Patent EvaluationTransgenic animal model for Alzheimer’s disease Transgenic animal model for Alzheimer’s

disease

Novartis AG: WO9803644

This patent describes a new transgenic animal model of Alzheimer’s disease(AD). The transgene consists of the human amyloid precursor protein(APP) gene containing one or more of the mutations known to co-segregatewith autosomal dominant familial AD (Swedish mutation K670N/M671L;London mutation V717I) linked to a rodent Thy-1 promoter. Qualitative(neuropathological) and quantitative (behavioural, cognitive) outcomemeasures are reported for transgenic animals aged 6 - 15 months. Neuropa-thological changes include neuronal and synaptic loss, and senile plaqueswith associated dystrophic neurites. Furthermore, immunostaining for themicrotubule-associated protein tau, which accumulates in a hyperphos-phorylated form in the neurofibrillary pathology of AD, is reported to bepositive in the dystrophic neurites, the first time that this has been found inan APP transgenic animal model. Hence, it is claimed that this transgenicanimal may replicate more faithfully and accurately the neuropathologicaland clinical phenotype of AD and, thus, supersede previous animal models.

Keywords:Alzheimer’s disease, amyloid, animal model, neurofibrillarypathology, transgenic

Exp. Opin. Ther. Patents (1998)8(6):729-732

1. Introduction

The lack of a valid animal model of AD is viewed by many as a majorimpediment to research into both the pathogenesis and the treatment of thiscondition. Various strategies have been used to produce animal models ofAD, including chronic inhibition of protein phosphatases 1 and 2A withokadaic acid [1] and injection of human A68 protein (phosphorylated tauprotein) into rodent brain [101]. However, because of the key role ofamyloid b-peptides (Ab) in the pathogenesis of AD [2], manipulation of theAPP gene is the method that has attracted most attention for the productionof a valid animal model. Generally, the protocol has been to linkpathogenic human APP mutations to various neurone-specific promoters incDNA, genomic DNA, or hybrid constructs which are then microinjectedinto pronuclei or targeted to early embryos using retroviral technology, forexample [3].

Although early experiences with APP transgenesis were somewhatchequered (see [3] for review), the report from Athena Neurosciences byGames et al. [4] has provided the most promising animal model for AD todate. This model combined the V717F APP mutation (codon numberingaccording to APP770 isoform) with the platelet-derived growth factor(PDGF)-b promoter. The resulting animals expressed the transgene mRNA,the holo-APP protein and Ab peptide at very high levels, and were initiallyreported to have many of the characteristic neuropathological features ofAD, namely senile plaques with abnormal neurites, and neuronal and

7291998 © Ashley Publications Ltd. ISSN 1354-3776

Patent Evaluation

1. Introduction

2. Biology and action

3. Expert opinion

Bibliography

Patents

Patent details

http://www.ashley-pub.com

Expert Opinion on Therapeutic Patents

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synaptic loss with reactive gliosis [4]. However,neurofibrillary tangles (NFTs), which correlate moreclosely with the cognitive impairment in AD thansenile plaques [5], were not seen. Further follow-up ofthese animals confirmed the similarity of their plaquepathology to that in AD [6] and showed increasedconcentration of Ab42 in the hippocampus with age,as well as temporal and regional specificity of Ab42deposition [7]; however, modern stereologicaltechniques have failed to confirm neuronal loss [8].Nonetheless, the ‘Athena mouse’ has become the goldstandard against which other AD transgenics arejudged. The current patent reports a further APPtransgenic animal model of AD; how does thiscompare with previously reported APP transgenics[4,6-12,102-105]?

2. Biology and action

As pointed out by Hsiao [13], several parameters needto be specified when designing transgenic animalmodels of AD, namely:

· host strain

· primary structure of APP transgenes

· transgenic APP expression levels

· outcome measures

Only three of these parameters are given in thecurrent patent, the exception being the host strain.Since this can influence transgenic phenotype, andhence the type of outcome measures that may beobserved [13], this is an important omission.

The structure of APP transgenes comprised theSwedish double mutation (K670N/M671L), with oneor more additional mutation, including the ‘Londonmutation’ (V717I). This recombinant human DNAconstruct (cDNA and/or genomic DNA) was function-ally linked to a rodent Thy-1 promoter element. Genetransfer was by pronuclear microinjection or retroviralinfection of early embryos.

Since the Swedish mutation was the first AD-linkedmutation found to increase Ab production intransfected cell lines [14,15], it is a logical choice forthe construction of transgenic animals and has beenpreviously used for this purpose [10-12,103]. The‘London mutation’ leads to increased production ofthe longer Ab variant (Ab1-42) that accumulates in theAD brain first [16,17]; it has been used for transgenicwork by investigators at Cephalon but without

producing AD-type pathology in mice up to 12months of age [3].

Various promoters have been used in transgenic ADanimal models [3], including neurone-specific enolase(NSE) [9,103], PDGF-b, [4,6-8], and Thy-1 [3,105]; APPtransgenes have also been inserted into prion protein(PrP) cosmid vectors in which the PrP open readingframe is replaced with the variant APP [10-12]. Thy-1was chosen in the current case in the light of studiessuggesting only Thy-1 could yield APP transgeneexpression levels comparable to or above endoge-nous mouse levels [18].

Transgene expression in the current model is reportedas the level of transgene APP mRNA versus endoge-nous APP mRNA. For the Swedish mutation alone thisis 3 - 10 times more; smaller elevations are reportedwhen two or three mutations are present. There areno data on expression levels of holo-APP or Ab, butthe studies of Andra et al. [18] suggest that proteinlevels parallel mRNA levels in APP transgenic animalsdriven by a Thy-1 promoter, reaching or exceedingendogenous APP.

The outcome measures presented are both qualitative(neuropathology) and quantitative (measures ofbehavioural and cognitive parameters). Neuropa-thological examination of transgenic animals at 6months of age showed accumulation of senile plaqueswith surrounding dystrophic neurites and alsoevidence of neuronal and synaptic loss; some or all ofthese features have been seen in previous models[4,6-12]. Immunostaining for the microtubule-associated protein tau using the AT8 antibody (whichrecognises abnormally phosphorylated residues atpositions Ser202 and Thr205 of tau) was positive in thedystrophic neurites around senile plaques; this featurehas not been reported in previous transgenics. Theother tau-immunopositive features that are seen in ADbrain, namely NFTs and cortical dystrophic neurites[19], and the presence of which correlates better withcognitive impairment [5], are not mentioned, andhence presumably were not seen. Neuropathology isnot reported for later time points but Western blots ofbrain extracts stained with AT8 are positive in animalsaged 6 and 15 months.

Quantitative outcome measures of animal behaviourand cognition are also presented. However, these arenot related to levels of transgene expression so it isnot possible to plot ‘dose-response’ curves, asreported for other transgenic models [10,12,13].

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents(1998)8(6)

730 Transgenic animal model for Alzheimer’s disease

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On the basis of these observations, it is claimed thistransgenic animal may be a more faithful model of ADthan those previously reported [4].

3. Expert opinion

This transgenic animal model for AD looks promisingin view of the combination of increased APPtransgene expression with neuropathological,behavioural, and cognitive changes reminiscent of theAD phenotype. The presence of posi t ivetau-immunoreactivity within the plaque-associateddystrophic neurites, as in AD, does seem to be a first:tau-immunoreactivity was not reported even indoubly transgenic animals (bearing both APP[K670N/M671L] and presenilin 1 [M146L] mutations)with ‘accelerated Alzheimer’s’ [12], although antineu-rofilament immunoreactivity has been seen in theplaque-associated dystrophic neurites in V717F mice[6]. However, despite this unique finding, somereservations remain. For example, if this is indeed amore accurate model of AD, it would be desirable tosee the complete spectrum of AD tau immunopa-thological changes, especially NFTs, in the transgenicanimals; perhaps they may become apparent at latertime points. Moreover, it is not clear from the patentwhich features of the construct are responsible for thesuccess of this transgenic animal in reproducingAD-type pathology: host strain, transgene mutationand/or promoter, level of transgene expression, etc.Previous reports of transgenics combining theSwedish APP mutation with the Thy-1 promoter havenot been identified to allow comparison. Until theseissues are resolved by further studies, it would seemthe case for adopting this AD animal model in prefer-ence to those already available is not yet convincinglyestablished.

The availability of animal models faithful to both theneuropathological and behavioural/cognitive aspectsof the AD phenotype is especially desirable at thepresent time, in view of the increasing number ofagents being claimed for the treatment of AD thatinvolve manipulation of APP and/or Ab [20]. Althoughdirect clinical trials of some of these agents may provefeasible, as has been the case for cholinesteraseinhibitors, innovative compounds designed (orclaimed) to down-regulate APP expression, reduceAb production, or block Ab aggregation in in vitroparadigms will require animal trials to establish theirsafety and efficacy. One example of this may be thevacuolar H+-ATPase inhibitor bafilomycin A1, in vitro

studies of which have established that it differentiallyaf fects proteolyt ic processing of mutant(K670N/M671L) APP compared to wild-type APP[21,106]. It would seem an ideal candidate drug fortesting in a transgenic model such as the one reportedhere to ascertain whether it affects the neuropa-thological and behavioural/cognitive phenotype.Moreover, until such time as an efficacious drug forAD has been developed and licensed via such a route,questions will remain as to the exact utility of animalmodels for testing anti-AD compounds, howeveruseful they may prove in elucidating the pathogenesisof the disease.

Bibliography

Papers of special note have been highlighted as:• of interest•• of considerable interest

1. ARENDT T, HOLZER M, FRUTH R, BRÜCKNER MK, GART-NER A: Paired helical filament-like phosphorylation oftau, deposition of b/A4-amyloid and memory impair-ment in rat induced by chronic inhibition of phos-phatase 1 and 2A. Neuroscience (1995) 69:691-698.

2. SELKOE DJ: Cell biology of the amyloid b-protein pre-cursor and the mechanism of Alzheimer’s disease.Ann. Rev. Cell Biol. (1994) 10:373-403.

3. SAVAGE MJ, HOWLAND DS, ALI SM et al.: APP transgene-sis: approaches towards the development of animalmodels for Alzheimer’s disease neuropathology. In:Neurobiology of Alzheimer’s Disease. Dawbarn D, Allen SJ(Eds.), bios Scientific Publishers, Oxford (1995):149-192.

4. GAMES D, ADAMS D, ALESSANDRINI R et al.: Alzheimer-type neuropathology in transgenic mice overexpress-ing V717F b-amyloid precursor protein. Nature (1995)373:523-527.

• First convincing report of an APP transgenic mouse withAD-type amyloid plaques in an appropriate neuroanatomi-cal distribution.

5. ARRIAGADA PV, GROWDON JH, HEDLEY-WHTYE ET,HYMAN BT: Neurofibrillary tangles but not senileplaques parallel duration and severity of Alzheimer’sdisease. Neurology (1992) 42:631-639.

6. MASLIAH E, SISK A, MALLORY M, MUCKE L, SCHENK D,GAMES D: Comparison of neurodegenerative pathol-ogy in transgenic mice overexpressing V717F b-am-yloid precursor protein and Alzheimer’s disease. J.Neurosci. (1996) 16:5795-5811.

7. JOHNSON-WOOD K, LEE M, MOTTER R et al.: Amyloidprecursor protein processing and Ab42 deposition in atransgenic mouse model of Alzheimer disease. Proc.Natl. Acad. Sci. USA (1997) 94:1550-1555.

8. IRIZARRY MC, SORIANO F, MCNAMARA M et al.: Ab depo-sition is associated with neuropil changes, but notwith overt neuronal loss in the human amyloid

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents(1998)8(6)

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10. HSIAO K, CHAPMAN P, NILSEN S et al.: Correlative mem-ory deficits, Ab elevation, and amyloid plaques intransgenic mice. Science (1996) 274:99-102.

• K670N/M671L transgenic mice with deficits in learning andmemory tests which correlated with increased Ab concen-trations and AD-type neuropathalogical damage.

11. IRIZARRY MC, MCNAMARA M, FEDORCHAK K, HSIAO K,HYMAN BT: APPsw transgenic mice develop age-relatedAb deposits and neuropil abnormalities, but no neu-ronal loss in CA1. J. Neuropathol. Exp. Neurol. (1997)56:965-973.

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13. HSIAO KK: Understanding the biology and molecularpathogenesis of Alzheimer’s disease in transgenicmice expressing amyloid precursor proteins. In: Mo-lecular Mechanisms of Dementia. Wasco W, Tanzi RE (Eds.),Humana Press, Totawa, NJ (1997):39-43.

14. CITRON M, OLTERSDORF T, HAASS C et al.: Mutation ofthe b-amyloid precursor protein in familial Alz-heimer’s disease increases b-protein production. Na-ture (1992) 360:672-674.

15. CAI X, GOLDE TE, YOUNKIN SG: Release of excess amy-loid bprotein from a mutant amyloid bprotein precur-sor. Science (1993) 259:514-516.

16. SUZUKI N, CHEUNG T, CAI XD et al.: An increased per-centage of long amyloid b-protein secreted by familialamyloid b-protein precursor (APP717) mutants. Sci-ence (1994) 264:1336-1340.

17. SCHEUNER D, ECKMAN C, JENSEN M et al.: Secreted amy-loid b-protein similar to that in the amyloid plaques ofAlzheimer’s disease is increased in vivo by the preseni-lin 1 and 2 and APP mutations linked to familial Alz-heimer’s disease. Nature Med. (1996) 2:864-870.

18. ANDRA K, ABRAMOWSKI D, DUKE M et al.: Expression ofAPP in transgenic mice: a comparison of neurone-specific promoters. Neurobiol. Aging (1996) 17:183-190.

19. BRAAK H, BRAAK E, GRUNDKE-IQBAL I, IQBAL K: Occur-rence of neuropil threads in the senile human brainand in Alzheimer’s disease: a third location of pairedhelical filaments outside the neurofibrillary tanglesand neuritic plaques. Neurosci. Lett. (1986) 65:351-355.

20. LARNER AJ, ROSSOR MN: Alzheimer’s disease: towardstherapeutic manipulation of the amyloid precursorprotein and amyloid b-peptides. Exp. Opin. Ther. Patents(1997) 7:1115-1127.

21. HAASS C, CAPELL A, CITRON M, TEPLOW DB, SELKOE DJ:The vacuolar H+-ATPase inhibitor bafilomycin A1 dif-ferentially affects proteolytic processing of mutantand wild-type b-amyloid precursor protein. J. Biol.Chem. (1995) 270:6186-6192.

Patents

101. UNIV. OF PENNSYLVANIA: WO9535366 (1995).

102. CEPHALON, INC.: WO9412627 (1994).

103. ATHENA NEUROSCIENCE, INC.: WO9511968 (1995).

104. UNIV. OF MINNESOTA: WO9520666 (1995).

105. MERCK & CO., INC.: WO9606927 (1996).

106. ELI LILLY & CO.: EP-689839-A (1996).

Patent details

Title: Transgenic animal model for Alzheimer’s disease

Assignee: Novartis AG

Inventors: Sommer B, Staufenbiel M

Priority data: 24/07/96 96GB-15569

Filing date: 23/07/97

Publication date: 29/01/98

Publication no.: WO9803644

© Ashley Publications Ltd. All rights reserved. Exp. Opin. Ther. Patents(1998)8(6)

732 Transgenic animal model for Alzheimer’s disease

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