologies similar to those of Alzheimer's disease.

W O 96/40895 PCTrUS96/09679 It is a further object of the present invention to provide transgenic ~nim~l~ expressing one or more A,B-cont~ining proteins at high levels in brain tissue.
It is a further object of the present invention to provide a method of 5 screening potential drugs for the treatment of Alzheimer's disease using transgenic animal models.
Su~ of the Invention The construction of transgenic animal models for testing potential tre~tment~ for Alzheimer's disease is described. The models are 10 characterized by a greater sirnilarity to the conditions existing in naturally occurring Alzheimer's disease, based on the ability to control expression of one or more of the three major forms of the ,B-amyloid precursor protein (APP), APP695, APP751, and APP770, or subfragments thereof, as well as various point mutations based on naturally occurring mutations, such as the 15 FAD mutations at amino acid 717, and predicted mutations in the APP gene.
The APP gene constructs are prepared using the naturally occurring APP
promoter of human, mouse, or rat origin, efficient promoters such as human platelet derived growth factor ~ chain (PDGF-B) gene promoter, as well as inducible promoters such as the mouse metallothionine promoter, which can 20 be regulated by addition of heavy metals such as zinc to the animal's water or diet. Neuron-specific expression of constructs can be achieved by using the rat neuron specific enolase promoter.
The constructs are introduced into animal embryos using standard techniques such as microinjection or embryonic stem cells. Cell culture 25 based models can also be prepared by two methods. Cells can be isolated from the transgenic ~nim~l~ or prepared from established cell cultures using the same constructs with standard cell transfection techniques.
The constructs disclosed herein generally encode all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, 30 preferably an A~-cont~ining protein, as described herein. Examples of A~-cont~inin~ proteins are proteins that include all or a contiguous portion of APP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692, W ~9-'~C99S PCT/U5'9~9C79 and/or '717, APP751, APP751 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP695, and APP695 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, where each of Ihese A~-cont~ining ~roteills inrl~ s amino acids 672 to 714 of human APP. Some 5 specific constructs that are described employ the following protein coding sequences: the APP770 cDNA; the APP770 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the APP751 cDNA cont~ining the KPI protease inhibitor domain without the OX-2 domain in the construct; the APP751 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the APP695 cDNA; the APP695 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; APP695, APP751, or APP770 cDNA trl-nr~te~l at amino acid 671 or 685, the sites of ,~-secretase or o~-secretase cleavage, respectfully; APP
cDNA ~ r~t~l to encode arnino acids 646 to 770 of APP; APP cDNA
trrlnr~t-~cl to encode amino acids 646 to 770 of APP and including at least one intron; lhe APP leader sequence followed by the A~ region (amino acids 672 to 714 of APP) plus the rem~ining carboxy terminal 56 amino acids of APP;
the APP leader sequence followed by the A,~ region plus the rem~ining carboxy terminal 56 amino acids with the addition of a mutation at amino acid 717; the APP leader sequence followed by the A,~ region; the A,~ region plus the rem~ining carboxy terminal 56 amino acids of APP; the A~ region plus the rem~ining carboxy terminal 56 amino acids of APP with the addition of a mutation at amino acid 717; a combination cDNA/genomic APP gene construct; a combination cDNA/genomic APP gene construct with the addition of a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a combination cDNA/genomic APP gene construct truncated at amino acid 671 or 685; and an APP cDNA construct cont~ini1lg at least amino acids 672 to 722 of APP.
l hese protein coding sequences are operably linked to leader sequences specifying the transport and secretion of the encoded A,B related protein. A ~refell~d leader sequence is the APP leader sequence. These W O ~G/~8g5 PCTrUS96/09679 combined protein coding sequences are in turn operably linked to a promoter that causes high expression of A,B in transgenic animal brain tissue. A
preferred promoter is the human platelet derived growth factor ,B chain (PDGF-B) gene promoter. Additional constructs include a human yeast artificial chromosome construct controlled by the PDGF-B promoter; a human yeast artificial chromosome construct controlled by the PDGF-B
promoter with the addition of a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the endogenous mouse or rat APP gene modified through the process of homologous recombination between the APP gene in a mouse or rat embryonic stem (ES) cell and a vector carrying the human APP cDNA bearing a mutation at amino acid position 669, 670, 671, 690, 692, 717, or a combination of these mutations, such that sequences in the resident rodent chromosomal APP gene beyond the recombination point (the preferred site for recombination is within APP exon 9) are replaced by the analogous human sequences bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations. These constructs can be introduced into the transgenic ~nim~lc and then combined by mating of ~nim~lc e~,e~hlg the different constructs.
The transgenic ~nim~lc, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer's disease as measured by their effect on the amounts of APP, A~, and neuropathology in the ~nim~l.c, as well as by behavioral alterations.
Brief Des~ ,lion of the Diawill~s The boxed portions of the drawings intlir~te the amino acid coding portions of the constructs. Filled portions indicate the various dom~in.c of theprotein as in~lic~ttod in the Figure Legend. Lines in~lic~te sequences in the clones that are 5' or 3' untr~n.cl~t~d sequences, fl~nking genomic sequences, or introns. The break in the line to the left of the constructs in Figures 7 and8 indicates the presence of a long DNA sequence.
Figure la is a schematic of the APP770 cDNA coding sequence.
Figure lb is a schematic of the APP770 cDNA coding sequence bearing a mutation at position 717.

W O 96/40895 PCTnUS96/09679 Figure 2a is a sch~m~tic of the APP751 cDNA coding sequence.
Figure 2b is a sçh~m~ti~ of the APP751 cDNA coding sequence bearing a mutation at position 717.
Figure 3a is a sch~m~tic of the APP695 coding sequence.
iFigure 3b is a sc~om~tic of the APP695 cDNA coding sequence bearing a mutation at position 717.
lFigure 4a is a sch~m~ti~ of a coding sequence for the carboxy te"";.~1 portion of APP.
Figure 4b is a srll~m~tir of a coding sequence for the carboxy 10 terminal portion of APP bearing a mutation at position 717.
Figure 5 is a schPnl~ti~- of a coding sequence for the A,~ portion of APP.
~ igure 6a is a schematic of a combination cDNA/genomic coding sequence allowing alternative splicing of the KPI and OX-2 exons.
Figure 6b is a schematic of a combination cDNA/genomic coding sequence bearing a mutation at position 717 and allowing alL~Il~Live splicing of the KPI and OX-2 exons.
Pigure 7a is a sch~m~tic of a human APP YAC coding sequence.
Pigure 7b is a schematic of a human APP YAC coding sequence bearing a mutation at position 717.
Figures 8a and 8b are sch~m~tirs of genetic alteration of the mouse APP gene by homologous recombination between the mouse APP gene in a mouse ES cell and a vector carrying the human APP cDNA (either of the wild-type (Figure 8a) or FAD mutant form (Figure 8b)) directed to the exon ~5 9 portion of the gene. As a result of this recombination event, sequences inthe resident mouse chromosomal APP gene beyond the recombination point in exon 9 are replaced by the analogous human sequences.
Figure 9 is a schematic map of the PDAPP vector, a combination cDNA/genomic APP construct.
Figure 10 is a diagram of the genomic region of APP present in the PDAPP construct. The sizes of original introns 6, 7 and 8, as well as the sizes of the final introns are indicated on the diagram. The locations of the W O 96/40895 PCT~US96/09679 deletions in introns 6 and 8 present in the PDAPP construct are also inr1irz~t~
Figure 11 is a diagram of the intermlodi~te constructs used to construct the APP splicing c~.c~ette and the PDAPP vector.
S Figure 12 is a diagram of the PDAPP-wt vector and the plasmids used to make the PDAPP-wt vector.
Figure 13 is a diagram of the PDAPP-Sw/Ha vector and the plasmids and interm~ tto constructs used to make the PDAPP-Sw/Ha vector.
Figure 14 is a diagram of the PDAPP695~ vector and the plasmids and interm~ te constructs used to make the PDAPP695v, vector.
Figure 15 is a diagram of the PDAPP751v~ vector and the plasmids and intermto~ te constructs used to make the PDAPP751~. vector.
Detailed l~es~l, ti~n of the Invention The constructs and transgenic ~nim~ and animal cells are prepared using the methods and materials described below.
Sources of materials.
Restriction endonucleases are obtained from conventional commercial sources such as New Fngl~n~1 Biolabs (Beverly, MA.), Promega Biological Research Products (Madison, WI.), and Stratagene (La Jolla CA.).
Radioactive materials are obtained from conventional commercial sources such as Dupont/NEN or Amersham. Custom-~lesign~ oligonucleotides for site-directed mutagenesis are available from any of several commercial providers of such materials such as Bio-Synthesis Inc., Lewisville, TX. Kits for carrying out site-directed mutagenesis are available from commercial suppliers such as Promega Biological Research Products and Stratagene.
Clones of cDNA including the APP695, APP751, and APP770 forms of APP
mRNA were obtained directly from Dr. Dmitry Goldgaber, NIH. Libraries of DNA are available from commercial providers such as Stratagene, La Jolla, CA., or Clontech, Palo Alto, CA. PC12 and 3T3 cells were obtained from ATCC (#CRL1721 and #CCL92, respectively). An additional PC12 cell line was obtained from Dr. Charles Marotta of Harvard Medical School, Massachusetts General Hospital, and McLean Hospital. Standard cell culture =~

W ~ ~ 3S PCT~US96/09679 media appropriate to the cell line are obtained from conventional commercial sources such as Gibco/BRL. Murine stem cells, strain D3, were obtained from Dr. Rolf Kemler (Doet~hm~n et al., J. Embryol. EJCP. Morphol. 87:27 (1985)). Lipofectin for DNA transfection and the drug G418 for selection of S stable Ll~,nsro~ ants are available from Gibco/BRL.
Definition of APP cDNA clones.
The cDNA clone APP695 is of the form of cDNA described by Kang et al., ~i'ature 325:733-735 (1987), and represents the most predominant form of APP in the brain. The cDNA clone APP751 is of the form described by Ponte et al., Nature 331:525-527 (1988). This form contains an insert of 168 nucleotides relative to the APP695 cDNA. The 168 nucleotide insert encodes the KPI domain. The cDNA clone APP770 is of the form described by Kitaguchi et al. Nature 331:530-532 (1988). This form contains an insert of 225 nucleotides relative to the APP695 cDNA. This insert includes the 168 nucleotides present in the insert of the APP751 cDNA, as well as an addition 57 nucleotide region that does not appear in APP751 cDNA. The 225 nucleotide insert encodes for the KPI domain as well as the OX-2 dom~in All three forms arise from the same precursor RNA ~,ans.;,i~ by alternative splicing. The 168 nucleotide insert is present in both APP751 cDNA and APP770 cDNA.
The sequence encoding APP695 is shown in SEQ ID NO:1. This sequence begins with the first base of the initiation codon AUG and encodes a 695 amino acid protein. The region from nucleotide 1789 to 1917 of SEQ
ID NO: 1 encodes the A,B. The amino acid sequence of APP695 is shown in SEQ ID NO:2. Amino acids 597 to 639 of SEQ ID NO:2 form the A,~. The amino-acid composition of the APP695 is A57, C12, D47, E85, F17, G31, H25, I23~ K38, L52, M21, N28, P31, Q33, R33, S30, T45, V62, W8, Y17 resulting in a calculated molecular weight of 78,644.45. These sequences are derived from Kang et al. (1988).
The sequence encoding APP751 is shown in SEQ ID NO:3. This sequence begins with the first base of the initiation codon AUG and encodes a 751 amino acid protein. Nucleotides 866 to 1033 of SEQ ID NO:3 do not appear in APP695 cDNA. The region from nucleotide 1957 to 2085 of SEQ
ID NO:3 encodes the A,B. The amino acid sequence of APP751 is shown in SEQ ID NO:4. Amino acids 289 to 345 of SEQ ID NO:4 do not appear in APP695. This 57 amino acid region includes the KPI do m~in. Amino acids 653 to 695 of SEQ ID NO:4 form the A~. These sequences are derived from Ponte et al. (1988).
The sequenre encoding APP770 is shown in SEQ ID NO:S. This sequence begins with the first base of the initiation codon AUG and encodes a 770 amino acid protein. Nucleotides 866 to 1090 of SEQ ID NO:5 do not 10 appear in APP695 cDNA. Nucleotides 1034 to 1090 of SEQ ID NO:S do not appear in APP751 cDNA. The region from nucleotide 2014 to 2142 encodes the A~. The amino acid sequence of APP770 is shown in SEQ ID
NO:6. Amino acids 289 to 364 of SEQ ID NO:6 do not appear in APP695.
This 76 amino acid region includes the KPI and OX-2 domains. Amino acids 15 345 to 364 of SEQ ID NO:6 do not appear in APP751. This 20 amino acid region includes the OX-2 domain. Amino acids 672 to 714 form the A,l~. A
probable membrane-sp~ il.g region of the APP occurs from amino acid 700 to 723. Unless otherwise stated, all references herein to nucleotide positions refer to the numbering of SEQ ID NO:5. This is the numbering derived 20 from the APP770 cDNA. Unless othenvise stated, all references herein to amino acid positions refer to the numbering of SEQ ID NO:6. This is the numbering derived from APP770. According to this numbering convention, for example, amino acid position 717 refers to amino acid 717 of APP770, amino acid 698 of APP751, and amino acid 642 of APP695. The above 25 sequences are derived from Kang et al. (1988) and Kitaguchi et al. (1988).
Unless otherwise noted, all forrns of APP and fragments of APP, including all forms of A,~, referred to herein are based on the human APP
amino acid sequence. For example, A~ refers to the human A,B, APP refers to human APP, and APP770 refers to human APP770. As used herein, the 30 term cDNA refers not only to DNA molecules actually prepared by reverse transcription of mRNA, but also any DNA molecule encoding a protein where the coding region is not in~e~ ed, that is, a DNA molecule having a W O 96~ 9S ~CT~US96~9679 continuous open reading fraIne encoding a protein. As such, the ter~n cDNA
as used herein provides a convenient means of l~fellh~g to a protein encoding DNA molecule where the protein encoding region is not interrupted by intron sequences (or any other sequences not encoding protein).
S DPfinitil~n of the APP g~ locus.
Chara~;leli;G~lion of phage and cosmid clones of human genomic DNA
clones listed in Table 1 below originally established a m;~ size of at least 100 kb for the Alzheimer's gene. There are a total of 18 exons in the APP gene (Lemaire et al., Nucl. Acid Res 17:517-522 (1989); Yoshikai et al.
10 (1990); Yoshikai et al., Nucleic Acids Res 102:291-292 (1991)). Yoshikai et al. (1990) describes the sequences of the exon-intron boundaries of the APP
gene. These results taken together indicate that the mil~;.. ~" size of the Alzheimer's gene is 175 kb.
I'able 1. AI~L.~ . 's Cosmid and Lambda Clones.

Name of Insert Library Clone Size (kb) ~ APP Region 1 GPAPP47A 35 25 kb promoter & 9 kb intron 1 Cosmid 2 GPAAP36A 35 12 kb promoter & 22 kb intron 1 3 GAPP30A 30-35 5' coding region 4 GAPP43A 30-35 exons 9, 10 and 11 1 GAPP6A 12 exon 6 2 GAPP6B 18 exons 4 and 5 3 GAPP20A 20 exon 6 4 GAPP20B 17 exons 4 and 5 Lambda 5 GAPP28A 18 exons 4 and 5 6 GAPP3A 14 exon 6 7 GAPP4A 19 exon 6 8 GAPPlOA 16 exons 9, 10 and 11 9 GAPP16A 21 exon 6 T,able 2 indicates where the 17 introns interrupt the APP coding sequence. The numbering refers to the nucleotide positions of APP770 - cDNA as shown in SEQ ID NO:5. The starting nucleotide of exon 1 represents the first transcribed nucleotide. It is negative because the + 1 5 nucleotide is the first nucleotide of the AUG initiator codon by convention CA 02221986 1997-ll-24 W O 96/~C89S PCTrUS96/09679 (Kang et al. (1988)). The ending nucleotide of exon 18 represents the last nucleotide present in the mRNA prior to the poly(A) tail (Yoshikai et al.
(1990)). It has been discovered that Yoshikai et al. (1990) and Yoshikai et al. (1991) contain an error in the location of exon 8. Figure 1 of Yoshikai et S al. (1991) includes an EcoRI fragment between EcoRI fragments cont~ining exon 7 and exon 8. In fact, this intervening EcoRI fragment is actually located imm~ tely after exon 8, so that the EcoRI fragment cont~ining exon 7 and the EcoRI fragment cont~ining exon 8 are adjacent to each other.
Table 2. Location of I~ ,-s in APP Gene Sequence.
Starting Ending Following nucleotide nucleotide Intron Exon 1 -146 57 Intron 1 Exon 2 58 225 Intron 2 Exon 3 226 355 Intron 3 Exon 4 356 468 Intron 4 Exon 5 469 662 Intron 5 Exon 6 663 865 Intron 6 Exon 7 866 1033 Intron 7 Exon 81034 1090 Intron 8 Exon 91091 1224 Intron 9 Exon 101225 1299 Intron 10 Exon 111300 1458 Intron 11 Exon 121459 1587 Intron 12 Exon 131588 1687 Intron 13 Exon 141688 1909 Intron 14 Exon 151910 1963 Intron 15 Exon 161964 2064 Intron 16 Exon 172065 2211 Intron 17 Exon 182212 3432 APP Gene Mutations.
Certain families are genetically predisposed to Alzheimer's disease, a condition referred to as familial Alzheimer's disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full len~th protein (Goate et al . (1991); Murrell el al . (1991); Chartier-Harlin etal. (1991)). These mutations co-segregate with the disease within the families. For example, Murrell et al. (1991) described a specific mutation found in exon 17 (which Murrell et al. refers to as exon 15) where the valine of position 717 is replaced by phenyl~l~ninP.

WO 9614~8,95 PCT~US96~0~67 ~nother FAD mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al. (1992)). In one form of this mutation, the lysine at position 670 is replaced by asparagine and the methionine at position 671 is replaced by leucine. The effect of this mutation is to increase the production of A~ in cultured cells approximately 7-fold (Citron et al., Nature 360: 672-674 (1992); Lai et al., Science 259:514-516 (1993)). Replacement of the methionine at position 671 with leucine by itself has also been shown to increase production of A,~.
Additional mutations in APP at amino acids 669, 670, and 671 have been shown to reduce the amount of A~ processed from APP (Citron et al., Neuron 14:661-670 (1995)). The APP construct with Val at amino acid 690 produces an increased amount of a tnmr.~tc-cl form of A~.
APP expression clones can be constructed that bear a mutation at amino acid 669, 670, 671, 690, 692, or 717 of the full length protein.
The mutations from Lys to Asn and from Met to Leu at amino acids 670 and 671, respectively, are sometimes referred to as the Swedish mutation.
Additional mutations can also be introduced at amino acids 669, 670, or 671 which either increase or reduce the amount of A,B processed from APP.
Mutations at these amino acids in any APP clone or transgene can be created by site-directed mutagenesis (Vincent et al., Genes & Devel. 3:334-347 (1989)), or, once made, can be incorporated into other constructs using standard genetic engineering techniclues. Some mutations at amino acid 717 are sometimes referred to as the Hardy mutation. Such mutations can include conversion of the wild-type Val717 codon to a codon for Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, or Gln. A preferred substitution for Val717 is Phe. These mutations predispose individuals expressing the mutant proteins to develop Alzheimer's disease. It is believed that the mutations affect the expression and/or processing of APP, shifting the balance toward Alzheimer's pathology. Mutations at amino acid 669 can include conversion of the wild-type Val669 codon to a codon for Trp, or deletion of the codon. Mutations at amino acid 670 can include conversion of the wild-type Lys670 codon to a codon for Asn or Glu, or deletion of the W O 96/40895 PCT/U~ 5679 codon. Mutations at amino acid 671 can include conversion of the wild-type Met671 codon to a codon for Leu, Val, Lys, Tyr, Glu, or Ile, or deletion of the codon. A l,lefelled substitution for Lys670 is Asn, and a preferred substitution for Met671 is Leu. These mutations predispose individuals 5 expressing the mutant proteins to develop Alzheimer's disease. The other listed mnt~tion.c to amino acids 669, 670, and 671 are known to reduce the amount of A~ processed from APP (Citron et al. (1995)). It is believed that these mutations affect proces~ing of APP leading to a change in A,~
production.
Trl-nr~t~d forms of APP can also be expressed from transgene constructs. For example, APP cDNA trl-nr~t~cl to encode amino acids 646 to 770 of APP. The APP cDNA construct tr~nr~t~cl to encode amino acids 646 to 770 of APP, and operatively linked to the PDGF-B promoter, is referred to as PDAPPc125.
Nucleic Acid Constructs Encoding A,~-conts~inin~ Proteins.
Constructs for use in transgenic ~nim~l~ include a promoter for expression of the construct in a m~mm~ n cell and a region encoding a protein that includes all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, with or without specific amino acid mutations as described herein. It is preferred that protein encoded is an A,B-cont~ining protein. As used herein, an A~-cont~ining protein is a protein that includes all or a contiguous portion of one of the three forms of APP:
APP695, APP751, or APP770, with or without specific amino acid mutations as described herein, where the protein includes all or a portion of amino acids 672 to 714 of human APP. Preferred A~-cont~ining ~ tehls include amino acids 672 to 714 of human APP. Preferred forms of such A,~-cont~ining proteins include all or a contiguous portion of APP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP751, APP751 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP695, and APP695 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717~ where each of these A~-cont~ining proteins includes amino acids 672 to 714 of human APP.

CA 0222l986 l997-ll-24 W O ~6l4CB3S PCTAUS96~09679 Preferred forms of the above A,~-cont~ining proteins are APP770;
APP770 bearing a mutation in tne codon encoding one or more amino acids selected from the group con.~i~ting of amino acid 669, 670, 671, 690, 692, 717; APP751; APP751 bearing a mllt~tion in tne codon encoding one or more amino acids selected from the group con~ ting of amino acid 669, 670, 671, 690, 692, 717; APP695; APP695 bearing a mutation in the codon - encoding one or more amino acids selected from t'ne group consisting of amino acid 669, 670, 671, 690, 692, 717; a protein consisting of amino acids 646 to 770 of APP; a protein consisting of amino acids 670 to 770 of APP; a protein con~ ting of amino acids 672 to 770 of APP; and a protein consisting of amino acids 672 to 714 of APP.
II1 the constructs disclosed herein, the DNA encoding the A,~-cont~ininl~ protein can be cDNA or a cDNA/genomic DNA hybrid, wl~ in the cDNA/genomic DNA hybrid includes at least one APP intron sequence wherein the intron sequence is sufficient for splicing.
P:referred constructs contain DNA encoding APP770; DNA encoding APP770 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA
encoding APP770 which encodes an amino acid sequence com~ g amino acids 672 to 714 of APP770; DNA encoding APP751; DNA encoding APP751 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA
encoding APP751 which encodes an amino acid sequence comprising amino acids 672 to 714 of APP770; DNA encoding APP695; DNA encoding APP695 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692., 717, or a combination of these mutations; a fragment of DNA
encoding APP695 which encodes an amino acid sequence comprising amino - acids 672 to 714 of APP770; APP cDNA trllnr~t~-~1 to encode amino acids 646 to 770 of APP; a combination cDNA/genomic DNA hybrid APP gene construct; a combination cDNA/genomic DNA hybrid APP gene construct bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; or a combination W O 96140895 PCT/U~,Gl'u~579 cDNA/genomic DNA hybrid APP gene construct t~lnr~te~l at amino acid 671 or 685.
Plert;l~cd forms of such constructs are APP770 cDNA; APP770 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 5 690, 692, 717, or a combination of these mutations; a fragment of APP770 cDNA encoding an APP amino acid sequence, the amino acid sequence COlllp~iSillg amino acids 672 to 714 of APP770; APP751 cDNA; APP751 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP751 10 cDNA encoding an APP amino acid sequence, the amino acid sequence comprising amino acids 672 to 714 of APP770; APP695 cDNA; APP695 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP695 cDNA encoding an APP amino acid sequence, the amino acid sequence 15 comprising amino acids 672 to 714 of APP770; APP cDNA trllnr~t~d to encode amino acids 646 to 770 of APP; a combination cDNA/genomic DNA
hybrid APP gene construct; a combination cDNA/genomic DNA hybrid APP
gene construct bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, and a combination of these mutations; and a combination cDNA/genomic DNA hybrid APP gene construct tr~ n~tt~(~ at amino acid 671 or 685.
Construction of Tral,sg~l~es.
Construction of various APP transgenes can be accomplished using any suitable genetic engineering technique, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1989). Regions of APP clones that have been engineered or mutated can be interchanged by using convenient restriction enzyme sites present in APP cDNA clones. A NruI site starts at position -5 (relative to the first nucleotide of the AUG initiator codon). A KpnI and an Asp718 site both start at position 57 (these are isoschizomers leaving differentsticky ends). A XcmI site starts at position 836 and cuts at position 843. A
ScaI site starts at position 1004. A XhoI site starts at position 1135. A

CA 0222l986 l997-ll-24 W O ~CI4~B9S PCTAU596~09679 BamHI ,site starts at position 1554. A BglII site starts at position 1994. An EcoRI site starts at position 2020. A SpeI site starts at position 2583.
Another EcoRI site starts at position 3076.
1'he clones bearing various portions of the human APP gene sequence 5 shown in Figures 1 to 5 can be constructed in a common manner using standard genetic engineering techniques. For example, these clones can be constructed by first cloning the polyA addition signal from SV40 virus, as a 253 base pair Bcl~ to BamHI fragment (Reddy et al., Science 200:494-502 (1978), .into a modified vector from the pUC series. Next, the cDNA coding sequences (APP770, APP751, or APP695) can be inserted. Correct orientation and content of the fragmPntc inserted can be determined through restriction endonuclease mapping and limited sequencing. The clones bearing various carboxy terminal portions of the human APP gene sequence shown in Figures 4 and 5 can be constructed through several steps in addition to those in-lir~t~?d above. For example, an APP770 cDNA clone (SEQ ID NO:5) can be digesl:ed with ~sp718 which cleaves after nucleotide position 57. The reslllting 5' extension is filled in using the Klenow enzyme (Sambrook et al.
(1989)) cmd ligated to a hexanucleotide of the following sequence: AGATCT, the recognition site for BglII. After cleavage with BglII, which also cuts after position 1994, and re-ligation, the translational reading frame of the protein is preserved. The trllnr~tr~l protein thus encoded contains the leader sequence, followed by approximately 6 amino acids that precede the A,B, followed by the A,B, and the 56 terminal amino acids of APP. The clone in Figure 5 is created by converting the nucleotide at position 2138 to a T by site directed mutagenesis in the clone of Figure 4a, thus creating a termination codon directly following the last amino acid codon of the A~.
APP cDNA clones naturally contain an NruI site that cuts 2 nucleotides upstream from the initiator methionine codon. This site can be used for attachment of the different promoters used to complete each construct.
APP transgenes can also be constructed using PCR cloning techniques. Such techniques allow precise coupling of DNA fragments in the transgenes.

W O ~6/~9S PCT~US96/09679 Combination cDNA/Genomic DNA Clones.
Endogenous APP expression results from Lldllscli~tion of precursor mRNA followed by alternative splicing to produce three main forms of APP.
It is believed that this alternative splicing may be important in producing the pattern of APP expression involved in Alzheimer's disease. It is also believed that the presence of introns in expression constructs can influence the level and nature of expression by, for example, targeting precursor mRNA to mRNA processing and transport pathways (Huang et al., Nucleic Acids Res. 18:937-947 (1990)). Accordingly, transgenes combining cDNA
and genomic DNA, which include intron sequences, are a preferred type of construct.
The RNA splicing mfch~ni.~m requires only a few specific and well known consensus sequences. Such sequences have been identified in APP
genomic DNA by Yoshikai et al. (1990). The disclosed transgenes can be constructed using one or more complete and intact intron sequences.
However, it is prerelled that the transgenes are constructed using trllnr~,te-l intron sequences that contain an effective amount of intron seqllen~e to allow splicing. In general, trllnr~terl intron sequences that retain the splicing donor site, the splicing acceptor site, and the splicing branchpoint sequence will co,~liLuLe an effective amount of an intron. The sufficiency of any trl-n~te~l intron sequence can be determined by testing for the presence of correctly spliced mRNA in transgenic cells using methods described below.
Other intron sequences and splicing signals which are not derived from APP gene sequences may also be used in the transgene constructs.
Such intron sequences will enhance expression of the transgene construct. A
preferred heterologous intron is a hybrid between the adenovirus major late region first exon and intron junction and an IgG variable region splice acceptor. This hybrid intron can be constructed, for example, by joining the 162 bp PvuII to HindIII fragment of the adenovirus major late region, cont~ining 8 bp of the first exon and 145 bp of the first intron, and the 99 bp HindIII to PstI fragment of the IgG variable region splice acceptor clone-6, as described by Bothwell et al., Cell 24:625-637 (1981). A similar splice W O 96/40895 PCT~U~,5,'~S79 signal has been shown to enhance ~A~Iession of a construct to which it was ~chPd, as described by Manley et al., Nucleic Acids Res. 18:937-947 (1990). It is ~ler~ ed that the heterologous intron be placed between the promoter and the region encoding the APP.
~ pler~,.led APP combination cDNA/genomic ~Al~lcssion clone includes an effective amount of introns 6, 7 and 8, as shown in Figure 6.
Such a transgene can be constructed as follows. A pl~Çe-l~d method of construction is described in Example 5. A plasmid cont~inin~ the cDNA
portion of the clone can be constructed by first converting the TaqI site at position 860 in an APP770 cDNA clone to an XlloI site by site-directed mutagenesis. Cleavage of the resnlting plasmid with XlloI cuts at the new XhoI site and a pre-existing XlloI site at position 1135, and releases the KPI
and OX-2 coding sequence. The plasmid thus generated serves as the acceptor for the KPI and OX-2 alternative splicing c~sett~.
I'he alLe~ Live splicing cassette can be created through a series of cloning steps involving genomic DNA. First, the TaqI site at position 860 in a genomic clone cont~ining exon 6 and the adjacent dowl~llc~un intron can be converted to an XlloI site by site-directed mutagenesis. Cleavage of the resllltinp plasmid with X7~oI cuts at the new XlloI site and an XlloI site within ~0 either in~tron 6 or 7. This fragment, cont~ining a part of exon 6 and at least a part of adj~ent intron 6, can then be cloned into the X7~oI site in a plasmid vector. Second, a genomic clone cont~ining exon 9 and the adjacent upstreaml genomic sequences is cleaved with X7~oI, cleaving the clone at the XlloI site at position 1135 (position 910 using the numbering system of Kang et al. (1987)) and an XlloI site in either intron 7 or 8. This fr~gmf nt7 cont~ining a part of exon 9 and at least a part of adjacent intron 8, can then be cloned into the X71oI site of another plasmid vector. These two exon/intron junction fragments can then be released from their respective plasmid vectors by cleavage with XhoI and either BamHI or BglII, and cloned ~0 together into the XhoI site of another plasmid vector. It is ~cfe~l~d that the exon/intron junction fragments be excised with BamHI. It is most preferable that BamHI sites are engineered in the intron portion of the exon/intron W O 961~-95 PCTrUS96/09679 junction fragments prior to their excision. This allows the elimin~tion of lengthy extraneous intron sequences from the cDNA/genomic clone.
The XlloI fragment resl-lting from cloning the two exon/intron junction fragments together can be cleaved with either BamHI or BglII, depending on which enzyme was used for excision step above, and the genomic 6.8 kb BamHI segment, cont~ining the KPI and OX-2 coding region along with their fl~nking intron sequences, can be inserted. This fragment was identified by Kitaguchi et al. (1988) using Southern blot analysis of BamHI-digested Iymphocyte DNA from one normal individual and eight Alzheimer's disease 10 patients using a 212 bp TaqI-AvaI fragment, nucleotides 862 to 1,073, of APP770 cDNA as the hybridization probe. Genomic DNA clones cont~ining the region of the 225 bp insert can be isolated, for example, from a human leukocyte DNA library using the 212 bp TaqI-AvaI fragment as a probe. In the genomic DNA, the 225 bp sequence is located in a 168 bp exon (exon 7) 15 and a 57 bp exon (exon 8), separated by an intron of approximately 2.6 kb (intron 7), with both exons flanked by intron-exon consensus seq~lenres. The exon 7 corresponds to nucleotides 866 to 1,033 of APP770, and the exon 8 to nucleotides 1,034 to 1,090. Exon 7 encodes the highly conserved region of the Kunitz-type protease inhibitor family domain.
After cleavage with Xk,oI, this alternative splicing cassette, cont~ining both exon and intron sequences, can then be excised by cleavage with ~hoI
and inserted into the XlloI site of the modified APP770 cDNA plasmid (the acceptor plasmid) constructed above. These cloning steps generate a combination cDNA/genomic expression clone that allows cells in a transgenic 25 animal to regulate the inclusion of the KPI and OX-2 domains by a natural alternative splicing mechanism. An analogous gene bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations, can be constructed either directly by in vitro mutagenesis. A
mutation to amino acid 717 can also be made by using the mllt~tecl form of 30 APP770 cDNA described above to construct an acceptor plasmid.

_ W O ~ a~5 PCT~US96~09679 Promoters.
Dirrc~ promoter se~len~çs can be used to control expression of nucleotide sequences encoding A~B-cont~inin~ pl~L~ s. The ability to regulate expression of the gene encoding an A~B-cont~ining protein in S transgenic ~nim~lc is believed to be useful in ev~ ting the roles of the different APP gene products in AD. The ability to regulate expression of the gene encoding an A~-cont~ining protein in cultured cells is believed to be useful in ev~ ting expression and proce~sing of the different A~-cont~ining gene products and may provide the basis for cell cultured drug screens. A
preferrecl promoter is the human platelet derived growth factor ,B (PDGF-B) chain gene promoter (~S~h~ra et al., Cell 64:217-227 (1991)).
Preferred promoters for the disclosed APP constructs are those tlnat, when operatively linked to the protein coding sequences, m~ t~ expression of one or more of the following expression products to at least a specific level in brain tissue of a two to four month old animal transgenic for one of the disclosed APP constructs. The products and their expression levels are A,B to a level of at least 30 ng/g (6.8 pmoles/g) brain tissue and preferably atleast 40 ng/g (9.12 pmoles/g) brain tissue, A~,~ to a level of at least 8.5 ng/g(1.82 pmloles/g) brain tissue and preferably at least 11.5 ng/g (2.5 pmoles/g) brain tissue, full length APP (FLAPP) and APPcY combined (FLAPP+APP~) to a level of at least 150 pmoles/g brain tissue, APP~B to a level of at least 42 pmoles/g brain tissue, and mRNA encoding human A~-cont~ining protein to a level at least twice that of mRNA encoding the endogenous APP of the transgenic animal. A,B is the total of all fornns of A~. A~,~ is a form of A~
having amino acids 1 to 42 of A~B (corresponding to amino acids 672 to 714 of APP). FLAPP+APPcY refers to APP forms cont~ining the first 12 amino acids of the A,B region (corresponding to amino acids 672 to 684 of APP).
Thus, FL,APP+APP~ represents a mixture of full length forms of APP and APP cleaved at the ~-secretase site (Esch et al., Science 248:1122-1124 (1990)). APP,~ is APP cleaved at the ,B-secretase site (Seubert et al., Nature 361:260-263 (1993)).

W O gf/~89J PCTrUS96/09679 It is intended that the levels of expression described above refer to amounts of expression product present and are not limited to the specific units of measure used above. Thus, an expression level can be measured, for example, in moles per gram of tissue, grams per grams of tissue, moles per S volume of tissue, and in grams per volume of tissue. The equivalence of these units of measure to the measures listed above can be determined using known conversion methods.
The levels of expression described above need not occur in all brain tissues. Thus, a promoter is considered plc~rell~d if at least one of tne levels10 of expression described above occurs in at least one type of brain tissue.
Where expression is tissue-specific, it is understood that if the expression level is sufficient in the specific brain tissue, the promoter is considered preferred even though the expression level in brain tissue as a whole may not, and need not, reach a threshold level. It is preferred that this level of 15 expression is observed in hippoc~mp~l and/or cortical brain tissue. The promoter can m~ te expression of the above ~ ssion products to tne levels described above either constitutively or by induction. ~nrlurtion can be accomplished by, for example, a~lmini.ctration of an activator molecule, by heat, or by expression of a protein activator of transcription for the promoter 20 operatively linked to the gene encoding an A,B-cont~ining protein. Many inducible expression systems which would be suitable for this purpose are known to those of skill in the art.
It is preferred that, in making the above measurements, the brain tissue is prepared by the following method. A brain from a transgenic test 25 animal is ~ sect~d and the tissue is kept on ice throughout the homogenization procedure except as noted. The brain tissue is homogenized in 10 volumes (w/v) of 5 M g-~ni~line-HCl, 50 mM Tris-HCl, pH 8.5. The sample is then gently mixed for 2 to 4 hours at room temperature.
Homogenates are then diluted 1:10 in cold casein buffer #1 (0.25%
30 casein/phosphate buffered saline (PBS) 0.05% sodium azide, pH 7.4, lX
protease inhibitor cocktail) for a final 0.5 M gll~ni~lin~ concentration and kept on ice. 100X protease inhibitor cocktail is composed of 2 mg/ml aprotinin, . CA 02221986 1997-11-24 WO ~G14CB~S PCT~US96~09679 0.5 M EDTA, pH 8.0, 1 mg/ml leupeptin. Diluted homogenates are then spun in an Eppendorf microfuge at 14,000 rpm for 20 minllt~s at 4~C. If further dilutions are required, they can be made with cold gll~ni-lin~ buffer #2 (1 part guanidine buffer #1 to 9 parts casein buffer #1).
S It is ~l~re.led that the following assay be used to identify pler~lled promoters for their ability to me~ te expression of A~B to the levels described above. Antibody 266 (Seubert et al., Nature 359:325-327 (1992)) is dissolved at 10 ,ug/ml in buffer (0.23 g/L NaHPO.-HO, 26.2 g/L NaHP0.-7HO, 1 g/L sodium azide adjusted to pH 7.4) and 100 ~I/well is coated onto 96-well immnno~ y plates (Costar) and allowed to bind overnight. The plate is then aspirated and blocked for at least 1 hour with a 0.25% human serum alibumin solution in 25 g/L sucrose, 10.8 g/L Na..HPO~-7H!O, 1.0 g/L
NaHPO.-H!O, 0.5 g/L sodium azide adjusted to pH 7.4. The 266 coated plate is then washed lX with wash buffer (PBS/0.05% Tween 20) using a Skatron plate washer. 100 ~l/well of A~1~0 standards and brain tissue samples are added to the plate in triplicate and incubated overnight at 4~C.
A,Bl~0 standards are made from 0.0156, 0.0312, 0.0625, 0.125, 0.250, 0.500, and 1.000 ~4g/ml stocks in DMSO stored at ~0~ C as well as a DMSO only control for background determination. A,B standards consist of 1:100 dilution of each standard into guanidine buffer #3 (1 part BSA buffer to 9 parts gu~ni~1in~o buffer #1) followed by a 1:10 dilution into casein buffer#1 (Note: the final A,B concentration range is 15.6 to 1000 pg/ml and the final gl-~ni~lin~ concentration is 0.5 M). BSA buffer consists of 1 % bovine serum albumin (BSA, immllnoglobulin-free)/pBs/o.o5% sodium azide. The plates and casein buffer #2 (0.25% casein/PBS/0.05% Tween 20/pH 7.4) are then brought to room temperature (RT). The plates are then washed 3X with wash buffer. Next, 100 ~Ll/well of 3D6-biotin at 0.5 ~g/ml in casein buffer #2 is added to each well and incubated at 1 hour at RT.
Monoclonal antibody 3D6 was raised against the synthetic peptide DAEFRGGC (SEQ ID NO:10) which was conjugated through the cysteine to sheep anti-mouse imml~noglobulin. The antibody does not recognize secreted APP but does recognize species that begin at A,~ position 1 (Asp). For W O 96/40895 PCT~US96/09679 biotinylating 3D6, follow Pierce's NHS-Biotin protocol for labeling IgG (cat.
#20217X) except use 100 mM sodium bicarbonate, pH 8.5 and 24 mg NHS-biotin per ml of DMSO.
The plates are then again washed 3X with wash buffer. Then, 100 ,ul/well of horseradish peroxidase (HRP)-avidin (Vector Labs, cat. # A-2004) diluted 1 :4000 in casein buffer #2 is added to each well and incubated for 1 hour at RT. The plates are washed 4X with wash buffer and then 100 ,ul/well of TMB substrate (Slow TMB-ELISA (Pierce cat. # 34024)) at RT is added to each well and incubated for 15 minutes at RT. Finally, 25 ~I/well of 2 N HSO. is added to each well to stop the enzymatic reaction, and the plate is read at 450 nm to 650 nm using the Molecular Devices Vmax reader.
It is preferred that the relative levels of mRNA encoding human A,B-cont~ining protein mRNA encoding the endogenous APP of the L~ sg~ C
animal be measured in the manner described by Bordonaro et al., Biotechniques 16:428-430 (1994), and Rockenstein et al., J. Biol. Chem.
270:28257-28267 (1995). Preferred methods for measuring the expression level of A,B,~, FLAPP+APPcY, and APP,l~ are described in Example 8.
Yeast Artif~lcial Chrom(!so~nPc.
The constructs shown in Figure 7 can be constructed as follows.
Large segments of human genomic DNA, when cloned into certain vectors, can be propagated as autonomously-replicating units in the yeast cell. Such vector-borne segments are referred to as yeast artificial chromosomes (YAC;
Burke et al. Science 236:806 (1987)). A human YAC library is commercially available (Clontech, Palo Alto, CA) with an average insert size of 250,000 base pairs (range of 180,000 to 500,000 base pairs). A YAC
clone of the Alzheimer's gene can be directly isolated by screening the library with the human APP770 cDNA. The inclusion of all of the essential gene regions in the clone can be confiIIned by PCR analysis.
The YAC-APP clone, shown in Figure 7a, can be established in embryonic stem (ES) cells by selecting for neomycin resistance encoded by the YAC vector. ES cells bearing the YAC-APP clone can be used to produce transgenic mice by established methods described below under WO ~6~4~a3s PCT~US96/09679 "Transgenic Mice" and "Embryonic Stem Cell Metnods". The YAC-APP
gene bearing a mutation at amino acid 717 (Figure 7b) can be produced through the generation of a YAC libraIy using genomic DNA from a person affected by a mutation at amino acid 717. Such a clone can be iflerltifie~l and S established in ES cells as described above.
Genetic Alteration of the Mouse APP Gene.
The nucleotide sequence homology between the human and murine Alzheimer's protein genes is approximately 85%. Within the A,~-coding region, there are three amino acid differences between the two sequences.
Amino acids Lys 670, Met671, and Val717,which can be mllt~te(l to alter APP procescing, are conserved between mouse, rat, and man. Wild-type rodents do not develop Alzheimer's disease nor do they develop deposits or plaques in their central nervous system (CNS) analogous to those present in human Alzheimer's patients. Therefore, it is possible that the human but not the rodent form of A,~ is capable of causing disease. Homologous recombination (Capecchi, Science 244:1288-1292 (1989)) can be used to convert the mouse Alzheimer's gene in situ to a gene encoding the human A~
by gene replacement. This recombination is directed to a site downstream from the KPI and OX-2 domains, for example, within exon 9, so that the natural alternative splicing mech~nicmc a~ ol~liate to all cells within the transgenic animal can be employed in expressing the final gene product.
Both wild-type (Figure 8a) and mutant (Figure 8b) forms of human cDNA ran be used to produce transgenic models expressing either the wild-type or mutant forms of APP. The recombination vector can be constructed from a human APP cDNA (APP695, APP751, or APP770 form), either wild-type, mutant at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations. Cleavage of the recombination vector, for example. at the XhoI site within exon 9, promotes homologous recombination within the directly adjacent sequences (Capecchi (1989)). The endogenous APP gene res~ ing from this event would be normal up to the point of recombination, within exon 9 in this example, and would consist of the human cDNA
sequence thereafter.

CA 0222l9X6 l997-ll-24 W O 9C/1~35 PCTrUS96/09679 Preparation of Constructs for Transfections and Microinjectionc.
DNA clones for microinjection are cleaved with enzymes appl~,pliat~
for removing the bacterial plasmid sequences, such as SalI and NotI, and the DNA fragments electrophoresed on 1 % agarose gels in TBE buffer 5 (Sambrook et al. (1989)). The DNA bands are vi.cl-~li7ed by st~inin~ with et~ m bromide, and the band cont~inin~ the APP ~>rt;ssion sequences is excised. The excised band is then placed in dialysis bags cont~inin~ 0.3 M
sodium acetate, pH 7Ø DNA is electroeluted into the dialysis bags, extracted with phenol-chloroform (1:1), and precipitated by two volumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-D~ column.
The column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM
Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DNA solutions are passed through the column for three times to 1~ bind DNA to the colurnn matrix. After one wash with 3 ml of low salt buffer, the DNA is eluted with 0.4 ml of high salt buffer and precipitated by two volumes of ethanol. DNA concentrations are measured by absorption at 260 nm in a W spectrophotometer. For microinjection, DNA concentrations are adjusted to 3 ,ug/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA. Other 2C methods for purification of DNA for microinjection are also described in Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1986); in Palmiter et al., Nature 300:611 (1982); in The Qiagenologist, Application Protocols, 3rd edition, published by Qiagen, Inc., Chatsworth, CA.; and in Sambrook et al., 25 Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
Construction of Transgenic ~nim~lc.
A. Animal Sources.
Anirnals suitable for transgenic experiments can be obtained from 30 standard commercial sources such as Charles River (Wilmington, MA), Taconic (Gerrn~ntown, NY), and Harlan Sprague Dawley (Tn~i~n~polis, IN).
Many strains are suitable, but Swiss Webster (Taconic) female mice are W 09r'4~9s PCTnUS96~g67 preferred for embryo retrieval and transfer. B6D2F, (Taconic) males can be used for mating and vasectomized Swiss Webster studs can be used to stimnl~te pseudop,~ all~y. Vasectomized mice and rats can be obtained from the supplier.
]~. Microinjection Procedures.
The procedures for manipulation of the rodent embryo and for microinjjection of DNA are described in detail in Hogan et al., Manipulating the Mouse Embr~o (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 19~6), the te~ching.c of which are generally known and are incorporated herein.
C. T~ sg~,ic Mice.
Female mice six weeks of age are intl~lced to superovulate with a S IU
injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma) followecl 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadob~opin (hCG; Sigma). Females are placed with males immP~ tely after hCG injection. Twenty-one hours after hCG injection, the mated females are sacrificed by CO, asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5 % bovine serum albumin (BSA; Sigma).
Surrounding cuml~h-s cells are removed with hyaluronidase (1 mg/ml).
Pronuclear embryos are then washed and placed in Earle's b~l~n~ec~ salt solution cont~ining 0.5 % BSA (EBSS) in a 37.5~C incubator with a hnmitlified atmosphere at 5% CO, 95 % air until the time of injection.
Embryos can be implanted at the two cell stage.
~5 Randomly cycling adult female mice are paired with vasectomized males. Swiss Webster or other comparable strains can be used for this purpose. Recipient females are mated at the same time as donor females. At ~ the time of embryo transfer, the recipient females are anesthPti7p~l with an intraperiltoneal injection of 0.015 ml of 2.5 % avertin per gram of body 30 weight. The oviducts are exposed by a single miclline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with w~t~hm~kers forceps. Embryos to be W O ~611~-9S pcT~uss6lo9679 ~rell~d are placed in DPBS (Dulbecco's phosphate buffered saline) and in the tip of a L.a-~,rel pipet (about 10 to 12 embryos). The pipet tip is insertedinto the infundibulum and the embryos Lldl~,r~llcd. After the transfer, the incision is closed by two sutures.
S D. T~ gwlic Rats.
The procedure for generating transgenic rats is similar to that of mice (~mmPr et al., Cell 63:1099-112 (1990)). Thirty day-old female rats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female placed with a proven male. At the same time, 40-80 day old females are placed in cages with vasectomized males. These will provide the foster mothers for embryo ~ ,r~l. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer. Donor females that have mated are sacrificed (COs asphyxiation) and their oviducts removed, placed in DPBS
(Dulbecco's phosphate buffered saline) with 0.5% BSA and the embryos collected. Cumulus cells surrounding the embryos are removed with hyaluronidase (1 mg/ml). The embryos are then washed and placed in EBSS
(Earle's bal~nre~l salt solution) cont:~ining o.s% BSA in a 37.5~C incubator until the time of microinjection.
Once the embryos are injected, the live embryos are moved to DPBS
for ll~l~re~ into foster mothers. The foster mothers are ~npsthpti7p(l with ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip). A dorsal mi~1linP
incision is made through the skin and the ovary and oviduct are exposed by an incision through the muscle layer directly over the ovary. The ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip ofthe transfer pipet is inserted into the infundibulum. Approximately 10 to 12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the foster mothers are housed singly.
E. Embryonic Stem (ES) Cell Methods.
1. Introrll-cti~ln of cDNA into ES cells.
Methods for the culturing of ES cells and the subsequent production of transgenic ~nimz~l~, the introduction of DNA into ES cells by a variety of _ WO ~)'/lû~95 PCT/US96/0967g methods such as electroporation, calcium phosphate/DNA precipitation, and direct injection are described in detail in Teratocarcinomas and Embryonic Stem Ce~'ls, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987), the te~chings of which are generally known and are incorporated herein.
5 Selection of the desired clone of transgene-cont~ining ES cells can be accomplished through one of several means. For random gene integration, an APP clone is co-l,leci~iLal~d with a gene encoding neomycin re~ict~n~e.
Transfection is carried out by one of several methods described in detail in Lovell-Badge, in Teratocarcinomas and Embryonic Stem Cells, A Practical 0 Approac,ll, ed. E.J. Robertson, (IRL Press 1987), or in Potter et al., Proc. Natl. Acad. Sci. USA 81:7161 (1984). Lipofection can be performed using reagents such as provided in comrnercially available kits, for example DOTAP (Boehringer-Mannheim) or lipofectin (BRL). Calcium phosphate/DNA precipitation, lipofection, direct injection, and 15 electroporation are the preferred methods. In these procedures, 0.5 X 10 ES cells are plated into tissue culture dishes and transfected with a mixture ofthe linearized APP clone and 1 mg of pSV2neo DNA (Southern and Berg, J.
Mol. Appl. Gen. 1:327-341 (1982)) precipitated in the presence of 50 mg lipofectin (BRL) in a final volume of 100 ,ul. The cells are fed with selection 20 m~ m cont~ining 10% fetal bovine serum in DMEM supplemented with G418 (between 200 and 500 ~g/ml). Colonies of cells resistant to G418 are isolated using cloning rings and expanded. DNA is extracted from drug resistant clones and Southern blots using an APP770 cDNA probe can be used to identify those clones carrying the APP sequences. PCR detection 25 methods may also used to identify the clones of interest.
DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination, described by Capecchi (1989). Direct injection results in a high efficiency of integration. Desired clones can be identified through PCR of DNA prepared 30 from pools of injected ES cells. Positive cells within the pools can be identified by PCR subsequent to cell cloning (Zinnmer and Gruss, Nature 338:150-153 (1989). DNA introduction by electroporation is less efficient W O 9G/lCe95 PCT~US96/09679 and requires a selection step. Methods for positive selection of the recombination event (for example, neo resistance) and dual positive-negative selection (for example, neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Joyner et al., Nature 338:153-156 (1989), and Capecchi (1989), the teachings of which are generally known and are incorporated herein.
2. Embryo Recovel~ and ES cell Injection.
Naturally cycling or superovulated female mice mated with males can be used to harvest embryos for the implantation of ES cells. It is desirable to 10 use the C57BL/6 strain for this purpose when using mice. Embryos of the a~ iate age are recovered approximately 3.5 days after sl1rces.sful mating.
Mated females are sacrificed by CO asphyxiation or cervical dislocation and embryos are flushed from excised uterine horns and placed in Dulbecco's modified essential medium plus 10% calf serum for injection with ES cells.
15 Approximately 10 to 20 ES cells are injected into blastocysts using a glass microneedle with an internal diameter of approximately 20 ,um.
3. Transfer of Embryos to Pseudopregnant Females.
Randomly cycling adult female mice are paired with vasectomized males. Mouse strains such as Swiss Webster, ICR or others can be used for 20 this purpose. Recipient females are mated such that they will be at 2.5 to 3.5 days post-mating when required for implantation with blastocysts cont~ining ES cells. At the time of embryo transfer, the recipient females are anesthloti7~cl with an intraperitoneal injection of 0.015 ml of 2.5% avertin pergram of body weight. The ovaries are exposed by making an incision in the 25 body wall directly over the oviduct and the ovary and uterus are externalized.
A hole is made in the uterine horn with a 25 gauge needle through which the blastocysts are L~ relled. After the transfer, the ovary and uterus are pushed back into the body and the incision is closed by two sutures. This procedure is repeated on the opposite side if additional transfers are to be 30 made.
Identification, Characterization, and Utilization of Transgenic Mice and Rats.

W O~Ct~9S PCTflUS96~ag67g Transgenic rodents can be j(lentifie-l by analyzing their DNA. For this purpose, tail samples (1 to 2 cm) can be removed from three week old anim~l~. DNA from these or other samples can then be prepared and analyzed by Southern blot, PCR, or slot blot to detect transgenic founder (F.) ~nim~l~ and their progeny (F, and F).
A. P~th-,lo~ l Studies.
The various F., F,, and F ~nim~l.c that carry a transgene can be analyzed by immllnohistology for evidence of A,B deposition, expression of APP or APP cleavage products, neuronal or neuritic abnormalities, and 10 infli~ tQly responses in the brain. Brains of mice and rats from each transgenic line are ~lxed and then sectioned. Sections are stained with antibodies reactive with the APP and/or the A,B. Secondary antibodies conjuga~ed with fluorescein, rhodamine, horse radish peroxidase, or ~lk~lin.o phosphatase are used to detect the primary antibody. These methods permit 15 identifi~tion of amyloid plaques and other pathological lesions in specific areas of the brain. Plaques ranging in size from 9 to ~ 50 ~Lm characteristically occur in the brains of AD patients in the cerebral cortex, but also may be observed in deeper grey matter including the amygdaloidl nucleus, corpus striatum and diencephalon. Sections can also be stained with 20 other antibodies diagnostic of Alzheimer's plaques, recognizing antigens such as APP, Alz-50, tau, A2B5, neurofilaments, synaptophysin, MAP-2, ubiquitin, complement, neuron-specific enolase, and others that are characteristic of Alzheimer's pathology (Wolozin et al., Science 232:648 (1986); Hardy and Allsop, Trends in Pharm. Sci. 12:383-388 (1991); Selkoe, 25 Ann. Rev. Neurosci. 12:463-490 (1989); Arai et al., Proc. Natl. Acad. Sci.
USA 87:2249-2253 (1990); Majocha et al., Amer. Assoc. Neuropathology Abs 99:72 (1988); Masters et al., Proc. Natl. Acad. Sci. 82:4245-4249 (1985); Majocha et al., Can J Biochem Cell Biol 63:577-584 (1985)).
Staining with thioflavin S and Congo Red can also be carried out to analyze 30 the presence of amyloid and co-loc~1i7~tion of A~ deposits within neuritic plaques and NFTs.
B. Analysis of APP and A,~ Expression.

W O 96/40895 PCTrUS96/09679 1. mRNA.
Messenger RNA can be isolated by the acid gll~nidinillm thiocyanate-phenol:chloroform extraction method (Chomaczynski and Sacchi, Anal Biochem 162:156-159 (1987)) from cell lines and tissues of transgenic ~nim~l~ to deLe,l~ le expression levels by Northern blots, RNAse and nuclease protection assays.
2. I'loleill.
APP, A,(~, and other fr~m~ont~ of APP can and have been detectecl by using polyclonal and monoclonal antibodies that are specific to the APP
10 extra-cytoplasmic domain, A,(~ region, A~,~, A~,~, APP~, FLAPP+APPcY, and C-terminus of APP A variety of antibodies that are human sequence speci~lc, such as 10D5 and 6C6, are very useful for this purpose (Games et al. (1995)).
3. Western Blot Analysis.
Protein fractions can be isolated from tissue homogenates and cell Iysates and subjected to Western blot analysis as described by, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor, NY, 1988); Brown et al., J. Neurochem. 40:299-308 (1983); and Tate-Ostroff et al., Proc Natl Acad Sci 86:745-749 (1989).
Briefly, the protein fractions are denatured in Laemmli sample buffer and electrophoresed on SDS-Polyacrylamide gels. The proteins are then transferred to nitrocellulose filters by electroblotting. The filters are blocked, incubated with primary antibodies, and finally reacted with enzyme conjugated secondary antibodies. Subsequent inr~lb~tion with the a~pr.,pliate 25 chromogenic substrate reveals the position of APP derived proteins.
C. Pathological and Behavioral Studies.
1. Pathological Studies.
Immunohistology and thioflavin S staining are conducted as described elsewhere herein.
In situ Hybridi7 ~tionc: Radioactive or enzymatically labeled nucleic acid probes can be used to detect mRNA in situ. The probes are de~raded or prepared to be approximately 100 nucleotides in length for better penetration . CA 02221986 1997-11-24 W O 96/40895 PC~US96~09679 of cells. The hybridization procedure of Chou et al., J. Psych. Res. 24:27-50 (1990), for fixed and paraffin embedded samples is briefly described below a]though similar procedures can be employed with samples sectioned as frozen material. Paraffin slides for in situ hybridization are dewaxed in 5 xylene and rehydrated in a graded series of ethanols and finally rinsed in phosphate buffered saline (PBS). The sections are post-fixed in fresh 4%
paraformaldehyde. The slides are washed with PBS twice for 5 minutes to remove paraformaldehyde. Then the sections are permeabilized by tre~tmt?nt with a 20 ,ug/ml l~rol~ ase K solution. The sections are re-fixed in 4%
10 paraformaldehyde, and basic molecules that could give rise to background probe binding are acetylated in a 0.1 M triethanolamine, 0.3 M acetic anhydride solution for 10 minutes. The slides are washed in PBS, then dehydrated in a graded series of ethanols and air dried. Sections are hybridized with antisense probe, using sense probe as a control. After 15 a~plo~liate washing, bound radioactive probes are ~let~cte~l by autoradiography or enzymatically labeled probes are detected through reaction with the a~r~liate chromogenic subslldlt;s.
2. Behavioral Studies.
Behavioral tests ~lesign~-l to assess learning and memory deficits are 20 employed. An example of such as test is the Morris water maze (Morris, Learr. Motivat. 12:239-260 (1981)). In this procedure, the animal is placed in a circular pool filled with water, with an escape platform submerged just below the surface of the water. A visible marker is placed on the platform so that the animal can find it by navigating toward a proximal visual cue.
25 Alternatively, a more complex form of the test in which there are no local cues to mark the platform's location will be given to the ~nim~l.c. In this form, the animal must learn the platform's location relative to distal visual cues, and can be used to assess both reference and working memory. A
learning deficit in the water maze has been demonstrated with PDAPP
30 transgenic mice. An example of behavioral analysis for assessing the effect of transgenic expression of A,B-cont~ining proteins is described in Example 9.
The procedures applied to test transgenic mice are similar for transgenic rats.

, W O 96/4-~5 PCTAJS96/09679 D. Preferred Characteristics.
The above phenotypic characteristics of the disclosed transgenic ~nim~l.c can be used to identify those forms of the disclosed transgenic ~nim~l.c that are preferred as animal models. Additional phenotypic S characteristics, and assays for measuring these characteristics, that can also be used to identify those forms of the disclosed transgenic animals that are p.erell~d as animal models, are described in Example 6. These characteristics are preferably those that are similar to phenotypic characteristics observed in Alzheimer's disease. APP and A~B markers which 10 are also useful for idenLiryil1g those forms of the disclosed transgenic ~nim~l.c that are preferred as animal models are described below. Any or all of the these markers or phenotypic characteristics can be used either alone or in combination to identify pl~rell~d forms of the disclosed transgenic animals.
For example, the presence of plaques in brain tissue that can be stained with 15 Congo red is a phenotypic characteristic which can identify a disclosed transgenic animal as preferred. It is intended that the levels of expression of certain APP-related proteins present in preferred transgenic ~nim~lc (~liccucced above) is an independent characteristic for identifying preferred transgenic ~nimzll.c. Thus, the most preferred transgenic animals will exhibit 20 both a disclosed expression level for one or more of the APP-related proteins and one or more of the phenotypic characteristics flicc~csed above.
Especially plef~ d phenotypic characteristics (the presence of which identifies the animal as a ~lere,l~d transgenic animal) are the presence of amyloid plaques that can be stained with Congo Red (Kelly (1984)), the 25 presence of extracellular amyloid fibrils as identified by electron microscopy by 12 months of age, and the presence of type I dystrophic neurites as identified by electron microscopy by 12 months of age (composed of spherical neurites that contain synaptic proteins and APP; Dickson et al., Am J Pathol 132:86-101 (1988); Dickson et al., Acta Neuropath. 79:486-493 30 (1990); Masliah et al., J Neuropat~ol Exp Neurol 52: 135-142 (1993);
Masliah et al., Acta Neuropathol 87: 135-142 (1994); Wang and Munoz, J
Neuropathol Exp Neurol 54:548-556 (1995)). Examples of the detection of ~0 9G/~ S PCT~US96~0967 these characteristics is provided in Example 6. It is most preferred that the transgenic ~nim~lc have arnyloid plaques that can be stained with Congo Red as of 14 months of age.
S~ of C(~mr~ol-nrl~ for Tr~ of ~l~h~im~r's Disease.
1'he transgenic ~nim~lc, or animal cells derived from transgenic ~nim~1.c, can be used to screen compounds for a potential effect in the treatment of Alzheimer's disease using standard methodology. In such AD
screenin,g assays, the compound is ~llminict~red to the ~nim~lc, or introduced into the culture media of cells derived from these ~nim~l.c, over a period of time and. in various dosages, then the ~nim~lc or anirnal cells are ex~min~
for alterations in APP expression or procecsing, histopathology, and/or, in the case of ~nim~lc, behavior using the procedures described above and in the examples below.. In general, any improvement in behavioral tests, reduction in the severity of histopathology characteristic of AD present in the 15 transgeniic ~nim~l.c, and/or reduction in the levels of A,B or APP cleavage products observed in treated ~nim~l.c, relative to ullLl~at~d ~nim~l.c, is indicative of a compound useful for treating Alzheimer's disease.
Expression of the various forms of APP and A,~ can be directly measurecl and compared in treated and ullllcaLed transgenic ~nim~l.s both by 20 immunohistochemistry and by ql-~"~ live ELISA or immllnoblot measurernents as described above and in the examples. Currently, it is known that two forms of APP products are found, APP and A~B (Haass and Selkoe, (,ell 75:1039-1042 (1993)). They have been shown to be intrinsically associated with the pathology of AD in a tirne dependent manner. Therefore, 25 preferred assays compare age-related changes in APP and A,~ expression in the transgenic mice. As described in Example 6, increases in A,B have been demonstrated during aging of the PDAPP mouse.
- Targets for expression measurements can be selected based on changes in the expression or location of specific markers in Alzheimer's disease.
30 Preferred targets for assay measurement are A~ markers known to increase in individuals with Alzheimer's disease are total A,~ (A,~), A,l~ 1-42 (A,~,~.; A~Bwith amino acids 1-42), A,~, (A~B with amino acids 1-40), A,~ N3(pE) CA 0222l986 l997-ll-24 W O 9G/1C99S PCTrUS96/09679 (A~(pE));A~ X-42(A~.;A~ forms ending at amino acid 42);A~'X40 (A,~;A,~ forms ending at amino acid 40); insoluble A~ (A,B~); and soluble A~(A~'~;Kuo et al., J. Biol. Chem. 271(8):4077-4081(1996)). A~(pE) has pyroglutamic acid at position 3 (Saido, Neuron 14:457466(1995)). A~
refers to any of the C-~el,nillal forms of A~such as A~,~.. A~5~ refers to forms of A,B that are recovered as described in Gravina, J. Biol. Chem.
270:7013-7016(1995). APP~ can also be specifically measured to assess tne amount of ~-secretase activity (Seubert et al., Nature 361:260-263(1993)).
Several of these A,6' forms and their association with Alzheimer's disease are 10 described by Haass and Selkoe (1993). Detection and measurement of A,~, A~B~, and A,~ are described in Example 6. Generally, specific forms of A~B
can be assayed, either q~l~ntit~tively or qualitatively using specific antibodies, as described below. When referring to amino acid positions in forms of A~, the positions correspond to the A,6' region of APP. Amino acid 1 of A,B
corresponds to amino acid 672 of APP, and amino acid 42 of A~ corresponds to amino acid 714 of APP.
Also preferred as targets for assay measurement are APP markers.
For example, dirre~ t forms of secreted APP (termed APP~ and APP,B) can also be measured (Seubert et al., Nature 361:260-263(1993)). Other APP
forms can also serve as targets for assays to assess the potential for compounds to affect Alzheimer's disease. These include FLAPP+APPc~, full length APP, C-terminal fr~gm~nt~ of APP, especially C100 (the last 100 amino acids of APP) and C57 to C60 (the last 57 to 60 amino acids of APP), and any forms of APP that include the region corresponding to A~
APP forms are also preferred targets for assays to assess the potential for compounds to affect Alzheimer's disease. The absolute level of APP and APP transcripts, the relative levels of the different APP forms and their cleavage products, and loc~li7~tion of APP expression or processing are all markers associated with Alzheimer's disease that can be used to measure the effect of treatment with potential therapeutic compounds. The loc~li7~tion of APP to plaques and neuritic tissue is an especially preferred target for these assays.

W O ~G/IC~95 PCTAUS96~09679 Quantitative measurement can be accomplished using many standard assays. For example, LlallSC~ t levels can be measured using RT-PCR and hybridization methods including RNase protection, Northern analysis, and R-dot analysis. APP and A,B levels can be assayed by ELISA, Western S analysis, and by comparison of immunohistochemic~lly stained tissue sections. Tmmnnt~histochemical staining can also be used to assay loc~li7~tiion of APP and A,~ to particular tissues and cell types. Such assays were described above and specific examples are provided below.
A.. Screening assays using cultured cells.
Screening assays for determinin~ the therapeutic potential of compoun.ds can also be performed using cells derived from ~nim~l.s transgenic for the disclosed APP constructs and cell cultures stably .recLt,d with the disclosed constructs. For example, such assays can be performed on cultured cells in the following mall.lel. Cell cultures can be 15 transfected generally in the manner described in International Patent Application No. 94/10569 and Citron et al. (1995). Derived transgenic cells or transfected cell cultures can then be plated in Corning 96-well plates at 1.5to 2.5 x l~ cells per well in Dulbecco's minim~.l essential media plus 10%
fetal bovine serum.
Following overnight incubation at 37~C in an incubator equilibrated with 10~ carbon dioxide, media are removed and replaced with media cont~ining a compound to be tested for a two hour pretreatment period and cells were in~lb~ted as above. Stocks cont~ining the compound to be tested are first prepared in 100% dimethylsulfoxide such that at the final 25 concentration of compound used in the treatment, the concentration of dimethylsulfoxide does not exceed 0.5%, preferably about 0.1%.
At the end of the pretreatment period, the media are again removed - and replaced with fresh media cont,-ining the compound to be tested as above and cells ,are incubated for an additional 2 to 16 hours. After treatment, 30 plates are cenl~iruged in a Beckman GPR at 1200 rpm for five minutes at room temperature to pellet cellular debris from the conditioned media. From each well, 100 ,uL of conditioned media or appropriate dilutions thereof are PCT~US96/09679 transferred into an ELISA plate precoated with antibody 266 (an antibody directed against amino acids 13 to 28 of A,B) as described in International Patent Application No. 94/10569 and stored at 4~C overnight. An ELISA
assay employing labelled antibody 6C6 (against amino acids 1 to 16 of A~B) S can be run to measure the amount of A~ produced. Dirr~le.lt capture and detPction antibodies can also be used.
Cytotoxic effects of the compounds are measured by a modification of the method of Hansen et al., J. Immun. Method. 119:203-210 (1989). To the cells r~m~ining in the tissue culture plate, 25 ,uL of a 3,(4,5-dimethylthiazol-10 2-yl)2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/mL) is added to a final concentration of 1 mg/mL. Cells are incubated at 37~C for one hour, and cellular activity is stopped by the addition of an equal volume of MTT lysis buffer (20% w/v sodium dodecylsulfate in 50%
dimethylform~3mi~1e, pH 4.7). Complete extraction is achieved by overnight 15 ~h~king at room temperature. The dirre.e,lc~ in the OD~ and the OD~ is measured in a Molecular Device's UV_ microplate reader, or equivalent, as an in~lic~tor of the cellular viability.
The results of the A,~ ELISA are fit to a standard curve and expressed as ng/mL A,~. In order to normalize for cytotoxicity, these results are 20 divided by the MTT results and expressed as a percentage of the results from a control assay run without the compound.
B. A,B toxicity assays.
The effect of potential therapeutic compounds on the toxicity of A~ to neuronal cells can be determined in the following manner.
Primary Rat Cortical Cell Cultures: Primary rat cortical cultures are established from 18 day rat fetuses. Cortical tissue is dissociated by incubation in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; GIBCO Laboratories, Grand Island, New York, USA) for 20 minutes at 37~C. The trypsin is then inactivated by resuspending the cells in serum-30 cont~inin~ medium (DMEM/FBS): Dulbecco's Modified Eagles' Medium (DMEM) cont~inin~ 4.5 g/L glucose, 1 mM sodium pyruvate, 1 mM
glut~minto, 100 Units/mL penicillin, 100 ,ug/mL streptomycin, and . CA 02221986 1997-11-24 W O ~C/IC~ PCTMS96~09679 supplen-~nt~ with 10% heat-inactivated fetal bovine serum (GIBCO
Laboratories, Grand Island, New York, USA). Cells are then pelleted by cenllirugation and resuspended in a ch~ lly-defined m~dillm (DMEM/B27:DMEM cont~inin,~ B27 supplement; GIBCO Laborator.ies, 5 Grand Island, New York, USA) in place of FBS. Polyethyleneimine (PEI;
Sigma Chemical Company, St. Louis, Missouri, USA)-coated 6.4 mm (96-well) dishes are rinsed once with DMEM/FBS, and then seeded at 0.75 to 1.25 x 10 cells per well in 0.1 mL DMEM/B27. Cultures are m~int~in~(l in a H!O-saturated incubator with an atmosphere of 90%air/10%CO at 37~C.
10 Cell viability is visually assessed by phase contrast microscopy and q-l~ntifiecl by measuring the reduction of alamarBluen (Alamar Biosciences, Inc., Sacramento, California, USA) as described below. Serum replacement with B27 supplement yields nearly pure neuronal cultures as judged by immllnocytochemistry for glial fibrillary acidic protein and neuron-specific enolase (Brewer et al., J. Neuro. Sci. Res. 35(5):567-577 (1993)). This procedure can also be used to l.le~ale cell cultures from the disclosed transgenic anim~
A.ll publications cited herein are hereby incorporated by reference.
The information contained in these publications for which the publications are cited is generally known.
FY~mrle 1: Expression of pMTAPP-1 in NIH3T3 and PC12 Cells.
The clone pMTAPP-l is an example of an APP770 expression construct as shown in Figure la where the promoter used is the metalloth~ionine promoter. Stable cell lines were derived by transfecting NIH3T3 and PC12 cell lines (ATCC #CCL92 and CRL1721). Five hundred thousand NIH3T3 or PC12 cells were plated into 100 mm dishes and transfected with a mixture of S mg of the SalI fragment and 1 mg of pSV2neo DNA (Southern and Berg (1982)) precipitated in the presence of 50 mg lipofectin (Gibco, BRL) in a final volume of 100 ,L41. Polylysine-coated plates were used for PC12 cells, which normally do not adhere well to tissue culture dishes. The cells were fed with selection medium cont~ining 10%
fetal bovine serum in DMEM or RPMI and supplemented with G418. Five , W O ~6/~9~ PCT~US96/09679 hundred mg/ml (biological weight) and 250 mg/ml of G418 were used to select colonies form NIH3T3 and PC12 cells, respectively. Fifteen days after transfection, colonies of cells resistant to G418 were isolated by cloning ringsand exr~n~ l in T flasks. The presence of APP cDNA in the cells was 5 tlet~cte~l by PCR using the procedure of Mullis and Faloona, Methods En~ymol. 155:335-350 (1987), the teachings of which are generally known and are incorporated herein.
Expression of APP in 25 colonies from each cell line was analyzed by imml-nost~inin~ (Majocha et al. (1988)). Cells were grown to subconfluence 10 and fixed in a solution cont~ining 4% paraformaldehyde, 0.12 M NaCl, and 20 mm NaPO~, pH 7Ø They were inrllb~t~l overnight with a primary monoclonal antibody against a synthetic A,~ sequence (Masters et al. (1985);
Glenner and Wong) provided by Dr. Ronald Majocha, l~c~chllcett~ General Hospital, Boston, MA, followed by a generalized anti-mouse antibody 15 conjugated to biotin (Jackson ImmunoResearch Labs, PA). Tmmllnost~ining was then performed by adding avidin-horseradish peroxidase (HRP) (Vector Labs, Burling~m~, CA) and ~ minobenzidine as the chromogen (Majocha et al. (1985)). The results in~1ir.~t~d that the pMTAPP-1 vector was explessillg APP in both NIH3T3 and PC12 cells.
20 Example 2: Expression of pEAPP-1 in PC12 Cells.
pEAPP-lis an example of an APP770 expression construct as shown in Figure la where the promoter used is the 2~ kb human APP gene promoter. DNA from this construct was transfected into PC12 cells as described above. Certain clones of pEAPP-l transfected cells exhibited a 25 differentiation phenotype morphologically similar to that exhibited by PC12 cells treated with nerve growth factor (NGF). PC12 cells normally are fairly round and flat cells. Those transfected with pEAPP-l have cytoplasmic extensions resembling neurites. PC12 cells treated with NGF
extend very long neuritic extensions. Thirteen PC12 cell clones transfected 30 with pEAPP-l were selected and propagated. Eight of these cell clones exhibited the spontaneous dirre~ iation phenotype with clones 1-8~ 1-1, and 1-4 exhibiting the strongest phenotypes. Staining of pEAPP-l transfected W O~ 95 PCTAUS96/09679 PC12 cells with antibody against the A~ as described in Example 1 in~ tPd that those cells exhibiting the differentiation were also ~lC~Sillg APP.
Because PC12 cells transfected with the pMTAPP-1 clone did not exhibit this phenotype even though the APP770 cDNA is expressed, these results suggest 5 that expression of APP770 from the human promoter has novel properties regarding the physiology of the cell.
F,~mrl~ 3: Expression of pMTA4 in PC12 Cells.
p~TA4 is an example of the type of construct shown in Figure 4a where the promoter used is the metallothionine promoter. The protein 10 encoded by this construct differs slightly from that depicted in Figure 4a. An APP770 ~,DNA clone was digested with Asp718 which cleaves after position 57 (number system of Kang et al. (1987)). The resl-lting 5' extension was filled in using the Klenow enzyme (Sambrook et al. (1989)). The same DNA
alation was also cleaved with EcoRI which also cuts after position 2020 1~ and the resllltin~ 5' extension was filled in using the Klenow enzyme (Sambrook et al. (1989)). Self-ligation of this molecule results in an expression clone in which the trlln~atPcl protein thus encoded contains the leader sequence, followed by a shortened version of the A,B starting with the sequence Phe-Arg-Val-Gly-Ser-of the A~ followed by the 56 terminal amino 20 acids of APP. DNA from this construct was transfected into PC12 cells as described above.
~mrle 4: Generation of T~ s~ ic ~ice expressing APP under the control of the MT-l promoter.
Tr~msgenic mice were made by microinjecting pMTAPP-1 vector 25 DNA into pronuclear embryos. pMTAPP-1 is an example of the type of construct shown in Figure la in which the APP770 coding sequence is operably linked to the metallothionine promoter. The procedures for microinjection into mouse embryos are described in Manipulating the Mouse Embryo b~ Hogan et al. (1986). Only a brief description of the procedures is 30 described below.
Mice were obtained from Taconic Laboratories (German Town, New York). Swiss Webster female mice were used for embryo retrieval and CA 0222l986 l997-ll-24 W O ~C,'1C6~5 PCTrUS96/09679 implantation. B6D2F, males were used for mating and vasectomized Swiss websLel studs were used to .~im~ t~ pseudopregnancy.
A. Embryo Rec~
Female mice, 4 to 8 weeks of age, were in-lllced to superovulate with 5 5 IU of pregnant mare's serum gonadotropin (PMSG; Sigma) followed 48 hours later by 5 IU of human chorionic gonadotropin (hCG; Sigma).
Females were placed with males imm~ tely after hCG injection. Embryos were recovered from excised oviducts of mated females 21 hours after hCG
in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin 10 (BSA; Sigma). Surrounding cumulus cells were removed with hyaluronidase (1 mg/ml). Pronuclear embryos were then washed and placed in Earle's balanced salt solution cont, ining 0.4% BSA (EBSS) in a 37.5~C in~ hator with a hl-mitlified atmosphere at 7% CO, s~ o, and 88% N until the time of injection.
B. Microinjection.
Elutip-DD purified SalI DNA was dissolved in 5 mM Tris (pH 7.4) and 0.1 mM EDTA at 3 ,ug/ml concentration for microinjection.
Microneedles and holding pipettes were pulled from Fisher coagulation tubes (Fisher) on a DKI model 720 pipette puller. Holding pipettes were then broken at approximately 70 ~m (O.D.) and fire polished to an I.D. of about 30 ~Lm on a Narishige microforge (model MF-83). Pipettes were mounted on Narishige mi~;lulna~ ulators which were ~tt~hPd to a Nikon Diaphot microscope. The air-filled injection pipette was filled with DNA solution through the tip after breaking the tip against the holding pipette. Embryos, in groups of 30 to 40, were placed in 100 ,ul drops of EBBS under paraffin oil for micromanipulation. An embryo was oriented and held with the holding pipette. The injection pipette was then inserted into the male pronucleus (usually the larger one). If the pipette did not break through the membrane immt-fli, ttoly the stage was tapped to assist in penetration. The nucleus was then injected and the injection was monitored by swelling of the nucleus. Following injection, the group of embryos was placed in EBSS until transfer to recipient females.

W O ~C/~C~95 PCTnUS96~09679 C. Transfer.
l~n~lomly cycling adult female mice were paired with vase~;lomi~ed Swiss Webster males. Recipient females were mated at the same time as donor females. At the time of ~ , the females were anesthetized with 5 avertin. The oviducts were exposed by a single mitllin~ dorsal incision. An incision was then made through the body wall directly over the oviduct. The ovarian bursa was then torn with watch makers forceps. Embryos to be transferred were placed in DPBS and in the tip of a transfer pipet (about 10 to 12 embryos). The pipet tip was inserted into the infundibulum and 10 embryos were ~ .rell~d. After the transfer, the incision was closed by two sutures.
D. Analysis Of Mice For Transgene Integration.
At three weeks of age or older, tail samples about 1 cm long were excised for DNA analysis. The tail samples were digested by inrllb~ting with ~h~king overnight at 55~C in the presence of 0.7 ml 5 mM Tris, pH 8.0, 100 mM EDl'A, 0.5% SDS and 350 ,~lg of proteinase K. The digested material was extracted once with an equal volume of phenol and once with an equal volume of phenol:chloroform (1:1 mixture). The ~u~elllatants were mixed with 70 ~cl 3 M sodium acetate (pH 6.0) and the DNA was precipitated by 20 adding equal volume of 100% ethanol. The DNA was spun down in a microfuge, washed once with 70% ethanol, dried and dissolved in 100 ,ul TE
buffer (10 mM Tris pH 8.0 and 1 mM EDTA).
Ten to twenty microliters of DNA from each sample was digested with BamHI, electrophoresed on 1% agarose gels, blotted onto nitrocellulose 25 paper, and hybridized with ~P-labeled APP cDNA fragment. Transgenic ~nim~l~ were identified by autoradiography of the hybridized nitrocellulose filters. Ihe DNAs were also analyzed by PCR carried out by synthetic primers to generate an 800 bp fragment of APP DNA.
A total of 671 pronuclear embryos were microinjected out of which 30 73 live and 6 dead pups were born. DNA analysis identified 9 transgenic mice (5 females and 4 males) which were bred to generate F, and F
transgenics. These ~nim~l~ can be analyzed for expression of mRNA and PCT~US96/09679 W O ~G/1D~95 protein of APP in different tissues and for analysis of behavioral and pathological abnorrn~1ities as described above. Transgenic mice with this construct express transgenic RNA.
F~r~mpl~ 5: Construction of APP co ~ u~l cont~;nin~ a combination S cDNA/genomic coding sequence.
A cDNA/genomic APP construct cont~ining introns 6, 7 and 8 was ~paled by combining APP cDNA encoding exons 1-6 and 9-18 with genomic APP sequences encoding introns 6, 7 and 8, and exons 7 and 8 (see Figure 6). In order to create a splicing c~ette small enough for convenient 10 insertion in a pUC vector, two deletions in intronic sequences were made. A
deletion was made in intron 6 from position 143 of intron 6 to the BamHI
site located upstream of the beginning of exon 7 (1658 bp before the beginning of exon 7). Another deletion was made in intron 8 from the first BamHI site in intron 8 to a site at 263 bp before the beginning of exon 9.
15 To avoid confusion, these trl-nr?.te~l forms of APP introns 6 and 8 are referred to herein as intron ~6 and intron ~8. BamHI sites were engineered at the sites of these deletions, so that they are m~rkçr1 by the presence of BamHI sites. In this constr~ct, referred to as PDAPP, exons 7 and 8 and intron 7 are intact genomic sequences, except that the unique XhoI site in 20 intron 7 was destroyed.
DNA fragments cont~ining the trunr~te-l introns were generated as follows: a BamHI site was enginPçred 143 bp into intron 6 nucleotide by PCR mutagenesis ("Mutagenesis by PCR" in PCR Technology: Current Innovations (Griffith and Griffith, eds., CRC Press, 1994) pages 69-83) and 25 another BamHI site was engineered by PCR mutagenesis 263 bp prior to the beginning of exon 9. These sites were engineered into separate APP genomic DNA clones cont~ining the junctions of exon 6 and intron 6, and intron 8 and exon 9, respectively, resulting in modified APP genomic DNA clones.
The entire cassette was assembled in the APP cDNA clone as follows 30 (Figure 11). The 889 bp BamHI to XcmI fragment of APP cDNA conr~ining exons 1 through 5 and part of exon 6 (including nucleotides 1 to 843 of SEQ
ID NO:5) was cloned into a vector cont~ining BamHI and XhoI sites W O 9"_CB95 PCTAUS96/09679 downstream from the insertion site to make APP770x-oligo-x. APP770x-oligo-x was then cut with XcmI and BamHI. Then two fr~gmtqnt~ were obtained from the modi~led APP genomic DNA clone cont~inin~ the junction of exon 6 and intron 6 described above by cutting with XcmI and BamHI.
S The reslllting 34 bp fragment from the XcmI in exon 6 to the XcmI in intron 6, and 131 bp fragment from the XcmI in intron 6 to the artificially created BamHI site at position 143 bp of intron 6 were ligated into APP770x-oligo-x in a three-way ligation step to make APP770x-E6O1igo-x. The orientation of the fragrnents was confirrn~l by sequencing. APP770x-E6O1igo-x was then cut with BamHI and X71oI. Then the 313 bp BamHI and X71oI fragment from the modified APP genomic DNA clone cont~ining the junction of intron 8 and exonl 9 was ligated into APP770x-E6O1igo-x to make APP770xE6E9x.
APP770xE6E9x was then cut with BamHI and the 6.8 kb BamHI
fragment of APP genomic DNA encoding the KPI and OX-2 domains (exons 7 and 8) was inserted at this site. This fragment starts at the BamHI site 1658 bp upstream of the start of exon 7 and extends to the first BamHI site in intron 8. This BamHI fragment was obtained from a lambda phage genomic clone encoding this portion of the APP gene, that was obtained from a Human Placental genomic library in the Lambda FIXII vector obtained from Stratagene. This BamHI fragment originally contained an X71oI site which was destroyed by cutting, filling in, and religation. The locations of the deletions are diagramed in Figure 10. This clone, cont~ining exons 1-8 and part of 9, and introns 6, 7 and 8, was termed the "APP splicing cassette."
The APP splicing cassette was cut out with NruI and X7zoI and used to replace the Nn~I to X7loI cDNA fragment of APP cDNA bearing the Hardy mutation. This mutant form of APP cDNA was produced by converting the G at nucleotide position 2145 to T by site directed mutagenesis. This changes the encoded amino acid from Val to Phe. The resulting construct is a combination cDNA/genomic APP "minigene."
Sequencing of the 6.8 kb BamHI fragment cont~ining APP exons 7 and 8 derived from the APP genomic clone used to generate this construct showed that intron 7 is 2.6 kb long, and that the first BamHI site in intron 8, W O ~G/~0~3~ PCT~US96/09679 the upstream site of the deletion in intron 8 engineered into the APP
minigene construct, is 2329 bp dowl~L.eal,l from the end of exon 8. This does not coincide with the restriction map of the APP gene published by Yoshikai et al. (1990) and Yoshikai et al. (1991). Comparison of their map 5 to our sequence intli~t~c that Yoshikai et al. switched the order of two EcoRI fragments in their restriction mapping. The 1.60 kb EcoRI fragment cont~ining exon 8 is actually upstream of the 1.48 kb EcoRI fragment and the 1.48 kb Eco~ fragment Yoshikai et al. mapped in intron 7 is actually in intron 8. We have confirmed this location for the Eco~l fragment cont~ining 10 exon 8 by sizing of PCR generated fragments from human DNA.
This APP minigene was operatively linked to the PDGF-B promoter to provide expression of the APP cDNA/genomic construct in m~mm~ n cells. The PDGF ~B-chain 5' fl~nking sequence was inserted ul,~Llc;alll of the NruI site at the beginning of the APP minigene. This fragment includes 1.3 15 kb upstream of the lldnsclipLion initiation site, where the PDGF-B promoter resides, and approximately 70 bp of 5' untrancl:~ted region, ending at the AurII site (Higgins et al. (1994)). The late SV40 polyadenylation signal, carried on a 240 bp BamHI to BclI fragment, was added downstream of the APP minigene. This construct, combining the PDGF-B promoter, the APP
20 splicing cacsettP, the Hardy mutation, and the SV40 polyadenylation signal is referred to as PDAPP (Figure 9).
FY~mrle 6: Tla~ ellic mice cont~inin~ the PDAPP construct.
Transgenic mice were gelleldt~d using the PDAPP construct described in Example 5. Transgenic mice were generated by microinjection using 25 standard techniques as described above. PDAPP DNA was microinjected into the embryos at the two-cell stage. Plasmid sequences (pUC) were removed by SacI and NotI digestion before microinjection. Seven founder mice were generated and line 109 was used for extensive analysis. Only heterozygous anim~l.c were used. Southern analysis of 104 animals from four 30 generations showed that approximately 40 copies of the transgene were inserted at a single site and tr~ncmittPd in a stable manner. Human APP
messenger RNA was produced in several tissues of the transgenic mouse, but w ogc,~4ca35 PCT~US96/09679 at especially high levels in brain. RNase ~lotecLion assays revealed at least 20-fold more APP expression in the brains of line 109 ~nim~l.c than in the mouse lines expressing neuron-speci~lc enolase (NSE)-promoter-driven APP
gelles previously described by Quon et al. (1991), Mucke et al., Brain S Res. 666: 151-167 (1994), McConlogue et al., Neurobiol. Aging 15:S12 (1994), and Higgins et al., Ann Neurol. 35:598-607 (1994).
A.. Expression of APP Transcripts and Protein.
RNA was isolated from brain tissue as described by Chomaczynski and Sacchi, Analyt. Biochem. 162:156-159 (1987), and subjected to RT-PCR
as described by Wang et al., Proc. Natl. Acad. Sci. U.S.A. 86:9717-9721 (1989), using human-specific APP primers (5'-CCGATGATGACGAGGACGAT-3', SEQ ID NO:7;
5'-TGAACACGTGACGAGGCCGA-3', SEQ ID NO:8) using 40 cycles of 1 minute a~ 94~C, 40 seconds at 60~C, and 50 seconds at 72~C. RT-PCR
analysis demonstrated the presence of transcripts encoding the 695, 751 and 770 isofarms of human APP in transgenic animal brains but not in brains from non-transgenic litterm~trs. The i~lçlltitif~s of the human APP RT-PCR
bands from the transgenic mouse RNA were verified by subcloning and sequencing.
The relative levels and alternative splicing of APP transcripts in brains of PDAPP transgenic mice, NSE-APP transgenic mice, non-transgenic mice, and hnm~nc with and without AD were compared in RNase protection assays (Rockens~ein et al., J. Biol. Chem. 270:28257-28267 (1995). PDAPP mice expressed approximately 5-fold higher total APP mRNA levels than non-transgenic controls, and at least 20-fold higher human APP mRNA levels than most NSE-APP transgenic mice. While NSE-driven human APP
expression does not affect the levels of murine APP mRNA, PDAPP
~ transgenic mice showed a significant 30% decrease in murine APP
transcripts. While the relative ablln-l~nres of murine APP770:751:695 mRNAs in non-transgenic mouse brains were roughly 1:1:35, the corresponding human APP mRNA levels in PDAPP transgenic mouse brains were 5:5:1.

W O 96/40895 PCTrUS96/09679 Analysis of holo-APP was performed by brain homogenization in 10 volumes of PBS cont~ining 0.5 mM EDTA, 10 ~g ml leupeptin and 1 mM
PMSF. Samples were spun at 12,000g for 10 min and the pellets resuspended in RIPA (150 mM NaCI, 50 mM Tris, ph 8.0, 20 rnM EDTA, 1.0% deoxycholate, 1.0% Triton X-100, 0.1% SDS, 1 mM PMSF and 10 ~g ml-~ leupeptin). Samples (each cont~ining 30 ~g total protein) were analyzed by SDS-PAGE, Lldl~ d to Immobilon membranes and reacted with either the holo-APP antibody, anti-6 (anti Bx 6), described by Oltersdorf et al., J.
Biol. Chem. 285:4492-4497 (1990), or 8E5 monoclonal antibody. 8E5 was prepared against a bacterial fusion protein encompassing human APP residues 444-692 (Oltersdorf et al. (1990)) and is human-specific, showing essentially no crossreactivity against mouse APP. Immunoblot analysis of total APP
expression (human and mouse) in transgenic mouse line 109 and control littermate brain tissue using C-terminal APP antibody anti-6 showed much higher levels of expression in the transgenic mice. Immunoblot analysis of brain homogenates using either the holo-APP polyclonal antibody anti-6 or the human-specific APP monoclonal antibody 8E5 revealed human APP over-expression in the transgenic mouse at levels at least 3-fold higher in hippocampus than either endogenous mouse APP levels or those in AD brain.
For immnnoblot analysis of A,~, a 9-month-old mouse brain was homogenized in 5 ml 6 M gll~niclin~ HCI, 50 mM Tris, pH 7.5. The homogenate was centrifuged at 100,000g for 15 min and the supernatant was dialyzed against H0 overnight adjusted to PBS with 1 mM PMSF and 25 ~g ml leupeptin. This material was immunoprecipitated with antibody 266 resin, and immunoblotted with the human-specific A~ antibody, 6C6, as described by Seubert et al., Nature 359:325-327 (1992). Using this human-specific A~ antibody (6C6), a 4 kD ~B amyloid-immllnoreactive peptide was isolated from the brains of the transgenic ~nim~l.c, which corresponds to the relative molecular mass of A~. Brain levels of A,~ were at least 10-fold higher in line 109 ~nim~lc than in the previously described human APP
transgenic mice. Embryonic day 16 cortical cell cultures from transgenic ~nim~l.c constitutively secreted human A,~, including a substantial fraction of ~YO 961l~ e3s PCT~US96JV9679 42 (5 ng ml total A~; 0.7 ng ml A~ 42), as ~l~t~cte~l in media by human-s,peci~lc A,~ enzyme-linked immllnosorbent assays, as described b~
Seubert et al. (1992) and McConlogue et al. (1994), and as described in Example 8. Thus, line-109 ~nim~l.c greatly ove~ essed human APP
rnRNA, holo-APP and A~B in their brains.
B. HistoI ~th~lc~y of PDAPP Transgenic Mice.
Brains from 180 ~ Sgt;l~iC and 160 age-m~trhe~i non-transgenic age-matched controls (4 to 20 months old) representing five generations of the line 109 pedigree were e~le~L~ively ex~min~d histopathologically. Some 10 mouse brains were removed and placed in alcohol fixative (Arai et al., Proc.
Natl. Acad. Sci. U.S.A. 87:2249-2253 (1990)) for 48 hours before paraffin embedding. Other mice were perfused with saline followed by 4%
paraformaldehyde in 0.1 M sodium phosphate. For paraffin embedded brains, 6 ~m coronal or parasaggital sections from transgenic and non-15 transgenic mice were placed adjacent to each other on poly-L-lysine coated slides. The sections were deparaffinized, rehydrated and treated with 0.03%
H0 for 30 min before overnight incubation at 4~C with a 1:1,000 dilution of the A,B antibody, R1280 (Tamaoka et al., Proc. Natl. Acad. Sci. U.S.A.
89:1345-1349 (1992)). For absorption studies, synthetic human A,~ 140 Z0 peptide (Games er al., Neurobiol. Aging 13:569-576 (1992)) in 10% aqueous dimethylsulphoxide was added to a final concentration of 7.0 ~M to the diluted antibody and incubated for 2 hours at 37~C. The diluent was applied to the sections and processed under the same conditions as the standard antibody solution. Peroxidase rabbit IgG kit (Vector Labs) was then used as 25 recommended, with 3,3'~ minc)benzidine (DAB) as the chromogen.
Similarly fixed human AD brain was processed simnlt~n~ously under identical conditions.
Before 4 months of age, no obvious A~ deposition was ~çtçcted.
However, by approximately 4 months of age, the transgenic ~nim~ls began to 30 exhibit deposits of human A~ in the hippocampus, corpus callosum and cerebral ,cortex. These A~ plaques increased with age, and by eight months many deposits of 30 to 200 ,um were seen. As the ~nim~l~ aged beyond 9 W O 96/40895 PCTnJS96/09679 months, the density of the plaques increased until the A,B-st~inin~ pattern resembled that of AD. Vascular amyloid, another feature of AD pathology, developed in older mice. Robust pathology was also seen in anotner transgenic line generated from the PDAPP vector (line 35).
A~ deposits of varying morphology were clearly evident as a result of using a variety of A,~ antibodies, including well characterized human-specific A,B antibodies and antibodies specific for the free amino and carboxy termini of A,B 1-42. Antibody 9204, described by Saido et al., J. Biol. Chem.
289:15253-15257 (1994), is specific to A,~ 1-5 and was used at a 10 concentration of 7.0 ~g ml . Antibody 277-2, specific for A~B 1-42, was ~lepaled by immnni~ing New 7e~1~n~1 white rabbits with the peptide cysteine-aminoheptanoic acid-A~ 33-42 conjugated to cationized BSA ('Super Carriers'; Pierce) using a standard i"""llni~tion protocol (500 ~g per injection). Specific antibodies were affinity-purified from serum against the 15 immunogen immobilized on agarose beads. Before incubation with antibody 277-2, sections were treated for 1 to 2 min with 80% formic acid. For detection, the antibody was reacted using the peroxidase rabbit IgG kit (Vector Labs). The product was then visualized using DAB as the chromogen, Some sections were then inrubatr~ overnight at 4~C with a 20 1:500 dilution of polyclonal anti-GFAP (Sigma). The GFAP antibody was reacted using the s~lk~linr phosphatase anti-rabbit IgG kit and ~lk~linr phosphatase substrate kit 1 (Vector Labs; used according to the m~nll~cturer's recommendations). Additional sections were inr~ t~?tl overnight with the F480 antibody (Serotec) used at a 1:40 dilution to 25 visualize microglial cells. The mouse peroxidase kit (Vector Labs) was then used according to the m~mlf~cturer's recommendations. Some sections were stained with thioflavin S using standard procedures (Dickson et al., Acta Neuropath. 79:486-493 (1990)) and viewed with ultraviolet light through an FITC filter of m~ximllm wavelengtn 440 nm.
Serial sections demonstrated many plaques were positively stained witn both the 9204 and 277-2 antibodies. The forms of the A~ deposition ranged from diffuse irregular types to compacted plaques witn cores.

CA 0222l986 l997-ll-24 W O 96/4,0895 PCTAUS96/09679 Roughly spherical, and wispy, irregular deposits, were labelled with antibody 9204 specific for the free amino terminus of A,B. Astrocytic gliosis associated with A,B deposition was evident after double immlm~labelling with antibodies to glial fibrillary acidic protein (GFAP) and human A,B. A
S comp~ct~t1 A,B core and 'halo' was evident in several plaques. Non-transgenic littermates showed none of these neuropathological changes.
Immunost~inin~ was fully absorbable with the relevant ~yll~le~ic peptide, and was apparent using a variety of processing conditions, including fixation with paraformaldehyde and Trojanowski methods. Many plaques were stained with thiol~lavin S, and some were also stained using the Bielschowsky silver method a;nd were birefringent with Congo Red, indicating the true amyloid nature of these deposits.
The majority of plaques were intim~t~ly surrounded by GFAP-positive reactive astrocytes, similar to the gliosis found in AD plaques. The neocortices of the lldllsgenic mice contained diffusely activated microglial cells, as ciefined by their amoeboid appearance, shortened processes, and staining vt~ith Mac-1 antibody. Staining by antibodies recognizing phosphorylated neurofil~m~nt~i and phosphorylated tau inrlir~t~l that aberrant phosphorylation occurred in PDAF~P brain that was similar to AD. These phosphorylations are seen in AD and are thought to preclude formation of neurofibrillary tangles. Although paired helical filaments (PHF) have not yet been tlett~ctecl in PDAPP mice, the detection of abnormally phosphorylated neurofilaments and tau are thought to be associated with, and the initial step in, the fo~mation of PHF in AD.
Clear evidence for neuritic pathology was apparent using both conventional and confocal immllnnmicroscopy. Forty ,um thick vibratome sections were incubated overnight at 4~C with R1280 (1:1,000) in ~ combination with polyclonal anti-synaptophysin (l:lS0; Dako) or 8E5 (7.0,ug 1). Some sections were incubated with anti-synaptophysin or monoclonal anti-MAP 2 (1:20, Boehringer-Mannheim), and then reacted with a goat anti-rabbit biotinylated antibody (1:100) followed by a mixture of FITC-conjugated horse anti-mouse IgG (1:75) and avidin D Texas red (1:100) CA 0222l986 l997-ll-24 W O 9G/1~835 PCT/U'.3~ 5679 (Vector Labs). The double-immlmolabelled sections were viewed on a Zeiss Axiovert 35 microscope with attached laser confocal sc~nning system MRC
600 (Bio-Rad). The Texas red charmel collected images of the R1280 or synaptophysin labelling, and the FITC channel collected synaptophysin, 8E5, 5 or MAP 2 labelling. Optical z-sections 0.5 ,~bm in thickness were collected from each region, similar to the image processing and storage described by Masliah et al., J. Neuropath. Exp Neurol. 52:619-632 (1993).
Many A~ plaques were closely associated with distorted neurites that could be ~letect~ ~1 with human APP-specific antibodies and with anti-10 synaptophysin antibodies, suggesting that these neurites were derived in partfrom axonal sprouts, as observed in the AD brain. The plaques compressed and distorted the surrounding neuropil, also as in the AD brain. Synaptic and dendritic density were also reduced in the molecular layer of the hippoc~mp~l dentate gyrus of the transgenic mice. This was evident by 15 reduced immunostaining for the presynaptic marker synaptophysin and the dendritic marker MAP-2 in AD brain (Masliah et al., Am. J. Path. 138:235-246 (1991)).
Confirmation of the presence of extracellular A~B was obtained using immllnoelectron microscopy. For immun~ electron microscopy, mice were 20 perfused with saline followed by 2.0% paraformaldehyde and 1.0%
glutaraldehyde in cacodylate buffer. Forty ~4m thick vibratome sections were incubated with the R1280 antibody, and reacted using a peroxidase rabbit IgG
kit (Vector Labs). Immunolabelled sections with A~ deposits were then fixed in 1.0% ammonium tetraoxide and embedded in epon/araldite before viewing 25 ultrathin sections with a Jeol CX100 electron microscope (Masliah et al., Acta Neuropath . 81 :428433 (1991)) .

W O ~G/~C e ss PCTrUS96109679 Table 3. Ull~a~lluctural .~imil~rities and Dirre~ ces Between AD and PDAPP Tr~,s~ ic Plaques.

h~im~r's Disease PDAPP
Amyloid fibrils size 9-11 nm 9-11 nm electron density ml ~er~tP high pinocytic vesicles abundant ocr~ci~7n~1 Dy~ p~ eurites dense laminar bodies abundant abundant synaptic vesicles and contacts yes yes neurofilatnent a~e-lm--l~ion yes yes 'lYPE Il[
paired helical fil~m~n~c yes none?
Cells &.~ with amyloid f~
microglia abundant OCC:ICiQn~
neurons occ~ion~l abundant n~ usc~ ory granules abundant ahllnrl~nt rough enrlopl~cmi-~ reticulum abundant abundant coated pits yes yes Tables 3 and 4 present a snmm~ry of the above results, showing cytologic,al and pathological similarities between AD and PDAPP mice. For every feature ex~min.o-l7 with the exception of paired helical filaments, the PDAPP mice exhibited pathology characteristic of AD. These fin~1ing.~ show s that production of human APP in transgenic (TG) mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD.

W O ~G/~EgS PCTAUS96/09679 Table 4. Pathology in Al;~ ,e,'s Disease and the PDAPP Mouse.
Alzheimer's PDAPP
Disease A~ Deposition into Plaques Diffuse + +
s Neuritic + +
Vascular + +
Brain Region Specificity + +
Neuritic Dystrophy + +
Synaptic Loss + +
Tnfl~mm~tory Response Astrocytosis + +
Microgliosis + +

Cytoskeletal Alterations Phosphorylated Neurofil~ment~ + +
Phosphorylated Tau + +
PHF/Tangles + -(?) The most notable feature of these transgenic mice is their Alzheimer-like neuropathology, which includes extracellular A~B deposition, dystrophic neuritic components, gliosis, and loss of synaptic density with regional specificity resembling that of AD. Plaque density increases with age in these transgenic mice, as it does in hllm~n.~ (Selkoe, Rev. Neurosi. 17:489-517 (1994)), implying a progressive A,B deposition that exceeds its clearance, as also proposed for AD (Maggio et al., Proc. Natl. Acad. Sci. U.S.A. 89:5462-5466 (1992)). The PDAPP transgenic mice provide strong new evidence for the primacy of APP expression and A~ deposition in AD neuropathology.
Such mice also provide a sufficiently robust AD model in which to test whether compounds that lower A,B production and/or reduce its neurotoxicity in vitro can produce beneficial effects in an animal model prior to advancing such drugs into human trials.

W O ~6/~9S PCT/U~,CJ~679 m~ 7: Construction APP Ir~ls7~ es ~ g APP from the PDGF-B ~
PDAPP transgenic mice contain a splicing c~c.cette that permits expression of all three major human APP isoforms, where expression is s driven by the PDGF-B promoter, and which inrlllclec a mutation in amino acid 717, the site of famili~l AD mutations. It is expected that these features, and others described above, can be used independently to produce transgenic mice useful as models of Alzheimer's disease. Some specific examples of such constructs are described below.
o A. Const~uction of PDAPP-wt.
A wild type version of the cDNA/genomic clone PDAPP was constructed in which the mutation to amino acid 717 was replaced with the wild type. This was accomplished by replacing the 1448 bp XlloI to SpeI
fragment of PDAPP, which includes the part of the APP cDNA seq~len~e that encodes the Hardy mutation in which Val717 is replaced by Phe, with the 1448 bp ,XhoI to SpeI fragment of a wild type APP clone. This fragment corresponds to the region from position 1135 to 2588 of SEQ ID NO:S.
None of the intron sequences of PDAPP are replaced or removed by this substitution. This construct is referred to as PDAPP-wt. A schem~tic of PDAPP-~;vt and its construction is shown in Figure 12.
B. Construction of PDAPP-SwHa.
Another version of the cDNA/genomic clone PDAPP was constructed in which the Swedish mutant at amino acids 670 and 671 was introduced.
Plasmid pNSE751.delta3'spl.sw contains cDNA of the human APP751 which 2s includes the Swedish mutation of Lys to Asn and Met to Leu at amino acids 670 and 671, respectively. A 563 bp EcoRI to SpeI fragment from this plasmid was replaced with the corresponding 563 bp EcoRI to SpeI fragment of PDAPP, which includes an identical part of the APP cDNA sequence with the exception of Phe717 of the Hardy mutation. This fragment corresponds to the region from position 2020 to 2588 of SEQ ID NO:5. This results in pNSE.delta3'spl.sw/ha, which contains both the Swedish mutation at amino acids 670 and 671, and the Hardy mutation at amino acid 717.

W O 96/40895 PCTrUS96/09679 The 1448 bp ~oI to SpeI fragment of PDAPP was then replaced with the 1448 bp XhoI to SpeI fragment of pNSE752.delta3'spl.sw/ha, which contains both the Swedish mutation and the Hardy mutation, to form PDAPP-Sw/Ha. A schematic of PDAPP-Sw/Ha and its construction is shown in Figure 13.
C. Construction of PDAPP695~.
A construct encoding only APP695, but ret~ining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP, can be made. This can be accomplished by lig~ting the 6.6 kb X71oI to N~7uI fragment from PDAPP, lo which contains the C-terminal part of the APP sequences, and the polyadenylation, pUC, and PDGF-B promoter sequences, to the 1.2 kb X~oI
to BclI fragment of pCK695, which contains a hybrid splice signal and the rem~ining N-terminal portion of the APP sequences (on a 911 bp X7toI to Nrv~I fragment of APP695 cDNA). The hybrid splice signal is the same as 15 was described earlier and is also present in vector pohCK751, which is described by Dugan et al., JBiological Chem. 270:10982-10989 (1995).
pCK695 is identical to pohCK751 except that the herpes simples virus replication and packaging sequences of pohCK751 were removed, and the plasmid encodes APP695 instead of APP751.
~o In this vector the PDGF-B promoter drives the expression of APP695 cont~ining the mutation of Val717 to Phe. The hybrid splice signal is included to potentially enhance expression. Additional vectors derived from this may be constructed which lack any splice signals, or into which other splice signals have been added to obtain this same function.
2s D. Construction of PDAPP751~,.
A construct encoding only APP751, but retaining the Hardy mutation.
PDGF-B promoter, and vector sequences of PDAPP, can be made. This can be accomplished by li~ting the 6.65 kb X71oI to KpnI fragment of PDAPP, including part of the APP sequences, the polyadenylation signals, pUC and 30 PDGF-B promoter sequences to the 1.0 kb KpnI to XhoI fragment cont;~ininE
the remainder of the human APP751 cDNA sequences (nucleotides 57 to 1084 of SEQ ID NO:3) to make the intermerli~te plasmid PDAPP~sp751v~.

W O ~61~C~9S PCTAUS96/0967g The 1.0 kb KpnI to X7zoI fragment encoding a portion of human APP7~1 can be obtained from the plasmid poCK751, which is iclenti~l to pohCK751 except that the herpes simplex viral sequences were removed.
l'o introduce splicing sequences, the first intron from PDAPP, which s is intron ~6,iS then inserted into PDAPP~sp751vF to make PDAPP751~,. To accomplish this, the 2,758 bp Asp718 to ScaI fragment of PDAPP cont~ining exons 2 through 6, intron /~6, and part of exon 7, is ligated to the 6,736 bp fragment obtained by complete digestion of PDAPP~sp751v~ with Asp718 and partial digestion with ScaI. This 6,736 bp fragment provides the rem~ining additional APP sequences (part of exon 1, the rest of exon 7, and exons 9 through 18), polyadenylation signals, pUC and PDGF-B promoter sequences.
The resulting construct is referred to as PDAPP751v,.
II1 this vector the PDGF-B promoter drives the expression of APP751 cont~inin~ the mutation of Val717 to Phe. One splice signal (derived from intron 6) is included to potentially enhance expression. Additional vectors derived from this may be constructed which lack any splice signals, or into which ot]her splice signals have been added to obtain this same function.
E. Construction of PDAPP770 A construct encoding only APP770, but ret~ining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP, can be made. This can be accomplished by replacing the KpnI to X71oI fragment of PDAPP751~, conr~ining APP exons 2-7 and a part of exon 9, with the KpnI to X7~oI
fragment of APP770 cDNA, which contains exons 2-8 and a part of exon 9.
This fragment corresponds to nucleotides 57 to 1140 of SEQ ID NO:5. The 2~ resulting construct is referred to as PDAPP770~.
In this vector the PDGF-B promoter drives the expression of APP770 cont~ining the mutation of Val717 to Phe. PDAPP770~, contains the same ~ intron sequences present in PDAPP751~ . Additional vectors derived from this may be constructed into which a splice signals have been added to obtain enhanced expression.

CA 02221986 1997-ll-24 WO 96/40895 PCT~US96/09679 ExaInple 8: E~.e~ion Levels of APP Expression Productsin Brain T~sue of PDAiPP Mice.
The PDAPP mouse line described in Example 6 was e~min~l for the levels of several derivatives of the APP in hippocampal, cortical, and s cerebellar brain regions of mice of various ages. Levels of APP cleaved at the beta-secretase site (APP~B) and APP cont~ining at least 12 amino acids of A,~ (FLAPP+APPcY; a mixture of APP~ and full length APP (FLAPP)) were found to be nearly constant within a given brain region at all ages evaluated.
The hippocampus expressed the highest level of all APP forms. In contrast, 10 gn~ni~lin-- extractable levels of A,~ showed rem~rk~ble age-dependent increases in a manner that mirrored the amyloid plaque deposition observed immunohistochPmir~lly. Specific~lly, A,B levels in hippocampus increased 17-fold by 8 months of age and 106-fold by 1 year of age, compared to that found in 4 month old ~nim~l~. At 1 year of age A,~ con~tit--ttos 15 approximately 1% of the total protein in hippocampus. The cerebral cortex also showed large increases in A,~' with age. In contrast, the mean level of A~ in cerebellum across all age groups was comparatively low and unch~n~ing .
Further analysis of the A,B in these brains using an ELISA specific for 20 A~ . showed that this longer version made up 27% of the 19 pmoles/g of the A,~ present in the brains of young ~nim~; this percentage increased to 97%
of the 690 pmoles/g in 12 month old ~nim~l.c. The selective deposition of A~,~ and the spacial distribution of the A~ deposits are further evidence that the pathological processes ongoing in the PDAPP transgenic mice parallel the 2s human Alzheimer's diseased condition.
Levels of A~B-cont~ining proteins were measured through the use of ELISAs configured with antibodies specific to A~, A~, APP cleaved at the ~-secretase site (Seubert et al. (1993)), and APP cont~ining the first 12 amino acids of A~ (FLAPP+APPcY; a mixture of full length APP and ~-30 secretase cleaved APP (Esch et al.)). Striking similarities in both theregional variation and depositing form of A~' are noted between the mouse model and the human AD condition. The results also show that, because of . CA 02221986 1997-11-24 WO ~Gt4'1855 PCT/U:j,G~ 679 the m~nitll(ie and temporal predictability of A~ deposition, the PDAPP
mouse is a practical model in which to test agents that either inhibit the processin~ of APP to A,~ or retard A~ amyloidosis.
A. Materials and Methods.
s 1. Brain Tissue Preparation.
The heterozygote transgenic (Line 109, Games et al.; Rockenstein et al.) andl non-transgenic ~nim~l~ were anestht-ti7~ocl with Nembutol (1:5 solution in 0.9% saline) and perfused intracardially with ice cold 0.9%
saline. The brain was removed and one hemisphere was ~ ~alcd for immunohistoch~omir~l analysis, while four brain regions (cerebellum, hippocampus, th~l~mn~, and cortex) were fii~.sect~(i from the other hemisphere and used for A,~ and APP measures.
To prepare tissue for ELISAs, each brain region was homogenized in 10 volwmes of ice cold g~l~ni-lin~ buffer (5.0 M gu~ni~lin.o-HCI, 50 mM Tris-Cl, pH 8.0) using a motorized pestle (Kontes?. The homogenates were gently mixed on a Nutator for three to four hours at room temperature, then either assayed directly or stored at -20~C prior to qll~ntit~tion of A~B and APP. Preliminary experiments showed the analytes were stable to this storage condition.
2. ~,~ Measurements.
The brain homogenates were further diluted 1:10 with ice-cold casein buffer (0.25% casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20 ~g/ml aprotinin, 5 mM EDTA pH 8.0, 10 ,ug/ml leupeptin), reducing the final concentration of gll~ni~linl- to 0.5 M, before centrifugation (16,000 x g 2s for 20 minutes at 4~C). The A,~ standards (1-40 or 1-42 amino acids) were prepared such that the final composition included 0.5 M guanidine in the presence of 0.1% bovine serum albumin (BSA).
~ The "total" A~ sandwich ELISA consists of two monoclonal antibodies (mAb) to A,B. The capture antibody, 266, is specific to amino acids 13-28 of A,B (Seubert et al. (1992)); while the antibody 3D6, which is specific to amino acids 1-5 of A,~, was biotinylated and served as the reporter antibody. The 3D6 biotinylation procedure employs the m~mlf~cturer's W O 96/40895 PCTrUS96/09679 (Pierce) protocol for NHS-biotin labeling of immunoglobulins except 100 mM
sodium bicarbonate, pH 8.5 buffer was used. The 3D6 antibody does not recognize secreted APP or full-length APP but detects only A,l~ species with amino terminal aspartic acid. The assay has a lower limit of sensitivity of approximately 50 pg/ml (11.4 pM) and showed no cross-reactivity to the endogenous murine A~B peptide at concentrations up to 1 ng/ml.
The configuration of the A,B,~-specific sandwich ELISA employs the mAb 21F12, which was gellelaLed against amino acids 33-42 of A,~. The antibody shows less than 0.4% cross-reactivity with A~ in either ELISA or 0 competitive radioil-,,,,l,,,o~c~y (RIA). Biotinylated 3D6 is also the reporter antibody in this assay which has a lower limit of sensitivity of approximately 125 pg/ml (28.4 pM).
The 266 and 21F12 mAbs were coated at 10 ~g/ml into 96-well immllno~c~y plates (Costar) overnight at room telllpel~LIllc. The plates were 15 then aspirated and blocked with 0.25% human serum albumin in PBS buffer for at least 1 hour at room temperature, then stored desiccated at 4~C until use. The plates were rehydrated with wash buffer prior to use. The samples and standards were added to the plates and in~lh~ted at room temperature for 1 hour. The plates were washed at least 3 times with wash buffer (Tris 20 buffered saline, 0.05% Tween 20) between each step of the assay.
The biotinylated 3D6, diluted to 0.5 ,ug/ml in casein assay buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4), was in~ub~te~l in the well for 1 hour at room telllpeldLul~. Avidin-HRP (Vector, Burling~mP, CA), diluted 1:4000 in casein assay buffer, was added to the wells and incubated 25 for 1 hour at room temperature. The colorimetric substrate (100 ~l), Slow TMB-ELISA (Pierce), was added and allowed to react for 15 minutes, after which the enzymatic reaction is stopped with 25 ~l of 2 N HSO.. Reaction product was quantified using a Molecular Devices Vmax measuring the difference in absorbance at 450 nm and 650 nm.
3. APP ELISAs.
Two different APP assays were utilized. The first recognizes APP~
and full length forms of APP (FLAPP+APPo~), while the second recognizes =

. CA 02221986 1997-11-24 W O ~61~.?.5 P ~ AUS96/09679 APP,~ (APP ending at the metnionine precerling the A~ domain (Seubert et al. (1~993)). The capture antibody for both the FLAPP+APPc~ and APP~B
assays is 8E5, a monoclonal antibody raised to a bacterially ~ cssed fusion protein co.l~onding to human APP amino acids 4~ 592 (Games et al.).
5 The reporter mAb (2H3) for the FLAPP+APPc~ assay was gel~ld~d against amino acids 1-12 of A,B. The lower limit of sensitivity for the 8E5/2H3 assay is approximately 11 ng/ml (150 pM). For the APP,B assay, the polyclonal antibody 192 was used as the reporter. This antibody has the same specificity as antibody 92 (Seubert et al. (1993)), that is, it is specific10 to the carboxy-terminus of the ,~-secretase cleavage site of APP. The lower limit of sensitivity for tne ~-secretase 8ES/192 assay is approximately 43 ng/ml (600 pM).
For both APP assays, the 8E5 mAb was coated onto 96-well Costar plates as described above for 266. Purified recombinant secreted APPc~ (the 15 AP 751 form) and APP596 were the reference standards used for the FLAPP+APPcY and APP,~ assays, respectively. APP was purified as described previously (Esch et al.) and APP concellL~lions were dt~ lllined by amino acid analysis. The S M guanidine brain homogenate sainples were diluted 1:10 in specimen diluent for a final buffer composition of 0.5 M
20 NaCl, 0.1% NP-40, 0.5 M gll~ni-linf . The APP standards for the respective assays were diluted into buffer of the same final composition as for the samples. The APP standards and samples were added to the plate and incubated for 1.5 hours at room temperature. The plates were thoroughly washed between each step of the assay with wash buffer. Reporter antibodies 25 2H3 and 192 were biotinylated following the same procedure as for 3D6 and were i;ncubated with samples for 1 hour at room temperature. Streptavidin-~lk~linP phosphatase (Boehringer Mannheim), diluted 1:1000 in specimen ~ diluent, was in~ub~tecl in the wells for 1 hour at room temperature. The fluorescent substrate 4-methyl-umbellipheryl-phosphate, was added, and the 30 plates read on a Cytofluor~ 2350 (Millipore) at 365 nm excitation and 450 nm emission.

W O 96/40895 PCTrUS96/09679 4. Monoclonal Antibody Pro~ cti~.
The immunogens for 3D6, 21F12, and 2H3 were separately conjugated to sheep anti-mouse immunoglobulin (Jackson Immunoresearch Labs) using maleimidohexanoyl-N-hydroxysuccinimi-le (Pierce). A/J mice s (Jackson Laboratories) were given intraperitoneal injections (IP) of 100 ,ug of the applupliate immllnogen em~ ified in Freund's complete adjuvant (Sigma) and two subsequent IP injections of 100 ~g immunclgen were given on a biweekly basis in Freund's incomplete adjuvant (Sigma). Two to three weeks after the third boost, the highest titer mouse of a given immlmogen was 0 injected intravenously and intraperitoneally with 50-100 ,ug of immllnogen inPBS. Three days post injection, the spleen was removed, splenocytes were isolated and fused with SP2/0-Agl4 mouse myeloma cells. The hybridoma ~ .e.llalallL~, were screened for high affinity monoclonal antibodies by RIA as previously described (Seubert et al. (1992)). Purified monoclonal antibodies 15 were pl~al~d from ascites.
5. l.. u.. ohistorh~
The tissue from one brain hemisphere of each mouse was drop-fixed in 4% paraformaldehyde and post-fixed for three days. The tissue was mounted coronally and 40 ,~4m sections were collected using a vibratome.
20 The sections were stored in anti-freeze at -20~C prior to staining. Every sixth section, from the posterior part of the cortex through the hippocampus, was irnmunostained with biotinylated 3D6 at 4~C, overnight. The sections were then incubated with horseradish peroxidase avidin-biotin complex (Vector) and developed using 3,3'-diaminoben7idin~ (DAB) as the 2s chromagen.
B. Results.
1. A,~ and APP Assays.
The FLAPP+APP~ assay recognizes secreted APP including the first 12 amino acids of A~. Since the reporter antibody (2H3") is not specific to 30 the alpha clip site occurring between A,B amino acids 16 and 17 (Esch et al.), this assay also recognizes full length APP. Preliminary experiments using immobilized APP antibodies to the cytoplasmic tail of full length APP to wo 9~/ ~a 9.9S PC~AUS96J~9679 deplete the mixture suggest that approximately 30 to 40% of the FLAPP+APPcY is full length. The APP~ assay recognizes only the APP
clipped imm~ tely amino-terminal to the A,~ region due to the specificity of the polyclonal reporter antibody 192 (Seubert et al. (1993)).
s The specific nature of the A~ olcactivity was further charactcrized as follows. Gn~ni~linlo homogenates of brain (excluding cerebellum and brain stem) were subjected to size exclusion chromatography (Superose 12) and the res--lting fractions analyzed using the total A~ assay.
Comparisons were made of 2, 4, and 12 month transgenic mouse brain homogenates and a non-transgenic mouse brain homogenate to which A,B,~
had been spiked at a level roughly equal to that found in the 12 montn old transgenic mice. The elution profiles of the transgenic brain homogenate were similar in that the peak fractions of A~' immlmoreactivity occurred in the same position, a single broad symmetric peak which was coincident with the imnnllnoreactive peak of spiked A~ . Attempts were then made to immlmodeplete the A,B immllnoreactivity using resin bound antibodies against A,~ (mAb 266 against A,~,~.), the secreted forms of APP (mAb 8E5 against APP~ of the APP695 form), the carboxy-terminus of APP (mAb 13G8 against APP~ of the APP695 form), or heparin agarose. Only the 266 resin captured A~ immnnoreactivity, demon~ d~ g that full length APP or carboxy-terminal fragments of APP are not contributing to the A,~
measurement. The A~ ELISA employs a capture antibody that recognizes A~,~ but not A,~,~. The A,~, assay, like the total A~B assay, is not affected bythe full length or carboxy-terminal forms of APP cont~ining the A,~ region in the hormogenates as shown by similar immunodepletion studies.
2. Total A,5 and APP Measures.
Table 5 shows the levels of total A~, FLAPP+APP~, and APP~ in the hippocampus, cortex, cerebellum, and th~l~mns of transgenic mice as a function of age. Each data point represents the mean value for each age group. The relative levels of FLAPP+APP~ and APP~ in all four brain regions remain relatively constant over time. The hippocampus expresses the highest levels of FLAPP+APPa! and APP,B followed by the th~l~ml-c, cortex.

W O 96/40895 PCT~US96/09679 and cerebellum, respectively. In the hippocarnpus, the levels of FLAPP+APPo~ are approximately 3.5 to 4.5-fold higher than APP~B at all ages. The mean value of all ages for FLAPP+APP~ and APP~B assays in the hippocampus were 674 (+465) pmoles/gram and 175 (+11) pmoles/grarn, s respectively. From this it can be estim~t~l that the pool of brain APP
consis~, of approximately 50% APP~, 30% full length APP, and 20% APP,B.
TABLE 5. PDAPP Tr~ulsg~l1e Cohort Animal Data Total A~ & APP Mea~,ures in pmoles/gram of Brain Tissue.

AGE INA~ & APP CEREBELLUM HIPPOCAMPUSCORTEX THALAMUS
MONTHSFORM
2 A,~ 4.03 il.O8 35.41 i6.3814.25 6.41 il.59 (n = 8) (n = 8)i Z .27 (n = 8) (n=8) 2 FLAPP~ ND ND ND ND
APPa 2 APP,~ ND ND ND ND
4 A~B 4.10 iO.61 38.08 i6.5115.95 7.60 il.52 n= 14)(n = 14) i2.60(n = 14) (n= 14) 4 FLAPP+ 395 il20703 ilO6 446 i706.37 il66 APPa (n=14)(n=14) (n=14) (n=14) 4 APP,~ 78 i38 (n=14) 198 i30 (n-14) 126 i23 70 il7 (n= 14)(n= 14) 6 A,6 4.55 il.38 87.48 i30.3330.19 8.34 i2.40 (n=10)(n=10) i8.33 (n=10) (n= lo) 6 FLAPP+ 403 i77694 ilO7 506 i97670 il56 APPa (n = 10) (n = 10)(n = 10) (n = 10) 6 APP,B 51 i87 (n=10)194 i35 (n=10) 129 i25 56 i33 (n= 10)(n= 10) 6.5 A,B 5.42 il.O8 133.63 i57.10 33.27 8.83 il.l9 (n = 10) (n = 10)i 12.19 (n = 10) (n= 10) 6.5FLAPP+ 346 i74580 ill5 436 i63553 :~123 APPa (n=10)(n=10) (n=10) (n=10i 6.5 ~PP,~ 27 i77 (n=10) 169 i41 (n=10) 108 il6 58 i2' (n= 10)(n= 10) 7 A,B 4.44 iO.56 200.77 i94.68 60.55 8.94 il.l9 (n=l~)(n=10) i27.13 (n=10) (n= 10) W09''4~9S PCT~US96/09679 7 FLAPP+ 378 i70 656 i73 (n=10) 469 ~62 604 il07 APP~r(n=10) (n=10) (n=10) 7 APP,t~56 i52 (n=10) 176 :t27 (n=10) 101 i20 56 i28 (n= 10)(n= 10) 7.5 A~5.14 il.39461.35 i345.9581.83910.84 +5.22 (n= 10) (n= 10) i53.00 (n= 10) (n=10) 7.5 FLAPP+362 iS4554 i77 (n=10) 409 i44503 ~:80 APPc~(n = 10) (n = 10)(n = 10) 7.5 APP,~20 i58 (n=10) 168 +27 (n=10) 118 i21 57 i22 (n = 10)(n = 10) 8 A,~4.42 iO.73635.52 i302.45128.6810.87 i3-39 (n=13) (n=13) i62.80 (n=13) (n=13) 8 FLAPP+386 iS2 660 ilO2 494 i87672 il50 APP~ (n=13) (n=13) (n=13) (n=13) 8 APP,~64 i77 (n=13) 174 i27 (n=13) 102 i26 57 i30 (n=13) (n=13) 8.5 A,~5.54 :tl.ll633.11 ~t363.14 118.39 13.96 i7.34 (n=10) (n=10) i59-91 (n=10) (n= 10) 8.5 FLAPP+439 i~79 764 ill4 558 i80750 il32 APPcY(n=10) (n=10) (n=10) (n=10) 8.5 APP,I~28 iS9 (n=10) 185 i34 (n=10) 108 i42 47 i28 (n= 10)(n = 10) 9 A,B5.52 il.ll 1512.39 254.8319.46 ::8.99 (n= 10) i624.286 i 105.927(n= 10) (n= 10) (n = 10) 9 FLAPP+500 ~112 763 il25 549 i78815 il67 APPcr(n=10) (n=10) (n=10) (n=10) 9 APP,~4 i83 (n=10)169 i25 (n=10) 121 i32 49 i26 (n= 10)(n= 10) A,64.04 il.O2 2182.21 343.49 15.46 (n=ll) ill944g il65.531il3.38 (n=ll) (n=ll) (n=ll) FLAPP+452 il30678 i93 (n=ll)491 ilO2693 il66 APP~ (n=ll) (n=ll) (n=ll) APP,652 i32 (n=ll) 159 i22 (n=ll) 87 i:15 46 ilO
~ (n=ll) (n=ll 12 A,B3.26 iO.35 4356.23 691.17 18.08 (n=9)il666.44 (n=9) i360.93il3.50 (n = 9)(n = 9) 12 FLAPP+385 il66 638 i272 444 ~171708 i278 APP~ (n=10) (n=10) (n=10) (n=10) W O 96/40895 PCT~US96/09679 12 APP~ 41 ~29(n=10) 134 ~47(n=10) 76 ~31 35 il9 (n=10) (n=10) = not~ illed In contrast to APP levels, A,B levels increased dr~m~ti~ ly with age in the hippocampus and cortex. However, no such increase was noted in the cerebellum of the PDAPP transgenic mice, and only a moderate increase was seen in th~l~ml-c (Table 5). The increase of A,~ is greater in the hippocampus relative to the cortex, which also correlates with the 3D6 imml-n~ histochPmi~l results (see discussion below). Compared to the cortex levels of 4 month old mice, A,~ levels increase 10-fold by 8 months of age and 41-fold at 12 months old (660 i 380 pmoles A,B/gram tissue at age 12 months). The corresponding increases in A~ observed in hippocampus are lO even more i~ ,lc~s~ive, as the 8 month value is 15 times that at 4 months oldand increases to 106-fold at 12 months old (4,040 ~ 1750 pmoles A,l~/g tissue at 12 months). At 12 months of age, A~ co,~ s approximately 1%
of protein in hippocampus of the PDAPP mice.
To see if the dramatic rise in brain A~ concentration is due to 15 amyloid deposition, we next vicl~li7P~l A,~ deposits imml-nohistochPmi~ally, using the opposite hemisphere of the same mice used for A~ measurements.
Notably~ a parallel increase in A,B plaque burden and A,~ level exists. These fin~ling~ strongly argue that the rise in brain A~ concentration determined by ELISA is due to the age-dependent amyloidosis.
3. A,~,~ Measures in Transgenic Mouse Brain.
Concentrations of A~, in the cortex of transgenic mice were evaluated at different ages. As shown in Table 6, the percentage of A~ which is A~, in the cortex of transgenic mice, also increases with age. The ELISA data suggest that A~,. is ~l~rel~nlially depositing in the transgenic mice, and that 25 the deposits clPtçctçcl by mAb 3D6 immlmostaining are primarily A,B,~.
Table 6. A~ Levels in the Cortex of Transgenic Brain.
Age (months) A,B~ (pmoles/g) 4 4.71 8 75.65 247.43 CA 0222l986 l997-ll-24 W O~G/~ as~ PCT/US96J09679 12 614.53 4. A,~ Tm~ nosl~;..;..~r in PDAPP Transgenic Brain.
Transgenic ~nim~l.c with A~ values representing the mean A,B value of the age group were used for 3D6 immllnostaining. A progression of A~
depositio~n is seen in the 4, 8, 10, and 12 months old anim~lc. At four months of age, transgenic brains contained small, rare punctate deposits, 20 ,um in diameter, that were omy infrequently observed in the hippocampus and frontal and cingulate cortex. By eight months of age, these regions contained a number of thioflavin-positive A~' aggregates that formed plaques as large as o 150 ~m iin ~ mPter. At ten months of age, many large A,~ deposits were found throughout the frontal and cingulate cortex, and the molecular layers of the hippocampus. The outer molecular layer of tne dent te gyrus receiving perforant pathway arr~lenl~ from the entorhinal cortex was clearly delinP~tto~
by A,B deposition. This general pattern was more pronounced by heavier A~' deposition at one year of age. The anatomical loc~li7~tion of A,~ deposition is remarkably similar to that seen in Alzheimer's disease.
C. Di.cc~ n.
A,~ amyloidosis is an established diagnostic criteria of Alzheimer's disease (]\~irra et al., Neurology 41 :479-486 (1991)) and is consistently seen in higher cortical areas as well as the hippocampal formation of the brain in affected subjects. It is believed that A,B amyloidosis is a relatively early event in the pathogenesis of AD that subsequently leads to neuronal dysfunction and dementia through a complex cacc~e of events (Mann et al., Neurodegeneration 1:201-215 (1992); Morris et al., Neurology 46:707-719 2s (1996)). Various pathways of APP processing have been described (reviewed in Schenk et al., J. Med. Chem. 38:4141-4154 (1995)) including the major cY-secretase pathway where cleavage of APP occurs with A~ (Esch et al.) and the amyloidogenic or ,B-secretase pathway where cleavage of APP occurs at the N-tenninus of A,B (Seubert et al. (1993)). Further cleavage of APP leads to the constitutive production of A~ forms including those ending at position 40 (A~B, ) or 42 (A~ ). ELISAs that detect specific APP products arising from these individual pathways in the PDAPP mouse brain allow W O 96/40895 PCT~U~G~ 679 de~ ination of whether differential procecsing of APP contributes to the regional or temporal specificity of amyloid formation and deposition.
A~ amyloid deposition seen in the PDAPP mouse brain is high~y age and region specific. Amyloid deposition begins at around 7 months of age, 5 and by 12 months of age, amyloid deposition is very profound throughout the hippocampus and in the rostral region of the cortex. The age dependent increases in amyloid deposition correlate well with the dramatic rise in A~
levels in these brain regions as measured by ELISA assay. An increase in A,B is measurable by 7 months of age and by 10 months the hippocampus as lO 2180 pmoles/g of A,l~, a concentration equivalent to that of my cytoskeletal proteins and comparable to the levels found in the cortex of human AD brain (Gravina et al., J. Biol. Chem. 270:7013-7016 (1995)). A~ levels in the cerebellum, an unaffected brain region, still are at 4 pmoles/g -- essentially unchanged relative to the levels at 4 months of age -- again correlating with amyloid deposition measured by histological analyses. These results in-1ic~t~
that in aged PDAPP mice, brain A,B levels reflects amyloid burden and therefore direct immllnl~assay measurement of brain A~ levels can be used to test for compounds that reduce amyloid plaque burden.
In the PDAPP mouse, individuals suffering Down's Syndrome, and individuals with certain forms of FAD, overproduction of A~B is almost certainly an accelerating factor not only in A~ deposition but in subsequent neuropathology (Citron et al., Mann et al., Miller et al., Archives of Biochem. Biophys. 301:41-52 (1993)). A comparison of the A,~
measurements seen in the PDAPP mouse with those reported for AD brain tissue reveals several striking similarities. For example, in the PDAPP
mouse, the relative levels of A~ peptide in hippocampus from young (2 months of age) versus old (10 months of age) mice is nearly a hundred fold.
Similar findings were noted by Gravina et al. in comparing control brain tissue relative to that of AD. The rise in brain A,(~ levels in the PDAPP
mouse is rather pronounced between the ages of six to nine months of age.
Again, this timecourse parallels, in an accelerated manner, that seen in Down's Syndrome brain tissue, where amyloid deposition begins at . CA 02221986 1997-11-24 approximately 30 years of age and increases subst~nti~lly until approximately age 60 (Mann).
In sllmm~ry, the above results show that a reproducible increase in measurable A,~ occurs in the brain tissue of the PDAPP mice and that this increasle correlates with the severity of amyloid deposition. These fin~ling.c indicate that these mice can be used to identify agents or compounds that pharmacologically reduce A,B peptide production or affect its deposition.
Ex~~ lc 9: Behavioral Differences in PDAPP Transgenic Mice.
Alzheimer's disease is characterized by cognitive deficits inrlll-ling o memory loss, and impairment of memory functions. To determine if the disclosed transgenic mice exhibit similar deficits, transgenic (TG) and non-transgemic (nTG) mice were evaluated for task performance in three types of maze apparatus used to test working and reference memory; the Y maze, the radial arm maze (RAM), and the water maze. The transgenic mice tested ese~ilL the fifth generation derived from the PDAPP lmice described in Example 6. The Y maze and the radial arm maze are used to assess spontaneous alternation which is a function of working memory. For the Y
maze task, the mouse is placed in the stem of a Y maze twice, each time allowing a choice entry into one of the arms. Entering both arms is a successr~ul alternation, requiring memory of the previously entered arm, while entering the same arm on both trials is a failure. Chance performance is 50% alternation, that is, 50% of the mice alternate.
For the radial arm maze task, the mouse is placed at the center of a maze with multiple arms r~ ting from the center. In the testing described below, a radial eight-arm maze was used. Alternation performance is measured by allowing only eight entries, with the number of different arms entered being the measure of performance. The number of different-arm ~ entries can be compared to the number of different-arm entries expected by chance, which is 5.25 (Spetch and Wilkie, "A program that stimulates 30 random choices in radial arm mazes and similar choice situations" Behavior Research Methods & Instrumenlation 12:377-378 (1980)). Performance w o 96/~ce95 PCTrUS96/09679 above chance, that is, above 5.25, requires memory of the previously entered arms.
The water maze used for the tests described below consists of a pool of water in which a submerged platform is placed. This hidden platform (HP) can be found by swimming mice either by chance (first trial) or through memory of positional clues visible from the tank (subsequent trials). Subject mice were trained in the hidden platform task according to standard procedures. Briefly, mice were first pretrained in a small pool (47 cm m~ter, 20 cm platform), which teaches them how to navigate in water, o that the platform is the goal, that there is no other escape, and that to find it they must resist their natural inclination to stay along the sides of the pool.
They were then trained to find a single platform position in the hidden platform task using a larger pool and smaller platform (71 cm pool, 9 cm platform).
During the HP task, visual cues were located inside the pool (hlLldlllaze cues; black pieces of cardboard - circle, plus, or horizontal lines -located in three quadrants at the top of the wall, which was 38 cm high above water level), and various room cues were visible outside the pool (extramaze cues).
The mice assigned to the characterization cohort study were tested on the behavioral tasks described above over 3 days during the week or two before euth~n~ . Their transgenic status was not known to the tester. Non-transgenic littermates were used for comparison. Each morning the subject mice were run in the Y maze and RAM as described above. They were then 25 tested for general strength on the inclined plane (INP) test. For this, mice were placed in a 10-cm-wide runway lined with ridged plastic and elevated with the head up at 35. The angle was then increased until the mouse slid off, and the angle was recorded. This was repeated three times each day.
The average scores for the three days were calculated for each mouse for the 30 Y maze (0=repeat, 1=alternate), RAM (number of different arms and time to finish, 10 minute limit), and INP (average of all nine trials). General activity was also rated on the f1rst day of testing. Each mouse was observed =

WO 9GI~9J PCT/USg6~0~767S~

in the cage, and picked up and held. A mouse that ren-~in~od calmly in the hand was scored 1, with progressively greater activity and reaction to h~n~lling scored up to 4.
Following the above tests each day, mice were tested in the water maze a,s described above. Briefly, mice were pretrained in a small pool to climb on a large submerged platform as their only means of escape from the water. They were then given six blocks of four trials each to learn the locatiorn of a small platform in a large pool. For analysis, all four trials within each block were averaged. The exception was the first hidden o platform block, for which only the last three trials were averaged. The first trial was analyzed separately, because it is the only one for which platform locatiom could not be known, and thus did not relate to spatial learning. It is thus used as a control for non-spatial factors, such as motivation and swimming speed. The performance effects between blocks were analyzed as a repeated measure for the hidden platform task. Standard analysis of variance (ANOVA) calculations were used to assess the si~nifi~nre of the results.
Results in the RAM show that TG performed si~nifi~ntly worse than nTG across all ages (Group effect: p=0.00006). The time to finish was also significantly different between TG and nTG mice (Group effect: p=0.005).
The correlation between the time to finish and the number of arms chosen was small (R= -0.15, p=0.245 in each group). This suggests that the consistent impairment in the RAM is not accounted for by the increased time to complete the task taken by TG mice. Results in the Y maze were also 2~ significantly different for TG and nTG mice (Group effect: p=0.011).
Validation studies performed on non-transgenic mice indicate that the Y maze is a less sensitive measure than the RAM.
~ Measures of strength (INP) and activity indicate no differences between TG and nTG mice. These are considered very rough measures, with only large differences being detectable. There was, however, a decrease in the activity score for all mice over time (Age effect: p=0.070). There was a difference in body weight, with TG weighing 8% less than controls (Group W O 96/40895 PCTrUS96/09679 effect: p=0.0003), primarily in female TG mice. However, this does not seem to have an effect on the results, as shown by the lack of any dirrelellce in strength (see above) or swimming speed (see below) between TG and nTG
mice.
s Results of the hidden platform task, considered here a test of reference memory, show a consistent difference between TG and nTG mice.
ANOVA reveals that the effects of transgenic status (Group effect:
p=0.00016) and trial blocks (Block effect: p<0.00001) are .signific~nt. The effect of transgenic status on performance is accounted for by slower performance by TG mice across all trial blocks and ages. Analysis of Trial 1 reveals an effect of transgenic status (Group effect: p=0.018), suggesting a difference in performance before learning has occurred. However, an analysis of covariance, with trial 1 as the covariate, still yields a signific~nt deficit in TG mice (p=0.00051).
It was also possible that some physical dirre~ ces between TG and nTG mice, rather than cognitive differences, could have been responsible for some of the performance differences seen in the water maze tasks. However, no significant difference in strength or activity was observed (see above).
Another possibility considered was the effect of swimming speed on perforrnance since a slower swimmer with equivalent cognitive ability would take longer to reach the platform. To test this, video tracking was used in the hidden platform task to measure the ~ t~n~e travelled to reach the platform (a measure of the amount of searching done by the mice which is related to cognitive ability), the swimming speed (a measure of physical ability unrelated to cognitive ability), and the amount of time need to find theplatform (a measure of the combination of both the distance travelled and the swimming speed). This was done in older and younger mice than reported above, using three trials per block and no pretraining. The time needed to find the platform was significantly different in TG and nTG mice (Group effect: p<0.0005), with the TG mice taking longer. However, the swimming speed was not signific~ntly different between TG and nTG mice (Group effect: p=0.879). Thus, the difference in time needed to find the W O 96/40895 PCTAUS96~09679 platforrn is likely to be due to a cognitive dirr~le.lce between TG and nTG
mice. This is col~hnled by measures of the ~ t~nre travelled to find the platforrn. The TG mice travelled ~ignific~ntly further t'nan the nTG mice before reaching the platform (Group effect: p<0.0005). These results 5 in-lic~te that the differences seen between TG and nTG mice in the time to reach the platform in the water maze tasks are due to differences in cognitive ability.
To test whether nTG mice retain a beKer memory of the platform location than TG mice, a probe trial was given immP~ tely following hidden o platform training in which the platform was removed. Video tracking was used to determine the number of crossings of the former platform location made b~y the mice relative to crossings of non-platform locations. There was a signific~nt difference seen between the relative crossings of TG and nTG
mice (Group effect: p=0.006). This is evidence that the nTG mice s remember the former location of the platform better than TG mice.
]:t was also possible that the difference observed between TG and nTG
mice in the time needed to reach the platform could have been influenced by differences in perception of the cues or motivational differences. To test this, TG and nTG mice were subjected to visible platform tasks in the water maze. ~or these tasks, a platform was placed in the pool so that it was visible above the water. Three different platforms were tested, a dark platform 25 mm above the surface (most visible), a gray platform 25 mm above tne surface, and a dark platform 5 mm above the surface (both less visible). The results show no difference in the time to find the most visible platform between TG and nTG mice (Group effect: p=0.403). There was not any greater decrease in performance in TG mice when less visible platforrns were used, suggesting that their vision was as good as nTG mice.
~ These results in-lic~te that perceptual and motivational differences do not influence the time to reach the platform in the water maze tasks described above.
Performance differences between TG and nTG mice were shown for RAM, Y maze, and water maze cognitive tasks in mice aged 4 to 8 months W O 96/40895 PCT~.96~'0~M9 (2 to 12 months for the water maze). All of these ~irrt;~ellces in~icate, and are consistent with, cognitive deficits in the transgenic mice as a group. The various tasks combined to test working memory and reference memory, both of which are implicated in cognitive impairment observed in Alzheimer's victims.
Modifications and variations of the making and testing of transgenic animal models for testing of Alzheimer's disease will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.

. CA 02221986 1997-11-24 W 0~61~89S PCT~US96~09679 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Athena Neurosciences, Inc.
(ii) TITL]3 OF INVENTION: Method For Identi~ying Al~he;m~'s Disease Therapeutics Using Transgenic Animal Models (iii) NUMB~R OF SEQUENCES: 10 (iv) CORRESP~V~N~ ~n~.ss:
(A) ADDRESSEE: Patrea L. Pabst (B) STREET: 2800 One Atlantic Center ~ 1201 West Peachtree Street (C) CITY: Atlanta (D) STATE: GA
(E) COUNTRY: USA
(F) ZIP: 30309-3450 (v) COMPIJTER READABLE FORM:
(A) MEDIUM TYPE: Floppy diSk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION N~MBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/486,538 (B) FILING DATE: June 7, 1995 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pabst, Patrea L.
(B) REGISTRATION NUMBER: 31,284 (C) REFERENCE/DOCKET NUMBER: ANSlOOCIP
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404)-873-8794 (B) TELEFAX: (404)-873-8795 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2085 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOI'HETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1-2085 (D) OTHER INFORMATION: /function= "coding region for APP695."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln AAT GGG AAG TGG GAT TCA GAT CCA TCA GGG ACC A~A ACC TGC ATT GAT 192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu W O 96/40895 PCT~uS96/09679 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Ary Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Ary Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp As.. Val A~p Sel Ala Asp Aia Glu Glu Asp Asp Ser Asp Val TGG TGG GGC GGA GCA GAC ACA GAC TAT GCA GAT GGG AGT GAA GAC A~A 672 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu GAG ACA CCT GGG GAT GAG AAT GAA CAT GCC CAT TTC CAG A~A GCC AAA 960 Glu Thr Pro Gly Asp Glu Asn Glu ~is Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg GAA TGG GAA GAG GCA GAA CGT CAA GCA AAG AAC TTG CCT A~A GCT GAT 1056 , W~ 9~1C~95 PCT~U~6,'0~79 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro hys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys H.is Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg ~is Asp Ser Gly Tyr Glu Val W O 96/4089~ PCT~US96/09679 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg CAC CTG TCC AAG ATG CAG CAG AAC GGC TAC GAA AAT CCA ACC TAC A~G 2064 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 695 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg ~la Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu ~ln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn ~rp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu ~ys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile ~sp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu W O ~G/4~9S PCT~US9C/09679 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu ~lu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu ~lu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile ~la Thr Thr l'hr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys ~lu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg ~lu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp ~ys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn ~yr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe ~sn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His ~hr Leu Lys H:is Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala ~lu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn ~yr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser ~yr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn CA 0222l986 l997-ll-24 PCT~US96/09679 ~lu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr ~hr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val ~le Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile ~is His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg ~is Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2253 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
~A) NAME/KEY: CDS
~B) LOCATION: 1-2253 ~D) OTHER INFORMATION: /~unction= "coding region for APP751.'' ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn . CA 02221986 l997-ll-24 W O 96/~855 PCTAUS96~09679 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu GTT CCT GAC A~G TGC A~A TTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys GAA ACT CAT CTT CAC TGG CAC ACC GTC GCC A~A GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu A~G AGT ACC AAC TTG CAT GAC TAC GGC ATG TTG CTG CCC TGC GGA ATT 528 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 1~0 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cy,5 Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu W O 9k'4~g95 PCTAJS96/09679 His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Ary Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Ary Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr . CA 02221986 1997-11-24 W O ~6/~0~9~ PCT~US96~09679 Asn Ile Lys T:hr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe GCA GAA GAT GTG GGT TCA AAC A~A GGT GCA ATC ATT GGA CTC ATG GTG 2064 ~ Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu AAG AAG A~A CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG GAG GTT GAC 2160 Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn GGC TAC GAA A~T CCA ACC TAC AAG TTC TTT GAG CAG ATG CAG AAC 2253 Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn 7~L0 745 750 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 751 amino acids ~B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOI.ECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro ~0 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu ~ Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile W O 9G/~99S PCT~US96/09679 165 .170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu ~lu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu ~lu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe ~yr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr ~ys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln ~la Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe ~ln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg ~la Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val ~rg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met ~hr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu CA 0222l986 l997-ll-24 W O 96/4~895 PCTAJS96/0g679 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn ~et Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro ~er Leu Thr Glu Thr ~yS Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala ~sp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr ~sn Ile Lys l'hr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe ~rg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp V'al Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp ~la Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn ~ly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn ~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2310 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEAT~RE:
(A) NAME/KEY: CDS
(B) LOCATION: 1-2310 (D) OTHER INFORMATION: /~unction= ~coding region ~or APP770."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln PCT/U~G/~5679 40 . 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala A~n Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu GTT CCT GAC AAG TGC A~A TTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cy9 Arg Ala Met Ile TCC CGC TGG TAC TTT GAT GTG ACT GA~ GGG AAG TGT GCC CCA TTC TTT 960 CA 0222l986 l997-ll-24 PCT~US96/09679 W O 96l40895 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly C'ys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala AAG CAC CGA G.~G AGA ATG TCC CAG GTC ATG AGA GAA TGG GAA GAG GCA 1248 Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 770 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Ary ~la Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro ~ln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln W ~ 96140~95 PCT~US96/09679 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu ~ln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn ~rp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr A:rg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu Hls Gln Glu Arg Met Asp Val Cys Glu Thr Xis Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu ~ys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile ~sp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 1~0 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gl.y Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu ~lu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu ~lu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe ~yr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr G1U GlU Tyr ~ys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 34~ 345 350 Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala CA 0222l986 l997-ll-24 WO 9''1C~95 PCT~US96/09679 Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu ~ln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys ~yr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp ~lu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val ~eu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val ~sp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser ~ly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu ~al Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val ~lu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met ~ln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met wo g ~ a~s PCTrUS96/09679 Gln Asn (2) INFORMATXON FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) sTR~n~nN~rs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
~iii) HYPOTHETICAL: NO
(iv) ANTI:-SENSE: NO
(Xi ) S~'~U~N~ DESCRIPTION: SEQ ID NO:7:
CCGATGATGA CC,AGGACGAT 20 !2 ) INFORMATION FOR SEQ ID NO:8:
( i ) ~r~'~UI~N~'~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) S~2u~N~ DESCRIPTION: SEQ ID NO:9:
Phe Arg Val Gly Ser (2) INFORMATION FOR SEQ ID NO:10:
(i) SE52UENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SE52UENCE DESCRIPTION: SEQ ID NO:10:
Asp Ala Glu Phe Arg Gly Gly Cys

Claims (34)

We claim:

1. A non-human transgenic mammal wherein the transgenic mammal has a nucleic acid construct stably incorporated into the genome, wherein the construct comprises a promoter for expression of the construct in a mammalian cell and a region encoding the A.beta.-containing protein, wherein the promoter is operatively linked to the region, wherein the region comprises DNA encoding the A,.beta.-containing protein, wherein the A.beta.-containing protein consists of all or a contiguous portion of a protein selected from the group consisting of APP770, APP770 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP751, APP751 bearing a mutation in one or more of the amino acids selected from. the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP695, and APP695 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, wherein the A.beta.-containing protein includes amino acids 672 to 714 of human APP, wherein the promoter mediates expression of the construct such that A.beta.~ is expressed at a level of at least 30 nanograms per gram of brain tissue of the mammal when it is two to four months old, A.beta.~~ is expressed at a level of at least 8.5 nanograms per gram of brain tissue of the mammal when it is two to four months old, APP and APP.alpha. combined are expressed at a level of at least 150picomoles per gram of brain tissue of the mammal when it is two to four months old, APP.beta. is expressed at a level of at least 40 picomoles per gram of brain tissue of the mammal when it is two to four months old, and/or mRNA encoding the A.beta.-containing protein is expressed to a level at least twice that of mRNA
encoding the endogenous APP of the transgenic mammal in brain tissue of the mammal when it is two to four months old.

2. The mammal of claim 1 wherein the A.beta.-containing protein is selected from the group consisting of APP770; APP770 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP751; APP751 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP695; APP695 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; a protein consisting of amino acids 646 to 770 of APP; a protein consisting of amino acids 670 to 770 ofAPP; a protein consisting of amino acids 672 to 770 of APP; and a protein consisting of amino acids 672 to 714 of APP.

3. The mammal of claim 2 wherein the DNA encoding the A.beta.-containing protein is cDNA or a cDNA/genomic DNA hybrid, wherein the cDNA/genomic DNA hybrid includes at least one APP intron sequence wherein the intron sequence is sufficient for splicing.

4. The mammal of claim 1 wherein the promoter is the human platelet derived growth factor .beta. chain gene promoter.

5. The mammal of claim 1 wherein the region further comprises DNA
encoding a second protein, wherein the DNA encoding the A.beta.-containing protein and the DNA encoding the second protein are operative linked such that the region encodes an A.beta.-containing fusion protein comprising a fusion of the A.beta.-containing protein and the second protein.

6. The mammal of claim 5 wherein the second protein is a signal peptide.

7. The mammal of claim 1 produced by introduction of the construct into an embryo, insertion of the embryo into a surrogate mother, and allowing the embryo to develop to term.

8. The mammal of claim 1 wherein the mammal is a rodent.

9. The mammal of claim 1 produced by mating transgenic mammals expressing different constructs.

10. The mammal of claim 1 wherein the codon encoding amino acid 717 is mutated to encode an amino acid selected from the group consisting of Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, and Gln.

11. The mammal of claim 10 wherein the codon encoding amino acid 717 is mutated to encode Phe.

12. The mammal of claim 1 wherein the codon encoding amino acid 670 is mutated to encode an amino acid selected from the group consisting of Asn andGlu, or the codon encoding amino acid 670 is deleted, and/or wherein the codon encoding amino acid 671 is mutated to encode an amino acid selected from the group consisting of Ile, Leu, Tyr, Lys, Glu, Val, and Ala, or the codon encoding amino acid 671 is deleted.

13. The mammal of claim 12 wherein the codon encoding amino acid 670 is mutated to encode Asn, and/or the codon encoding amino acid 671 is mutated to encode Leu or Tyr.

14. The mammal of claim 1 wherein the construct further comprises an effective amount of at least one intron, wherein the effective amount of at least one intron is located in the region of the construct encoding a human amyloid precursor protein.

15. The mammal of claim 14 wherein the intron is an APP gene intron.

16. The mammal of claim 1 wherein the promoter mediates expression of the construct such that A.beta.~ is expressed at a level of at least 30 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, A.beta.~~~ is expressed at a level of at least 8.5 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP and APP.alpha. combined are expressed at a level of at least 150picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP.beta. is expressed at a level of at least 40 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two tofour months old, and/or mRNA encoding the A.beta.-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in hippocampal or cortical brain tissue of the mammal when it is two to four months old.

17. The mammal of claim 1 wherein amyloid plaques that can be stained with Congo Red are present in brain tissue of the mammal.

18. A method for testing compounds for an effect on expression or processing of an A.beta.-containing protein comprising a) administering the compound to be tested to a non-human transgenic mammal, or mammalian cells derived from the transgenic mammal, wherein the transgenic mammal has a nucleic acid construct stably incorporated into the genome, wherein the construct comprises a promoter for expression of the construct in a mammalian cell and a region encoding the A.beta.-containing protein, wherein the promoter is operatively linked to the region, wherein the region comprises DNA encoding the A.beta.-containing protein, wherein the A.beta.-containing protein consists of all or a contiguous portion of a protein selected from the group consisting of APP770, APP770 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP751, APP751 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP695, and APP695 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, wherein the A.beta.-containing protein includes amino acids 672 to 714 of human APP, wherein the promoter mediates expression of the construct such that A,.beta.~ isexpressed at a level of at least 30 nanograms per gram of brain tissue of the mammal when it is two to four months old, A.beta.~ is expressed at a level of at least 8.5 nanograms per gram of brain tissue of the mammal when it is two to four months old, APP and APP.alpha. combined are expressed at a level of at least 150picomoles per gram of brain tissue of the mammal when it is two to four months old, APP.beta. is expressed at a level of at least 40 picomoles per gram of brain tissue of the mammal when it is two to four months old, and/or mRNA encoding the A.beta.-containing protein is expressed to a level at least twice that of mRNA
encoding the endogenous APP of the transgenic mammal in brain tissue of the mammal when it is two to four months old; and either b1) measuring the amount of an APP marker or a A.beta. marker produced by the transgenic animal, or by mammalian cells derived from the transgenic mammal, and comparing the amount measured to the amount of the APP marker or the A.beta. marker produced by a transgenic mammal, or by mammalian cells derived therefrom, to which the compound has not been administered, wherein a change in the amount of the APP marker or the A.beta. marker indicates that expression or processing of APP has been altered; or b2) determining the histopathology of an APP marker or a A.beta. marker in the transgenic animal, and comparing the histopathology to the histopathology ofthe APP marker or the A.beta. marker in a transgenic mammal to which the compound has not been administered, wherein a change in the histopathology of the APP marker or the A.beta.
marker indicates that expression or processing of APP has been altered.

19. The method of claim 18 wherein the A.beta.-containing protein is selected from the group consisting of APP770; APP770 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP751; APP751 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP695; APP695 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; a protein consisting of amino acids 646 to 770 of APP; a protein consisting of amino acids 670 to 770 ofAPP; a protein consisting of amino acids 672 to 770 of APP; and a protein consisting of amino acids 672 to 714 of APP.

20. The method of claim 19 wherein the DNA encoding the A.beta.-containing protein is cDNA or a cDNA/genomic DNA hybrid, wherein the cDNA/genomic DNA hybrid includes at least one APP intron sequence wherein the intron sequence is sufficient for splicing.

21. The method of claim 18 wherein the promoter is the human platelet derived growth factor .beta. chain gene promoter.

22. The method of claim 18 wherein the region further comprises DNA
encoding a second protein, wherein the DNA encoding the A.beta.-containing protein and the DNA encoding the second protein are operative linked such that the region encodes an A.beta.-containing fusion protein comprising a fusion of the A.beta.-containing protein and the second protein.

23. The method of claim 22 wherein the second protein is a signal peptide.

24. The method of claim 18 wherein the A.beta. marker is selected from the group consisting of A.beta.~, A.beta.~, A.beta.~, A.beta.~(pE), A.beta.~, A.beta.~, A.beta.~, A,B~, and the APP marker is selected from the group consisting of full length APP, APP.alpha.,APP.beta., FLAPP+ APP.alpha., the last 100 amino acids of APP, and the last 57 to 60 amino acids of APP.

25. The method of claim 18 wherein the APP marker is selected from the group consisting of APP695, APP751, and APP770, and wherein the change in histopathology is a reduction in the amount of APP marker localized in plaques and neuritic tissue.

26. The method of claim 18 wherein the mammals are rodents.

27. The method of claim 18 wherein the codon encoding amino acid 717 is mutated to encode an amino acid selected from the group consisting of Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, and Gln.

28. The method of claim 27 wherein the codon encoding amino acid 717 is mutated to encode Phe.

29. The method of claim 18 wherein the codon encoding amino acid 670 is mutated to encode an amino acid selected from the group consisting of Asn andGlu, or the codon encoding amino acid 670 is deleted, and/or wherein the codon encoding amino acid 671 is mutated to encode an amino acid selected from the group consisting of Ile, Leu, Tyr, Lys, Glu, Val, and Ala, or the codon encoding amino acid 671 is deleted.

30. The method of claim 29 wherein the codon encoding amino acid 670 is mutated to encode Asn, and/or the codon encoding amino acid 671 is mutated to encode Leu or Tyr.

31. The method of claim 18 wherein the construct further comprises an effective amount of at least one intron, wherein the effective amount of at least one intron is located in the region of the construct encoding the A.beta.-containing protein.

32. The method of claim 31 wherein the intron is an APP gene intron.

33. The method of claim 18 wherein the promoter mediates expression of the construct such that A.beta.~ is expressed at a level of at least 30 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, A,.beta.~ is expressed at a level of at least 8.5 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP and APP.alpha. combined are expressed at a level of at least 150picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP.beta. is expressed at a level of at least 40 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two tofour months old, and/or mRNA encoding the A.beta.-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in hippocampal or cortical brain tissue of the mammal when it is two to four months old.

34. The method of claim 18 wherein amyloid plaques that can be stained with Congo Red are present in brain tissue of the mammal.