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WO2008049073A2 - Compositions pour le traitement d'une inflammation dans les tissus cérébraux ou autres - Google Patents

Compositions pour le traitement d'une inflammation dans les tissus cérébraux ou autres Download PDF

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WO2008049073A2
WO2008049073A2 PCT/US2007/081826 US2007081826W WO2008049073A2 WO 2008049073 A2 WO2008049073 A2 WO 2008049073A2 US 2007081826 W US2007081826 W US 2007081826W WO 2008049073 A2 WO2008049073 A2 WO 2008049073A2
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tnfα
patient
brain
predisposed
raav
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WO2008049073A3 (fr
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William J. Bowers
Howard J. Federoff
Michelle C. Janelsins
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University of Rochester
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University of Rochester
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

  • This invention relates to compositions and methods for inhibiting the development of inflammation, and more particularly to viral vectors for inhibiting inflammation in tissues such as the brain.
  • AD Alzheimer's Disease
  • AD has multiple proximate initiating mechanisms. Those that are best described are inherited in an autosomal dominant fashion. These relatively rare but nonetheless mechanistically illustrative examples indicate that one major potential cause of AD is related to the accumulation of aberrant proteo lyrically processed amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • a ⁇ small amyloid beta
  • ⁇ -, ⁇ -, and ⁇ -secretase cleavages are referred to as the ⁇ -, ⁇ -, and ⁇ -secretase cleavages.
  • Available evidence suggests that when ⁇ -secretase cleaves the APP molecule, this precludes the pathological generation through ⁇ -secretase activity of A ⁇ fragments 1-40 and 1-42. Under circumstances where ⁇ -secretase cleavage is enhanced or ⁇ -secretase cleavage is diminished, pathological A ⁇ accumulation is augmented.
  • Other non- amyloid models of AD pathogenesis are also supported by extensive cell biologic, molecular biologic, and clinical data. Particularly noteworthy in this regard is the biochemical basis of the neuronal tangle which, to a large extent, reflects the abnormal and increased phosphorylation of the microtubule protein tau.
  • Tau is a microtubule-associated protein with numerous functions within the neuron.
  • One such cellular role is to stabilize and promote the polymerization of microtubules. This function has led to the hypothesis that the inability of tau to adequately bind and promote polymerization of microtubules would result in diminished transport within a neuron.
  • the abnormal morphologic entity in AD patients' brains known as the neurofibrillary tangle is comprised primarily of tau
  • abnormalities of tau directly or indirectly, play a central role in the pathogenesis of AD by progressively leading to a loss of fast axonal transport.
  • tau has been detected within all three neuronal compartments of the neuron.
  • a number of abnormalities of tau have been identified or suggested in AD neurons. These abnormalities include formation of tau into abnormal straight filaments or paired helical filaments, aggregations of paired helical filaments into larger entities known as the neurofibrillary tangles, hyperphosphorylated tau, truncated tau, and the inability of tau to bind microtubules due to phosphorylation of key epitopes within the binding domain. Ongoing studies in numerous laboratories are examining potential links between extracellular A ⁇ and its effect on neuronal cell signaling mechanisms that may regulate tau phosphorylation.
  • the present invention is based, in part, on our discovery that the proinflammatory cytokine tumor necrosis factor-alpha (TNF ⁇ ) is upregulated in the entorhinal cortex of 3xTg- AD mice (a mouse model of AD) at a time that suggests a role for this cytokine in the etiology of AD.
  • the present anti-inflammatory compositions including inhibitors of TNF ⁇ , can be used to treat patients who are predisposed to AD and those believed to have progressed to having AD, albeit at an early stage.
  • the present compositions can also be used to treat patients who are predisposed to other diseases and disorders associated with inflammation.
  • an anti-inflammatory agent is administered intraparenchymally to an affected site
  • the present methods can be used to treat patients who have been diagnosed with AD, including mid- or late-stage AD, or who have been diagnosed with a disease or disorder associated with inflammation. Examples of such diseases and disorders are provided below.
  • a disease or disorder associated with inflammation e.g., a disease or disorder of the nervous system (e.g., the CNS)
  • the patient must be one who is simply predisposed to a disease or disorder associated with inflammation (e.g., a disease or disorder of the nervous system (e.g., the CNS)) or who is just beginning to exhibit symptoms or develop other signs upon which a diagnosis can be made.
  • the patient population includes not only patients who are pre-symptomatic or in the very early stages of disease onset, but also patients who are more readily diagnosed due to overt symptoms or other indications that the disease or disorder is present (e.g. , patients who are more profoundly and clearly affected by an established condition).
  • a particular route of administration e.g., intraparenchymal injection to a targeted region of the nervous system
  • a particular vector e.g., an rAAV vector
  • the patient population includes not only patients who are pre-symptomatic or in the very early stages of disease onset, but also patients who are more readily diagnosed due to overt symptoms or other indications that the disease or disorder is present (e.g. , patients who are more profoundly and clearly affected by an established condition).
  • the invention features methods of treating a patient who is predisposed to AD.
  • the methods include providing (e.g., by identifying) a patient determined to be predisposed to AD and administering to the patient a therapeutically effective amount of an anti-inflammatory agent.
  • the administration when carried out in the event of a determined or suspected predisposition, can be oral or parenteral (e.g., intravenous or intraparenchymal (e.g., into a region of the brain)).
  • the patient can be apparently healthy or have no symptoms of a disease or disorder associated with inflammation (e.g., AD).
  • Embodiments may include one or more of the following features. Physicians are trained to diagnose illness and recognize that some diagnoses are easier to make than others. Physicians can also recognize or diagnose patients who are predisposed to a given condition, including those described herein. For example, a patient can be determined to be predisposed to AD. One or more of the following findings may be considered and may contribute to this determination: advanced age; family history and/or genetic profile; prior head injury, particularly of a severe nature; cardiovascular disease (e.g. , atherosclerosis; particularly of a severe nature); poor and/or progressively declining results in cognitive tests; and memory impairment in the absence of dementia, delirium, brain tumor, cerebrovascular accident, or vitamin B 12 deficiency.
  • cardiovascular disease e.g. , atherosclerosis; particularly of a severe nature
  • the anti-inflammatory agent used in the event of a predisposition to AD, AD, or predisposition or development of another CNS-related disorder associated with inflammation can be a TNF ⁇ inhibitor (e.g. , a soluble TNF ⁇ receptor or a fragment or other variant thereof that binds TNF ⁇ and/or an anti-TNF ⁇ antibody and/or an agent that inhibits the expression of TNF ⁇ (e.g. , a nucleic acid that mediates RNA interference (RNAi) with respect to TNFa)).
  • TNF ⁇ inhibitor e.g. , a soluble TNF ⁇ receptor or a fragment or other variant thereof that binds TNF ⁇ and/or an anti-TNF ⁇ antibody and/or an agent that inhibits the expression of TNF ⁇ (e.g. , a nucleic acid that mediates RNA interference (RNAi) with respect to TNFa)
  • RNAi RNA interference
  • the anti-inflammatory agent is administered by an intraparenchymal injection to a region of the patient'
  • the anti-inflammatory agent can also be administered via a recombinant adenovirus associated viral (rAAV) vector. Regardless of the precise agent used or the route of its administration, the patient can be a human.
  • the invention features methods of treating a patient who is diagnosed as having AD (e.g., mid- to late-stage AD) or who, as described above, is predisposed to AD.
  • the methods include providing the patient and administering to the patient, by an intraparenchymal injection to a region of the brain, a therapeutically effective amount of an anti-inflammatory agent.
  • Embodiments may include one or more of the following features.
  • a patient can be determined to be predisposed to AD in consideration of one or more of the following: advanced age; family history and/or genetic profile; prior head injury, particularly of a severe nature; cardiovascular disease (e.g., atherosclerosis; particularly of a severe nature); poor and/or progressively declining results in cognitive tests; and memory impairment in the absence of dementia, delirium, brain tumor, cerebrovascular accident, or vitamin B 12 deficiency.
  • the anti-inflammatory agent can be a TNF ⁇ inhibitor (e.g., a soluble TNF ⁇ receptor or a fragment or other variant thereof that binds TNF ⁇ and/or an anti-TNF ⁇ antibody and/or an agent that specifically or selectively inhibits the expression of TNF ⁇ (e.g., a nucleic acid that mediates RNA interference (RNAi) with respect to TNFa)).
  • TNF ⁇ inhibitor e.g., a soluble TNF ⁇ receptor or a fragment or other variant thereof that binds TNF ⁇ and/or an anti-TNF ⁇ antibody and/or an agent that specifically or selectively inhibits the expression of TNF ⁇ (e.g., a nucleic acid that mediates RNA interference (RNAi) with respect to TNFa)
  • RNAi RNA interference
  • the region of the patient's brain can include the entorhinal cortex and/or the hippocampus, and the anti-inflammatory agent can be administered via an rAAV vector.
  • the patient can be a human
  • the patient can be determined to be predisposed to AD, and the determination can be made on the basis of the findings described above.
  • the anti-inflammatory agent can be an agent that inhibits the expression or activity of TNF ⁇ .
  • the agent can be a soluble TNF ⁇ receptor or a fragment or other variant thereof that binds TNF ⁇ and/or an anti-TNF ⁇ antibody and/or an agent that specifically or selectively inhibits the expression of TNF ⁇ (e.g., a nucleic acid that mediates RNA interference (RNAi) with respect to TNF ⁇ ).
  • RNAi RNA interference
  • the rAAV vector can be administered by various routes, including by an intraparenchymal injection into a region of the brain (e.g., a region including the entorhinal cortex or the hippocampus).
  • the patient can be a human.
  • the invention features an rAAV vector that includes a nucleic acid sequence encoding a protein having anti-inflammatory properties.
  • the vector can further include a nucleic acid sequence encoding a protein that increases the circulating half- life of an anti-inflammatory protein to which it is fused. That is, the vector can encode a chimeric or fusion protein including an anti-inflammatory protein and a protein that increases the circulating half- life of the anti-inflammatory protein relative to that of the anti-inflammatory alone.
  • the protein that increases the circulating half-life can be, for example, an immunoglobulin or a portion thereof (e.g., the Fc region of an immunoglobulin (IgG)) or an albumin (e.g., human albumin) or a portion thereof sufficient to increase the circulating half- life.
  • the protein is a TNF ⁇ inhibitor (e.g., a soluble TNF ⁇ receptor; a fragment or other variant thereof that selectively binds TNF ⁇ ; or any of the other inhibitors known and/or described herein).
  • the invention features an rAAV vector that includes a nucleic acid sequence encoding an inflammatory agent, such as TNF ⁇ (e.g., human TNF ⁇ ).
  • TNF ⁇ e.g., human TNF ⁇
  • Such a vector can be administered to an animal, and non-human animals (e.g., transgenic mice including the 3xTg-AD mice described herein) bearing an rAAV vector expressing an inflammatory or antiinflammatory agent (e.g., TNF ⁇ or a soluble TNF ⁇ receptor) are within the scope of the present invention.
  • TNF ⁇ e.g., human TNF ⁇
  • TNF ⁇ e.g., human TNF ⁇
  • an inflammatory or antiinflammatory agent e.g., TNF ⁇ or a soluble TNF ⁇ receptor
  • TNF ⁇ -expressing animals are therefore useful to understand how TNF ⁇ influences the early stages of AD and will further define the role of this cytokine on pathological outcomes.
  • the present invention can also be characterized in terms of use.
  • the invention features the use of an anti-inflammatory agent, including the TNF ⁇ inhibitors known in the art and/or described herein as well as other anti-inflammatory agents, in the preparation of a medicament for the treatment of a patient who is predisposed to, but has not yet developed or fully developed, a disease or disorder associated with inflammation in the nervous system (e.g., the CNS).
  • a disease or disorder can be any of those described herein, and the inhibitor, when configured as a protein, can be delivered via expression from an rAAV vector.
  • Figure 1 is a Table (Table 1) listing various immune markers and their currently understood major functions. These proinflammatory markers were investigated in the temporal and spatial progression of early AD pathogenesis.
  • Figure 2 is a Table (Table 2) summarizing the results of expression analyses of the listed immune markers in the entorhinal cortices of 3xTg-AD mice at three and six months of age and in age-matched non-transgenic mice. The results are expressed as the fold change relative to levels measured at two months of age. TNF ⁇ and MCP-I mRNA levels are selectively elevated in the entorhinal cortex of 3xTg-AD mice prior to overt amyloid plaque pathology.
  • Figure 3 is a Table (Table 3) summarizing the results of expression analyses of the listed immune markers in the hippocampi of 3xTg-AD mice at three and six months of age and in age-matched non-transgenic mice. The results are expressed as the fold change relative to levels measured at two months of age. Inflammation-related transcript levels remained stable in the hippocampus of 2, 3, and 6 month-old 3xTg-AD and control mice.
  • Figure 4 is a bar graph showing the number of F4/80-positive cells in the entorhinal cortex of 3xTg-AD mice and non-transgenic mice at two and six months of age.
  • the 3xTg-AD entorhinal cortex harbors an increased number of macrophages/microglia at six months of age. Error bars indicate standard deviation.
  • N 4 per genotype per time point. "*" indicates p ⁇ 0.008.
  • Figure 5 is a bar graph showing the number of F4/80-positive cells in the hippocampus of 3xTg-AD mice and non-transgenic mice at two and six months of age.
  • the 3xTg-AD hippocampus does not have an increased number of macrophages/microglia at six months of age. Error bars indicate standard deviation.
  • N 4 per genotype per time point.
  • compositions and methods can be used to treat patients who are predisposed to a central nervous system (CNS) disease or disorder associated with inflammation (e.g. , AD) and, in certain embodiments, patients believed to have progressed to manifest signs or symptoms of such diseases or disorders (e.g., mid- or late-stage AD).
  • CNS disease or disorder e.g., AD
  • the methods applicable to patients who are predisposed to a CNS disease or disorder include the step of administering to a patient a therapeutically effective amount of an anti-inflammatory agent (e.g., gene-based or small-molecule based modulator of TNF ⁇ activity, e.g., a TNF ⁇ inhibitor).
  • the agent can be administered by an intraparenchymal injection to a region of the brain, e.g., to the entorhinal cortex of the brain.
  • the agent can be administered via rAAV.
  • CNS diseases or disorders associated with inflammation Inflammation and inflammatory mediators contribute to acute, chronic and psychiatric CNS disorders, which can be treated by administration of an anti-inflammatory agent as described herein.
  • the present methods and compositions can be administered to subjects who are predisposed to or who have progressed into a CNS disease or disorder associated with inflammation.
  • disorders are diverse and include, for example, acute brain injury (as may result from a surgical procedure performed on the brain), acute spinal cord injury, stroke and cerebral ischaemia, epilepsy, multiple sclerosis, motor neuron disease, movement disorders, disorders of related systems of the retina and of muscle, including optic neuritis, macular degeneration, diabetic retinopathy, and dermatomyositis, AD, Parkinson's disease, Huntington's disease, Guillain Barre syndrome, myasthenia gravis, amyotropic lateral sclerosis, Creutzfeldt- Jakob disease, progressing motor neuron disease, depression, anxiety, bipolar disorder, and schizophrenia (see, Lucas et al, Br. J. Pharmacol. I47:S232-S240, 2006).
  • Factors to be considered in determining predisposition to AD include, for example, acute brain injury (as may result from a surgical procedure performed on the brain), acute spinal cord injury, stroke and cerebral ischaemia, epilepsy, multiple sclerosis, motor neuron disease
  • Age The greatest known risk factor for AD is increasing age. Most individuals with the disease are 65 or older, and the likelihood of developing AD doubles about every five years after age 65. After age 85, the risk reaches nearly 50 percent.
  • Family history Another risk factor is family history. A person who has a parent, brother or sister, or child with AD is more likely to develop AD. The risk increases if more than one family member has the illness. Genetic factors, environmental factors, or both can play a role.
  • APOE-e4 apoliprotein E-e4
  • APOE-e4 is one of three common forms of the APOE gene. Inheriting one copy of APOE-e4 predisposes one to AD, and inheriting two copies increases the risk further. In addition, APOE-e4 may tend to make symptoms appear at a younger age than usual. There may be as many as a dozen other AD "risk" genes.
  • Some proposed candidate risk genes include, for example, butyryl-cholinesterase, angiotensin- 1 converting enzyme, the human leucocyte antigen (HLA) complex, ⁇ 2 -macroglobulin, the low-density lipoprotein-like receptor, the serotonin transporter, ubiquilin-1, interleukin-1, and perhaps VLDL receptor, and ⁇ i- antichymotrypsin (Holmes, Br. J. Psych. 180:131-34, 2002).
  • HLA human leucocyte antigen
  • ⁇ 2 -macroglobulin the low-density lipoprotein-like receptor
  • the serotonin transporter ubiquilin-1, interleukin-1, and perhaps VLDL receptor
  • ⁇ i- antichymotrypsin Holmes, Br. J. Psych. 180:131-34, 2002.
  • AD Deterministic genes are believed to directly cause disease. With respect to AD, these genes have been found in only a few hundred extended families worldwide, and these patients are said to have "familial” AD. Genetic tests are available for both APOE-e4 and the rare genes that directly cause AD.
  • Head injury There appears to be a strong link between serious head injury and future risk of AD (see, e.g., Fleminger et ah, J. Neurol. Neurosurg. Psych. 74:857-62, 2003). There seems to be a gender difference in this link, with males who have had head injury developing AD at a higher rate than females. A possible explanation for this is the role of female hormones, estrogen and progesterone (Fleminger et al ).
  • Cardiovascular disease The risk of developing AD or vascular dementia appears to be increased by many conditions that damage the heart or blood vessels. These include high blood pressure, heart disease, stroke, diabetes and high cholesterol.
  • vascular cognitive impairment is a term that takes into account the spectrum of severity of cognitive impairment associated with vascular disease (e.g., mild, moderate and severe - also called vascular dementia). AD and vascular dementia share some common risk factors.
  • Risk factors for vascular dementia include: demographic (e.g., age, male sex), atherosclerotic (e.g., history of hypertension, diabetes mellitus, hyperlipedemia), genetic (familial vascular encephalopathies, apoE e4), and stroke-related (e.g., white matter disease, volume of cerebral tissue loss) (Gore lick, Stroke 35_[suppI]:2620-22, 2004).
  • Risk factors for AD include many atherosclerotic risk factors, e.g., hypertension, diabetes mellitus, smoking, lipids, hyperinsulemia, homocysteine, physical inactivity, and fat consumption (Gorelick infra). Dietary factors (e.g., fish consumption, vitamins E and C) may protect against AD.
  • vascular risk factors can have negative effects on the brain and cognitive abilities.
  • Dietary factors play major roles in development of neurodegenerative disorders. Dietary factors may interact with AD-causing or predisposing genes. In particular, it known that high-calorie diet and folic acid deficiency increase the risk for AD. Deficiency in Vitamin B 12 may also impact development of AD .
  • Anti-inflammatory agents Some key inflammatory mediators in CNS diseases and disorders include, e.g., interleukin-1, TNF ⁇ , interleukin-6, prostaglandins, nitric oxide, complement, microglial activation, acute phase proteins, interleukin-8, soluble interleukin-2 receptor, and interferon- ⁇ (Lucas et al., infra). Anti-inflammatory agents that target these and other CNS inflammatory mediators are encompassed by the methods and compositions of this invention.
  • Potential anti-inflammatory agents include, e.g., interleukin-1 receptor antagonists, caspase-1 inhibitors, soluble receptor TNF ⁇ converting enzyme, COX inhibitors, iNOS inhibitors, complement antagonists, statins, interferon ⁇ , NSAIDs, minocycline, naloxane, and others (see, e.g., Lucas et al., infra).
  • TNF ⁇ is one of the central mediators of tissue inflammation and has been implicated in the pathogenesis of many neurological conditions. Specific inhibitors of TNF ⁇ provide the possibility of prevention and therapeutic intervention in TNF -mediated CNS diseases and disorders, e.g., in AD. For example, thalidomide, 4 (IV) inhibitor thalidomide analogues, and other phosphodiesterase IV inhibitors can function as anti-TNF agents. TGF ⁇ has also been shown to inhibit TNF ⁇ (see U.S. Patent No. 5,650,396). Further, anti-TNF therapies may include gene therapy, including RNAi, and the development of selective inhibitors of the TNF ⁇ converting enzyme.
  • TNF ⁇ inhibitors can also include peptides that bind to TNF ⁇ (see, e.g., U.S. Patent No. 5,698,195). Some TNF ⁇ inhibitors are described in U.S. Patent No. 6,177,077, the contents of which are hereby incorporated in their entirety by reference.
  • TNFa Receptors or TNFa-binding fragments thereof Proteins that inhibit TNF ⁇ have been detected in normal human urine and in the sera of patients with cancer or endotoxemia. These proteins have since been identified as the extracellular domains of TNF receptors, and may be referred to as soluble TNF ⁇ receptors. Any naturally occurring or non-naturally occurring receptor that binds and thereby inhibits TNF ⁇ can be used in the present methods. These receptors include those described by Aderka et al, Isrl. J. Med. Sci. 28:126-130, 1992 (sTNF-Rs); Seckinger et al. , J. Exp. Med. 167: 1511-1516, 1988; Engelmann et al. , J. Biol.
  • TNF-binding proteins originally purified from human urine, that have an approximate molecular weight of 30 kDa and bind TNF ⁇ more effectively than TNF ⁇ can also be used (Engelmann et al., J. Biol. Chem. 265:1531-1536, 1990; see also Engelmann et al., J. Biol. Chem. 264:11974-11980, 1989).
  • TNF inhibitors TNF-I
  • European Patent publication number 0 433 900 Al WO 92/13095
  • European Patent Publication number 0 526 905 A2 disclosing, inter alia, multimers
  • WO 92/07076 disclosing, inter alia, receptors lacking the fourth cysteine-rich subdomain of the extracellular binding domain of the 55 kDa TNF receptor
  • European Patent Publication number 0 398 327 Al European Patent Publication 0 308 378 A2.
  • the contents of these references are hereby incorporated by reference in their entirety.
  • Anti-TNFa antibodies and fragments thereof Some therapeutic success has been demonstrated with infliximab (REMICADETM from Centocor), a chimeric anti-TNF monoclonal antibody (mAb), in treating Crohn's Disease and rheumatoid arthritis; and with etanercept (ENEBRELTM from Immunex Corporation), a recombinant fusion protein consisting of two soluble TNF receptors joined by the Fc fragment of a human IgGl molecule, in treating rheumatoid and psoriatic arthritis.
  • infliximab REMICADETM from Centocor
  • mAb chimeric anti-TNF monoclonal antibody
  • ENEBRELTM etanercept
  • Adalimumab is a humanized IgGl monoclonal antibody for use in rheumatoid arthritis (Nash et al, MJA 183:205-208, 2005).
  • Polyclonal murine antibodies to TNF are disclosed by Cerami et al. (EPO Patent Publication 0212489, Mar. 4, 1987). Such antibodies were said to be useful in diagnostic immunoassays and in therapy of shock in bacterial infections. Rubin et al.
  • EPO Patent Publication 0218868, Apr. 22, 1987 discloses murine monoclonal antibodies to human TNF, the hybridomas secreting such antibodies, methods of producing such murine antibodies, and the use of such murine antibodies in immunoassay of TNF.
  • Yone et al. discloses anti-TNF murine antibodies, including mAbs, and their utility in immunoassay diagnosis of pathologies, in particular Kawasaki's pathology and bacterial infection.
  • Other investigators have described rodent or murine mAbs specific for recombinant human TNF which had neutralizing activity in vitro (Liang, et al. Biochem. Biophys. Res. Comm.
  • Some of these mAbs were used to map epitopes of human TNF and develop enzyme immunoassays (Fendly et al., infra; Hirai et al., infra; Moller et al., infra) and to assist in the purification of recombinant TNF (Bringman et al., infra).
  • Neutralizing antisera or mAbs to TNF have been shown in mammals other than man to abrogate adverse physiological changes and prevent death after lethal challenge in experimental endotoxemia and bacteremia. This effect has been demonstrated, e.g., in rodent lethality assays and in primate pathology model systems (Mathison, et al, J. Clin. Invest. 81:1925-1937, 1988; Beutler, et al, Science 229:869-871, 1985; Tracey, et al, Nature 330:662-664, 1987; Shimamoto, et al, Immunol. Lett. 17:311-318, 1988; Silva, et al, J. Infect. Dis. 162:421-427, 1990; Opal, et al, J. Infect. Dis. 161:1148-1152, 1990; Hinshaw, et al, Circ. Shock 30:279-292, 1990).
  • Putative receptor binding loci of hTNF has been disclosed by Eck and Sprang, J. Biol. Chem. 264(29): 17595-17605, 1989, who identified the receptor binding loci of TNF ⁇ as consisting of amino acids 11-13, 37-42, 49-57 and 155-157.
  • TNF ligands which can bind to monoclonal antibodies having the following epitopes: at least one of 1-20, 56-77, and 108-127; at least two of 1-20, 56-77, 108-127 and 138-149; all of 1-18, 58-65, 115-125 and 138-149; all of 1-18, and 108-128; all of 56-79, 110- 127 and 135- or 136-155; all of 1-30, 117-128 and 141-153; all of 1-26, 117-128 and 141-153; all of 22-40, 49-96 or 49-97, 110-127 and 136-153; all of 12-22, 36-45, 96-105 and 132-157; both of 1-20 and 76-90; all of 22-40, 69-97, 105-128 and 135-155; all of 22-31 and 146-157; all of 22-40 and 49-98; at least one of 22-40, 49
  • the antibody can have a non-human variable region or a TNF antigen-binding portion thereof and a human constant region.
  • the antibody can also be humanized or fully human.
  • the antibody e.g., a chimeric antibody
  • TNF ⁇ inhibitors of the present invention can downregulate and/or inhibit TNF ⁇ signaling pathways. Soluble TNF ⁇ signals predominantly through TNFRl , resulting in recruitment of TRADD, RIPl, TRAF2, TRAFl, clAPl and 2 and TRAF2 and activation of the IKK/NF- ⁇ B pathway. This cascade initiates the production of other proinflammatory cytokines and genes involved in adaptive immunity, although it can also attract FADD, resulting in a caspase 8-mediated apoptosis.
  • the transmembrane form of TNF ⁇ typically signals through TNFR2, recruiting TRAF2, TRAFl, and clap 1 and 2, activating the JNK/cJun pathway that stimulates pro- apoptotic gene expression.
  • Therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a therapeutic result.
  • a therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of an anti-inflammatory agent ⁇ e.g., binding protein of TNF ⁇ ) to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter, e.g., inflammation or a sign or symptom of a CNS disorder or disease associated with inflammation.
  • an agent to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in human patients.
  • this property of the agent can be evaluated by examining the ability of the agent to function in in vitro by assays known to the skilled practitioner.
  • Treating a patient who is predisposed to a condition is not limited to instances where the risk of progression to a disease state is reduced to zero. "Treating" encompasses a reduction in risk, a prolonging of the period before progression to disease, a prolonging of the advancement of the disease, which may be especially noticeable in steadily progressive disorders, prophylaxis, or a reduction in the frequency or severity of the symptoms that may eventually develop.
  • Anti-inflammatory agents e.g., TNF ⁇ inhibitors
  • TNF ⁇ inhibitors can be administered either as individual therapeutic agents or in combination with one another and/or other therapeutic agents and/or therapeutic regimes ⁇ e.g., changes in diet and exercise routines and memory- strengthening exercises).
  • Proteinaceous inhibitors can be administered as proteins or their delivery can be facilitated by any suitable vector construct such as a plasmid or rAAV vector.
  • Pharmaceutical carriers can be selected on the basis of the chosen route of administration and standard pharmaceutical practice. The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight.
  • 1.0 to 5, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • treatment of the conditions described herein, including AD in humans can be provided as a daily dosage of anti-inflammatory agent, e.g., TNF ⁇ inhibitor, at 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • anti-inflammatory agent e.g., TNF ⁇ inhibitor
  • the inhibitor may be administered only a limited number of times ⁇ e.g., one, two, or three times).
  • the rAAV vectors can produce long-term, chronic production of an anti-inflammatory agent and may, therefore, provide long-term benefit following a single administration (which may, of course, be repeated as necessary).
  • the anti-inflammatory agents of the present invention can be administered by any of the following methods, with the stipulations provided otherwise herein, to treat the diseases and disorders describe herein: subcutaneous, intravenous, intrathecal, intramuscular, intranasal, oral, transepidermal, parenteral, by inhalation, intracerebroventricular, or intraparenchymal.
  • a preferred embodiment of administration is intraparenchymal injection.
  • the intraparenchymal injection can be carried out to the exclusion of intrathecal and intracerebroventricular injections.
  • a patient may be assessed over time for the appearance or progression of disease symptoms (e.g., weekly, monthly, annually, or semi-annually), and the treatment regime can be adjusted accordingly.
  • the treatment regime can be adjusted accordingly.
  • Recombinant adeno-associated virus vector The anti-inflammatory agents of the present invention, e.g., TNF ⁇ inhibitors, can be administered via an rAAV vector e.g., rAAV2.
  • AAV is a replication-deficient parvovirus native to humans and nonhuman primates that exists in nature in over 100 distinct variants. Many of the AAV serotypes have distinct cell and tissue affinities.
  • AAV has a number of properties that support its potential role in gene therapy, for example, it is generally non-pathogenic, capable of persistent infection, and generally elicits mild innate cytokine response. In addition, its genome is readily modified in proviral plasmids, and recombinant production and purification methods are already in place (see, e.g., Flotte Pediatric Res. 58:1143-1147, 2005).
  • Adeno-associated virus is an integrating human DNA parvovirus which has been proposed for use as a gene delivery vehicle for somatic gene therapy.
  • This small non- enveloped virus contains a 4.6 kb single stranded DNA genome that encodes sets of regulatory and capsid genes called rep and cap.
  • Rep polypeptides rep78, rep68, rep62 and rep40
  • the cap proteins VPl, VP2 and VP3 form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures.
  • AAV life cycle of AAV is characterized by both lytic and latent components (B. J. Carter, in Handbook of Parvoviruses , ed., P. Tijsser, CRC Press, pp.155-168, 1990).
  • ss single stranded
  • the lytic phase of the life cycle begins when a cell harboring an AAV pro virus is challenged with a secondary infection by a herpesvirus or adenovirus which encodes helper functions that are recruited by AAV to aid in its excision from host chromatin (Carter, supra).
  • the infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner.
  • the rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or - ss DNA genomes following cell lysis.
  • Recombinant forms of AAV have been developed as vectors by replacing all viral open reading frames with a therapeutic minigene, while retaining the necessary cis elements contained in the ITRs.
  • rAAV Recombinant forms of AAV
  • U.S. Patent Nos. 4,797,368; 5,153,414; 5,139,941; 5,252,479; and 5,354,678; and International Publication Nos. WO91/18088 published Nov. 28, 1991; WO93/24641 published Dec. 9, 1993 and WO94/13788 published Jun. 23, 1994 See also O.S. Patent Nos. 5,756,283; 7,282,199; 7,241,447; and 7,208,315.
  • Transduction with rAAV has been demonstrated in a wide variety of cell types including differentiated, non-dividing cells, suggesting the potential of this vector system for in vivo gene delivery to organs such as muscle, liver, central nervous system and lung.
  • Inflammatory processes have been intimately associated with classic AD pathology in the post-mortem human brain, where evidence of astrogliosis and activated microglia in the vicinity of amyloid plaques has been readily observed (Vehmas et al. , Neurobiol. Aging 24:321-331, 2003. Implication of inflammatory mediators in early pathogenic events during pre-symptomatic stages of AD, however, has not been clearly defined at present due to limited availability of early-stage human clinical samples and a lack of animal models that faithfully recapitulate the human disorder.
  • triple-transgenic AD mouse (xTg- AD) presently represents the most advanced animal model available in that it harbors three AD-relevant genetic alterations, which result in spatial distribution and progression of amyloid and tau pathologies strikingly similar to human AD.
  • 3xTg- AD triple-transgenic AD mouse
  • the entorhinal cortex and hippocampus were the regions chosen because of the evidence implicating these regions in the earliest stages of disease (Braak and Braak, Neurobiol. Aging 16:271-278, 1995; Pennanen et al., Neurobiol. Aging 25:303-310, 2004; de Toledo-Morrell et al, Neurobiol. Aging 25: 1197-1203, 2004; Braak and Braak, Int. Psychogeritr. 9 Suppl 1 :257-261, 1997).
  • We have observed that expression of the pro-inflammatory cytokine TNF ⁇ is enhanced at periods prior to the appearance of symptoms and pathology of AD .
  • RNA was isolated from microdissected hippocampus- or entorhinal cortex-enriched tissue from 2, 3, and 6 month-old 3xTg-AD and non-transgenic mice with TRIzol solution (Invitrogen, Carlsbad, CA). RNA was treated with RQ DNAse I (Promega, Madison, WI) to selectively degrade any contaminating genomic DNA, followed by phenol: chloroform extraction and ethanol precipitation. One microgram of total RNA was reverse transcribed using Applied Biosystems High-Capacity cDNA Archive Kit.
  • RNA served as the control to which all samples were normalized (Applied Biosystems, Foster City, CA).
  • ⁇ C T method normalizing the 3 and 6 month-old 3xTg-AD and control mouse samples to the 2 month-old 3xTg-AD and non-transgenic samples, respectively.
  • Sections were washed four times for 3 minutes each in PB to remove cyroprotectant. To quench endogenous peroxidase activity, sections were incubated for 25 minutes with 3% H 2 O 2 (Sigma). Sections were mounted onto slides and allowed to dry. Slides were incubated in 0.15 M PB + 0.4% Triton-XIOO for 5 minutes at room temperature (RT; 22°C) to permeabilize the tissue. The slides were then incubated with blocking solution containing 3% normal goat serum, 3% bovine serum albumin, and 0.4% Triton-X 100 in 0.15 M PB for 1 hour. Slides were incubated with rat monoclonal anti-F4/80 antibody (Serotec, 1 : 100) overnight in blocking solution.
  • slides were washed eight times for 3 minutes each with 0.15 M PB prior to incubation with Vectastain biotinylated goat antiimmunoglobulin (Vector Laboratories, Burlingame, CA) for 2 hours at RT. Excessive secondary antibody was washed in 0.15 M PB and incubated with A and B reagents (Vector Laboratories, Burlingame, CA) to conjugate HRP. Slides were developed using a DAB peroxidase kit, according to manufacturer's instructions for nickel enhancement (Vector Laboratories, Burlingame, CA).
  • Positively stained F4/80-expressing cells were visualized using an Olympus AX-70 microscope equipped with a motorized stage (Olympus, Melville, NY) and the MCID 6.0 Elite Imaging Software (Imaging Research, Inc.). Sections were analyzed under 4x magnification. Five equal sections of entorhinal cortex and seven equal sections of hippocampus from each mouse (4 mice total) per time point were analyzed. Fifty percent of the defined region of interest in the entorhinal cortex or hippocampus was assessed, under 60 x magnification. The coordinates from which sections were chosen for the entorhinal cortex were 2.92 mm to 4.04 mm posterior from Bregma.
  • the sections counted in the hippocampus were from 1.70 mm to 3.40 mm posterior from Bregma.
  • Qualitative immunohistochemical analysis of amyloid deposition in 3xTg-AD and non- transgenic mice Sections were washed three times for 5 minutes each, then twice for 30 minutes in PBS to remove cryoprotectant. To quench endogenous peroxidase activity, sections were incubated with 3% H 2 O 2 and 3% methanol for 25 minutes. Sections were then washed twice for 5 minutes each with PBS, followed by epitope retrieval treatment with 90% formic acid for 5 minutes at RT. Next, sections were washed twice for 5 minutes each with PBS.
  • Tissue was permeabilized with PBS + 0.1% PBS/Triton-X 100. Sections were then incubated for 1 hour at RT with PBS + 0.1% PBS/Triton-X 100 + 10% normal goat serum. Sections were incubated overnight at 4°C with primary 6E 10 antibody (Signet, 1 : 1000) in PBS 0.1 % PBS/Triton-X 100 + 1% normal goat serum. Samples were washed twice for 10 minutes each with PBS + 0.1% Triton-X 100 + 1% normal goat serum prior to addition of secondary antibody.
  • the mouse HRP ABC kit was used according to manufacturer's protocol (Vector Laboratories, Burlingame, CA). Excessive secondary antibody was washed in PBS and developed using a DAB peroxidase kit, according to manufacturer's instructions for nickel enhancement (Vector Laboratories, Burlingame, CA) and mounted on slides. We obtained the following results.
  • Age-dependent accumulation of human APPswe in 3xTg-AD Mice We performed 6E10 immunohistochemistry on brain sections derived from 2, 3, and 6 month-old 3xTg-AD mice to establish time sequence of APPswe gene expression. At two months of age, 3xTg-AD did not exhibit immunocytochemically detectable levels of APPswe, but at subsequent time points, a robust staining for human APPswe was readily apparent in the entorhinal cortex and hippocampus. This pattern of staining precedes the appearance of extracellular amyloid plaque deposition and mirrors our results using the APP-specific antibody 22Cl 1, suggesting that the species being detected by the 6E10 antibody in this experiment is most likely the human
  • APPswe transgene-derived precursor form of the protein did not exhibit any detectable APPswe in the brain regions examined.
  • Pro-inflammatory transcript profiling of SxTg-AD and non-transgenic mouse entorhinal cortex and hippocampus reveals temporal and spatial expression of TNFa and MCP-I: We predicted that if inflammation was involved at the earliest stages of the disease process, we would observe the coordinate expression of immunomodulatory molecules between 3 and 6 months of age, when intracellular A ⁇ begins to accumulate in the entorhinal cortex and hippocampus of 3xTg-AD mice.
  • AD is a complex chronic disorder with varying pathologic sequelae from which the underlying causative mechanisms are unknown.
  • Activation of microglia and astrocytes, and the presence of many inflammatory mediators, including cytokines, chemokines and complement proteins have been only identified in the post-mortem AD brain in the vicinity of senile plaques and NFTs (Vehmas et al., Neurobiol. Aging 24:321-331, 2003; Afagh et al, Exp. Neurol. 138:22- 32, 1996).
  • cytokines cytokines
  • chemokines chemokines
  • inflammatory processes precede significant extracellular amyloid plaque deposition in the 3xTg-AD brain, substantiated by increased TNF ⁇ and MCP-I transcript levels, coincident temporally with the production of intracellular A ⁇ accumulation.
  • the expression of these molecules is spatially localized to the entorhinal cortex but not hippocampus at the early time-points assessed.
  • TNF ⁇ can be expressed by astrocytes, microglia and neurons in response to various stimuli in the CNS (Hanisch, Glai 40:140-155, 2002). Initially, TNF ⁇ is an innate mediator, promoting chemokine and cytokine expression and extravasation of other immune cells. One possible mechanism that may implicate TNF ⁇ in contributing to AD pathogenesis is evidence that it can increase A ⁇ peptide production (Blasko et al., Neurobiol. Dis. 7:682-689, 2000).
  • inflammatory molecule signaling may cause increased cleavage of APP by the ⁇ - secretase complex, whereby TNF ⁇ , IL-I ⁇ , and IFN- ⁇ have been shown to enhance production of A ⁇ peptides via a ⁇ -secretase-dependent mechanism in vitro.
  • antagonizing TNFRl signaling can lead to diminished ⁇ -secretase activity (Liao et ah, J. Biol. Chem. 279:49523-49532, 2004).
  • Further evidence supporting pathogenic effects of TNF ⁇ -mediated signaling is TNFRl and TRADD, a TNF receptor adaptor protein that allows for NF- ⁇ B and JNK activation, are both increased in AD tissue and APPs we mice.
  • MCP-I is a chemokine that is expressed by microglia and astrocytes that facilitates extravasation of immune cells expressing its cognate receptor, CCR2, to cross the blood brain barrier and guides them to the site of damage.
  • CCR2 cognate receptor
  • TNF ⁇ and MCP-I were the only two that changed significantly over time, possibly signifying their importance during nascent stages of AD pathogenesis. Perhaps, the other inflammatory targets are triggered at later stages of the disease in response to further neurodegenerative events.
  • Exogenously applied A ⁇ can trigger the expression of cytokines in vitro and when injected directly into the mouse brain (Tuppo and Arias, Int. J. Biochem. Cell Biol. 37:289-305, 2005; Perry et ah, Neurobiol. Aging 22:873-883, 2001).
  • TNF ⁇ and MCP-I was detected specifically within the entorhinal cortex and not the hippocampus, despite the fact that immunocytochemically detectable intraneuronal A ⁇ increased over time in both brain regions.
  • TNF ⁇ and MCP-I transcript levels were elevated at 3 months of age in 3xTg-AD mouse entorhinal cortex, and were increased to statistical significance by 6 months of age. This suggests a state of chronic up-regulation and positive-feedback for the expression of both of these inflammatory molecules. Therefore, we are unable to conclude, as detected by the methodology employed in this study, that intracellular A ⁇ accumulation is the sole contributing factor promoting TNF ⁇ and MCP-I transcript expression specifically in the entorhinal cortex.
  • these molecules are neuronally expressed within the entorhinal cortex because they are inherently more sensitive to accumulating A ⁇ , as other neurons were shown previously to express both of these molecules during times of stress (Hanisch, GHa 40:140-155, 2002).
  • the entorhinal cortex elaborates inflammatory molecule expression owing to the structure of A ⁇ elaborated.
  • oligomeric A ⁇ is posited to be the more toxic structural intermediate arising during A ⁇ fibrillogenesis, and this form has been shown to readily induce cytokine expression in vitro (Lindberg et al., J. MoI. Neurosci. 27:1-12, 2005).
  • APPswe/CCL2 mice demonstrate enhanced microglial numbers that are concurrent with increased extracellular A ⁇ deposition, possibly due to an inability to effectively clear A ⁇ (Yamamoto et al., Am. J. Pathol. 166:1475-1485, 2005). This may relate partially to the increased ApoE levels observed in APPswe/CCL2 mice produced by microglia and macrophages. If a similar mechanism is at play in the 3xTg-AD mouse model, this finding suggests a pathogenic role for these cells in initiating degeneration within the entorhinal cortex. In summary, our results indicate an early role for inflammatory processes in the temporal and spatial evolution of AD pathogenesis.
  • TNF ⁇ and MCP-I are produced specifically within the entorhinal cortex where human AD has been shown to arise, these molecules are likely playing an instrumental role in disease perpetuation.
  • This work provides insight into the involvement of TNF ⁇ and MCP-I mediated inflammation in the temporal and spatial progression of early AD pathogenic events.
  • Our use of the 3xTg-AD model to assess these early events is unique, as all previous studies have examined inflammatory processes in the context of either amyloid or tau pathology, but not both. This transgenic mouse allows us to directly examine the dynamic interplay of inflammation, A ⁇ pathology, and tau dysfunction.
  • Intrahippocampal infusion of r AAV-TNFa leads to marked changes in intraneuronal A ⁇ immunopositivity, microglial activation and enhancement of TNFa downstream targets:
  • rAAV vector expressing human TNF ⁇ to create a localized, chronic inflammatory environment in the 3xTg-AD brain at a time prior to pathology to reveal downstream effects in the CAl region of the hippocampus.
  • Verification of TNF ⁇ production from the rAAV2-TNF ⁇ construct was carried out by transfecting HEK293A cells with the construct or transducing the cells with 109 particles of rAAV2-TNF ⁇ .
  • the culture supernatants were measured for presence of TNF ⁇ by ELISA (R&D Systems) by manufacturer's instructions.
  • Quantitative real-time PCR analysis of downstream targets of TNF ⁇ was carried out by microdissecting hippocampi of 6 month 3xTg-AD mice injected with rAAV2-TNF ⁇ or rAAV2-GFP.
  • RNA was isolated with Trizol (Invitrogen) and DNaseI treated with RQl DNase (Promega).
  • cDNA was produced using Applied Biosystems High Capacity cDNA Archive kit. lOOng of cDNA was used in a qRT-PCR reaction using primers/probes in Micro fluidic cards (Applied Biosystems). 18s RNA served as the control to which all samples were normalized.
  • rAAV vector expressing soluble TNF receptor antagonist (s. TNF-Rl -Ig) is being administered to AD mice prior to the appearance of pathological features. This vector will be administered within the entorhinal cortex to assess whether inhibition of TNF ⁇ signaling, prior to initial endogenous TNF ⁇ expression, will diminish onset and severity of AD.
  • a rAAV vector harboring a mouse intestinal alkaline phosphatase (MIAP) transcription unit will serve as a non-immunogenic negative control.
  • MIAP mouse intestinal alkaline phosphatase
  • Recombinant AAV-lac particles can be prepared using a baculovirus-based method of packaging and purified by CsCl ultracentrifugation (Urabe et al, Hum. Gene Ther. 13:1935-1943, 2002). Titering can be performed on HT-1080 cells.
  • a manufacturer's kit and protocol can be used to detect the insert ⁇ e.g., sTNFRl-Ig; R&D systems).
  • Particle numbers for injection can vary.
  • stereotactic injections were made with 5 x 10 9 particles of rAAV virus into the entorhinal cortex (Bregma: -3 mm; Lateral: 3.5 mm; Ventral: -4.25 mm).

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Abstract

La présente invention concerne des procédés permettant de traiter un patient prédisposé à la maladie d'Alzheimer (MA). Les procédés selon l'invention consistent à diagnostiquer la prédisposition d'un patient à la maladie d'Alzheimer, et à administrer à ce patient une quantité thérapeutiquement efficace d'un agent anti-inflammatoire.
PCT/US2007/081826 2006-10-18 2007-10-18 Compositions pour le traitement d'une inflammation dans les tissus cérébraux ou autres Ceased WO2008049073A2 (fr)

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