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Title:
A NOVEL ANIMAL MODEL OF ALZHEIMER'S DISEASE USING GENETICALLY ALTERED STEM CELLS
Document Type and Number:
WIPO Patent Application WO/2002/013837
Kind Code:
A1
Abstract:
Disclosed herein is a neural stem cell (NSC) engineered to overexpress $g(b)-amyloid. This NSC has been transfected with a $g(b)-amyloid construct comprising DNA of APP695 gene with Swedish and Indiana mutations, a general or tissue-specific promoter, and a selection marker. Also disclosed is an in vitro method of screening for chemical activity against the development of β-amyloid, whose steps include providing a plurality of NSC engineered to overexpress $g(b)-amyloid; contacting the $g(b)-amyloid-producing NSCs with a chemical substance; and determining the presence of $g(b)-amyloid. A method of producing a nonhuman Alzheimer's disease animal includes introducing sufficient NSCs expressing a $g(b)-amyloid construct into the nonhuman animal, such that the $g(b)-amyloid so expressed causes an Alzheimer's disease model. A transformed NSC cell line is transfected with a $g(b)-amyloid construct, such that the NSC express $g(b)-amyloid.

Inventors:
SNABLE GARY L (US)
ABOODY KAREN S (US)
HARTLEY DEAN (US)
NOLL ELIZABETH (US)
Application Number:
PCT/US2001/025630
Publication Date:
February 21, 2002
Filing Date:
August 14, 2001
Export Citation:
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Assignee:
LAYTON BIOSCIENCE INC (US)
SNABLE GARY L (US)
ABOODY KAREN S (US)
HARTLEY DEAN (US)
NOLL ELIZABETH (US)
International Classes:
A01K67/027; A61K35/30; C07K14/47; C12N15/85; A61K35/12; (IPC1-7): A61K35/00; C12N5/00; C12N5/02; C12N15/63; C12N15/85; C12N15/87
Foreign References:
US5750376A1998-05-12
Other References:
"Progress report on Alzheimer's disease", NATIONAL INSTITUTE ON AGING, 1999, pages 1 - 50, XP001055020
BLESCH ET AL.: "Ex vivo gene therapy for Alzheimer's disease and spinal cord injury", CLINICAL NEUROSCIENCE, vol. 3, 1996, pages 268 - 274, XP001055021
LENDON ET AL.: "Exploring the etiology of Alzheimer's disease using molecular genetics", JAMA, vol. 277, no. 10, 12 March 1997 (1997-03-12), pages 825 - 831, XP001055022
MORRISON-BOGORAD ET AL.: "Alzheimer disease research comes of age", JAMA, vol. 277, no. 10, 12 March 1997 (1997-03-12), pages 837 - 840, XP001055023
SELKOE ET AL.: "Alzheimer's disease: Genotypes, phenotype and treatments", SCIENCE, vol. 275, 31 January 1997 (1997-01-31), pages 630 - 631, XP001055024
Attorney, Agent or Firm:
Luther, Barbara J. (Ltd. P.O. Box 6149 Stateline, NV, US)
Download PDF:
Claims:
Claims
1. A neural stem cell (NSC) engineered to overexpress ßamyloid.
2. The NSC of claim 1 in which the NSC has been transfected with a (3amyloid construct comprising DNA of APP695 gene with Swedish and Indiana mutations, a general or tissuespecific promoter, and a selection marker.
3. An in vitro method of screening for chemical activity against the development of ßamyloid, the method comprising the steps of a. providing a plurality of NSC engineered to overexpress ßamyloid; b. contacting the (3amyloidproducing NSCs with a chemical substance ; and c. determining the presence of ßamyloid.
4. Method of producing a nonhuman Alzheimer's disease animal which comprises introducing sufficient NSCs expressing a (3amyloid construct into the nonhuman animal, such that the (3amyloid so expressed causes an Alzheimer's disease model.
5. A transformed NSC cell line transfected with a ßamyloid construct, such that the NSC express ßamyloid.
Description:
A Novel Animal Model of Alzheimer's Disease Using Genetically Altered Stem Cells Technical Field This invention is in the field of cell models or animal models. More particularly, this invention provides a new model for Alzheimer's disease using transfected human stem cells.

Background Alzheimer's disease (AD) is due to a degenerative process characterized by progressive loss of cells from the basal forebrain, cerebral cortex, hippocampus and other brain areas. The mechanism underlying the initiation of this progressive pathophysiology is thought to be age-related accumulation of amyloid beta-protein (Aß). Acetylcholine- transmitting neurons and their target nerves are particularly affected, although the lost cells produce a variety of neurotransmitters. Senile plaques and neurofibrillary tangles are present.

Pick's disease has a similar clinical picture to AD but a somewhat slower clinical course and circumscribed atrophy mainly affecting the frontal and temporal lobes.

Analysis of the post-mortem brains of Alzheimer's disease victims has led to several theories about the underlying causes of pathology. Prominent among these theories are 1) chronic inflammation (Eikelenboom and Verhius. Exp gerontol 34 : 453-61,1999 ; Gahtan and Overmier, Neurosci Biobehav rev 272: 32321-3238, etc.), 2) oxidative stress (Smith et al. J Neurosci 17: 2653-2657,1997), 3) mitochondrial defects (Beal. Biochim Biophys Acta 1366: 211-23,1998), 4) aberrant phosphorylation (Trojanowski and Lee. Am JPath 144: 449-53, 1994), 5) and inappropriate cell cycle signals (Raina et al. Int JExp Pathol 80 : 71-6, 1999).

The diversity of theories and proposed mechanisms indicates that AD may involve multiple physiological changes.

Animal models for AD and other dementias display hereditary tendency toward the formation of such plaques composed of Ap. Transgenic animal lines have been produced, including two human genes known to be involved in AD pathology: amyloid precursor protein (APP) (the protein cleaved to generate A (3) and the E4 isoform of apolipoprotein E (apoE4). The mutated human APP gene also was added to the transgenic animal. It is thought that if a drug has an effect in the model, it also may be beneficial in at least some forms of AD and Pick's disease. At present there are palliative treatments but no means to restore function.

Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson's and other neurodegenerative diseases, however it has not been exploited to develop a specific models of AD by over-expressing human Ap. The experimental replacement of neurons by direct grafting of fetal tissue into the brain has been accomplished suggesting that a stem cell model of AD is feasible.

The experimental grafting of human fetal neurons has been limited by scarcity of appropriate tissue sources, logistic problems, legal and ethical constraints and poor survival of grafted neurons in the human host brain. A source of implantable neurons, which is the most ethically controversial, is that of human fetal tissue. U. S. Pat. No. 5,690,927 issued November 25,1997, also utilizes human fetal tissue. Human fetal neuro-derived cell lines are implanted into host tissues. The methods allow for treatment of a variety of neurological disorders and other diseases. A preferred cell line is SVG.

U. S. Pat. No. 5,753,491 issued May 19,1998, is an invention that generally relates to methods for treating a host by implanting genetically unrelated cells in the host. More particularly, the present invention provides human fetal neuro-derived cell lines, and methods of treating a host by implantation of these immortalized human fetal neuro-derived cells into the host. One source is the mouse, which is included in the U. S. patent number 5,580,777 issued December 3,1996. This patent encompasses a method for the in vitro production of lines of immortalized neural precursor cells, including cell lines having neuronal and/or glial cell characteristics, comprises the step of infecting neuroepithelium or neural crest cells with a retroviral vector carrying a member of the myc family of oncogenes.

U. S. Pat. No. 5,753,506 issued May 19,1998 reveals an in vitro procedure by which a homogeneous population of multi-potential precursor cells from mammalian embryonic neuroepithelium (CNS stem cells) was expanded up to 109 fold in culture while maintaining their multi-potential capacity to differentiate into neurons, oligodendrocytes, and astrocytes.

Chemically defined conditions are presented that enable a large number of neurons, up to 50% of the expanded cells, to be derived from the stem cells. In addition, four factors- PDGF, CNTF, LIF, and T3-have been identified which, individually, generate significantly higher proportions of neurons, astrocytes, or oligodendrocytes. These defined procedures permit a large-scale preparation of the mammalian CNS stem cells, neurons, astrocytes, and oligodendroctytes. These defined procedures permit a large-scale preparation of the mammalian CNS stem cells, neurons, astrocytes, and oligodendrocytes under chemically

defined conditions with efficiency and control. These cells are proposed as an important tool for developing animal models of particular diseases by over-expressing specific proteins, including AD.

Another source of stem cells is that of primate embryonic stem cells. U. S. Pat. No.

5,843,780 issued December 1,1998, utilizes these stem cells. A purified preparation of stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (-); SSEA-3 (+) ; TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation have normal karyotypes and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cells lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed in the patent.

Yandava et al (PNAS USA 95: 7029-34, June 1999) demonstrated that neural stem cells implanted at birth resulted in widespread engraftment throughout the shiverer mouse with oligodendrocytes capable of myelinating up to 52% of host neuronal processes with better compacted myelin of a thickness and periodicity more closely approximating normal.

Some mice even shivered less.

In summary, there is substantial evidence that transplantation of stem cells over- expressing APP could produce AD-like pathology, allowing regional and age-dependent alteration to be studied. Additionally, by using other transgenic mice this model allows the rapid testing of various genetic backgrounds.

Summary of Invention Disclosed herein is a neural stem cell (NSC) engineered to overexpress (3-amyloid.

This NSC has been transfected with a P-amyloid construct comprising DNA of APP695 gene with Swedish and Indiana mutations, a general or tissue-specific promoter, and a selection marker. Also disclosed is an in vitro method of screening for chemical activity against the development of P-amyloid, whose steps include providing a plurality of NSC engineered to overexpress (3-amyloid ; contacting the (3-amyloid-producing NSCs with a chemical substance; and determining the presence of p-amyloid. A method of producing a nonhuman Alzheimer's disease animal includes introducing sufficient NSCs expressing a (3-amyloid construct into the nonhuman animal, such that the (3-amyloid so expressed causes an

Alzheimer's disease model. A transformed NSC cell line is transfected with a P-amyloid construct, such that the NSC express (3-amyloid.

Detailed Description of Invention In one embodiment of this invention, there is a neural stem cell transfected with an Alzheimer's gene, which can be used as an animal model to study, or to test possible treatments. Examples of DNA, which can be transfected alone or in combination, include, but are not limited to, ApoE4, ApoES, ApoE Pittsburgh, Indiana and London mutations, a portion thereof, and a combination thereof. The NSC also can be transfected with other appropriate DNA, including but not limited to that for presenilin.

Definitions: "Neuronal cells"are those having at least an indication of neuronal phenotype, such as staining for one or more neuronal markers. Examples of neuronal markers include, but are not limited to, microtubule associated protein (MAP-2), neuron-specific nuclear protein, tyrosine hydroxylase, and calbindin. Neuronal stem cells are those that can adopt the phenotype of a variety of nerve cells, as well as the phenotype of, for example, astrocytes and dopaminergic cells.

"Non-tumerogenic"refers to the fact that the source has not been known to cause tumors.

General Methods Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Springs Harbor Laboratory, New York, 1989 and 1992; and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland, 1989.

Polymerase chain reaction (PCR) is carried out generally as in PCR Protocols : A Guide to Methods And Applications, Academic Press, San Diego, CA, 1990. Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, are performed as generally described in Sambrook, et al., 1989, Molecular Cloning : A Laboratory Manual, Cold Springs Harbor Laboratory Press, and methodology as set forth in United States Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5, 192, 659; and 5,272,057 and incorporated herein by reference. In-situ PCR in combination with Flow Cytometry can be used for

detection of cells containing specific DNA and mRNA sequences (Testoni et al., Blood 87: 3822, 1996).

Gene Therapy Gene therapy as used herein refers to the transfer of genetic material (e. g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition.

The genetic material of interest encodes a product (e. g. a protein, polypeptide, peptide, functional RNA, antisense) whose in vivo production is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Alternatively, the genetic material of interest encodes a suicide gene. For a review see, in general, the text"Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997).

Alzheimer's Disease Models In the last decade there has been an explosion of research directed toward developing AD animal models. Patents have issued for a number of transgenic animals, namely, U. S.

Pat. Nos. 5,387,742 to Scios Nova (mouse with nerve-tissue specific promoter and DNA sequence encoding for (3-amyloid precursor protein A751 and A770); 5,602,299 to Mount Sinai School of Medicine of the City University of New York (mouse producing human neurofilament subunit M); 5,767,337 to Duke University (mouse with human apolipoprotein E with inactivated endogenous mouse apoE gene; also method of screening by detecting effect of compound on activity of apoE isoforms and physiology); 5,811,633 to Athena Neurosciences, Inc./Elan (mouse with cDNA-genomic DNA hybrid sequence; cDNA encodes APP770 or mutated APP770 for which is substituted genomic DNA consisting of exon 6 and an amount of the adjacent downstream intron sufficient for splicing, the KI and OX-2 coding region and an amount of each of their upstream and downstream introns sufficient for splicing and exon 9 with adjacent upstream intron sufficient for splicing; the construct is transcribed and differentially spliced in mouse brain cells); 5,849,999 to McLean Hospital and Wellesley College (mouse with DNA for. 1) the Flag peptide fused to the amino terminus of APP-C100 polypeptide directly or with < 5 intervening amino acids and 2) brain- active expression control sequences, such that the mouse exhibits degeneration of neurons and synapses in the hippocampal formation and cognitive impairment; also methods for enhancing neurodegeneration and cognitive impairment in the same mouse; also screening

methods for identifying compounds that inhibit AD neurodegeneration and cognitive impairment); 5,850,003 to Athena Neurosciences (rodent with transgene heterologous APP containing the Swedish mutation of 595 asparagine and 596 leucine and producing detectable ATF-ßAPP) ; 5,877,399 to University of Minnesota and Johns Hopkins (mouse whose genome has an APP transgene with regulatory sequences from a prion gene promoter linked to coding sequence for APP695. WT, APP695. SWE or APP695. TRI; whose performance in memory and learning is impaired; and whose cortico-limbic pathology is abnormal; also the mouse's progeny; also methods for screening using the mouse for compounds to ameliorate or treat AD symptoms); 5,898,094 to University of South Florida (a transgenic mouse with the DNA sequence for mutant presenilin M146L and the DNA sequence for APP K670N, M671L and having accelerated p-amyloid deposition compared to mice with one of the two transgenes or non-transgenic mice); 5,912,410 to Scios Inc. (mouse with nerve-specific promoter and DNA sequence for amino acid nos. 289-345 of ßAPP) ; 6,025,183 to Yissum Research Development Co. of Hebrew University of Jerusalem (cells with recombinant vectors encoding human acetylcholinesterase and human promoter); 6,037,521 to Hoechst Japan Ltd. (mouse with p-actin promoter operably linked to the sequence for the 99-103 amino acid carboxy terminus of ßAPP and to an enhancer; also method of screening for drug activity against AD by administering the drug to the mouse); 6,046,381 to the University of California (mouse with both alleles of endogenous apoE ablated and human apoE3 with regulatory sequences for neuron expression; method of screening for active compounds in the same mouse which analyzes apoE3-mediated behavior).

Also separately patented are methods of screening for compounds with an effect on AD, including U. S. Patent Nos. 5,604,102 to Athena Neurosciences, Inc. (method for monitoring (3-amyloid in vivo detects the presence of Swedish variant amino terminal in a specimen from a rodent transformed to express the Swedish mutation of (3APP wherein the amino terminal fragment has been cleaved between Leu596 and Asp597; method for determining inhibition of (3APP production in a transformed rodent; method for detecting (3APP in any biological specimen); and 5,720,936 to Athena Neurosciences, Inc. (method of screening for altered expression or processing of APP using a particular transgenic mouse).

The NSC expressing APP protein containing the Swedish and/or Indiana mutations would be desirable, as they could overproduce Ap causing pathologic changes characteristic of AD. NSC with human genetic mutations are more likely to assure properly synthesized, secreted and folded proteins. The availability of such cells may obviate the need to maintain

colonies of AD mice and rodents and to perform experiments on them at just the right time.

This transplantation model would allow other variables to be tested, including regional and aged-dependent expression, as well as testing in various genetic backgrounds by using transgenic and knock-out mice. Additionally, standard rodents, or any other species (non- human primates), can be administered the Alzheimer-transfected cells, allowing species variations to be identified. Such animals can readily be tested for the safety and effectiveness of anti-Alzheimer therapy.

The construct synthesis can be varied in ways known to those skilled in the art. For example, the antibiotic used for selection can be not only puromycin, but also neomycin or blasticidin. Likewise, different promoters, etc. can be used.

Delivery of Cells The cells of the present invention are administered and dosed in accordance with typical procedures used for the experimental animal, taking into account the current condition, the site and method of administration, scheduling of administration, age, sex, body weight and other factors. The cell dose must be effective to decrease function or cause symptoms as are selected as appropriate measures by those skilled in the art.

In the method of the present invention, the cells of the present invention can be administered in various ways as would be appropriate to implant in the central nervous system. Preferably the transfected NSCs are administered by stereotactic injection to produce regional expression.

The transfected-NSC dose with which to provide Alzheimer-like symptoms in a small animal model is in the range of about 20,000 cells to about 500,000 cells ; and for a primate test, the dose is about 150,000 to 2,000,000 cells. The doses can be divided among different locations. Additional doses can be given for greater effect.

Examples Example 1 Neural stem cells (NSCs) are genetically engineered in vitro to overproduce human B- amyloid protein. A plasmid using the retroviral pBabePuro backbone (Morgenstern and Land, Nucl Acids Res 18,3587-96,1990) was constructed to include the human APP gene or a portion thereof under the LTR promoter (kindly provided by Dr. Michael Black).

Retrovirus vectors were packaged by co-transduction of the Cdpuro plasmid with amphotropic (M12YA) or ecotropic (MV12) envelope-coding plasmid cDNA (Sena-Estees et

al, JVirol73, 10426-39,1999) into 293T/17 cells (Pear et al, Proc Natl Acad Sci USA 90, 8392-96,1993). Cdpuro retroviral supernatant was harvested as previously described (Sena- Esteves et al. ibid.) and used for multiple infections of any of several lacZ-positive NSC clonal lines (e. g., human H1, H6 and murine C17. 2). All have the p-galactosidase reporter gene and are neo (G418) resistant.

The p-amyloid component is the full-length familial p-amyloid sequence (approx 2 kb) or the truncated 42-bp p-amyloid sequence. Particularly preferred is APP 695 with Swedish and Indiana mutations. Following a standard procedure for lipofectamine infection, cells are placed under puromycin (or hygromycin) selection for two weeks, expanded, and stock frozen. Expression of p-amyloid from transduced NSCs is confirmed by antibody staining in cell culture and Western blot analysis. Such p-amyloid-producing NSCs are called NSC-BA4.

Example 2 NSC-BA4 cells (H1-BA4 or C17.2-BA4 cells) are transplanted into nude rats and Fisher rats. Such cells overproduce amyloid in the brain and induce surrounding neuronal damage and/or apoptosis, similar to human pathology. A dose of at least 200,000 NSC-BA4 is stereotactically engrafted into each rat's right frontal lobe. The cells are injected via 30- gauge Hamilton syringe at a concentration of 50,000 cells/. l PBS. The Fisher rats receive daily ip injections of cyclosporine. At specified time points (days 7,14,28,42, or 56), animals are sacrificed by transcardial perfusion with 4% paraformaldehyde. Their brains are post-fixed overnight and cryoprotected in 30% sucrose. The tissue is cryostat-sectioned. The 10-micron serial sections are collected on glass slides in sets of 10 as per standard protocol.

A small subset of animals receive an injection of non-p-amyloid NSC in the opposite hemisphere one week prior to sacrifice to examine the ability of NSCs to migrate to areas of amyloid deposition in the brain.

A panel of stains and antibodies is used to identify the distribution of NSCs and amyloid in the rat brain. Histologic analysis is performed using light microscopy and fluorescent microscopy. Image analysis is optionally used for quantification of cells.

Examples of the stains and antibodies include but are not limited to X-gal counterstained with neutral red/methyl green or cresyl violet, H&E, double immunofluorescent staining (0-gal antibody conjugated to Texas Red and/or B-amyloid antibody conjugated to FITC), nestin, GFAP, NeuN, NF and vimentin.

Example 3 NSCs (H1 and/or C17.2, both with Alzheimer transfection) are implanted into the hippocampus of wt mice to produce localized injury. The mice are tested for behavioral effects at 12 and 32 weeks, just prior to sacrifice. After sacrifice, one hemisphere of each brain is gently shaken in 0.9% saline for 30 minutes and immersion-fixed in 4% paraformaldehyde for 24 hours, and cryoprotected in 15% sucrose/PBS followed by overnight immersion in 30% sucrose/PBS.

At least some of the following methods are used to stain for amyloid deposits: primary antibodies to AB residues 17-24 (4G8, Senetek, Maryland Heights, MO, 1: 1000), GFAP (Boehringer Mannheim, Indianapolis, IN, 1: 1000); counterstaining with Congo red and evaluations using cross-polarized illumination; and staining with 1% thioflavin S after 10 min in Mayer's hematoxylin to mask nuclear fluorescence. NSCs are identified by the presence of lac-Z and by antibodies specific only to human epitopes, including anti-human mitochondria (hMit), anti-human nuclei (hNuc ; Chemicon), and anti-human nuclear matrix antigen (NuMA ; Calbiochem). The differentiation fate of the engrafted NSCs also will be determined.

Example 4 The same NSCs (as above, expressing APP) are used in this experiment are injected and tested for behavior. The Morris water maze tests spatial reference learning and memory.

Mice are placed into a large circular pool of water from which they escape onto a hidden platform. The platform is hidden, first by arranging its top surface just beneath the water surface, and second by rendering the water opaque so that the platform is invisible. The latency and directionality to escape from the water to the platform are measured. Passive avoidance measures cognitive function. In the acquisition (learning) phase, the rat is placed on a small acrylic platform in the corner of the test box. When the animal steps down from the platform, it receives a low level shock (0.2 mA) until it returns to the platform. The latency to the initial step down, the number of descents and total trial time until rat remains on the platform for 3 min are measured. In the retention phase, we measure whether the animal steps off the platform. Normal mice learn the task quickly with few step-downs and maximal retention; mice affected with AD are less successful; mice treated with a useful AD drug candidate improve. Spontaneous Activity Monitoring starts with placing the animals in a square acrylic box overnight. The box has two infrared beams to measure direction and

movement of the animal. When the animal moves in the cage, the beams are broken and a signal is sent to the computer, which provides a tabulation.

There are numerous other uses for these AD-transfected NSCs, all of which are encompassed by this patent application. One such use is in vitro screening for drug efficacy.

The cells are plated and allowed to grow to a certain density. The drug candidate is applied and incubated. There are positive and negative controls (a positive control being a drug known to have a beneficial effect in AD). The number of surviving cells and their character are compared.

Such a drug screening test also can be done in animals receiving AD-transfected NSCs. The NSCs would be appropriately injected into test animals. After an appropriate time, the animals are given the drug candidate. Behavioral improvements by the animal or lack of decreased capacity indicate success. Histological studies also can be performed on the animal to test the persistence of the AD-transfected NSCs.