WO2013016826A1 - Transgenic non-human animal model of neurodegenerative disease - Google Patents

Abstract

Transgenic non-human mammals expressing human Caspase-6 are described, as well as methods of preparation thereof and uses thereof, for example for the study of and for assessing compounds for therapy and prevention of neurodegenerative diseases, such as Alzheimer's disease.

Description

TRANSGENIC NON-HUMAN ANIMAL MODEL OF NEURODEGENERATIVE DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application serial No. 61/514,532, filed on August 3, 201 1 , which is incorporated herein by reference in its entirety. TECHNICAL FIELD

The present invention generally relates to a transgenic non-human animal model of neurodegenerative disease and more particularly relates to a transgenic non-human animal model of neurodegenerative disease based on the presence of a human Caspase-6 encoding transgene, as well as methods of preparation thereof and uses thereof. BACKGROUND ART

Neurodegenerative diseases affect millions worldwide, becoming more and more prominent with an increasingly aging population. Alzheimer's disease (AD) in particular is very common amongst elderly subjects, and is characterized by a progressive decline in memory loss and other cognitive functions. Neuropathologies of the disease include the accumulation of tangles, β-amyloid-containing plaques, dystrophic neurites, and loss of synapses and neurons (Selkoe, D. et a/., 1999, Alzheimer's Disease, 2^nd Ed., Terry R. et a/., eds. pg. 293-310. Philadelphia: Lippincott, Williams and Wilkins).

While significant research efforts have been dedicated to the amyloid plaques and neurofibrillary tangles in Alzheimer Disease (AD), the underlying molecular mechanism for neuronal degeneration and dysfunction remains unknown. Yet, the identification of a protein initiating neuronal degeneration could provide a novel target to stem the progressive dementia of AD.

Although transgenic mouse models have been developed in an effort to study AD, they do not exactly reproduce the known order of pathological features of AD. Furthermore, since only a single point mutation is required to cause AD in humans, the mouse model with 3 or more mutant genes (including several mutations within one gene) raises concerns as to whether the pathology is the result of AD-associated mutations or whether the pathology arises by severely stressing neurons.

There is thus a need for novel animal model systems for the study of neurodegenerative diseases such as AD.

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. SUMMARY OF THE INVENTION

The present invention generally relates to a transgenic non-human animal model of neurodegenerative disease and more particularly relates to a transgenic non-human animal model of neurodegenerative disease based on the presence of a human Caspase-6 encoding transgene, as well as methods of preparation thereof and uses thereof.

In an aspect, the present invention provides a non-human transgenic animal whose genome comprises a transgene comprising a sequence encoding a human Caspase-6 polypeptide.

In an embodiment, the above-mentioned animal is a mammal, in a further embodiment a rodent, in yet a further embodiment the rodent is a mouse.

In an embodiment, the above-mentioned transgene is inserted into chromosome X, in a further embodiment into the hypoxanthine-guanine phosphonbosyltransferase (HPRT) locus of chromosome X.

In an embodiment, the above-mentioned human Caspase-6 polypeptide is a self-activated form of Caspase-6, in a further embodiment a self-activated form of Caspase-6 lacking the pro- domain of pro-caspase-6, in yet a further embodiment the self-activated form of Caspase-6 comprises residues 24 to 293 of SEQ ID NO:2.

In an embodiment, the above-mentioned transgene further comprises a promoter sequence, in a further embodiment the promoter sequence is a CMV immediate early enhancer/chicken β-actin (CAG) promoter sequence.

In an embodiment, the above-mentioned transgene further comprises a control sequence allowing conditional expression of said human Caspase-6 polypeptide. In a further embodiment, the conditional expression is tissue-specific expression in neural tissue.

In another embodiment, the above-mentioned control sequence comprises a STOP sequence flanked by two Cre recombinase target sequences.

In another aspect, the present invention provides a cell line derived from the above- mentioned non-human transgenic animal.

In another aspect, the present invention provides a method of determining whether a compound may be used for preventing or treating neurodegenerative disease, comprising treating the above-mentioned non-human transgenic animal with the compound and determining whether symptoms and/or pathology of the neurodegenerative disease are improved, reduced, or their onset delayed, relative to an untreated transgenic mammal, wherein the improvement, reduction or delayed onset is indicative that the compound may be used for preventing or treating neurodegenerative disease. In an embodiment, the above-mentioned neurodegenerative disease is Alzheimer's disease.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows conditional knock-in of p20p10Casp6 in mice. A: Design of the transgene inserted into the HPRT locus of chromosome X. B: RT-PCR of human Casp6 from brain mRNA extracted from Kl/Cre+/-, Kl/Cre-/-, and WT/Cre+/- male mice. The primers specifically amplify human Casp6 cDNA. Primer dimers are observed under the lowest marker. C: Western blot analysis with anti-human Casp6 from the Kl/Cre+/- and control mice. D: Taken from Tsien et al., Cell 1996¹¹. Sagital section of the mouse brain in T29.1 CaMKII promoter-Cre recombinase cross with CAG-STOP-lacZ reporter yields β-galactosidase activity almost specifically in the CA1 of the mouse hippocampus (blue). E: PCR reaction of ACL1 locus (CAG-loxP-STOP-loxP-Casp6p20p10) from KI+/-,Cre-/(KI/WT), KI+/-,Cre+/- (Kl/Cre), and WT/WT, Cre+/- (Wt/Cre) mouse brain genomic DNA shows that the loxed STOP sequence has been excised from the genome.

Figure 2 shows Morris water maze probe day 9 of male mice between 10 and 18 months of age. A. Diagram of water maze protocol. B. Results of probe day expressed as % time spent in each quadrant. The KICasp6 expressing mice (Kl/Cre) spend less time in the target quadrant than the WT/Cre and the KI/WT control mice on the probe day indicating that they do not remember as well.

Figure 3 shows Morris water maze of 16-17 month old male Casp6p20p10 Kl (knock-in). Controls represent n=7 Kl/Cre-/- and Kl (Kl/Cre+/-) n=6 males of age 16-17 months. A. The 3 day pre-training with a visible platform in the Morris water maze shows that the Casp6 Kl mice do not have any vision problems as there is no significant difference between controls and Kl. Note that the error bar is so small that it is obscured by the symbol. The training with a hidden platform in the opposite quadrant and by changing the cues on the wall was done for 5 consecutive days at a rate of 3 trials per day. While the controls learned the platform position within 15 seconds in 5 days of training, the Casp6 Kl mice showed delayed learning (results skewed by one mouse that never found the platform within 90 seconds). B. Swim speed is identical in controls and Casp6 Kl indicating no motor impairment in Casp6 Kl. C. Morris water maze probe trial. Statistical analysis (95% confidence interval) of the difference between controls and Kl mice was performed with a two-tailed unpaired t-test [samples have a normal distribution (Gaussian) and equal SD]. In the probe trial, all Kl mice showed deficits. For A-C, data represents the mean and the error bars represent the SEM.

Figure 4 shows analysis of learning and memory in female Caspase-6 knock-in mice. Because the Kl Casp6 transgene is knocked in the X chromosome and only one X chromosome is expressed, all females carry 2 copies of the Kl so that there is assurance of expression of at least one allele. We refer to KI/KI;Cre+/- as Kl/Cre, Ki/KI:Cre-/- as KI/WT (wild type) and WT/WT;Cre+/- as WT/KI. A. Graphs showing the % time spent by each mouse of the indicated genotype in the target quadrant on the probe day. B. Bar diagram showing the average % time spent in the target quadrant on the probe day by younger (3-9 months) and older (14-18 months) female mice separated by genotype.

Figure 5: shows the performance of male mice cohorts of 4-6 months and 8-11 months on the Morris water maze. Test was performed with a 3 day pre-training with a visible platform followed by 5 days of training. Probe day in the absence of a platform was performed 24 hrs after the last training trial. Numbers of mice for the 4-6 months old males are: 13 Kl/Cre, 16 KI/WT and 1 1 WT/Cre. Numbers of mice for the 8-11 months old males are: 14 Kl/Cre, 12 KI/WT and 10 WT/Cre.

Figure 6 shows the performance of male and female mice cohorts of 5, 9 or 17-18 months of age on the Morris water maze test. Mice were tested with a visible platform with visible cues on the platform for three consecutive days at three trials per 45 minutes (NSW, SWN, and WNS). The fourth day, the platform was hidden under 1 cm of water and cues on the wall were switched. Mice were given 90 seconds to find the platform and were tested for 5 consecutive days. The time to find the platform was measure with a HSV image software and CCD tracking camera in seconds and reported as latency to find the platform.

Figure 7 shows the performance of male and female mice cohorts of 4-8, 8-16 or 16-20 months of age on the Morris water maze probe day 9 test. The first 8 days of the test were performed as described above for Figure 2. On the ninth day, the platform was removed and mice given 60 seconds to swim in the pool. The % time and % distance swam in the target quadrant (where the platform was in the previous 5 days) was recorded as well as the number of times the mice crossed exactly where the platform was present (Figure 7A). Mice showing difficulty to reach the visible platform in less than 15-20 seconds were removed from the analyses. In each graph, the darker grey bars (left) represent the target quadrant and the lighter grey bars (right) represent an average of all three other quadrants. Figure 7B shows the results expressed relative to the age of the mice.

Figure 8 shows the expression of human Casp6 in the CA1 of the Kl/Cre hippocampus. Micrographs of (A) Kl/Cre, KI/WT, and WT/Cre hippocampal tissue sections immunostained with a specific anti-human Casp6 antibody. Magnified areas of the Kl/Cre hippocampal CA1 region (B) showing that few neurons of the pyramidal cell layer (PCL) contain human Casp6 in the cell soma but axons and dendrites projecting from the pyramidal neurons into the statum oriens (SO), stratum radiatum (SR), and stratum lacunosum molecular (SLM) contain human Casp6. CC indicates corpus callosum.

Figure 9 shows the specificity of the anti-human Casp6 antibody used in Figure 8. A shows immunopositive reactivity only in the Kl/Cre mouse hippocampus and not in the control KI/WT and WT/Cre mice. B. Adsorption of the LSB 477 anti-human Casp6 antibody with recombinant human Caspase-6 protein eliminates immunoreactivity. C. Neither Caspase-6 null nor wild type control mouse hippocampus is immunostained by the anti-human Caspase-6 LSB-477 antibody. D. The anti-Caspase-6 antibody LSB-477 immunostains cotton wool plaques in a familial AD case similarly than the anti-Tau Casp6 or anti-active Caspase-6 antisera.

Figure 10 shows that Tau is cleaved by Casp6 as demonstrated by immunostaining of Kl/Cre mouse brain using a TauACasp6 antiserum. A shows hippocampus area where some fine neuritic staining is observed from the dendate gyrus projections into the CA3 (arrow) in the Kl/Cre but not in the KI/WT. B and C show higher magnification of strongly stained neuropil threads in the cortex. D and E show neurons with strong intracellular TauACasp6 intracellular aggregates.

Figure 11 shows a PHF-1 Tau immunostaining of 20 month old Kl/Cre hippocampus (panels A-E) and cortex (panels F-l).

Figure 12 shows Αβ immunostaining of 20 month old Kl/Cre hippocampus.

Figure 13 shows an immunostaining of astrocytes with anti-GFAP in Control or Kl/Cre mouse brains. Mouse brain tissue sections of 5μηι were immunostained with anti-mouse GFAP on the automated Dako immunostainer and imaged digitally with the Mirax Scanner. Pictures show a representative example of control (n=6) and Kl/Cre (n=3) indicating gliosis of the stratum radiatum of the hippocampus in the Kl/Cre but not in the controls KI/WT or WT/Cre. Indicated on the right of the high magnification panel is an area of increased gliosis. Antibody used at 1/8000.

Figure 14 shows an immunostaining of astrocytes with anti-lbal microglial marker in Control or Kl/Cre mouse brains. Mouse brain tissue sections of 5μηι were immunostained with anti- mouse GFAP on the automated Dako immunostainer and imaged digitally with the Mirax Scanner. Pictures show a representative example of control (n=6) and Kl/Cre (n=3) indicating slightly increased microgliosis of the stratum radiatum of the hippocampus in the Kl/Cre but not in the controls KI/WT or WT/Cre. Indicated on the right of the high magnification panel is an area of increased gliosis. Anti-lbal used at 1/2000. Figure 15 shows details of human Caspase-6 polypeptide (Accession P55212, SEQ ID

NO:2).

Figure 16 shows details of human Caspase-6 encoding gene (Accession NG_019187, SEQ ID NO:3).

Figure 17 shows the nucleotide (upper sequence, SEQ ID NO: 4) and amino acid (lower sequence, SEQ ID NO: 5) sequence of the p20p10Casp6 construct used herein. The amino sequence corresponding to residues 24-293 of the human Caspase-6 polypeptide is underlined. The C-terminal end comprises a His-tag.

Figure 18 shows the nucleotide sequence encoding human Caspase-6 (Accession NM_001226; SEQ ID NO: 1). The coding sequence corresponds to nucleotides 79-960.

DISCLOSURE OF INVENTION

The present invention provides a non-human transgenic animal model for studying neurodegenerative disease, such as Alzheimer's disease (AD).

Prior to Applicant's studies described herein, earlier mouse models of Alzheimer's disease have targeted expression of proteins associated with familial AD such as over-expression of mutant amyloid precursor protein, presenilin I and presenilin II. In addition, mutations of Tau associated with fronto-temporal dementia are added to the mouse to study the formation of tangles. For AD, mouse models often contain several mutant genes in order to show the AD-associated pathology. These models do not exactly reproduce the known order of pathological features of Alzheimer disease. Furthermore, since only a single point mutation is required to cause Alzheimer disease in humans, the mouse model with 3 or more mutant genes (including several mutations within one gene) raises concerns as to whether the pathology is the result of AD-associated mutations or whether the pathology arises by severely stressing neurons. The amyloid hypothesis has now been clearly tested with these mouse models, showing that amyloid vaccines can prevent cognitive decline in mice. However, in humans, complete elimination of the amyloid peptide in the brains of AD patients has not improved cognition or affected the duration, severity or progression of Alzheimer disease.

The model described herein follows exactly the order of events that occur in AD, starting with the activation of Caspase-6, glial inflammatory reaction, and age-dependent memory impairment, formation of tangles and finally accumulation of amyloid beta peptide. Since Caspase-6 is activated in the brain of aged individuals with reduced cognition and in the entorhinal cortex layer II known to be first affected in Alzheimer disease, this model represents an event that precedes all other recognized pathological and cognitive events of AD and that leads to the development of all the pathologies observed in AD.

The present invention provides a non-human transgenic animal, such as a non-human transgenic mammal, harboring a transgene comprising a nucleotide sequence encoding a human Caspase-6 polypeptide, or an ancestor of said animal, at an embryonic stage.

"Transgenic" refers to any animal, e.g. a mammal, in which one or more of the cells of the animal contain heterologous nucleic acid encoding human Caspase-6, which has been introduced by way of human intervention, such as by transgenic/genetic engineering techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical crossbreeding, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. Although transgenic mice represent a preferred embodiment of the invention, other transgenic mammals including, without limitation, transgenic rodents (for example, hamsters, guinea pigs, rabbits, and rats), and transgenic pigs, chickens, cattle, sheep, goats, non-human primates (e.g., marmosets) may be constructed by standard techniques and are included in the invention. The term "transgene" means a nucleic acid sequence (encoding, e.g., human Caspase-6) that has been introduced into a cell by way of human intervention such as by way of the described methods herein. A transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced. As described in more detail below, a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

Uses of the non-human transgenic animal of the invention include:

- an in vivo model for the study of neurodegenerative disease, such as AD;

- in vivo testing/screening of compounds to assess their ability to treat neurodegenerative disease, such as AD (e.g., inhibitors of Caspase-6);

- in vivo testing/screening of compounds to assess their ability to treat Huntington's disease (e.g., inhibitors of Caspase-6);

- in vivo testing/screening of compounds to assess their ability to treat/inhibit axonal degeneration in the peripheral as well as in the central nervous system (e.g., inhibitors of Caspase-6);

Further, since the model developed herein can be used to achieve tissue-specific expression of Caspase-6, the model may similarly be used to determine the effect of Caspase-6 activation in other tissues of the body. Methods for generating transgenic animal, such as transgenic mice, are well known in the art (see "Manipulating the Mouse Embryo: A Laboratory Manual" by B. Hogan et al. (Ed. Cold Spring Harbor Laboratory, 1986; "Manipulating the Mouse Embryo: A Laboratory Manual" by B. Hogan et al. (Ed., Cold Spring Harbor Laboratory, 1994) and U.S. Pat. No. 4,736,866 for exemplary methods for the production of a transgenic mouse).

The DNA fragment can be integrated into the genome of a transgenic animal by any method known to those skilled in the art. The DNA molecule containing the desired gene sequence (Caspase-6) can be introduced into pluripotent cells, such as ES cells, by any method that will permit the introduced molecule to undergo recombination at its regions of homology. Techniques that can be used include, but are not limited to, calcium phosphate/DNA co-precipitates, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, and polycations, (e.g., polybrene, polyornithine, etc.) The DNA can be single or double stranded DNA, linear or circular. (See for example, Hogan et al., supra, U.S. Patent Nos. 5,602,299; 5,175,384; 6,066,778; 4,873, 191 and 6,037,521 ; retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA 82:6148-6152 (1985)); gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983)); and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989)).

For example, the zygote is a good target for microinjection, and methods of microinjecting zygotes are well known (see US 4,873, 191). Embryonal cells at various developmental stages can also be used to introduce transgenes for the production of transgenic animals. Different methods are used depending on the stage of development of the embryonal cell. Such transfected embryonic stem (ES) cells can thereafter colonize an embryo following their introduction into the blastocoele of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal (reviewed in Jaenisch, Science 240:1468-1474 (1988)). Prior to the introduction of transfected ES cells into the blastocoele, the transfected ES cells can be subjected to various selection protocols to enrich the proportion of ES cells that have integrated the transgene if the transgene provides a means for such selection. Alternatively, PCR can be used to screen for ES cells that have integrated the transgene.

In addition, retroviral infection can also be used to introduce transgenes into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenisch, Proc. Nati. Acad. Sci. USA 73: 1260-1264 (1976)). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al. supra, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927-6931 (1985); Van der Putten et al., Proc. Natl. Acad Sci. USA 82:6148-6152 (1985)). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra, 1985; Stewart et al., EMBO J. 6:383-388 (1987)). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner D. et al., Nature 298:623-628 (1982)). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of cells, which form the transgenic animal. Further, the founder can contain various retroviral insertions of the transgene at different positions in the genome, which generally will segregate in the offspring. In addition, transgenes may be introduced into the germline by intrauterine retroviral infection of the mid-gestation embryo (Jahner et al., supra, 1982). Additional means of using retroviruses or retroviral vectors to create transgenic animals known to those of skill in the art involves the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilized eggs or early embryos (WO 90/08832 (1990); Haskell and Bowen, Mai. Reprod. Dev. 40: 386 (1995)).

Any other technology to introduce transgenes into a non-human animal, e.g. knock-in or rescue technologies can also be used to prepare a transgenic non-human animal of the present invention. Knock-in technology is well known in the art as described, e.g., in Casas et al. (2004) Am. J. Pathol. 165, 1289-1300.

Once founder animals are produced, they can be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic mice to produce mice homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the transgene and the effects of expression.

The transgenic animals are screened and evaluated to select those animals having the phenotype of interest. Initial screening can be performed using, for example, Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals can also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase- PCR (rt-PCR). Samples of the suitable tissues can be evaluated immunocytochemically using antibodies specific for the transgene. The transgenic non-human mammals can be further characterized to identify those animals having a phenotype useful in methods of the invention. In particular, transgenic non-human mammals overexpressing the transgene (e.g. QPCT or QPCTL) can be screened using the methods disclosed herein. For example, tissue sections can be viewed under a fluorescent microscope, in systems where fluorescence indicates the presence of the reporter gene.

The term "human Caspase-6 polypeptide" as used herein refers to the full length Caspase-6 polypeptide precursor (SEQ ID NO: 2), or any fragment and/or variant thereof exhibiting caspase-6 activity. The human Caspase-6 polypeptide may be a constitutively active form of caspase-6, as described in Srinivasula et al In an embodiment, the human Caspase-6 polypeptide is a self- activated form of a human Caspase-6 polypeptide, such as a human Caspase-6 polypeptide lacking one or the two propeptides present in the precursor (corresponding to about residues 1 to 23 and 180-193 in SEQ ID NO: 2), in a further embodiment a human Caspase-6 polypeptide lacking the first propeptide present in the precursor (corresponding to about residues 1 to 23 in SEQ ID NO: 2). An example of a nucleic acid comprising a sequence encoding a human Caspase- 6 polypeptide is set forth in SEQ ID NO: 1 and Figure 18.

In an embodiment, the fragment and/or variant exhibits at least 70, 75, 80, 85, 90 or 95% identity with the full length Caspase-6 polypeptide precursor (SEQ ID NO: 2), or with a human Caspase-6 polypeptide lacking the first propeptide present in the precursor (i.e. comprising residues 24 to 293 of SEQ ID NO:2), and retains caspase-6 activity. In an embodiment, the fragment and/or variant is constitutively active or exhibits the capacity to self-activate.

In embodiments, the non-human transgenic mammal of the invention additionally harbors a further transgene or a mutation associated with neurodegenerative disease.

The present invention further provides a somatic cell line derived from a transgenic non-human animal (e.g., a mammal) according to the invention, wherein the cells of the cell line comprise a human Caspase-6 encoding nucleic acid integrated into its genome.

In an embodiment, the non-human transgenic animal (e.g., a mammal) is a Caspase-6 knock-in (Kl), having targeted insertion of a human Caspase-6 encoding nucleic acid in a selected chromosome (e.g., chromosome X) and at a selected locus (e.g., the HPRT locus) in its genome.

In an embodiment, the human Caspase-6 encoding nucleic acid can be engineered to be operatively linked to appropriate expression elements (transcriptional regulatory sequences) such as promoters or enhancers in the transgene to allow expression of the human Caspase-6 polypeptide in an appropriate cell or tissue. Accordingly, in an embodiment, the transgene further comprises one or more expression control elements (e.g., a promoter sequence). The use of the expression control mechanisms allows for the targeted delivery and expression of the Caspase-6 polypeptide. For example, the transgenes of the present invention may be constructed using an expression cassette which includes in the 5 '-3' direction of transcription, a transcriptional and translational initiation region associated with gene expression in brain tissue, DNA encoding a human Caspase-6 and a transcriptional and translational termination region functional in the host animal. One or more introns also can be present. The transcriptional initiation region can be endogenous to the host animal or foreign or exogenous to the host animal. The transgene can contain a regulatory element that provides tissue specific or inducible expression of an operatively linked nucleic acid. One skilled in the art can readily determine an appropriate tissue-specific promoter or enhancer that allows expression of human Caspase-6 in a desired tissue. It should be noted that tissue-specific expression as described herein does not require a complete absence of expression in tissues other than the preferred/targeted tissue. Instead, "cell-specific" or "tissue- specific" expression refers to a majority of the expression, or higher expression, of a particular gene of interest in the preferred/targeted cell type or tissue. Regulatory elements, including promoters or enhancers, can be constitutive or regulated, depending upon the nature of the regulation, and can be regulated in a variety of tissues, or one or a few specific tissues. The regulatory sequences or regulatory elements are operatively linked to the Caspase-6 sequence such that the physical and functional relationship between the Caspase-6 sequence and the regulatory sequence allows transcription of the Caspase-6 sequence. Regulatory elements useful for expression in eukaryotic cells can include, for example, regulatory elements including the CMV immediate early enhancer/chicken β-actin (CAG) promoter, the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, PGTF, Moloney marine leukemia virus (MMLV) promoter, thy-1 promoter and the like. The transgene may further comprise a sequence encoding one or more selectable markers. As used herein, a "selectable marker" refers to a genetic element that provides a selectable phenotype to a cell in which the selectable marker has been introduced. A selectable marker is generally a gene whose gene product provides resistance to an agent that inhibits cell growth or kills a cell. A variety of selectable markers can be used in the DNA constructs of the invention, including, for example, Neo, Hyg, hisD, Gpt and Ble genes, as described, for example in Ausubel et al. (Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)) and U.S. Patent No. 5,981 ,830. Drugs useful for selecting for the presence of a selectable marker include, for example, G418 for Neo, hygromycin for Hyg, histidinol for hisD, xanthine for Gpt, and bleomycin for Ble (see Ausubel et al, supra, (1999); U.S. Patent No. 5,981 ,830). DNA constructs of the invention can incorporate a positive selectable marker, a negative selectable marker, or both (see, for example, U.S. Patent No. 5,981 ,830).

In an embodiment, the transgenic non-human mammal provides conditional expression of human Caspase-6, for example tissue-specific expression (e.g, in neural tissue, such as the brain or a tissue thereof). Tissue-specific expression of the human Caspase-6 polypeptide may be achieved through the use of tissue-specific promoters. Non-limiting examples of suitable tissue- specific promoters include the albumin promoter (liver-specific; Pinkert et al., (1987) Genes Dev. 1 :268-277); lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al., (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741- 748), neuron-specific promoters (e.g., the neurofilament promoter, the Thy-1 promoter or the Bri- protein promoter; Sturchler-Pierrat et al., (1997) Proc. Natl. Acad Sci. USA 94: 13287-13292, Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al., (1985) Science 230:912-916), cardiac specific expression (alpha myosin heavy chain promoter, Subramaniam, A, et al., J Biol Chem 266: 24613-24620, 1991), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166).

In another embodiment, the transgene further comprises a control sequence allowing conditional or controlled expression of said human Caspase-6 polypeptide. For example, the expression of the transgene may also be controlled using the Cre/loxP system (e.g., as described in the Examples below) where all of the germ cells and somatic cells of the transgenic non-human mammal contain a nucleic acid sequence encoding human Caspase-6, and its expression in a particular tissue may be activated based on the expression of the Cre recombinase in that tissue. In such a system, the control sequence comprises a STOP sequence flanked by two Cre recombinase target sequences, such as loxP sites. The control sequence, which includes a STOP sequence, is located between the promoter and the human Caspase-6 nucleic acid, thus preventing the transcription of the human Caspase-6 gene. In the presence of the Cre recombinase (which may be expressed under the control of a tissue-specific promoter), the control sequence is excised (thus removing the STOP sequence), which results in expression of the Caspase-6 polypeptide. Another example of site-directed recombination system is the FLP-FRT recombination system (Schlake T, Bode J (1994). Biochemistry 33 (43): 12746-12751). One skilled in the art can readily determine other suitable conditional/controlled expression or recombination systems for controlling the expression of a transgene. In an embodiment, the non-human mammal provides inducible expression of Caspase-6. The present invention further provides a method of generating a transgenic non-human mammal harboring a transgene expressing human Caspase-6, the method comprising:

providing an embryonic stem (ES) cell comprising the transgene (which has been prepared for example by introducing a targeting vector comprising a Caspase-6 encoding nucleic acid sequence into the ES cell);

injecting the ES cell into a blastocyst of a non-human mammal;

transplanting the blastocyst into a foster (pseudopregnant) mother; and

allowing development of the non-human transgenic mammal.

An alternative method to generate a transgenic mammal harboring a transgene expressing human Caspase-6 comprises injecting a gene encoding Caspase-6 into the pronucleus of a fertilized egg of the animal, and allowing development of the transgenic non-human mammal.

The methods and compositions of the present invention are particularly useful in the evaluation of effectors of human Caspase-6 activity and for the development of drugs and therapeutic agents for the treatment and prevention of Caspase-6-associated diseases, more particularly Caspase-6-associated neurodegenerative diseases such as Alzheimer's disease (AD) and Huntington's disease (HD).

The transgenic non-human animal or the cells of the transgenic non-human animal of the invention can be used in a variety of screening assays. For example, any of a variety of potential agents suspected of affecting human caspase-6 activity (either directly or indirectly), as well as the appropriate antagonists and blocking therapeutic agents, can be screened by administration to the transgenic non-human animal and assessing the effect of these agents upon the function and phenotype of the cells and on the (neurological) phenotype of the transgenic non-human animals.

Behavioral studies may also be used to test potential therapeutic agents, such as those studies designed to assess motor skills, learning and memory deficits. An example of such a test is the Morris Water maze, as described below. Additionally, behavioral studies may include evaluations of locomotor activity such as with the rotor-rod and the open field.

Accordingly, the present invention further provides a method of determining whether a compound may be used for preventing or treating a neurodegenerative disease (e.g., AD, HD), comprising treating a non-human transgenic mammal harboring a transgene expressing human Caspase-6, as described above, with the compound and determining whether symptoms and/or pathology of the neurodegenerative disease are improved, reduced, or their onset delayed, relative to an untreated transgenic non-human mammal, wherein the improvement, reduction or delayed onset is indicative that the compound may be used for preventing or treating the neurodegenerative disease (e.g., AD, HD).

The present invention further provides a use of a non-human transgenic mammal harboring a transgene expressing human Caspase-6 for determining whether a compound may be used for preventing or treating neurodegenerative disease (e.g., AD, HD).

In embodiments, the symptoms and/or pathology of the neurodegenerative disease include age-dependent memory impairment, glial inflammatory reaction, formation of tangles and/or accumulation of amyloid beta peptide.

The above-noted screening method or assay may be applied to a single test compound or to a plurality or "library" of such compounds (e.g., a combinatorial library). Any such compounds may be utilized as lead compounds and further modified to improve their therapeutic, prophylactic and/or pharmacological properties for preventing and/or treating a neurodegenerative disease.

Test compounds (drug candidates) may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. MODE(S) FOR CARRYING OUT THE INVENTION

The present invention is illustrated in further details by the following non-limiting examples.

Example 1 : Materials and Methods

Preparation of human Caspase-6 expressing transgenic mice. To prevent expression of Casp6 during early development, a conditional expression mouse line expressing human Casp6 using Quick knock-in™ technology (Genoway, France) was developed. The human Casp6 cDNA lacks its pro-domain (p20p10Casp6) to promote self-activation upon expression in mammalian cells⁴. The sequence of the p20p10Casp6 construct is depicted in Figure 17. The p20p10Casp6 cDNA was placed under the control of the ubiquitous and strong CAG promoter (CMV immediate early enhancer/chicken β-actin promoter fusion) (FIG. 1A). A LoxP flanked STOP cassette was inserted between the promoter and the transgene to allow regulation of expression by Cre recombinase excision of the STOP sequence. The transgene has been knocked in the HPRT locus (without disrupting HPRT activity) of the chromosome X thereby avoiding problems caused by non- targeted insertion of the transgene throughout the genome. The Kl has been transfected in 129/Ola cells and these injected in C57BL/6 mice. The mice have been crossed with the Calmodulin kinase II (CaMKII) regulated-Cre recombinase expression mouse (T29.1)⁹ to verify expression of the p20p10Casp6 in the knock in (Kl) mice. The 29.1 (in C57BI/6 background) was reported to exclusively express Cre recombinase in the CA1 pyramidal neurons of the hippocampus and in scattered neurons of the forebrain approximately at 2-3 weeks after birth (FIG. 1 D). However, others report strong expression in the dentate gyrus, subiculum, and hypothalamus¹⁰. All mice are viable and reproduce well. The human p20p10Casp6 mRNA and protein is detected in the brain of KI+/-;Cre+/- (also called Kl/Cre) but not in KI+/-;Cre-/- (KI/WT) or KI-/-;Cre+/- (WT/Cre) mice at 4 months (FIGs. 1 B and C) and 8 months of age. The STOP sequence from the genomic DNA in brain is excised by Cre (FIG. 1 E).

Water maze experiments. Mice were bred in the pathogen-free Goodman transgenic research facility at McGill University and then sent to the Montreal Neurological Institute (MNI) animal care facility for water maze experiments. For the water maze test, we tested the mice with a visible platform with visible cues on the platform for three consecutive days at three trials per 45 minutes (NSW, SWN, and WNS). The fourth day, the platform was hidden under 1 cm of water and cues on the wall were switched. Mice were given 90 seconds to find the platform and were tested for 5 consecutive days. The time to find the platform was measure with a HSV image software and CCD tracking camera in seconds and reported as latency to find the platform. On the ninth day, the platform was removed and mice given 60 seconds to swim in the pool. The % time and % distance swam in the target quadrant (where the platform was in the previous 5 days) was recorded as well as the number of times the mice crossed exactly where the platform was present. Mice showing difficulty to reach the visible platform in less than 15-20 seconds were removed from the analyses.

Perfusion of mice and collection of brains for histology. Mice were anaesthetized by isoflurane inhalation (2: 1) prior to cardiac perfusion through the left ventricle with a 21 G needle, with 0.9% sodium chloride solution followed by 4% paraformaldehyde solution (pH 7.4). All solutions were administered and monitored from an IV pouch and flow rate was adjusted by catheter. After complete perfusion of the animal, the brain was removed and placed in vials containing 10% neutral buffered formalin (Fisher Scientific, Kalamazoo, US) at room temperature for 24 hours to facilitate fixation. Prior to sectioning, whole brains were changed into 70% ethanol and cut with a coronal acrylic matrix to isolate brain area of interest. Tissue was blocked approximately -0.94 mm to -4.04 mm posterior (-) to Bregma to isolate the CA regions of the hippocampus. The tissue block was paraffin embedded and three consecutive coronal sections sliced at 6 μηι thickness were placed on one slide by the Institut de Recherche en Immunologie et Cancerologie (IRIC, Universite de Montreal).

Immunostaining of tissue sections. Formalin-fixed, paraffin-embedded 4 μηι thick hippocampal tissue sections were deparaffinized and rehydrated. Sections were treated with antigen retrieval buffer (10mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9) for 20 min at 97°C in the Pascal Dako Cytomation apparatus and immunostained using the Dako Autostainer Plus automated slide processor and the EnVision™ Flex system (Dako, ON). Tissue sections were treated with peroxidase for 5 min., blocked with Serum-Free Protein Block (Dako, ON) for 30 min., submitted to primary antibodies diluted in EnVision™ Flex Antibody Diluent for 30 min. The LSB477 was developed against an internal sequence in human Caspase-6 (Lifespan Biosciences) was used at 1/5000 dilution. The Αβ F25276 antiserum (1/2000) was developed against Αβ peptide 1- 40¹¹. The PHF-1 antibody (1/1000) was generously provided by Peter Davies (Department of Neuroscience, Albert Einstein College of Medicine, New York, NY). Immunoreactivity was revealed with mouse/rabbit-HRP secondary antibodies for 30 min. and diaminobenzidine (DAB; Dako, ON) for 10 min. Slides were counterstained with hematoxylin. Sections were dehydrated and mounted in Permount mounting medium (Fisher Scientific, ON). The MIRAX SCAN was used to scan tissue sections and generate high-resolution digital images, which were analyzed using the MIRAX Viewer Program (Zeiss, DE).

Example 2: Characterization of human Caspase-6 expressing transgenic mice

Male mice were evaluated for learning and memory. No significant problems with learning (5 day training) or memory (probe day) were found in a Morris water maze (Protocol according to FIG. 2A) in cohorts of 4-6 months and 8-1 1 months (FIG. 5). In fact, the Kl/Cre of 4-6 months performed slightly better in the learning phase of the test (FIG. 5A). However, by 16-17 months of age, the KI+/-;Cre+/- spend less time in the target quadrant on the probe day indicating impaired memory compared to age-matched control mice (FIG. 2B). The Kl/Cre or control aged mice perform the same in pre-training with a visible platform (FIG. 3A), and in training phase with the hidden platform. The swim speed of Kl/Cre does not differ significantly from controls (FIG. 3B). These results confirm that the mice are visually and physically normal. However, the probe test on day 9 shows that the Kl/Cre perform at approximately 50% of control mice in both the % time and the % distance spent in the target quadrant (FIG. 3C). These results indicate that memory problems develop in the Kl/Cre mice by 16-17 months of age.

Female KI/KI: Cre+/- were examined versus control KI/KI:Cre-/- and WT/WT: Cre+/-. These experiments were done with a large number of mice (n=40 for KI/KI: Cre+/- or Cre-/- and n=20 for WT/WT:Cre+/-). The results show the same profile as in the males (FIG. 4A). Only the Kl/Cre mice show memory impairment as they grow older (14-18 months) (FIG. 4B).

FIGs 6 and 7 show results of Morris water maze experiments in which female and male were grouped. Monitoring of the mouse learning and memory function by the Morris water maze method revealed that the Kl/Cre mice expressing human Casp6 in the CA1 of the hippocampus undergo age-dependent memory decline. A 3 consecutive day pre-training trial with a visible platform, indicated that Kl/Cre, KI/WT or WT/Cre mice tested did not have any visual impairment at 5, 9, or 17-18 months of age (FIG. 6). The learning phase with a hidden platform did not show significant difference in performance across all three genotypes at any of the age. Swim speed also were almost identical in all three groups of mice indicating that these do not suffer from motor problems.

In the probe trial of the 4-8 month groups, where the platform is removed and % time, % distance or platform crossing is monitored, only the KI/WT group showed significant recognition of the position where the platform was present in the learning phase of the test since they significantly spent more time or crossed the exact position of the removed platform than in the other quadrants (FIG. 7A). The KI/WT mice also swam more distance in the target quadrant than in the other quadrant but this did not reach statistical significance. Neither the WT/Cre nor the Kl/Cre groups recognized the target quadrant in the probe test indicating that the expression of Cre recombinase in the CA1 of the WT/Cre mice affects their learning function at ages 4-8 months. By 8-16 months of age, the WT/Cre mice seem to perform better but they still do not achieve a statistically significant difference. In contrast, both the Kl/Cre and KI/WT groups significantly cross the target quadrant previous platform position more frequently than in the other quadrants. By 16-20 months, the WT/Cre mice recover their ability to memorize the position of the platform since they show a statistically significant ability to recognize the target quadrant by crossing the platform more frequently in the target quadrant than in the other quadrants. In contrast, the Kl/Cre perform less well in all three parameters at 16-20 months of age although this only reaches statistical significance in the % time and % distance measures.

The age-dependent deficit is more obvious when probe day measures are expressed relative to the age of the mice (Fig. 7B). The Kl/Cre mice show a statistically significant linear regression in the % time, % distance, and number of platform crosses in the target quadrant relative to the age of the mice. In contrast, neither KI/WT nor WT/Cre mice display this age- dependent memory defect. Consistent with reports that the T29.1 CaMKIIa-Cre on C57BI/6 background express Cre recombinase almost exclusively in the CA1 pyramidal neurons of the hippocampus 2-3 weeks after birth¹², human Casp6 expression was detected in the CA1 region of the Casp6 Kl/Cre mice brains (FIG. 8A). The relative sparcity of Casp6 in neuronal soma (2-3 per tissue section) and the presence of Casp6 in axons and dendrites of the stratum oriens (SO), stratum radiatum (SR), and stratum lacunosum molecular (SLM) (FIG. 8B) is consistent with the expected localization of active Casp6 in neurites¹³. No positive immunoreactivity was observed in WT/Cre, KI/WT, or Casp6 KO brains (FIG. 9). Human Casp6 was observed in the hippocampal CA1 pyramidal neurons in the brain of mice between 2 to 24 months of age.

The granule cells of the dentate gyrus project to the CA3. Interestingly, these projections are immunopositive to anti-Tau cleaved by Casp6 in the Kl/Cre indicating that Casp6 has cleaved Tau (FIG. 10A). Furthermore, strong TauACasp6 immunopositive neurites are observed in the cortex (FIG. 10B). Rarely, some cortical neurons show TauACasp6 positive neuronal intracellular aggregates (FIG. 10C). The Kl/Cre mice brains show PHF-1 positive tangles, a marker of Alzheimer disease pathology, in the cortex of Kl/Cre males at 4-6 months of age. In 12-17 months, staining for PHF-1 appears in the dentate gyrus granular layer and there is also an occasional positive CA1 neuron. In 20 month mice, there is strong accumulation of PHF-1 positive inclusions in neurons of the CA1 (FIGs. 11A and B), dentate gyrus (FIGs. 1 1C-E) and the cortex (FIGs. 1 1 F-I).

The results also suggest that there are also anti-Αβ immunopositive neurons in the dentate gyrus and CA1 of 20 month old Kl/Cre (FIG. 12). Only weak and diffuse immunoreactivity is observed at earlier time points. The Kl/Cre also present with more GFAP-positive astrocytes (FIG. 13) and lba-1 -positive microglia (FIG. 14) in dentate gyrus and the stratum radium indicating an inflammatory reaction in areas with abundant synaptic connections. The results indicate that early activation of the p20p10Casp6 in mouse brain causes age-dependent memory impairment and specific pathological lesions consistent with Alzheimer's disease brains.

Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise. REFERENCES

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Claims

WHAT IS CLAIMED IS:

A non-human transgenic animal whose genome comprises a transgene comprising a sequence encoding a human Caspase-6 polypeptide.

The non-human transgenic animal of claim 1 , wherein said animal is a mammal.

The non-human transgenic animal of claim 2, wherein the mammal is a rodent.

The non-human transgenic animal of claim 3, wherein the rodent is a mouse.

The non-human transgenic animal of any one of claims 1 to 4, wherein the transgene is inserted into chromosome X.

The non-human transgenic animal of claim 5, wherein the transgene is inserted into the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of chromosome X.

The non-human transgenic animal of any one of claims 1 to 6, wherein said human Caspase-6 polypeptide is a self-activated form of Caspase-6.

The non-human transgenic animal of claim 7, wherein said self-activated form of Caspase-6 lacks the pro-domain of pro-caspase-6.

The non-human transgenic animal of claim 8, wherein said self-activated form of Caspase-6 comprises residues 24 to 293 of SEQ ID NO:2.

The non-human transgenic animal of any one of claims 1 to 9, wherein said transgene further comprises a promoter sequence.

The non-human transgenic animal of claim 10, wherein said promoter sequence is a CMV immediate early enhancer/chicken β-actin (CAG) promoter sequence.

The non-human transgenic animal of any one of claims 1 to 1 1 wherein said transgene further comprises a control sequence allowing conditional expression of said human Caspase-6 polypeptide.

The non-human transgenic animal of claim 12, wherein said conditional expression is tissue-specific expression in neural tissue.

14. The non-human transgenic animal of claim 12 or 13, wherein said control sequence comprises a STOP sequence flanked by two Cre recombinase target sequences.

15. A cell line derived from the non-human transgenic animal of any one of claims 1 to 14.

16. A method of determining whether a compound may be used for preventing or treating neurodegenerative disease, comprising treating the non-human transgenic animal of any one of claims 1 to 14 with the compound and determining whether symptoms and/or pathology of the neurodegenerative disease are improved, reduced, or their onset delayed, relative to an untreated transgenic mammal, wherein the improvement, reduction or delayed onset is indicative that the compound may be used for preventing or treating neurodegenerative disease.

17. The method of claim 16, wherein the neurodegenerative disease is Alzheimer's disease.