GB2380196A

GB2380196A - Transgenic animals with mutant human amyloid precursor protein sequences

Info

Publication number: GB2380196A
Application number: GB0219386A
Authority: GB
Inventors: Alexander James Harper
Original assignee: SmithKline Beecham Ltd
Current assignee: SmithKline Beecham Ltd
Priority date: 2001-08-21
Filing date: 2002-08-20
Publication date: 2003-04-02
Also published as: GB0120344D0; GB0219386D0

Abstract

The present invention provides a recombinant DNA construct comprising a comprising a coding sequence encoding human APP with a mutation selected from V46F, V46I, V46G, V46Y, V46L, V46A, V46P, V46W, V46M, V46S, V46T, V46N, and V46Q and a brain-specific calcium calmodulin kinase promoter sequence which is operably linked to the coding sequence. Further provided is a transgenic non-human animal whose genome incorporates a polynucleotide comprising a coding sequence which encodes the human APP protein with a mutation selected from V46F, V46I, V46G, V46Y, V46L, V46A, V46P, V46W, V46M, V46S, V46T, V46N, and V46Q wherein said coding sequence is operably linked to a brain-specific calcium calmodulin kinase II promoter sequence. (Numbering is based upon the APP C-terminal fragment A4CT (C99), and corresponds to Val 717 of APP 770 ).

Description

COMPOUNDS This invention relates to transgenic animals and their use as models of Alzheimer's disease.

The main protein component of the amyloid plaques found in the brain of Alzheimer's disease (AD) patients is Ap, a 4 kDa peptide consisting of mainly forty and forty-two residues (Ap) o, Api) being derived from the amyloid precursor protein (APP). APP can exist in multiple forms generated by alternative mRNA splicing. The first form of APP identified by Kang, J., et al., (ref 1) from a foetal brain cDNA library contained 695 amino acids (so-called APP695). This includes a 17 residue signal peptide sequence for transport of the protein into the endoplasmic reticulum. Subsequently, a number of slightly longer cDNA clones were isolated by other workers. The 751 amino acid APP sequence (APP751) described by Ponte, P. , et al., (ref 2) contained an additional 57-amino acid insert encoding a Kunitz-type serine proteinase inhibitor (KPI). Kitaguchi, N. , et al., (ref 3) identified another precursor of 770 ammo acids (APP770) with both the KPI sequence and an additional 19 amino acid insert. These isoforms of APP arise as a result of alternative splicing of exons 7 and 8 during transcription of the APP gene.

Additional isoforms generated by alternative splicing of exon 15 have also been detected (ref 4).

Human APP herein refers to all isoforms including the 695 form and A4CT.

APP can be cleaved at the N-terminus of Ass by an enzyme called ss-secretase generating

a soluble APP and the C-terminal fragment A4CT (C99). This 99 residue long membrane protein A4CT (ref. 5) which is the direct precursor for Ap contains the entire Ap domain, the membrane domain and the cytoplasmic tail of APP. Alternative processing of APP in a post-Golgi- compartment by a protease termed a-secretase leads to the cleavage of APP within the Ap domain yielding secretory APP and the transmembrane fragment p3CT which is the direct precursor for p3.

Both C-terminal fragments of APP, A4CT and p3CT, are cleaved within the membrane domain by a y-cleavage activity, thereby releasing Ass and p3 into the medium (refs. 6,7). In cells expressing wild type APP the site of y-cleavage is mainly the peptide bond Val (40)-Ile (41) of A4CT and to a minor extent the bond Ala (42)-Thr (43). In cells expressing APP with the Familial AD linked mutations at Val 717 (based on APPyyo, Val 46 of A4CT) an increased y-cleavage occurs behind Val (42), thus producing larger amounts of AP142 (ref 8).

A number of Familial AD linked mutations have been observed, namely V46F, V461 and V46G (numbering relative to A4CT).

Transgenic mice expressing A4CT have been produced using the human APP promoter (ref. 9), the human thy-1 promoter (ref. 10) and the JC viral early region promoter (ref. 11).

Transgenic mice have also been produced expressing amino acids 591-695 of APP695 (Cterminal 105 amino acids of APP) under control of the human neurofilament NF-L transcriptional regulatory sequences (ref. 12). Numerous promoters have been used in conjunction with the full length APP cDNA (refs. 13-18). Generation of transgenic mammals bearing APP derived DNA sequences are also described in W093/14200 (TSI Corporation),

W091/19810 (California Biotechnology Inc), W093/02189 (University of Calfornia), W089/00689, W092/06187 (The Upjohn Company), EP0451700 (Miles Inc.), W092/13069 (Imperial College of Science Technology and Medicine) and W089/06689 (McClean Hospital Corporation). These include other APP mutations, namely V46Y, V46L, V46A, V46P, V46W, V46M, V46S, V46T, V46N and V46Q.

However in not all the above cases described to date has the nature of the APPimmunoreactive deposits resembled the clinical situation and, with the exception of the models described in references 14,16 and 18, such transgenic animals have not been found to be faithful model systems for the amyloid deposits characteristic of Alzheimer's disease. Results obtained depend upon the source of the promoter and the protein coding sequence used. We have found that the use of a specific neuronal promoter will increase the possibility of the development of pathology that resembles the clinical situation. W098/03643 and refs. 19 and 20 disclose constructs comprising mutant human APP or A4CT DNA sequences.

The mouse CaM-kmase-II promoter is described in ref. 21 and 22.

Transgenic animals of the invention may develop amyloid plaques and are therefore useful as a model of Alzhiemer's disease and other neurodegenerative disorders.

The present invention relies on the use of a nucleic acid construct to generate a transgenic animal model for screening agents for potential use in the treatment of neurological disorders, in particular Alzheimers disease. The construct comprises a polynucleotide sequence encoding the human APP or A4CT protein with a mutation selected from V46F, V46I, V46G, V46Y, V46L, V46A, V46P, V46W, V46M, V46S, V46T, V46N, and V46Q (numbering relative to A4CT), operatively linked to a mouse CaM-K-II promoter. Such polynucleotide sequence is herein termed a human APP encoding polynucleotide. In a preferred embodiment, the nucleic acid construct further comprises a Internal Ribosomal Entry Site (IRES) downstream (3') of the nucleotide encoding the human APP protein, and a nucleic acid sequence encoding a reporter gene downstream of the IRES. This type of construct is termed bi-cistronic, and enables separate expression of both the human APP protein, and the reporter protein. The reporter protein may be, for example, luciferase, green fluorescent protein or derivatives thereof, or ss galactosidase, but preferably it is p galactosidase. The advantage of such a construct is that it enables one to monitor human APP2 expression, by monitoring expression of the reporter gene.

It will be understood that references herein to A4CT, API-42 and Apt-40 include all N-and C-terminal variants produced by alternative cleavage during processing.

In a preferred aspect the mutation in the human APP or A4CT transgene is V46F. In a further preferred aspect the DNA sequence is full length human APP, more preferably APP695, preferably without the signal sequence.

In one embodiment the human APP encoding polynucleotide is carried on a construct which further encodes the APP signal sequence (APP residues 1 to 17) immediately upstream (5') of the APP or A4CT DNA sequence. Hydrophobic residue inserts such as Leu, Glu or Met promote processing of A4CT to Ap and may be be included between the signal peptide and A4CT coding regions and will remain attached to the processed A4CT.

A"transgene"comprises a polynucleotide, isolated from nature, which has been manipulated in-vitro and which can be subsequently introduced into the genome of the same or a different species in either the native or modified forms, such that it is stably and heritably maintained in that genome. The polynucleotide preferably encodes a protein of interest, and is generally operatively linked to a regulatory sequence. The term transgene is herein used to refer to the polynucleotide and the regulatory sequences to which it is operably linked. The transgene may further comprise other polynucleotides which encode, for example, reporter genes in order to monitor expression of the transgene. An organism into which a transgene has been introduced is termed a"transgenic organism". A"transgenic construct"is a vector construct comprising the transgene.

The term"transgenic DNA"as used herein refers to the polynucleotide comprised within the transgene, which polynucleotide encodes the protein of interest.

The assembly of the transgenic construct follows standard cloning techniques, that are well known in the art (for example see Sambrook et al, (23) ). The human APP encoding polynucleotide may consist of cDNA or genomic DNA. If it is cDNA, the cDNA to be overexpressed can be prepared from a mRNA extracted from a relevant tissue, preferably a tissue in which the APP protein is known to be expressed, or alternatively can be extracted by probing a human cDNA library. The transgenic DNA, along with the CaMKII promoter and any other desired components such as artificial introns, the IRES, or a reporter gene can then be inserted into a cloning vector by restriction digest and ligation. Suitable cloning vectors for the assembly of transgenic constructs are those which provide for acceptable yields of DNA.

Suitable cloning vectors for the assembly of transgenic constructs are those which provide for acceptable yields of DNA. Any commercially available plasmid vector or phage that can carry a cDNA and which can be manipulated to contain a selection marker is suitable.

The CaM-K-11 promoter is operably linked to the coding sequence of the APP encoding polynucleotide in a manner that will permit the required temporal and spatial expression of the polynucleotide. There may or may not be intervening sequences between the linked polynucleotide and the promoter, provided that the promoter directs expression of the APP encoding polynucleotide so linked to it. Methods of so linking regulatory sequences to cDNAs to facilitate their expression are widely known in the art.

Such methods include, for example, directly ligating the nucleic acid comprising the CaMK-II promoter to the coding region of the APP encoding polynucleotide. Additional nucleic acid sequences may be included that modulate expression in the required manner. Examples of additional sequences include enhancer elements, artificial intron and others. In addition the nucleotide sequence of the CaM-K-II promoter, or other regulatory sequence, may be modified to increase levels of expression. Such modifications can be achieved using, for example, site-directed mutagenesis methods well known in the art (see Sambrook et al, supra).

In addition to modifying the sequence of regulatory elements to enhance, or otherwise change, expression levels, the coding sequence of the APP encoding polynucleotide may be modified to enhance or otherwise affect expression levels. For example if the transgenic DNA is from a different species than the host, the codon usage of the transgenic DNA can be altered to match more closely that of the host. It is well known in the art that different organisms use the 64 coding and stop codons at different frequencies. Codons that are infrequently used in an organism are termed"rare codons". If a transgenic DNA includes a codon that is a rare codon in the host, expression levels may be severely reduced. One solution is to replace one or more rare codons in the transgenic DNA with codons that are frequently used in the host. Other modifications to the transgenic DNA sequence include modifying the polynucleotide sequence surrounding the start codon (the initiator methionin encoding codon) to make this more closely match the consensus"Kozak"sequence (A/G CCATGG, where the ATG in bold is the start codon; see for example Kozak, M (ref 24). In the transcribed mRNA molecule the Kozak sequence is believed to provide the optimal environment for initiation of translation of the polypeptide.

Preferably, prior to the introduction of the transgene into the host cell, the vector portions are removed by restriction enzyme digestion, for example by using restriction sites in the vector that flank the transgene. Thus the genetic material that is actually introduced into the host cell will preferably comprise the coding sequence of the APP encoding polynucleotide and the regulatory sequences to which it has been operably linked together with other potential components of the transgene, for example a reporter gene. An example of this technique is given in reference 25.

An inducible system such as described in Ref 26 may also be used, which has the

advantage of regulating the gene expression (induction/repression). For example, the tetracycline-inducible system (Refs. 27,28) uses two constructs: a minimal promoter (PhCMV*- 1) fused to seven tetracyclic operator sequences and the cDNA in question; and a transgene containing the tetracycline-controlled trans-activator protein (tTA) coding sequence under the control of a promoter, for example taken from the following list: Human APP (ref. 9); rat neuron specific enolase (neurons) (ref. 29); human ss actin (ref. 30); human PDGFss (ref. 31); mouse Thy 1 (ref. 32); mouse Prion protein promoter (PrP) (ref. 33); Syrian hamster Prion protein promoter (ref. 34 and 35); rat synapsin 1 (brain) (ref. 36); human FMR1 (brain) (ref. 37); human neurofilament low (ref. 38), middle (brain) (ref. 39); NEX-1 (brain) (ref. 40); mouse APLP2 (brain) (ref. 41); rat alpha tubulin (ref. 42); mouse transferrin (ref. 43); mouse HMGCR (3hydroxy-3-methylglutaryl coenzyme A reductase, oligodendrocytes) (ref. 44), mouse myelin basic protein (ref. 45), mouse CaM-kinase-II promoter (ref. 21 and 22), human thy-l promoter (ref. 10), JC viral early region promoter (ref. 11); or human neurofilament NF-L transcriptional regulatory sequences (ref. 12). Each construct is used to generate a transgenic mouse. Crossing the two homozygous mice generates a double transgenic line which expresses the tTA according to the chosen promoter. This tTA induces expression of the cDNA by activating the PhCMV*- !, but only in the absence of tetracycline. In the presence of tetracycline there is only basal expression.

Generation of transgenic non-human mammals of the invention may be carried out conventionally, for example as described in W093/14200, W091/19810, W093/02189, W089/00689, W092/06187, EP0451700, W092/13069 and W089/06689.

There are a number of techniques that permit the introduction of genetic material, such as a transgene, into the germline. The most commonly used, and preferred protocol comprises direct injection of the transgene into the male pronucleus of the fertilised egg (Hogan et al. (25), resulting in the random integration into one locus of a varying number of copies, usually in a head to tail array (Costantini and Lacy (46) ). The injected eggs are then re-transferred into the uteri of pseudo-pregnant recipient mothers. Some of the resulting offspring may have one or several copies of the transgene integrated into their genomes, usually in one integration site. These "founder"animals are then bred to establish transgenic lines and to back-cross into the genetic background of choice. It is convenient to have the transgene insertion on both chromosomes (homozygosity) as this obviates the need for repeated genotyping in the course of routine mouse husbandry.

Alternatively, for the production of transgenic mice, transgenes can be introduced via embryonic stem (ES) cells, using electroporation, retroviral vectors or lipofection for gene transfer. This is followed by the random insertion into the genome of the pluripotent embryonic stem (ES) cells, followed by the production of chimeric mice and subsequent germline transmission. Transgenes

of up to several hundred kilobases of rodentian DNA have been used to produce transgenic mice in this manner (for example Choi et al. (47); Strauss et al. (48) ). The latter approach can be tailored such that the transgene is inserted into a pre-determined locus (non-randomly, for example ROSA26 or HPRT) that supports ubiquitous as well as tissue specific expression of the transgene (Vivian et al. (49)).

In one aspect, the transgenic mammal is generated by introduction of the transgenic construct into an embryo, insertion of the embryo into a surrogate mother and allowing the embryo to develop to term.

The transgenic construct is prepared for transfer to the host animal by cleavage of vector containing the transgenic construct and purification of the DNA (ref. 25) The transfer is carried out conventionally preferably using microinjection as described in detail in reference 25.

In an alternative aspect the parent transgenic non-human mammal is produced by introduction of the construct into embryonic stem cells by conventional methods such as calcium phosphate/DNA precipitation, direct injection or electroporation (ref. 50) followed by injection of the transformed cells into blastocytes and insertion of the resulting embryo into a surrogate mother as described above.

Transgenic animals may be identified by testing to ensure the required genotypic change has been effected. This may be done in any suitable fashion, for example by detecting the presence of the transgene by PCR with specific primers, or by southern blotting, preferably of tail DNA, with a specific probe.

Once the desired genotype has been confirmed, the transgenic animal line can be subjected to various tests to determine the phenotype. The tests involved in this phenotypic characterisation depend on what genotypic change has been effected, and may include, for example, morphological, biochemical and behavioural studies. The transgenic animals of the present invention demonstrate an increase in APP processing leading to increased amyloid levels and other APP cleavage products in the brain. In addition, they are likely to demonstrate cognitive defects.

This invention also includes any cells cultured from the transgenic non-human animal. The cells are cultured in-vitro. The genome of the cells can thus comprise the construct of the invention. The human APP polypeptide is typically expressed in such cells in an amount at least 2, preferably at least 5, for example 6,7, 8,9, and particularly preferably at least 1O. times greater than the amount of endogenous APP polypeptide that is expressed in the cells.

Cells cultured in-vitro from a transgenic animal may be prepared by any suitable method.

The cells are typically rodent and preferably mouse cells. Cultures of neuronal cells can therefore

be provided, for example cultures of primary hippocampal cells. The cells may be used to introduce other genes of interest by any known method (including viral delivery).

The transgenic non-human mammal is preferably a rodent such as rat or mouse, more preferably a mouse.

The appropriate transgenic DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site (s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The use of non-human animal models which overexpress or lack more than one gene can provide important insights into the interaction of different genetic loci in particular diseases, for example Alzheimers Disease. Consequently the interbreeding of the APP transgenic non-human animals of the present invention with non-human animal models which overexpress or underexpress a different transgene may produce alternative and potentially superior animal models of diseases such as Alzheimers. For example, the APP transgenic non-human animals of the present invention may be crossed with mice bearing a transgene comprising a nucleotide encoding the human aspartyl protease 2 protein (one example of such mice is described in the examples of this specification). In another aspect, the APP transgenic non-human animals of the present invention may be crossed with mice bearing a transgene comprising a nucleotide encoding the human presenilin protein. (such mice are described, for example, in W098/17782).

In each aspect, the second transgene may comprise a nucleotide encoding a wild type protein, or may comprise a nucleotide encoding a mutated protein. Preferred mutations in the presenilin transgene are A246E, M 146V and M146L.

The promoter of the second transgene may be any promoter that will lead to expression of the transgenic DNA in the transgenic non-human animal. The promoter may be, for example, Human APP (ref. 9); rat neuron specific enolase (neurons) (ref. 29); human ss actin (ref. 30); human PDGFss (ref. 31) ; mouse Thy 1 (ref. 32); mouse Prion protein promoter (PrP) (ref. 33); Syrian hamster Prion protein promoter (ref. 34 and 35); rat synapsin 1 (brain) (ref. 36); human FMRI (brain) (ref. 37); human neurofilament low (ref. 38), middle (brain) (ref. 39) ; NEX-1 (brain) (ref. 40); mouse APLP2 (brain) (ref. 41); rat alpha tubulin (ref. 42); mouse transferrin (ref. 43); mouse HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase, oligodendrocytes) (ref. 44), mouse myelin basic protein (ref. 45), mouse CaM-kinase-II promoter (ref. 21 and 22), human thy-1 promoter (ref. 10), JC viral early region promoter (ref. 11); or human neurofilament NF-L transcriptional regulatory sequences (ref. 12).

It is, however, preferable that both of the precursor transgenic non-human animal lines comprise transgenes which are operably linked to a neuronal specific promoter, for example the

CamKII promoter or the Thy 1. This ensures that in the non-human animal produced by the cross, both transgenes are expressed in a neuronal specific manner. In a preferred embodiement, a non-human transgenic animal is generated by crossing the APP lines of the present invention with a non-human transgenic animal whose cells contain transgenic DNA encoding the human Aspartyl 2 Protease (asp2). Particularly preferred is when said Asp2 encoding transgenic DNA is operably linked to the CamKII promoter. In a further preferred embodiment, a non-human transgenic animal is generated by crossing the APP lines of the present invention with a nonhuman transgenic animal whose cells contain transgenic encoding the human Presenilin 1 protein (PS 1). Particularly preferred is when said PS 1 encoding transgenic DNA is operably linked to the human PDGFss promoter.

Such transgenic non-human animals containing more than one transgene wherein one of the transgenes comprises a nucleotide encoding the human APP protein operably linked to the CamKII promoter are also part of the invention, and may be used as described herein. The transgenic non-human animal or cells of the invention may be used to screen for drugs which inhibit deposit of Ass by administering test drug to the mammal or cell culture medium and observing changes in APP expression and processing, histopathology and/or behavioural changes. The invention extends to such method of screening.

Suitable techniques for making such observations are described in W093/14200.

A method of identifying a therapeutic agent for the treatment of a condition characterised by the deposition of amyloid plaques and decreased cognition can therefore be provided, comprising : administering to an animal of the invention a candidate test substance and determining whether the candidate substance (1) prevents or delays the onset of the condition or (ii) treats the condition.

Option (ii) may be tested by determining whether the candidate substance causes a decrease in any of the cellular or physiological changes caused by the condition. Such cellular or physiological changes may include changes in the deposition of amyloid plaques, or a decrease in cognition and awareness of the transgenic non-human mammal.

A method of identifying a therapeutic agent for the treatment of a condition characterised by deposition of amyloid plaques and decreased cognition can also be provided, comprising:

contacting a candidate substance with a cell of the invention, and - determining whether the candidate substance (i) prevents or delays the onset of cellular changes associated with the condition, or (ii) causes a decrease in any of the cellular changes caused by the condition (such as any of the cellular changes mentioned herein).

Suitable candidate substances which may be tested in the above methods include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR grafted antibodies). Furthermore, combinatorial libraries, defined chemical identities, small molecules, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display libraries (e. g. phage display libraries) may also be tested. The candidate substances may be chemical compounds. Batches of the candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substances of batches which show inhibition tested individually.

Agents which may reverse the phenotypic changes such as the deposition of amyloid plaques and a decrease in cognition seen in the transgenic animals of the invention may be tested in-vivo or in cells or tissue preparations in-vitro. Compounds can be tested using the assays and tests used to characterise the invention. For example, after administration of any potential therapeutic agent, the response of the transgenic animal may be assessed by, for example, looking for an improvement in cognition. Methods for screening potential therapeutic agents using cell lines or animals are established. ELISA or Homogenous Time Resolved Fuorescence (HTRF) methodologies can be used.

Agents identified in the screening methods of the invention may be used to prevent or treat the diseases discussed above, in particular Alzheimers Disease. The condition of a patient suffering from such a disease can therefore be improved by administration of such a product. The formulation of the product for use in preventing or treating the disease will depend upon factors such as the nature of the agent identified and the disease to be prevented or treated. Typically the agent is formulated for use with a pharmaceutically acceptable carrier or diluent. For example it may be formulated for intracranial, parenteral, intravenous, intramuscular, subcutaneous, transdermal or oral administration. A physician will be able to determine the required route of administration for each particular patient. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

The dose of product may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A suitable dose may however be from 0. 1 to 100 mglkg body weight such as 1 to 40 mg/kg body weight. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.

The construct, cells or therapeutic agents of the invention may be present in a substantially isolated form. It will be understood that they may be mixed with carriers or diluents which will not interfere with their intended purpose and still be regarded as substantially isolated.

Thus the construct, cell or therapeutic agent of the invention may also be in a substantially

purified form, in which case it will generally comprise more than 90%, e. g. more 95%, 98% or 99%, by weight of the relevant preparation.

Examples Generation of transgenic mice expressing APPV46F The APPV46F cDNA, with the sequence referred to in Ref33, was prepared by site directed mutagenesis of the 695 isoform of APP. To generate the APPV46F mis-expression transgenes, the APPV46F cDNA was cloned into the vector pCamKIRESbgal. This vector contains the mouse calcium-calmodulin-dependent kinase IIa (CamKIIa) promoter, which has previously been shown to direct transgene expression to both the hippocampus and neocortex of

the mouse forebrain (Ref 36). The promoter is isolated in the manner described in ref 36 (see in particular reference note 20 or ref 36). Then, an 8. 5kb Sail promoter fragment was isolated by restriction digestion. This was ligated into Arm3 vector (a derivative ofpBluescript with the sites indicated flanking the SacI-KpnI pBluescript polylinker) digested with XhoI and Sail. This generated Arm3-CamKII (the 5'Sail site is destroyed). The 5Isolate form AS210S (a derivative of Plasmid AS210 (obtained from Stem Cell Sciences, Melbourne) contains the IRES-bgalintro-poly fragment as an XbaI-XhoI restriction fragment. The Xbal site was converted to a

Sail site (to create AS21 OS), and the resultant SalI/XhoI fragment containing the IRES-bgal- intro-poly cassette was ligated into the SalI site of Arm3-CamKII to generate CamKII-IRESbgal. This contains unique NotI and SaIl sites for the insertion of cDNAs. In the resulting vector, the promoter is upstream of a cassette containing the picornaviral internal ribosome-entry site

(IRES) (Ref 52) and the gene for beta-galactosidase and SV40 transcription termination signals.

The APP V46F cDNA was then inserted between the CamKII promoter and the IRES using the NotI and Sail sites. Transcription which initiates at the CamKIIapromoter capsite and terminates at the SV40 polyadenylation signals therefore results in a dicistronic message encoding APPV46F and beta-galactosidase proteins. The latter can be easily detected to confirm that transgene expression has taken place. The inclusion of a beta-galactosidase reporter gene in the construct enables simple, accurate and sensitive detection of cellular sites of transgene expression. This cloning process is shown schematically in figure 1.

The above described construct is used to generate transgenic mice by the following procedures: The construct is prepared and purified.

Female mice are induced to superovulate and embryos are recovered.

DNA is microinjected into the pronucleus of embryos.

Embryos are transferred into pseudopregnant mice (female mice previously paired with vasectomised males).

Embryos are allowed to develop normally and mice are born.

Founder mice are identified by Southern blot and PCR and bred on.

Suitable mice lines are as follows; Donor mice (embryos for pronucleus injection): [C57Bl/6 x CBA] F2 hybrid Acceptor mice: [C57Bl/6 x CBA] F1 hybrid Mice for further breeding: C57Bl/6 Crossbreeding of transgenic mice to generate carriers of two transgenes The transgenic mice expressing APP and the further desired transgene, for example aspartyl 2 protease, or presenilin which may be wild type presenilin, or may contain a mutation such as M146V, M671L or M146V, are crossbred and the offspring genotyped to select those with both of the transgenes. Preferably, both transgenes are under the control of the CamKII promoter. If the APP transgene is'a'and the second transgene is'b', then the following mating pattern is possible from heterozygous parents with two independently segregating genes:

alleles b a ab a- - b One in four offspring would be a double overexpressor, two in four would be single overexpressors (one for each transgene) and one in four offspring would be a wild type mouse.

This cross can be used to calculate the number of parents required to produce an experimental cohort of double overexpressors.

Generation of transgenic mice expressing ASP2 The ASP2 cDNA, with the sequence referred to in Ref 53 was prepared according to convention techniques. To generate the Asp2 mis-expression transgenes, the Asp2 cDNA was cloned into the vector pCamKIRESbgal, prepared as above.

Female mice are induced to superovulate and embryos are recovered.

DNA is microinjected into the pronucleus of embryos.

Embryos are allowed to develop normally and mice are born.

Founder mice are identified by Southern blot and PCR and bred on.

Suitable mice lines are as follows; Donor mice (embryos for pronucleus injection): [C57Bl/6 x CBA] F1 hybrid Acceptor mice: [C57BI/6 x CBA]FI hybrid Mice for further breeding: C57BI/6 Screening of drugs using transgenic mice The transgenic mice described above may be used to screen for potential activity of test drugs in the treatment of Alzheimer's disease and other neurodegenerative disorders.

APP expression and processing may be examined using detection of mRNA by Northern blots and detection of polypeptides using polyclonal and monoclonal antibodies that are specific to the terminal regions of the target peptides.

Histopathological observations may be made using immunohistological techniques to permit identification of amyloid plaques and related deposits such as hyperphosphorylated tau and in situ hybridisation using labelled probes to target mRNA.

Analyses of mouse brain Animals are culled by overdose of anaesthetic. Brains are removed and hemisected down the midline. The halves are placed in either 10% formal saline or snap frozen.

Extraction of Ass and APP from mouse brain for immunoassay Snap frozen half brains are weighed and then homogenised using a Polytron homogeniser on maximum speed for 30 seconds. Homogenates are prepared at 20% wt/vol in 50mM TrisHCI, 150mM NaCl, pH 8 with complete TM protease inhibitor (Boehringer Mannheim) added on the day according to the manufacturer's instructions.. Homogenates are then diluted with an equal volume of 50mM Tris-HCI, 150mM NaCI pH 8 containing 1% Nonidet P40 (10% solution, Pierce), 1 % deoxycholate, 0.4% sodium dodecyl sulphate and complete TM (as above).

Homogenates are then sonicated in a water bath sonicator for 45 minutes and then boiled for 10 minutes in a large beaker of water. The homogenates are then transferred to 1. 5ml Eppendorf tubes and centrifuged at 12000 rpm for 10 minutes. The supernatants are removed and stored in aliquots at-20 C.

Immunocytochemical Assessment of Brains from Transgenic Mice Hemisected mouse brain, stored in formal saline, is embedded in paraffin wax and cut into 10 u. m sections on a microtome before mounting onto gelatin subbed slides.

For immunocytochemistry, sections are dewaxed and rehydrated by standard techniques.

Sections being assessed for Ap or APP content are treated with 80% formic acid after rehydration. For other sections, boiling for 10 minutes in 0. 1M citrate buffer in a microwave oven enhances immunoreactivity. Endogenous peroxidase is blocked by incubating with hydrogen peroxide (0.3% in phosphate buffered saline for 30 minutes). Primary antibodies for Ass (lE8 - monoclonal to Ass 13-27; 0210- monoclonal to ABeta 33-40; 20gel0 and 5G5monoclonals to Ap 35-42, APP (22Cll - Weidemann et al, 1989), glial fibrillary acidic protein (anti-GFAP, Boehringer Mannheim), neuronal markers (synaptophysin, MAP-2, choline acetyl transferase-Boehringer Mannheim) and normal and hyperphosphorylated tau (Innogenetics) are incubated with the sections overnight at 4 C. Sections are visualised using DAB (Vector Labs) and after dehydration are mounted in DPX aqueous mountant.

Protein Assay Mouse brain homogenates are assayed for protein before gel electrophoresis to ensure equal protein loading between samples for comparison of expression levels. The proteins are measured by the Bradford dye-binding procedure (Ref 28) using Bio-Rad (Herts, UK) dye reagent concentrate. The assay is carried out in a 96 well plate with the absorbance read at 595nm using a Spectramax plate reader with software to analyse the results. As various reagents, particularly detergents, interfere with the Bradford assay calibrations are carried out in the appropriate sample vehicle.

SDS-PAGE Proteins are fractionated using a Novex mini-gel system (R & D Systems Europe Ltd, Oxon, UK). An equal amount of protein is loaded for comparison of APP expression levels between animals. Samples are diluted in 2x reducing sample buffer containing 0. IM Tris pH6.8, 10% sodium dodecyl sulphate (SDS), 0. 1 % bromophenol blue in glycerol and 50% ss- mercaptoethanol. The proteins are separated on a 6% Tris-glycine polyacrylamide gel containing SDS (Ref. 29) for 90 minutes at 125 volts. Protein standards are included of known molecular weight (Sigma) as well as a secreted APP standard (Pichia expressed, see Ref 32).

Western Blot Analysis After SDS-PAGE the gels are washed for 5 minutes in transfer buffer containing 2.5mM Tris, 19. 2mM glycine, 20% methanol at pH 8.3. The proteins are transferred from the gel onto 0.45um nitrocellulose membrane (Schleicher & Schuell) using a Bio-rad semi-dry system for 2 hours at 0. 8mNcm2. After transfer the proteins are visualised on the membrane by staining with a 10% solution of Ponceau S (Sigma) for 2 minutes. This is detained by rinsing with distilled water.

The membrane is blocked in phosphate buffered saline (PBS) containing 10% dried milk powder for at least 1 hour at room temperature. W02, monoclonal antibody raised against human Ass 1-16, IS incubated at 1ug/ml in PBS overnight at 40C. This is followed by three 10 minute washes in PBS. Secondary antibody is then added, this was anti-mouse horseradish peroxidase (Amersham NA931) at 1/3000 dilution in PBS containing 4% bovine serum albumin and 0. 1 % Tween 20 incubated for 1 hour at room temperature. PBS washes are repeated as above and then the membrane transferred to a clean dish for addition of enhanced chemiluminescence substrate (Amersham RPN2106). The immunoreactive bands are detected using Hyperfilm-ECL (Amersham) which was developed using an automated processor (X-OGraph Compact X4). APP bands are scanned using a Fluor-S Multilmager (Bio-rad) linked to a computer with software for processing data.

Determination of the Concentration of Ap 140 and 1-42 by Immunoassay For the immunoassay of Ap 1-40, plates (Gibco BRL, flat bottom 96 well plate, catalogue # 1-67008-A) are coated (overnight at 4 C) with 2F12 capture antibody, raised to Ass 1-16 peptide, at 0. 8/lg/ml m PBS. After coating, antibody solution is aspirated and plates blocked by incubating for 60 minutes at 37 C with 1% gelatine v/v (Amersham RPN416) in assay buffer (50mM Tris HCI, 150mM NaCl, 0.5% bovine gamma globulins, 0.05% Tween 20, pH 7.4, passed through a 0.2um filter before use). Following blocking, plates are washed 4 x 250ul with phosphate buffered saline with Tween 20 (Sigma Cat No P3563). Samples of mouse brain extract prepared as described above are added to the plates together with the detection antibody, biotinylated G21 0 (1/lg/ml m assay buffer) raised to an Ap 40 C-terminal peptide (Ref 30). The 2F 12 coated plate containing brain extract and 150ul of detection antibody is incubated overnight at 4 C. Plates are subsequently washed (4 x 250ul) with phosphate buffered saline with Tween 20.

Quantitation of peptide-antibody complexation is achieved by the binding of strepatavidin-Europium. To each well is added 200ul streptavidin-Europium (Wallac, Catalogue # 1244-360) diluted 1: 500 in 0.5% BSA, 0.05% Globulin, 0.01% Tween 20, 20uM DTPA

(Sigma D 6518) in Tris buffered saline pH 7.4. Plates are incubated at room temperature for 60 minutes before washing with phosphate buffered saline. Finally, 200ul of enhancer solution (Wallac, Catalogue # 1244-105) is added to each well and the plate shaken for 5 minutes at room temperature before measurement of emission by time-resolved fluorescence on a Wallac 1234 Delfia Fluorometer.

For the determination of Ap 1-42, plates are coated with the monoclonal antibody 5G5 (1g/ml in phosphate buffered saline) raised to the Ap 35-42 peptide. Detection is with biotin- 6E10 antibody (Senetek PLC). All other details are exactly as for Ass 1-40 determination.

Ap 1-40 and 1-42 (California Peptide Research Inc) are used for the construction of standard curves. Peptides are dissolved in dimethyl sulphoxide and diluted in appropriate brain extract buffer. Standards of Ass 1-40 and 1-42 are assayed with each batch of mouse brain extract. The concentration of Ass 1-40 and 1-42 is calculated by reference to the immunoassay signal produced by known concentrations of peptide.

Determination of the Concentration of full length and alpha-secretase cleaved APP by Immunoassay For the immunoassay of full length plus alpha-secretase cleaved APP (FL+sAPPa),

plates (Gibco BRL, flat bottom 96 well plate, catalogue # 1-67008-A) are coated (overnight at 4 C) with W02 capture antibody (Ref37), raised to Ass 1-16 peptide, at 2. 7f. lg/ml in PBS. After coating, antibody solution is aspirated and plates blocked by incubating for 60 minutes at 37 C with 1% gelatine v/v (Amersham RPN416) in assay buffer (50mM Tris HCl, 150mM NaCI, 0.5% bovine gamma globulins, 0.05% Tween 20, pH 7.4, passed through a 0. 2um filter before use). Following blocking, plates are washed 4 x 250ul with phosphate buffered saline with Tween 20 (Sigma Cat No P3563). Samples of mouse brain extract prepared as described above are added to the plates together with the detection antibody, a polyclonal raised to APP (Ref 31).

The 2F12 coated plate containing brain extract and 150ul of detection antibody is incubated overnight at 4 C. Plates are subsequently washed (4 x 250ul) with phosphate buffered saline with Tween 20.

Quantitation of peptide-antibody complexation is achieved by the binding of Europiumantirabbit IgG (Wallac) diluted 1: 500 in 0.5% BSA, 0.05% Globulin, 0. 01% Tween 20, 20uM DTPA (Sigma D 6518) in Tris buffered saline pH 7.4. Plates are incubated at room temperature for 60 minutes before washing with phosphate buffered saline. Finally, 200ul of enhancer solution (Wallac, Catalogue # 1244-105) is added to each well and the plate shaken for 5 minutes at room temperature before measurement of emission by time-resolved fluorescence on a Wallac 1234 Delfia Fluorometer.

Alpha-secretase cleaved secreted APP prepared from Pichia pasioris (see Ref 32) is used for the construction of a standard curve. The concentration ofFL+sAPPa (this antibody format does not distinguish between full length and alpha-cleaved APP) is calculated by reference to the immunoassay signal produced by known concentrations of peptide.

Observation of behavioural changes Observation of behavioural changes may employ conventional tests used to assess learning and memory deficits (Ref 54).

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Claims

Claims 1. A recombmant DNA construct comprising a coding sequence encoding human APP with a mutation selected from V46F, V461, V46G, V46Y, V46L, V46A, V46P, V46W, V46M, V46S, V46T, V46N, and V46Q and a brain-specific calcium calmodulin kinase promoter sequence which is operably linked to the coding sequence.
2. A transgenic non-human animal whose genome incorporates a polynucleotide comprising a coding sequence which encodes the human APP protein with a mutation selected from V46F, V461, V46G, V46Y, V46L, V46A, V46P, V46W, V46M, V46S, V46T, V46N, and V46Q wherein said coding sequence is operably linked to a brain- specific calcium calmodulin kinase II promoter.
3. A transgenic non-human animal according to claim 2 wherein the said brain cells are cells of the hippocampus, cerebellum or cortex.
4. A transgenic non-human animal according to claim 2 or 3 which is a rodent.
5. A transgenic non-human animal according to claim 4 which is a mouse.
6. A transgenic non-human animal cell whose genome incorporates a nucleotide comprising a coding sequence which encodes the human APP protein and a brain-specific calcium calmodulin kinase II promoter sequence which is operably linked to the coding sequence.
7. A cell according to claim 6 which is a rodent cell.
8. A cell according to claim 7 which is a mouse cell.
9. A method of producing a transgenic non-human animal as defined in claim 2, which method comprises : (a) introducing a construct as defined in claim 1 into a cell or embryo; and (b) generating a said animal from said cell or embryo.
10. A method according to claim 9 wherein the construct is injected into the pronucleus of a fertilized oocyte of a non-human animal.
11. A method according to claim 9 or 10 wherein the founder animals that are obtained are bred.
12. A method of evaluating a therapeutic agent for use in the treatment of alzheimer disease, which method comprises: i) administering the said agent to a transgenic non-human animal as defined in any of claims 2 to 5, or a transgenic non-human animal cell as defined in any of claims 6 to 8, and ii) evaluating the effect of the said agent on the transgenic non-human animal or said non-human transgenic animal cell.
13. A kit for screening agents for use in the treatment of alzheimers disease, which kit comprises a transgenic non-human animal cell as defined in any one of claims 6 to 8 and a means

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for determining whether a test agent inhibits cellular changes associated with APP expression in said cell.