Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-10-13
2003-01-07
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S009000, C800S012000, C800S021000, C800S022000, C800S025000, C435S325000, C435S455000, C435S463000, C435S320100
Reexamination Certificate
active
06504080
ABSTRACT:
The present invention relates to an animal model useful for testing potential therapeutic agents for the treatment of neurodegenerative disorders, in particular disorders associated with the presence of Lewy pathology.
Lewy pathology is a defining hallmark of degenerating neurons and/or glial cells in post mortem brain tissue of patients with neurodegenerative disorders including idiopathic Parkinson's Disease (PD), dementia with Lewy bodies (DLB), a Lewy Body variant of Alzheimer's Disease (LBVAD) and multiple system atrophies (MSA). Lewy-type changes seem central and may contribute mechanistically to dysfunction and degeneration of neurons and/or glial cells in these diseases. The characteristic appearance of Lewy pathology is known to the skilled artisan and described, e.g. in Spillantini et al., Proc. Natl. Acad. Sci. USA 95:6469-6473 (1998). Lewy pathology in neurons includes the aberrant distribution pattern, e.g. in soma and dendrites, and the finding of aggregates of the presynaptic protein &agr;-synuclein in neurons as compared to a predominantly axonal and presynaptic localisation in normal cells. Two mutations (A53T or A30P) in the &agr;-synuclein gene are linked to early-onset familial PD with Lewy pathology.
The lack of an experimental animal model showing neuropathological changes as observed in PD, DLB, LBVAD and MSA and reflecting underlying pathological mechanisms is a major obstacle that hampers significant advance in both basic research and drug development.
It has now surprisingly been found that features of Lewy pathology are evident in the brain of transgenic or somatic recombinant animals expressing an exogenous &agr;-synuclein gene under the control of nervous tissue specific regulatory sequences. The animals provide a first and novel model which can be used to test neuroprotective treatments for diseases involving &agr;-synucleinopathy.
Accordingly in a first aspect the invention provides transgenic or somatic recombinant non-human animals which exhibit &agr;-synucleinopathy. More particularly the invention provides transgenic or somatic recombinant non-human animals expressing exogenous &agr;-synuclein. Preferably they express exogenous &agr;-synuclein under the control of a nervous tissue specific regulatory sequence. More preferably the transgenic or somatic recombinant non-human animals overexpress exogenous &agr;-synuclein, e.g. express exogenous &agr;-synuclein under the control of a regulatory sequence selected from the group comprising a Thy-1 gene-regulatory sequence or a Tyrosine Hydroxylase (TH) gene-regulatory sequence.
In a further aspect the invention provides a recombinant DNA construct comprising a poly-nucleotide encoding an &agr;-synuclein polypeptide functionally linked to a nervous tissue specific regulatory sequence, e.g. a Thy-1- or a TH-regulatory sequence.
In a still further aspect a transgenic cell expressing exogenous &agr;-synuclein is provided, in particular a transgenic cell comprising a recombinant DNA construct comprising a poly-nucleotide encoding an exogenous (&agr;-synuclein polypeptide functionally linked to a nervous tissue specific regulatory sequence, and expressing said exogenous &agr;-synuclein.
Transgenic or somatic recombinant non-human animals include animals into which a suitable construct has been introduced, and, in the case of transgenic animals, progeny of such animals still retaining said construct. Examples for useful animal lines include any animal line normally kept as laboratory animals, e.g. mouse and rat lines. Very useful mouse lines are the C57BL/6 line and B6CF1 line.
&agr;-synucleinopathy in the animals shows several striking features of Lewy pathology as it presents itself in post mortem brain tissue of PD, LBVAD, DLB and MSA patients. Like in diseased human PD, LBVAD, DLB and MSA brains, subsets of neurons in the animals show &agr;-synuclein-stained perikarya and Lewy-like neurites. A small subset of these cells may also stain for ubiquitin.
&agr;-synucleinopathy may be analysed applying a panel of immuno- and histochemical techniques routinely used to assess Lewy pathology in human brain and well known in the art. The level of &agr;-synuclein mRNA expression may be analysed e.g. by RNA blotting, S
1
nuclease protection techniques and/or RT/PCR (reverse transcription and polymerase chain reaction) technology, the expression pattern of the exogenous gene in the brain may be determined by in situ hybridization, and detection of &agr;-synuclein protein in the brain may be effected using immunoblotting (western blot analysis) and/or immunohistochemical staining techniques, and the effects of the expression may be studied by histology and immunohistology, as well as by using state-of-the-art high resolution protein technologies (e.g. proteomics) and/or high-resolution RNA expression profiling (gene chip technologies).
Typically observed heterogeneous changes involved in &agr;-synucleinopathy include sausage-like enlargements of proximal and distal neuritic segments, thick or fine thread-like inclusions, as well as beaded or spindle-shaped neurites. For example some of the most prominently involved cell groups and/or brain regions in the Thy-1-&agr;-synuclein transgenic or somatic recombinant non-human animals include the nucleus centralis oralis pontis, the nucleus vestibularis lateralis, the deep cerebellar nuclei, the deep aspects of the tectal plate, and motor nuclei in the spinal cord. In TH-&agr;-synuclein transgenic or somatic recombinant non-human animals, the potentially affected and involved cell groups include mainly the catecholaminergic neurons, including those located in the substantia nigra (that are also affected in patients with PD), and that have been shown to expess a TH- or a TH-driven transgene. Involved cell groups may further include those that have been shown to express TH transiently, e.g. in mouse, the Purkinje cells in cerebellum during postnatal development days P21-28, and/or cells in other brain regions.
In affected brain areas of the transgenic or somatic recombinant non-human animals, there is astrocytic gliosis and microglial activation. Spinal roots immunostain for &agr;-synuclein and axonal degeneration is apparent with nerve fibers showing breakdown and segmentation of their myelin sheaths into elipsoids. Skeletal muscle contains atrophic angular fibers indicating neurogenic muscular atrophy and they show loss of neuromuscular synapses. In agreement, the transgenic or somatic recombinant non-human animals show a progressive impairment of limb and motor function. Specific tests known in the art provide easy and also non-invasive read-outs for limb and motor function in animals.
&agr;-synuclein sequences include wildtype &agr;-synuclein, e.g. as disclosed in Maroteaux and Scheller, Brain Res. Mol. Brain Res. 11:335- (1991); Polymeropoulos et al., Science 276:2045-2047 (1997); Hong et al., Neuroreport 9:1239-1243 (1998); Genbank accession numbers L08850 (human &agr;-synuclein); AF007758 (rat &agr;-synuclein); and mutated (&agr;-synuclein, e.g. an &agr;-synuclein linked to early onset familiar PD, e.g. A53T, as disclosed in WO 98/59,050, and/or A30P, as disclosed in Krueger et al., Nature Genetics 18:106-108 (1998).
The transgenic or somatic recombinant non-human animals may be generated according to well established methods for introduction of a recombinant DNA construct allowing germ-line or somatic insertion including viral or non-viral vector-mediated gene transfer into fertilized eggs, zygotes or early embryos and/or a specific tissue (such as brain) in the adult animal, e.g. by gene transfer into embryonic stem cells, retroviral infection of early embryos or pronuclear microinjection. Further manipulation of resulting fertilized eggs, zygotes or early embryos and breeding of resulting transgenic founder animals follows established routes of breeding transgenic animals.
Recombinant DNA constructs useful in the present invention may be prepared according to procedures known in the art. For example a nervous tissue specific regulatory sequence may be i
Crouch Deborah
Hess Susan
Novartis AG
Ton Thai-an N.
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