Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
1999-12-30
2001-10-09
Martin, Jill D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C514S04400A, C514S045000, C514S049000, C800S014000, C800S021000
Reexamination Certificate
active
06300539
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an animal model for use in the study of human diseases, conditions and disorders, especially neurodegenerative diseases, that are characterized by the damage or dysfunction of the central nervous system. The present invention also relates to compositions used in the generation of said animal model.
BACKGROUND OF THE INVENTION
Several chronic and acute degenerative disorders of the central nervous system (CNS) show as part of their pathology the presence of reactive brain macrophages or microglial cells. These microglial cells are associated with the pathological lesions of the disease in question. It has been suggested that the presence of these cells is either the cause of these diseases, or that microglia play a major role in their progression and outcome. The presence of microglial cells in these disease states is suggested to indicate an inflammatory or immune response within the brain.
Many human neurodegenerative disorders are characterized by such physical and biochemical pathology. Of these, Alzheimer's disease (AD) is the most common, characterized by a progressively worsening and debilitating dementia.
Alzheimer's disease affects millions of individuals of all races and ethnic backgrounds. The number of sufferers is expected to expand markedly as the proportion of the aged in the population increases (Plum, 1979). While some intellectual dysfunction is a natural result of the aging process, the dementia caused by AlzheimerIs disease is by no means normal. After onset of the disease, life expectancy ranges from only five up to twenty years.
There is presently no treatment that will arrest the progression of Alzheimer's disease. Clinical trials have been instigated involving the use of agents from such diverse pharmaceutical classes such as cerebral vasodilators, CNS stimulants, neuroleptics, nootropics, receptor stimulants, neuropeptides, aminergic enhancers and cholinergic enhancers. The results of these trials have all been disappointing to date.
The brains of individuals suffering from AD are characterized by prominent neuropathologic lesions, such as neurofibrillary tangles (NFTS), neuropil threads (NT) and amyloid-rich senile plaques (SP). These lesions are associated with massive loss of populations of CNS neurones and their development invariably accompanies the clinical dementia associated with AD. Thus, the reproduction of such lesions in an animal would provide a valuable model for the study of the development and progression of AD. Equally, the model would also provide for the screening of putative prophylactic and therapeutic compositions. Szczepanik et al., 1996 have reported an acute inflammatory response and the formation of necrotic lesions upon intrahippocampal infusion of lipopolysaccharides. Such a system is, however, unsuitable as a model for the chronic inflammatory response observed in AD.
At present, the only source f or study of the physical manifestations of AD in the CNS is from cadaver specimens. Although useful from an anatomical perspective, such material can provide little indication of the early progression of AD and is of no use in the design of therapies to counter the development of the lesions. Thus, prospective models of NFT and SP development that may or may not form the basis for the targeting of prophylactic pharmaceuticals are impossible to validate or disprove.
There is thus a great need for animal models of CNS diseases, conditions and disorders. There is also a need for animals that develop or mimic similar pathology and symptoms to those manifested in human sufferers and for a method that identifies compounds useful in the generation of such a model.
Such a model would be invaluable in the evaluation, of prospective treatments for CNS disease, including the assessment of therapeutic or prophylactic pharmaceutical preparations, or alternative non-invasive strategies. Suitable diseases that could be studied by such a technique include any whose symptoms are manifested through chronic cerebral inflammation. Examples include Alzheimer's type senile dementia, Lewy Body dementia, Parkinson's disease, Multiple sclerosis, transmissible spongiform encephalopathies, motor neuron disease and viral encephalopathies.
SUMMARY OF THE INVENTION
The present invention relates to a method of producing an animal model of chronic cerebral inflammation, comprising the step of introducing into said animal's brain a polynucleotide.
By chronic cerebral inflammation is meant the time-extended inflammation for periods of more than four days duration which results in the presence of microglial cells, invading leucocytes, and resident astrocytes in a state of hyperactivity not present in the normal brain.
By microglial cell is meant a cell of mesenchymal origin found within the CNS and peripheral nervous system (PNS) and derived either from resident progenitor cells of the CNS or from cells of the monocyte/macrophage lineage entering the brain from the circulation. These cells have a characteristic morphology and can be demonstrated by various staining methods. They represent the macrophage component of the brain (Barron, 1995).
The method of introduction into the brain may comprise any physical method of introduction of material into the brain parenchyma, an anatomical region of the CNS or the cerebrospinal fluid. Such methods include injection by miniosmotic pump, by needle, syringe or similar mechanism, or introduction by microdialysis. Preferably the composition is introduced by means of a mini-osmotic pump.
The invention also relates to compositions useful in the generation of such a model. These compositions may be in liquid, gel or solid form and may comprise a polynucleotide or mixture of polynucleotides. Preferably the compound is in a liquid form.
The polynucleotide may comprise any polynucleotide capable of generating a chronic inflammatory response. Preferably, the polynucleotide is 50 to 10,000 base pairs in length, more preferably 100 to 10,000 base pairs in length. Such polynucleotides include double or single-stranded polynucleotides. The polynucleotide may comprise ribonucleic acid (RNA), deoxyribonucleic acid (DNA), a polynucleotide analogue or a mixture of two or more of these compounds. Polynucleotide analogues include polynucleotides comprising modified chemical groups. Modifications may be present in one or more of the saccharide, purine/pyrimidine base or phosphate linkage parts of the molecule. Polynucleotide analogues also include hybrid polynucleotides such as hybrid RNA-DNA molecules and hybrid peptide-nucleic acid molecules. Additionally, the polynucleotide may be either natural, recombinant or synthetic in derivation. Preferably, the polynucleotide is exogenous to the animal; more preferably exogenous to the animal species. Where a natural polynucleotide preparation is employed, the polynucleotide is preferably in substantially isolated form. Preferably the polynucleotide is substantially free of cellular proteins and other non-polynucleotide cellular material. Preferably the composition comprises double-stranded RNA. More preferably, the composition comprises double-stranded synthetic RNA.
Introduction into the animal brain of the polynucleotide is characterized by one or more of the following effects:
i) activation of microglia in the CNS, in particular in the hippocampus, cortex and thalamus;
ii) induction of Interleukin-1&bgr;;
iii) tissue becomes hyperaemic--;--
iv) deposition of ubiquitin;
v) deposition of amyloid precursor protein;
vi) deposition of &bgr;4 immunoreactive inclusions; and
vii) loss of neurons in the hippocampus and thalamus and other brain regions. As related above, the polynucleotide molecule itself produces one or more of the above effects. The polynucleotide used in the invention does not encode APP.
Activated microglial cells can be differentiated from inactive microglial cells by immunochemistry. Activated cells express the MHC class II antigen, upregulate the Complement protein 3 receptor and exhibit an increase
Martin Jill D.
Mathews, Collins Shepherd & Gould P.A.
Medical Research Council
Woitach Joseph T.
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