Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
2001-06-01
2004-08-03
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C800S003000, C800S014000, C800S018000
Reexamination Certificate
active
06770797
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to neurodegenerative conditions.
Alzheimer's Disease is a neurodegenerative illness characterized by memory loss and other cognitive deficits. The prevalence of Alzheimer's Disease increases with age, and the presence of the disease is difficult to determine without brain biopsy. Alzheimer's Disease is characterized by the presence of neuritic plaques, neurofibrillary tangles, and neuronal cell death. Post-mortem slices of brain tissue from Alzheimer's disease patients exhibit the presence of amyloid in the form of proteinaceous extracellular cores of the neuritic plaques. The amyloid cores of these neuritic plaques are composed of a protein called the amyloid-beta or amyloid-b. Amyloid-containing neuritic plaques are a prominent feature of selective areas of the brain in Alzheimer's Disease patients as well as patients afflicted with Downs Syndrome. However, little is known about the development of Alzheimer's Disease or the mechanisms, which contribute to the disease.
SUMMARY OF THE INVENTION
The invention features a non-transgenic animal model for Alzheimer's Disease and methods for inducing prolonged in vivo gene expression in the brain. Prolonged expression of an exogenous nucleic acid in a mammal is achieved by contacting a cells or tissue with a composition, which contains a nucleic acid, a histone, and an amphipathic compound. Preferably, the composition also contains a liposome. The tissue is not skeletal muscle tissue. Preferably, the tissue is neuronal tissue such as central nervous system (CNS) tissue. For example, the tissue contains a post-mitotic neuronal cell, a cortical neuronal cell, a cerebellar neuronal cell, a glial cell, a vascular endothelial cell, or a hippocampal neuronal cell.
Gene expression in the tissue is detected in vivo for at least 48 hours after contacting the tissue with the nucleic acid composition. Preferably, gene expression persists for at least 72 hours, more preferably for at least 96 hours, more preferably for at least one week, and even more preferably for at least two to four weeks after contacting the tissue with the composition. By expression is meant transcription of a nucleic acid molecule. Gene expression is measured by detecting nucleic acid transcripts, or by detecting a translation product. The gene delivery system described herein has been successfully used to achieve gene expression in a target tissue for at least two months (and longer) after contacting the tissue with the composition.
The nucleic acid composition is in the form of a liposome, e.g., a neutral, anionic, or cationic liposome. The composition contains histone proteins, e.g., H1, H2A, H2B, H3, or H4. The amphipathic compound is cationic. For example, the compound is a polyamine, such as, a non-natural polyamine having a hydrophobic moiety such as a C6-C24 alkane, C6-C24 alkene, sterol, steroid, lipid, fatty acid, or a hydrophobic hormone. Optionally, the composition contains a nuclear localizing signal.
The nucleic acid portion of the composition is any polypeptide-encoding DNA or antisense template. To induce Alzheimer Disease physiological conditions in vivo, the nucleic acid encodes an Alzheimer's Disease-associated neural thread protein (AD-NTP) such as Alzheimer's Disease-associated neural thread protein 7c (AD7c-NTP)
An antisense template is one that is transcribed into a nucleic acid molecule that has a sequence that is complementary to a specific mRNA. The transcribed nucleic acid molecule binds to the complementary sequence in the specific mRNA and inhibits translation of the mRNA. An antisense oligonucleotide has a sequence that is complementary to a specific mRNA and binds to the complementary sequence in the specific mRNA to inhibit translation of the mRNA. For example, the nucleic acid portion of the composition is AD7c-NTP antisense molecule or a nitric oxide synthase III (NOS-3) antisense molecule. An AD7c-NTP antisense template is transcribed into an RNA molecule, which binds to a portion of an endogenous AD7c-NTP mRNA in a target cell or tissue and inhibits translation of the AD7c-NTP mRNA into a AD7c-NTP polypeptide or gene product. Similarly, NOS-3 antisense template is transcribed into an RNA molecule, which binds to a portion of an endogenous NOS-3 mRNA in a target cell or tissue and inhibits translation of the NOS-3 mRNA into a NOS-3 polypeptide or gene product. Preferably, the antisense DNA contains a sequence, which is complementary to a DNA sequence in the AD7c-NTP or NOS-3 cDNA sequence. For example, the DNA is complementary to a 5′ untranslated region of the gene
The composition and method are useful for gene therapy applications or to produce non-human animal model of a human disease state. For example, the nucleic acid portion of the composition contains a sequence encoding a polypeptide such as a AD7c-NTP polypeptide for development of an animal model of Alzheimer's Disease.
The methods are useful to make a non-transgenic model for Alzheimer's Disease. The model is a non-human animal, which contains an exogenous AD7c-NTP nucleic acid in its brain tissue, e.g., in a neuronal cell, of the animal. AD7c-NTP gene product is over-expressed in brain tissues of the animal. The animal model of human Alzheimer's Disease recapitulates the neurodegenerative process and neuropathological changes which occur in the brains of human patients with Alzheimer's Disease. Utilizing the prolonged gene expression system described above, the animal expresses an exogenous AD7c-NTP polypeptide in a neuronal cell of the animal for at least 48 hours and up to and exceeding a period of 4 weeks. Expression has been detected in vivo for up to two months after contacting brain tissue with the exogenous nucleic acid. The animal is a rodent or a non-human primate. Cortical and/or hippocampal neuronal cells express the AD7c-NTP polypeptide over extended periods of time and exhibit physical conditions and symptoms, which mimic human Alzheimer's Disease, e.g., sporadic Alzheimer's Disease. Such conditions include neuritic plaque formation, neuronal cell death by apoptosis (as well as activation of pro-apoptosis genes), and increased levels of phospho-tau, APP and amyloid-b.
Alternatively, the model is a non-human animal, which contains an exogenous nitric oxide synthase nucleic acid, e.g., NOS-3, and which expresses an exogenous nitric oxide synthase polypeptide in a neuronal cell of the animal for at least 48 hours and longer. The physical condition and symptoms of the animal mimic those of human Alzheimer's Disease.
Also within the invention is a method of inhibiting Alzheimer's Disease-associated neuronal cell death, by contacting an AD7c-NTP-overexpressing cell with a composition, which contains an AD7c-NTP antisense nucleic acid (e.g., an antisense template or an antisense ologinucleotide). The composition also contains a histone polypeptide. Optionally, the composition contains an amphipathic compound described above. The AD7c-NTP antisense template (e.g., a DNA) is transcribed into an RNA molecule, which binds to a portion of an endogenous AD7c-NTP mRNA in the overexpressing cell and inhibits translation of the AD7c-NTP gene product. In this manner, AD7c-NTP protein production is regulated and the adverse effects of overproduction reduced. The method is useful as a genetic therapeutic tool to reduce the pathologic symptoms of Alzheimer's Disease, e.g., sporadic Alzheimer's Disease. For example, downregulating pathological overproduction of endogenous AD7c-NTP in this manner leads to decreased production of NOS-3, decreased neuronal cell death, decreased production of amyloid precursor protein (APP) and amyloid-b, as well as decreased formation of neuritic plaques. NOS-3 expression is directly reduced by administering a NOS-3 antisense oligonucleotide or a NOS-3 antisense template.
Alzheimer's Disease associated neuronal cell death is also inhibited by contacting an AD7c-NTP overexpressing neuronal cell wi
de la Monte Suzanne M.
Wands Jack R.
Beattie Ingrid A.
Crouch Deborah
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
Rhode Island Hospital
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