Classifications

• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders

Definitions

• PROTEINS GENES AND THEIR USE FOR DIAGNOSIS AND TREATMENT OF VASCULAR DEMENTIA
• the present invention relates to the identification of proteins and protein isoforms that are associated with Vascular Dementia and its onset and development, and of genes encoding the same, and to their use for e.g., clinical screening, diagnosis, prognosis, therapy and prophylaxis, as well as for drug screening and drug development.
• vascular dementia is the second most common cause of dementia in the US and Europe and a very heterogeneous disease with many factors contributing to the overall pathogenesis.
• Eight major types of vascular dementia have been identified: 1. Multi-infarct dementia secondary to large cerebral emboli, 2. Strategically placed infarctions causing dementia, 3. Multiple subcortical lacunar lesions secondary to atherosclerosis or degenerative arteriolar changes, 4. Binswanger's disease (arteriosclerotic subcortical leukoencephalopathy)., 5. Mixtures of types 1, 2 and 3, 6. Haemorrhagic lesions causing dementia, 7. Subcortical dementia secondary to hereditary factors, and 8. Mixtures of dementia of the Alzheimer's type and vascular dementia (Konno et al.
• VD vascular dementia
• HIS Hachinski Ischemic Score
• ADDTC - Alzheimer Disease Diagnostic and Treatment Centers
• NNDS-AIREN National Institute of Neurological Disorders and Stroke-Association Internationale pour labericht et 1' Enseignement en Neurosciences
• DSM-IV Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition
• Stroke versus nonstroke such as cerebral tumors and subdural hematoma
• cerebrovascular dementia often coexists with other causes of dementia (Erkinjuntti Int Psychogeriatr (1997) 9 Suppl 1:51-8; discussion 77-83) complicating a proper diagnosis and effective treatment strategies.
• the majority of vascular dementias are caused by both genetic and environmental factors (Plassman and Breitner J Am Geriatr Soc (1996) 44:1242-50), although an increased prevalence of vascular dementia has been demonstrated in the cerebral arteriopathy syndrome, a genetic form of vascular dementia (Salloway and Hong J Geriatr Psychiatry Neurol (1998) 11:71-7), apolipoprotein E gene polymorphism in Binswanger's disease and vascular dementia (Higuchi et al.
• HCAA hereditary cystatin C amyloid angiopathy
• vascular dementia Current treatments of vascular dementia include antithrombic therapies (Crowth and Ginsberg in Stroke, Pathophysiology, Diagnosis, and Management Eds. Barnett, Mohr et al. Year, Churchill Livingston, a division of Harcourt Brace & Company), thrombolytic and defibrinogenating agents (Brott and hacke in Stroke, supra), antiplatelet agents (Weksler in Stroke, supra) and neuroprotective agents (Gluckmann and Gunn in Neuroprotection in CNS diseases, Eds. Baer and Beal Year, Marcel Dekker, Inc. New York).
• thrombic therapies Crowth and Ginsberg in Stroke, Pathophysiology, Diagnosis, and Management Eds. Barnett, Mohr et al. Year, Churchill Livingston, a division of Harcourt Brace & Company
• thrombolytic and defibrinogenating agents Brott and hacke in Stroke, supra
• antiplatelet agents Weksler in
• DAPs disease associated proteins
• the present invention provides methods and compositions for clinical screening, diagnosis, prognosis, therapy and prophylaxis of Vascular Dementia, for monitoring the effectiveness of Vascular Dementia treatment, for selecting participants in clinical trials, for identifying patients most likely to respond to a particular therapeutic treatment and for screening and development of drugs for treatment of Vascular Dementia.
• a first aspect of the invention provides methods for diagnosis of Vascular Dementia that comprise analyzing a sample of cerebrospinal fluid (CSF) by two-dimensional electrophoresis to detect the presence or level of at least one Vascular Dementia-Associated Feature (VF), e.g., one or more of the VFs disclosed herein or any combination thereof.
• CSF cerebrospinal fluid
• VF Vascular Dementia-Associated Feature
• a second aspect of the invention provides methods for diagnosis of Vascular Dementia that comprise detecting in a sample of CSF the presence or level of at least one
• VPI Vascular Dementia-Associated Protein Isoform
• prognosis monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development, and identification of new targets for drug treatment.
• a third aspect of the invention provides antibodies, e.g. monoclonal and polyclonal antibodies capable of immunospecific binding to a VPI, e.g., a VPI disclosed herein.
• a fourth aspect of the invention provides a preparation comprising an isolated VPI, i.e., a VPI free from proteins or protein isoforms having a significantly different isoelectric point or a significantly different apparent molecular weight from the VPI.
• a fifth aspect of the invention provides methods of treating Vascular Dementia, comprising administering to a subject a therapeutically effective amount of an agent that modulates (e.g., upregulates or downregulates) the expression or activity (e.g. enzymatic or binding activity), or both, of a VPI in subjects having Vascular Dementia, in order to prevent or delay the onset or development of Vascular Dementia, to prevent or delay the progression of Vascular Dementia, or to ameliorate the symptoms of Vascular Dementia.
• an agent that modulates e.g., upregulates or downregulates
• the expression or activity e.g. enzymatic or binding activity
• a sixth aspect of the invention provides methods of screening for agents that modulate (e.g., upregulate or downregulate) a characteristic of, e.g., the expression or the enzymatic or binding activity, of a VPI, a VPI analog, or a VPI-related polypeptide.
• VPI analog refers to a polypeptide that possesses a similar or identical function as a VPI but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the VPI, or possess a structure that is similar or identical to that of the VPI.
• an amino acid sequence of a polypeptide is "similar" to that of a VPI if it satisfies at least one of the following criteria: (a) the polypeptide has an amino acid sequence that is at least 30% (more preferably, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of the VPI; (b) the polypeptide is encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding at least 5 amino acid residues (more preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at
• a polypeptide with "similar structure" to that of a VPI refers to a polypeptide that has a similar secondary, tertiary or quarternary structure as that of the VPI.
• the structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.
• VPN fusion protein refers to a polypeptide that comprises (i) an amino acid sequence of a VPI, a VPI fragment, a VPI-related polypeptide or a fragment of a VPI-related polypeptide and (ii) an amino acid sequence of a heterologous polypeptide (i.e., a non-VPI, non-VPI fragment or non-VPI- related polypeptide).
• VPI homolog refers to a polypeptide that comprises an amino acid sequence similar to that of a VPI but does not necessarily possess a similar or identical function as the VPI.
• VPI ortholog refers to a non-human polypeptide that (i) comprises an amino acid sequence similar to that of a VPI and (ii) possesses a similar or identical function to that of the VPI.
• VPI-related polypeptide refers to a VPI homolog, a VPI analog, an isoform of VPI, a VPI ortholog, or any combination thereof.
• derivative refers to a polypeptide that comprises an amino acid sequence of a second polypeptide which has been altered by the introduction of amino acid residue substitutions, deletions or additions.
• the derivative polypeptide possess a similar or identical function as the second polypeptide.
• fragment refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a second polypeptide.
• the fragment of a VPI may or may not possess a functional activity of the second polypeptide.
• fold change includes “fold increase” and “fold decrease” and refers to the relative increase or decrease in abundance of a VF or the relative increase or decrease in expression or activity of a polypeptide (e.g. a VPI) in a first sample or sample set compared to a second sample (or sample set).
• a VF or polypeptide fold change may be measured by any technique known to those of skill in the art, however the observed increase or decrease will vary depending upon the technique used.
• fold change is determined herein as described in the Examples infra.
• isoform refers to variants of a polypeptide that are encoded by the same gene, but that differ in their pi or MW, or both. Such isoforms can differ in their amino acid composition (e.g. as a result of alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation). As used herein, the term “isoform” also refers to a protein that exists in only a single form, i.e., it is not expressed as several variants.
• modulate when used herein in reference to expression or activity of a VPI or ⁇ VPI-related polypeptide refers to the upregulation or downregulation of the expression or activity of the VPI or a VPI-related polypeptide. Based on the present disclosure, such modulation can be determined by assays known to those of skill in the art or described herein.
• the percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions.
• the "best alignment” is an alignment of two sequences which results in the highest percent identity.
• the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
• An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA (1990) 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
• the NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol. (1990) 215:403-410 have incorporated such an algorithm.
• Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. (1991) 25:3389-3402.
• PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
• Figure 1 is an image obtained from 2-dimensional electrophoresis of human CSF, which has been annotated to identify twelve landmark features, designated CSF1 to CSF12.
• the invention described in detail below provides methods and compositions for clinical screening, diagnosis and prognosis of Vascular Dementia in a mammalian subject for identifying patients most likely to respond to a particular therapeutic treatment, for monitoring the results of Vascular Dementia therapy, for drug screening and drug development.
• the invention also encompasses the administration of therapeutic compositions to a mammalian subject to treat or prevent Vascular Dementia.
• the mammalian subject may be a non-human mammal, but is preferably human, more preferably a human adult, i.e. a human subject at least 21 (more preferably at least 35, at least 50, at least 60, at least 70, or at least 80) years old.
• the invention will be described with respect to the analysis of CSF samples.
• a body fluid e.g. blood, serum, plasma, saliva or urine
• a tissue sample from a subject at risk of having or developing Vascular Dementia e.g. a biopsy such as a brain biopsy
• the methods and compositions of the present invention are useful for screening, diagnosis and prognosis of a living subject, but may also be used for postmortem diagnosis in a subject, for example, to identify family members of the subject who are at risk of developing the same disease.
• cerebrospinal fluid refers to the fluid that surrounds the bulk of the central nervous system, as described in Physiological Basis of Medical Practice (J.B. West, ed., Williams and Wilkins, Baltimore, MD 1985). CSF includes ventricular CSF and lumbar CSF.
• serum refers to the supernatant fluid produced by clotting and centrifugal sedimentation of a blood sample.
• plasma refers to the supernatant fluid produced by inhibition of clotting (for example, by citrate or EDTA) and centrifugal sedimentation of a blood sample.
• blood as used herein includes serum and plasma.
• two-dimensional electrophoresis is used to analyze
• VFs Vascular Dementia- Associated Features
• two-dimensional electrophoresis means a technique comprising isoelectric focusing, followed by denaturing electrophoresis; this generates a two- dimensional gel (2D-gel) containing a plurality of separated proteins.
• the step of denaturing electrophoresis uses polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).
• SDS-PAGE sodium dodecyl sulfate
• the Preferred Technology provides efficient, computer- assisted methods and apparatus for identifying, selecting and characterizing biomolecules (e.g. proteins, including glycoproteins) in a biological sample.
• a two-dimensional array is generated by separating biomolecules on a two-dimensional gel according to their electrophoretic mobility and isoelectric point.
• a computer-generated digital profile of the array is generated, representing the identity, apparent molecular weight, isoelectric point, and relative abundance of a plurality of biomolecules detected in the two-dimensional array, thereby permitting computer-mediated comparison of profiles from multiple biological samples, as well as computer aided excision of separated proteins of interest.
• the Basiji thesis provides a phase-sensitive detection system for discriminating modulated fluorescence from baseline noise due to laser scatter or homogeneous fluorescence, but the scanner can also be operated in a non-phase-sensitive mode.
• This phase-sensitive detection capability would increase the sensitivity of the instrument by an order of magnitude or more compared to conventional fluorescence imaging systems. The increased sensitivity would reduce the sample-preparation load on the upstream instruments while the enhanced image quality simplifies image analysis downstream in the process.
• a more highly preferred scanner is the Apollo 2 scanner (Oxford Glycosciences, Oxford, UK), which is a modified version of the above described scanner.
• the gel is transported through the scanner on a precision lead-screw drive system. This is preferable to laying the glass plate on the belt-driven system that is described in the Basiji thesis, as it provides a reproducible means of accurately transporting the ,gel past the imaging optics.
• the gel is secured against three alignment stops that rigidly hold the glass plate in a known position. By doing this in conjunction with the above precision transport system, the absolute position of the gel can be predicted and. recorded. This ensures that co-ordinates of each feature on the gel can be determined more accurately and communicated, if desired, to a cutting robot for excision of the feature.
• the carrier that holds the gel has four integral fluorescent markers for use to correct the image geometry. These markers are a quality control feature that confirms that the scanning has been performed correctly.
• the optical components of the Apollo 2 scanner have been inverted.
• the laser, mirror, waveguide and other optical components are above the glass plate being scanned.
• the scanner described in the Basiji thesis has these components underneath.
• the glass plate is mounted onto the scanner gel side down, so that the optical path remains through the glass plate. By doing this, any particles of gel that may break away from the glass plate will fall onto the base of the instrument rather than into the optics. This does not affect the functionality of the, system, but increases its reliability.
• the Apollo 3 scanner in which the signal output is digitized to the full 16-bit data without any peak saturation or without square root encoding of the signal.
• a compensation algorithm has also been applied to correct for any variation in detection sensitivity along the path of the scanning beam. This variation is due to anomalies in the optics and differences in collection efficiency across the waveguide.
• a calibration is performed using a perspex plate with an even fluorescence throughout. The data received from a scan of this plate are used to determine the multiplication factors needed to increase the signal from each pixel level to a target level. These factors are then used in subsequent scans of gels to remove any internal optical variations.
• feature refers to a spot detected in a 2D gel, and the term
• Vascular Dementia-Associated Feature refers to a feature that is differentially present in a sample (e.g. a sample of CSF) from a subject having Vascular Dementia compared with a sample (e.g. a sample of CSF) from a subject free from Vascular Dementia.
• a feature or a protein isoform of VPI, as defined infra
• isoform or VPI gives a different signal when applied to the first and second samples.
• a feature, isoform or VPI is "increased" in the first sample with respect to the second if the method of detection indicates that the feature, isoform or VPI is more abundant in the first sample than in the second sample, or if the feature, isoform or VPI is detectable in the first sample and undetectable in the second sample.
• a feature, isoform or VPI is "decreased” in the first sample with respect to the second if the method of detection indicates that the feature, isoform or VPI is less abundant in the first sample than in the second sample or if the feature, isoform or VPI is undetectable in the first sample and detectable in the second sample.
• the relative abundance of a feature in two samples is determined in two steps.
• the signal obtained upon detecting the feature in a sample is normalized by reference to a suitable background parameter, e.g., (a) to the total protein in the sample being analyzed (e.g., total protein loaded onto a gel); (b) to an Expression Reference Feature (ERF) i.e., a feature whose abundance is invariant, within the limits of variability of the Preferred Technology, in the population of subjects being examined, e.g. the ERFs disclosed below, or (c) more preferably to the total signal detected from all proteins in the sample.
• a suitable background parameter e.g., (a) to the total protein in the sample being analyzed (e.g., total protein loaded onto a gel); (b) to an Expression Reference Feature (ERF) i.e., a feature whose abundance is invariant, within the limits of variability of the Preferred Technology, in the population of subjects being examined, e.g. the ERFs disclosed below, or (c) more preferably to the total signal detected from all proteins in
• the normalized signal for the feature in one sample or sample set is compared with the normalized signal for the same feature in another sample or sample set in order to identify features that are "differentially present" in the first sample (or sample set) with respect to the second.
• VFs disclosed herein have been identified by comparing CSF samples from subjects having Vascular Dementia against CSF samples from subjects free from Vascular Dementia.
• Subjects free from Vascular Dementia include subjects with no known disease or condition (normal subjects) and subjects with diseases (including neurological and neurodegenerative diseases) other than Vascular Dementia.
• the first group consists of VFs that are decreased in the CSF of subjects having Vascular Dementia as compared with the CSF of subjects free from Vascular Dementia. These VFs can be described by apparent molecular weight (MW) and isoelectric point (pi) as provided in Table I.
• MW apparent molecular weight
• pi isoelectric point
• the second group consists of NFs that are increased in the CSF of subjects having Nascular Dementia as compared with the CSF of subjects free from Nascular Dementia.
• These NFs can be described by MW and pi as follows:
• the signal obtained upon analyzing CSF from subjects having Nascular Dementia relative to the signal obtained upon analyzing CSF from subjects free from Nascular Dementia will depend upon the particular analytical protocol and detection technique that is used. Accordingly, the present invention contemplates that each laboratory will, based on the present description, establish a reference range for each VF in subjects free from Vascular Dementia according to the analytical protocol and detection technique in use, as is conventional in the diagnostic art.
• at least one control positive CSF sample from a subject known to have Vascular Dementia or at least one control negative CSF sample from a subject known to be free from Vascular Dementia are included in each batch of test samples analyzed.
• the level of expression of a feature is determined relative to a background value, which is defined as the level of signal obtained from a proximal region of the image that (a) is equivalent in area to the particular feature in question; and (b) contains no discernable protein feature.
• the reference range depending upon the method of detection used and the conditions under which detection is carried out, can include no feature or isoform present, or non- detectable levels of feature or isoform present. Proteins described by pi and MW provided in Tables I and II can be identified by searching 2D-PAGE databases with those pi and MW values.
• databases typically provide interactive 2D gels images for a given set of sample and preparation protocol, and the skilled artisan can obtain information relevant to a given feature by pointing and clicking the appropriate section of the image.
• the signal associated with a VF in the CSF of a subject is normalized with reference to one or more ERFs detected in the same 2D gel.
• ERFs may readily be determined by comparing different samples using the Preferred Technology. Suitable ERFs include (but are not limited to) that described in the following table.
• the measured MW and pi of a given feature or protein isoform will vary to some extent depending on the precise protocol used for each step of the 2D electrophoresis and for landmark matching.
• the terms "MW” and "pi" are defined, respectively, to mean the apparent molecular weight and the apparent isoelectric point of a feature or protein isoform as measured in exact accordance with the Reference Protocol identified in Section 6 below. When the Reference Protocol is followed and when samples are run in duplicate or a higher number of replicates, variation in the measured mean pi of a VF or VPI is typically less than 3% and variation in the measured mean MW of a VF or VPI is typically less than 5%.
• VFs can be used for detection, prognosis, diagnosis, or monitoring of Vascular
• CSF from a subject is analyzed by 2D electrophoresis for quantitative detection of one or more of the following VFs: VF-4, VF-5, VF-12, VF-13, VF- 14, VF-15, VF-16, VF-17, VF-18, VF-19, VF-20, VF-21, VF-22, VF-23, VF-24, VF-25, VF- 26, VF-27, VF-29, VF-30, VF-31, VF-32, VF-33, VF-34, VF-35, VF-36, VF-37, VF-38, VF- 41, VF-42, VF-43, VF-44, VF-45, VF-46, VF-47, VF-48, VF-50, VF-51,
• a decreased abundance of said one or more VFs in the CSF from the subject relative to CSF from a subject or subjects free from Vascular Dementia indicates the presence of Vascular Dementia.
• CSF from a subject is analyzed by 2D electrophoresis for quantitative detection of one or more of the following VFs: VF-92, VF-93, VF-94, VF-95, VF-96, VF-97, VF-98, VF-99, VF-100, VF-101, VF-102, VF-103, VF-104, VF-105, VF-106, VF-107, VF-108, VF-109, VF-110, VF-111, VF-112, VF-113, VF-114, VF- 115, VF-116, VF-118, VF-120, VF-121, VF-122, VF-123, VF-124, VF-125, VF-126, VF-127,
• VF-300 299, VF-300, VF-301, VF-302, VF-303, VF-304, VF-305, VF-306, VF-307, VF-308, VF-309, VF-310, VF-311, VF-312, VF-313, VF-314, VF-315, VF-316, VF-317, VF-318, VF-319, VF-
• VF-342 NF-343, NF-344, NF-345, NF-346, NF-347, VF-348, VF-349, VF-350, VF-351,
• An increased abundance of said one or more VFs in the CSF from the subject relative to CSF from a subject or subjects free from Vascular Dementia indicates the presence of Vascular Dementia.
• CSF from a subject is analyzed by 2D electrophoresis for quantitative detection of (a) one or more VFs or any combination of them, whose decreased abundance indicates the presence of Vascular Dementia, i.e., VF-4, VF-5, VF-12, VF-13, VF- 14, VF-15, VF-16, VF-17, VF-18, VF-19, VF-20, VF-21, VF-22, VF-23, VF-24, VF-25, VF- 26, VF-27, VF-29, VF-30, VF-31, VF-32, VF-33, VF-34, VF-35, VF-36, VF-37, VF-38, VF- 41, VF-42, VF-43, VF-44, VF-45, VF-46, VF-47, VF-48, VF-50, VF-51, VF-52, VF-53, VF- 54, VF-55,
• CSF from a subject is analyzed by 2D electrophoresis for quantitative detection of one or more of the following VFs: VF-4, VF-5, VF-12, VF-13, VF-14, VF-15, VF-16, VF-17, NF-18, NF-19, VF-20, VF-21, VF-22, VF-23, VF-24, VF-25, VF-26, VF-27, VF-29, VF-30, VF-31, VF-32, VF-33, VF-34, VF-35, VF-36, VF-37, VF-38, VF-41, VF-42, VF-43, VF-44, VF-45, VF-46, VF-47, VF-48, VF-50, VF-51, VF-52, VF-53, VF-54, VF-55, VF-57, VF-58, VF-60, VF-64, VF-66
• a decrease in one or more VF/ERF ratios in a test sample relative to the VF/ERF ratios in a control sample or a reference range indicates the presence of Vascular Dementia; VF-4, VF-5, VF-12, VF-13, VF-14, VF-15, VF-16, VF-17, VF-18, VF-19, VF-20, VF-21, VF-22, VF-23, VF-24, VF-25, VF-26, VF-27, VF-29, VF-30, VF-31, VF-32, VF-33, VF-34, VF-35, VF-36, VF-37, VF-38, VF-41, VF-42, VF-43, VF-44, VF-45, VF-46, VF-47, VF-48, VF-50, VF-51, VF-52, VF-53, VF-54, VF-55, VF-57,
• an increase in one or more VF/ERF ratios in a test sample relative to the VF/ERF ratios in a control sample or a reference range indicates the presence of Vascular Dementia; VF-92, VF-93, VF-94, VF-95, VF-96, VF-97, VF-98, VF-99, VF-100, VF-101, VF-102, VF-103, VF-104, VF-105, VF-106, VF-107, VF-108, VF- 109, VF-110, VF-111, VF-112, VF-113, VF-114, VF-115, VF-116, VF-118, VF-120, VF-121, VF-122, VF-123, VF-124, VF-125, VF-126, VF-127, VF-128, VF-129, VF-130, VF-131, VF- 132, VF-134,
• CSF from a subject is analyzed by 2D electrophoresis for quantitative detection of (a) one or more VFs, or any combination of them, whose decreased VF/ERF ratio(s) in a test sample relative to the VF/ERF ratio(s) in a control sample indicates the presence of Vascular Dementia, i.e., VF-4, VF-5, VF-12, VF-13, VF-14, VF-15, VF-16, VF-17, VF-18, VF-19, VF-20, VF-21, VF-22, VF-23, VF-24, VF-25, VF-26, VF-27, VF-29, VF-30, VF-31, VF-32, VF-33, VF-34, VF-35, VF-36, VF-37, VF-38, VF-41, VF-42, VF-43, VF-44, VF-45, VF-46, VF-47
• CSF from a subject is analyzed for quantitative detection of a plurality of VFs.
• Vascular Dementia-Associated Protein Isoforms In another aspect of the invention, CSF from a subject, preferably a living subject, is analyzed for quantitative detection of one or more Vascular Dementia-Associated Protein Isoforms (VPIs) for screening or diagnosis of Vascular Dementia, to determine the prognosis of a subject having Vascular Dementia, to monitor the effectiveness of Vascular Dementia therapy, for identifying patients most likely to respond to a particular therapeutic treatment or for drug development.
• a given protein may be expressed as variants (isoforms) that differ in their amino acid composition (e.g.
• Vascular Dementia- Associated Protein Isoform refers to a protein isoform that is differentially present in CSF from a subject having Vascular Dementia compared with CSF from a subject free from Vascular Dementia.
• isoform also refers to a protein that exists in only a single form, i.e., it is not expressed as several variants.
• VPIs Two groups of VPIs have been identified by amino acid sequencing of VFs. VPIs were isolated, subjected to proteolysis, and analyzed by mass specfrometry using the methods and apparatus of the Preferred Technology. One skilled in the art can identify sequence information from proteins analyzed by mass specfrometry and/or tandem mass specfrometry using various spectral interpretation methods and database searching tools. Examples of some of these methods and tools can be found at the Swiss Institute of Bioinformatics web site at http://www.expasy.ch , and the European Molecular Biology Laboratory web site at www.mann.embl-heidelberg.de/Services/PeptideSearch/. Identification of VPIs was performed primarily using the SEQUEST search program (Eng et al, J. Am. Soc.
• the first group consists of VPIs that are decreased in the CSF of subjects having Vascular Dementia as compared with the CSF of subjects free from Vascular Dementia, where the differential presence is significant.
• the amino acid sequences of tryptic digest peptides of these VPIs identified by tandem mass specfrometry and database searching as described in the Examples, infra are listed in Table IV in addition to the pis and MWs of these VPIs.
• VF-50 VPI-27 5.48 55124 VLSALQAVQGLLVAQGR, DPTFIPAPIQAK, SLDFTELDVAAEK, ALQDQLVLVAAK
• VF-230 VPI-187 5.82 50026 YEAAVPDPR, EPGEFALLR, TALASGGVLDASGDYR
• VF-234 VPI-189 5.58 63762 GECQAEGVLFFQGDR, NFPSPVDAAFR, VWVYPPEK, DYFMPCPGR, RLWWLDLK
• the second group comprises VPIs that are increased in the CSF of subjects having Vascular Dementia as compared with the CSF of subjects free from Vascular Dementia, where the differential presence is significant.
• the amino acid sequences of tryptic digest peptides of these VPIs identified by tandem mass specfrometry and database searching are listed in Table V in addition to the pis and MWs of these VPIs.
• VPI is a protein comprising a peptide sequence described for that VPI (preferably comprising a plurality of, more preferably all of, the peptide sequences described for that VPI) and has a pi of about the value stated for that VPI (preferably within 10%, more preferably within 5% still more preferably within 1% of the stated value) and has a MW of about the value stated for that VPI (preferably within 10%, more preferably within 5%, still more preferably within 1% of the stated value).
• Proteins comprising the peptide sequences provided in Table IV and V can be identified by searching sequence databases with those peptides using search tools known to those skilled in the art.
• search algorithm tools that can be used to identify proteins from peptide sequences include: • BLAST (Basic Local Alignment Search Tool) : BLAST is maintained at the National
• NCBI Center for Biotechnology Information
• BLASTP can be used to search a protein sequence against a protein database.
• TBLASTN can be used to search a Protein Sequence against a
• the nr protein database maintained at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). The nr protein database is compiled of entries from various sources including SwissProt, SwissProt updates, PIR, and PDB. The BLAST resource is available for sequence searching.
• the Protein Identification Resource is a division of the National Biomedical Research Foundation (NBRF) which is affiliated with Georgetown University Medical Center and can be found at http://www nbrf.georgetown.edu/pir/searchdb.html.
• NBRF National Biomedical Research Foundation
• the database can be searched using BLAST and FASTA search algorithm tools.
• CSF from a subject is analyzed for quantitative detection of one or more of the following VPIs: VPI-2, VPI-3, VPI-6, VPI-7, VPI-8, VPI-9, VPI-10, VPI-11,
• VPI-98 VPI-99, VPI-100, VPI-130, VPI-131, VPI-133, VPI-158, VPI-159, VPI-160, VPI-
• Vascular Dementia a decreased abundance of the VPI or VPIs (or any combination of them) in the CSF from the subject relative to CSF from a subject or subjects free from Vascular Dementia (e.g., a control sample or a previously determined reference range) indicates the presence of Vascular Dementia.
• CSF from a subject is analyzed for quantitative detection of one or more of the following VPIs: VPI-56, VPI-57, VPI-58, VPI-61, VPI-62, VPI-63, VPI-64, VPI-65, VPI-66, VPI-67, VPI-68, VPI-69, VPI-70, VPI-71, VPI-72, VPI-73, VPI-75, VPI-77, VPI-78, VPI-79, VPI-80, VPI-81, VPI-82, VPI-83, VPI-84, VPI-85, VPI-87, VPI-88, VPI-108, VPI-109, VPI-110, VPI-111, VPI-112, VPI-113, VPI-114, VPI- 115, VPI-116, VPI-117, VPI-118, VPI-119, VPI-120, VPI-121, VPI-122, VPI-123, VPI-124,
• VPI-171, VPI-172, VPI-173, VPI-174, VPI-175, VPI-176, VPI-177, VPI-178, VPI-179, VPI-180, VPI-181, VPI-182, VPI-183, VPI-184, VPI-185, VPI-186, VPI-187, VPI-188, VPI- 189, VPI-190, VPI-191, VPI-192, VPI-193, VPI-194, VPI-195, VPI-196, VPI-197, VPI-198, VPI-199, VPI-200, VPI-201, VPI-202, VPI-203, VPI-204, VPI-205, VPI-206, VPI-207, VPI- 208 formulate VPI-209, VPI-210, VPI-211, VPI-212, VPI-213; and (b) one or more VPIs, or any combination of them, whose increased abundance indicates the presence of Vascular Dementia, i.e., VPI-56,
• CSF from a subject is analyzed for quantitative detection of one or more VPIs and one or more previously known biomarkers of Vascular Dementia (e.g., candidate markers such as hypersensitive platelet glutamate receptors (Berk et al, Int Clin Psychopliarmacol (1999) 14:199-122)).
• the abundance of each VPI and known biomarker relative to a control or reference range indicates whether a subject has Vascular Dementia.
• the abundance of a VPI is normalized to an Expression Reference Protein Isoform (ERPI).
• ERPIs can be identified by partial amino acid sequencing of ERFs, which are described above, using the methods and apparatus of the Preferred Technology. The partial amino acid sequences of an ERPI, and the known proteins to which it is homologous is presented in Table VI.
• the VPIs described herein include previously unknown proteins, as well as isoforms of known proteins where the isoforms were not previously known to be associated with Vascular Dementia.
• the present invention additionally provides: (a) a preparation comprising the isolated VPI; (b) a preparation comprising one or more fragments of the VPI; and (c) antibodies that bind to said VPI, to said fragments, or both to said VPI and to said fragments.
• a VPI is "isolated" when it is present in a preparation that is substantially free of contaminating proteins, i.e., a preparation in which less than 10% (preferably less than 5%, more preferably less than 1%) of the total protein present is contaminating protein(s).
• a contaminating protein is a protein or protein isoform having a significantly different pi or MW from those of the isolated VPI, as determined by 2D electrophoresis.
• a "significantly different" pi or MW is one that permits the contaminating protein to be resolved from the VPI on 2D electrophoresis, performed according to the Reference Protocol.
• an isolated protein comprising a peptide with the amino acid sequence identified in Table IV or V for a VPI, said protein having a pi and MW within 10% (preferably within 5%, more preferably within 1%) of the values identified in Table IV or V for that VPI.
• the VPIs of the invention can be qualitatively or quantitatively detected by any method known to those skilled in the art, including but not limited to the Preferred Technology described herein, kinase assays, enzyme assays, binding assays and other functional assays, immunoassays, and western blotting.
• the VPIs are separated on a 2-D gel by virtue of their MWs and pis and visualized by staining the gel.
• the VPIs are stained with a fluorescent dye and imaged with a fluorescence scanner. Sypro Red (Molecular Probes, Inc., Eugene, Oregon) is a suitable dye for this purpose.
• a preferred fluorescent dye is Pyridinium, 4-[2-[4- (dipentylamino)-2-trifluoromethylphenyl] ethenyl]-l- (sulfobutyl)-, inner salt. See U.S. Application No. 09/412,168, filed on October 5, 1999, which is incorporated herein by reference in its entirety.
• VPIs can be detected in an immunoassay.
• an immunoassay is performed by contacting a sample from a subject to be tested with an anti-VPI antibody under conditions such that immunospecific binding can occur if the VPI is present, and detecting or measuring the amount of any immunospecific binding by the antibody.
• Anti- VPI antibodies can be produced by the methods and techniques taught herein; examples of such antibodies known in the art are set forth in Table VII. These antibodies shown in Table VII are already known to bind to the protein of which the VPI is itself a family member.
• the anti-VPI antibody preferentially binds to the VPI rather than to other isoforms of the same protein.
• the anti-VPI antibody binds to the VPI with at least 2-fold greater affinity, more preferably at least 5-fold greater affinity, still more preferably at least 10-fold greater affinity, than to said other isoforms of the same protein.
• VPIs can be transferred from the gel to a suitable membrane (e.g. a PVDF membrane) and subsequently probed in suitable assays that include, without limitation, competitive and non-competitive assay systems using techniques such as western blots and "sandwich" immunoassays using anti-VPI antibodies as described herein, e.g., the antibodies identified in Table VII, or others raised against the VPIs of interest.
• suitable assays include, without limitation, competitive and non-competitive assay systems using techniques such as western blots and "sandwich" immunoassays using anti-VPI antibodies as described herein, e.g., the antibodies identified in Table VII, or others raised against the VPIs of interest.
• the immunoblots can be used to identify those anti-VPI antibodies displaying the selectivity required to immuno-specifically differentiate a VPI from other isoforms encoded by the same gene.
• VPI-17 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-23 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-31 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Hurnan SCIENTIFIC
• VPI-56 C8 Complement, Goat anti- ACCURATE CHEMICAL & BMD- G35 Human SCIENTIFIC
• VPI-63 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-79 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-80 C8 Complement Goat anti- ACCURATE CHEMICAL & BMD- G35 Human SCIENTIFIC
• VPI-83 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-84 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-92 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-112 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-118 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-128 Apolipoprotein D, Clone: 36C6, ACCURATE CHEMICAL & MED- CLA457 Mab anti-Human, paraffin, SCIENTIFIC IH ⁇ /VB CORPORATION VPI# Antibody Manufacturer Cat. No.
• VPI-130 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-147 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-149 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-162 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-164 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-166 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-168 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-169 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-170 C3 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-001-02 Human SCIENTIFIC
• VPI-172 Prothrombin Rabbit anti-Human ACCURATE CHEMICAL & AXL- 448/2 VPI# Antibody Manufacturer Cat. No.
• VPI-188 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-194 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-215 Alpha-1-Acid Glycoprotein, ACCURATE CHEMICAL & BYA- 6189-1 Clone: AGP-47, Mab anti- SCIENTIFIC Human CORPORATION
• VPI-228 Albumin Human, Chicken anti- ACCURATE CHEMICAL & IMS- 01-026-02
• VPI-229 Transthyretin, Prealbuminm, ACCURATE CHEMICAL & MED- CLA 193 55kD, Rabbit anti-Human SCIENTIFIC
• VPI-231 Apolipoprotein D, Clone: 36C6, ACCURATE CHEMICAL & MED- CLA457 Mab anti-Human, paraffin, SCIENTIFIC IH/WB CORPORATION
• VPI-232 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• VPI-234 Gelsolin, plasma + cytoplasmic, ACCURATE CHEMICAL & YBG- 4628-6210 Sheep anti- SCIENTIFIC
• VPI-239 C7 Complement, Goat anti- ACCURATE CHEMICAL & BMD- G34 VPI# Antibody Manufacturer Cat. No.
• VPI-241 C4 Complement, Chicken anti- ACCURATE CHEMICAL & IMS- 01-032-02 Human SCIENTIFIC
• Tissue Inhibitor of Matrix ACCURATE CHEMICAL & MED- CLA498 Metalloproteinase 2 (TIMP2) SCIENTIFIC (NO X W/TIMP1), Clone: 3A4, CORPORATION Mab anti-Human, paraffin, IH
• VPI-258 Factor H (Complement), ACCURATE CHEMICAL & IMS- 01-066-02 Chicken anti-Human SCIENTIFIC
• VPI-262 Apolipoprotein D, Clone: 36C6, ACCURATE CHEMICAL & MED- CLA457 Mab anti-Human, paraffin, SCIENTIFIC IH/WB CORPORATION
• VPI-267 Apolipoprotein D, Clone: 36C6, ACCURATE CHEMICAL & MED- CLA457 Mab anti-Human, paraffin, SCIENTIFIC IH/WB CORPORATION
• VPI-268 Gelsolin, plasma + cytoplasmic, ACCURATE CHEMICAL & YBG- 4628-6210 Sheep anti- SCIENTIFIC
• binding of antibody in tissue sections can be used to detect aberrant VPI localization or an aberrant level of one or more VPIs.
• antibody to a VPI can be used to assay a tissue sample (e.g., a brain biopsy) from a subject for the level of the VPI where an aberrant level of VPI is indicative of Vascular Dementia.
• an "aberrant level” means a level that is increased or decreased compared with the level in a subject free from Vascular Dementia or a reference level. If desired, the comparison can be performed with a matched sample from the same subject, taken from a portion of the body not affected by Vascular Dementia.
• any suitable immunoassay can be used, including, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement- fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
• a VPI can be detected in a fluid sample (e.g., CSF, blood, urine, or tissue homogenate) by means of a two-step sandwich assay.
• a capture reagent e.g., an anti-VPI antibody
• the capture reagent can optionally be immobilized on a solid phase.
• a directly or indirectly labelled detection reagent is used to detect the captured VPI.
• the detection reagent is a lectin. Any lectin can be used for this purpose that preferentially binds to the VPI rather than to other isoforms that have the same core protein as the VPI or to other proteins that share the antigenic determinant recognized by the antibody.
• the chosen lectin binds to the VPI with at least 2-fold greater affinity, more preferably at least 5-fold greater affinity, still more preferably at least 10-fold greater affinity, than to said other isoforms that have the same core protein as the VPI or to said other proteins that share the antigenic determinant recognized by the antibody.
• a lectin that is suitable for detecting a given VPI can readily be identified by methods well known in the art, for instance upon testing one or more lectins enumerated in Table I on pages 158-159 of Sumar et al, Lectins as Indicators of Disease-Associated Glycoforms, In: Gabius H-J & Gabius S (eds.), 1993, Lectins and Glycobiology, at pp. 158-174 (which is incorporated herein by reference in its entirety).
• Lectins with the desired oligosaccharide specificity can be identified, for example, by their ability to detect the VPI in a 2D gel, in a replica of a 2D gel following transfer to a suitable solid substrate such as a nitrocellulose membrane, or in a two-step assay following capture by an antibody.
• the detection reagent is an antibody, e.g., an antibody that immunospecifically detects other post-translational modifications, such as an antibody that immunospecifically binds to phosphorylated amino acids. Examples of such antibodies include those that bind to phosphotyrosine (BD Transduction Laboratories, catalog
• a gene encoding a VPI, a related gene, or related nucleic acid sequences is a gene encoding a VPI, a related gene, or related nucleic acid sequences or
• hybridization assays 10 subsequences, including complementary sequences, can also be used in hybridization assays.
• a nucleotide encoding a VPI, or subsequences thereof comprising at least 8 nucleotides, preferably at least 12 nucleotides, and most preferably at least 15 nucleotides can be used as a hybridization probe.
• Hybridization assays can be used for detection, prognosis, diagnosis, or monitoring of conditions, disorders, or disease states, associated with aberrant expression of
• such a hybridization assay can be carried out by a method comprising contacting a subject's sample containing nucleic acid with a nucleic acid probe capable of hybridizing to a DNA or RNA that encodes a VPI, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
• Nucleotides can be used for therapy of subjects having Vascular Dementia, as described below.
• the methods and compositions for clinical screening, diagnosis and prognosis of Vascular Dementia in a mammalian subject may be diagnostic of Vascular Dementia or indicative of Vascular Dementia.
• VFs Vascular Dementia-Associated Protein Isoforms
• VPIs Vascular Dementia-Associated Protein Isoforms
• compositions which are directed against or lead to modulation of diagnostic markers may have therapeutic value particularly in Vascular Dementia.
• Indicative methods and compositions are based on Vascular Dementia-Associated Features (VFs) and Vascular Dementia-Associated Protein Isoforms (VPIs) which are associated with Vascular Dementia but may not be specific only for Vascular Dementia, and may be associated with one or more other diseases or conditions.
• VFs Vascular Dementia-Associated Features
• VPIs Vascular Dementia-Associated Protein Isoforms
• Such indicative VFs or VPIs which are associated with Vascular Dementia, but not only with Vascular Dementia, are useful in screening, diagnosis and prognosis as indicators of Vascular Dementia.
• Indicative methods and compositions are particularly useful in the initial or general screening, diagnosis and prognosis of an individual subject, whereby a first indication of a subset of conditions or diseases, including Vascular Dementia, is thereby provided.
• Additional assessment utilizing diagnostic or particular Vascular Dementia VFs or VPIs may then be undertaken to provide specific, diagnostic screening, diagnosis and prognosis of the individual subject.
• the administration of therapeutic compositions which are directed against or lead to modulation of indicative markers may have therapeutic value in Vascular Dementia and other disorders as well, or may be useful therapeutically in more than one disease or condition
• a diagnostic marker changes (increases, decreases or otherwise alters form or character) significantly in only a single disease or condition or in only a small number of conditions, particularly in related conditions.
• Two such diagnostic markers, VF-37 and VF- 50, are provided below in Table VIII.
• An indicative marker changes (increases, decreases or otherwise alters form or character) significantly in more than one condition, particularly in Vascular Dementia and one or more other distinct diseases or conditions.
• One such indicative marker, VF-149 is found to increase in Vascular Dementia and is provided in Table IX.
• This same marker, identified or characterised by the same pi and MW, is noted as SF-219 as similarly found to be increased in Schizophrenia.
• the VF-149/SF-219 marker is therefore indicative of Vascular Dementia and/or Schizophrenia.
• kits comprising an anti-VPI antibody.
• a kit may optionally comprise one or more of the following: (1) instructions for using the anti-VPI antibody for diagnosis, prognosis, therapeutic monitoring or any combination of these applications; (2) a labelled binding partner to the antibody; (3) a solid phase (such as a reagent strip) upon which the anti-VPI antibody is immobilized; and (4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any combination thereof.
• the anti- VPI antibody itself can be labelled with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.
• kits comprising a nucleic acid probe capable of hybridizing to RNA encoding a VPI.
• a kit comprises in one or more containers a pair of primers (e.g., each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides) that under appropriate reaction conditions can prime amplification of at least a portion of a nucleic acid encoding a VPI, such as by polymerase chain reaction (see, e.g., Innis et al, 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of Q ⁇ replicase, cyclic probe reaction, or other methods known in the art.
• primers e.g., each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides
• Kits are also provided which allow for the detection of a plurality of VPIs or a plurality of nucleic acids each encoding a VPI.
• a kit can optionally further comprise a predetermined amount of an isolated VPI protein or a nucleic acid encoding a VPI, e.g., for use as a standard or control.
• the uni-variate differential analysis tools are useful in identifying individual VFs or VPIs that are diagnostically associated with Vascular Dementia or in identifying individual VPIs that regulate the disease process.
• the disease process is associated with a combination of VFs or VPIs (and to be regulated by a combination of VPIs), rather than individual VFs and VPIs in isolation.
• the strategies for discovering such combinations of VFs and VPIs differ from those for discovering individual VFs and VPIs. In such cases, each individual VF and VPI can be regarded as one variable and the disease can be regarded as a joint, multi-variate effect caused by interaction of these variables.
• the following steps can be used to identify markers from data produced by the Preferred Technology.
• the first step is to identify a collection of VFs or VPIs that individually show significant association with Vascular Dementia.
• the association between the identified VFs or VPIs and Vascular Dementia need not be as highly significant as is desirable when an individual VF or VPI is used as a diagnostic. Any of the tests discussed above (fold changes, wilcoxon rank sum test, etc.) can be used at this stage. Once a suitable collection of VFs or
• VPIs has been identified, a sophisticated multi-variate analysis capable of identifying clusters can then be used to estimate the significant multivariate associations with Vascular Dementia.
• LDA Linear Discriminant Analysis
• VFs or VPIs Linear Discriminant Analysis
• a set of weights is associated with each variable (i.e., VF or VPI) so that the linear combination of weights and the measured values of the variables can identify the disease state by discriminating between subjects having Vascular Dementia and subjects free from Vascular Dementia.
• Enhancements to the LDA allow stepwise inclusion (or removal) of variables to optimize the discriminant power of the model.
• the result of the LDA is therefore a cluster of VFs or VPIs which can be used, without limitation, for diagnosis, prognosis, therapy or drug development.
• LDA Flexible Discriminant Analysis
• Other enhanced variations of LDA permit the use of non-linear combinations of variables to discriminate a disease state from a normal state.
• the results of the discriminant analysis can be verified by post-hoc tests and also by repeating the analysis using alternative techniques such as classification trees.
• a further category of VFs or VPIs can be identified by qualitative measures by comparing the percentage feature presence of a VF or VPI of one group of samples (e.g., samples from diseased subjects) with the percentage feature presence of a VF or VPI in another group of samples (e.g., samples from control subjects).
• the "percentage feature presence" of a VF or VPI is the percentage of samples in a group of samples in which the VF or VPI is detectable by the detection method of choice. For example, if a VF is detectable in 95 percent of samples from diseased subjects, the percentage feature presence of that VF in that sample group is 95 percent. If only 5 percent of samples from non-diseased subjects have detectable levels of the same VF, detection of that VF in the sample of a subject would suggest that it is likely that the subject suffers from Vascular Dementia.
• the diagnostic methods and compositions of the present invention can assist in monitoring a clinical study, e.g. to evaluate drugs for therapy of Vascular Dementia.
• candidate molecules are tested for their ability to restore VF or VPI levels in a subject having Vascular Dementia to levels found in subjects free from Vascular Dementia or, in a treated subject (e.g. after treatment with antiplatelet agents such as aspirin,
• cholinesterase inhibitors such as rivastigmine, galantamine (Kumar et al.
• cytoprotective agents currently under clinical evaluation such as the calcium antagonists Nimodipine and Nicadipine, NMDA antagonists such as Selfotel, Dextrorphan, Cerestat, Eliprodil, Lamortigine, GABA agonists, Kappa- selective opiod antagonists, Lubeluzole, Free radicalscavengers, anti-ICAM antibodies and GM-1 ganglioside, Abbokinase®, Activase®,Aggrenox®, Anti-ICAM- 1 antibody, Anti-beta- 2-integrin antibody, Arvin®, Atacand®, CerAxon®, Cerebyx®, Ceresine®, Cerestat®, Cervene®, Coumadin®, Fiblast®, Fraxiparine®, Freedox®, Innohep®, Kabikinase®, Klerval®, LeukArrest®, Lipitor®, Lovenox®, Neurogard®
• VFs or VPIs can be assayed.
• the methods and compositions of the present invention areused to screen candidates for a clinical study to identify individuals having vasculardementia; such individuals can then be excluded from the study or can be placed in aseparate cohort for treatment or analysis. If desired, the candidates can concurrently bescreened to identify individuals with Lewy Body disease and/or senile dementia;procedures for these screens are well known in the art (Harding and Halliday, Neuropathol. Appl. Neurobiol. (1998) 24:195-201).
• the invention provides isolated mammalian VPIs, preferably human VPIs, and fragments thereof which comprise an antigenic determinant (i.e., can be recognized by an antibody) or which are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing.
• "Functionally active” as used herein refers to material displaying one or more functional activities associated with a full-length (wild- type) VPI, e.g., binding to a VPI substrate or VPI binding partner, antigenicity (binding to an anti-VPI antibody), immunogenicity, enzymatic activity and the like.
• the invention provides fragments of a VPI comprising at least 5 amino acids, at least 10 amino acids, at least 50 amino acids, or at least 75 amino acids. Fragments lacking some or all of the regions of a VPI are also provided, as are proteins (e.g., fusion proteins) comprising such fragments. Nucleic acids encoding the foregoing are provided.
• the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, etc.
• VPIs identified herein can be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• chromatography e.g., ion exchange, affinity, and sizing column chromatography
• centrifugation e.g., centrifugation
• differential solubility e.g., differential solubility
• the entire amino acid sequence of the VPI can be deduced from the nucleotide sequence of the gene coding region contained in the recombinant nucleic acid.
• the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al, 1984, Nature 310:105-111).
• native VPIs can be purified from natural sources, by standard methods such as those described above (e.g., immunoaffinity purification).
• VPIs are isolated by the Preferred Technology described supra.
• a narrow-range "zoom gel” having a pH range of 2 pH units or less is preferred for the isoelectric step, according to the method described in Westermeier, 1993, Electrophoresis in Practice (VCH, Weinheim, Germany), pp. 197-209 (which is incorporated herein by reference in its entirety); this modification permits a larger quantity of a target protein to be loaded onto the gel, and thereby increases the quantity of isolated VPI that can be recovered from the gel.
• the Preferred Technology typically provides up to 100 ng, and can provide up to 1000 ng, of an isolated VPI in a single run.
• a zoom gel can be used in any separation strategy that employs gel isoelectric focusing.
• the invention thus provides an isolated VPI, an isolated VPI-related polypeptide, and an isolated derivative or fragment of a VPI or a VPI-related polypeptide; any of the foregoing can be produced by recombinant DNA techniques or by chemical synthetic methods.
• nucleotide sequences of the present invention including DNA and RNA, and comprising a sequence encoding a VPI or a fragment thereof, or a VPI-related polypeptide, may be synthesized using methods known in the art, such as using conventional chemical approaches or polymerase chain reaction (PCR) amplification.
• the nucleotide sequences of the present invention also permit the identification and cloning of the gene encoding a VPI homolog or VPI ortholog including, for example, by screening cDNA libraries, genomic libraries or expression libraries.
• oligonucleotides can be designed for all VPI peptide fragments identified as part of the same protein.
• PCR reactions under a variety of conditions can be performed with relevant cDNA and genomic DNAs (e.g., from brain tissue or from cells of the immune system) from one or more species.
• vectorette reactions can be performed on any available cDNA and genomic DNA using the oligonucleotides (which preferably are nested) as above.
• Vectorette PCR is a method that enables the amplification of specific DNA fragments in situations where the sequence of only one primer is known.
• Vectorette PCR may pe performed with probes that are, for example, anchored degenerate oligonucleotides (or most likely oligonucleotides) coding for VPI peptide fragments, using as a template a genomic library or cDNA library pools.
• Anchored degenerate oligonucleotides can be designed for all VPI peptide fragments. These oligonucleotides may be labelled and hybridized to filters containing cDNA and genomic DNA libraries. Oligonucleotides to different peptides from the same protein will often identify the same members of the library.
• the cDNA and genomic DNA libraries may be obtained from any suitable or desired mammalian species, for example from humans. Nucleotide sequences comprising a nucleotide sequence encoding a VPI or VPI fragment of the present invention are useful for their ability to hybridize selectively with complementary stretches of genes encoding other proteins.
• nucleotide sequences at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, or 100% identical, to the sequence of a nucleotide encoding a VPI.
• relatively stringent conditions are used to form the duplexes, such as low salt or high temperature conditions.
• “highly stringent conditions” means hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C, and washing in O.lxSSC/0.1% SDS at 68 °C (Ausubel F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
• hybridization conditions For some applications, less stringent conditions for duplex formation are required. As used herein "moderately stringent conditions” means washing in 0.2xSSC/0.1% SDS at 42 °C (Ausubel et al, 1989, supra). Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be chosen depending on the desired results.
• DNA fragments are generated, some of which will encode parts or the whole of a VPI.
• Any suitable method for preparing DNA fragments may be used in the present invention.
• the DNA may be cleaved at specific sites using various restriction enzymes.
• the DNA fragments can then be separated according to size by standard techniques, including but not limited to agarose and polyacrylamide gel electrophoresis, column chromatography and sucrose gradient cenfrifugation.
• DNA fragments can then be inserted into suitable vectors, including but not limited to plasmids, cosmids, bacteriophages lambda or T4, and yeast artificial chromosome (YAC).
• suitable vectors including but not limited to plasmids, cosmids, bacteriophages lambda or T4, and yeast artificial chromosome (YAC).
• YAC yeast artificial chromosome
• the genomic library may be screened by nucleic acid hybridization to labelled probe (Benton and Davis, Science (1977) 196:180; Grunstein and Hogness, Proc. Natl. Acad.
• the genomic libraries may be screened with labelled degenerate oligonucleotide probes corresponding to the amino acid sequence of any peptide of the VPI using optimal approaches well known in the art.
• Any probe used is at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, or at least 100 nucleotides.
• a probe is 10 nucleotides or longer, and more preferably 15 nucleotides or longer.
• VPIs disclosed herein were found to correspond to isoforms of previously identified proteins encoded by genes whose sequences are publicly known. (Sequence analysis and protein identification of VPIs was carried out using the methods described in Section 6.1.14). To screen such a gene, any probe may be used that is complementary to the gene or its complement; preferably the probe is 10 nucleotides or longer, more preferably 15 nucleotides or longer.
• SWISS-PROT and trEMBL databases (held by the Swiss Institute of Bioinformatics (SIB) and the European Bioinformatics Institute (EBI) which are available at http://www.expasy.ch/) and the GenBank database (held by the National Institute of Health (NIH) which is available at http://www.ncbi.nlm.nih.gov/GenBank/) provide protein sequences for the VPIs listed in Tables IV and V under the following accession numbers and each sequence is incorporated herein by reference:
• degenerate probes or probes taken from the sequences described above by accession number may be used for screening.
• they can be constructed from the partial amino sequence information obtained from tandem mass spectra of tryptic digest peptides of the VPI.
• any probe may be used that is complementary to the gene or its complement; preferably the probe is 10 nucleotides or longer, more preferably 15 nucleotides or longer.
• Hybridization of such oligonucleotide probes to genomic libraries is carried out using methods known in the art. For example, hybridization with one of the above-mentioned degenerate sets of oligonucleotide probes, or their complement (or with any member of such a set, or its complement) can be performed under highly stringent or moderately stringent conditions as defined above, or can be carried out in 2X SSC, 1.0% SDS at 50 °C and washed using the washing conditions described supra for highly stringent or moderately stringent hybridization.
• clones containing nucleotide sequences encoding the entire VPI, a fragment of a VPI, a VPI-related polypeptide, or a fragment of a VPI-related polypeptide any of the foregoing may also be obtained by screening expression libraries.
• DNA from the relevant source is isolated and random fragments are prepared and ligated into an expression vector (e.g., a bacteriophage, plasmid, phagemid or cosmid) such that the inserted sequence in the vector is capable of being expressed by the host cell into which the vector is then introduced.
• an expression vector e.g., a bacteriophage, plasmid, phagemid or cosmid
• Various screening assays can then be used to select for the expressed VPI or VPI-related polypeptides.
• the various anti-VPI antibodies of the invention can be used to identify the desired clones using methods known in the art. See, for example, Harlow and Lane, 1988 Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Appendix IV. Colonies or plaques from the library are brought into contact with the antibodies to identify those clones that bind antibody.
• colonies or plaques containing DNA that encodes a VPI, a fragment of a VPI, a VPI-related polypeptide, or a fragment of a VPI-related polypeptide can be detected using DYNA Beads according to Olsvick et al, 29th ICAAC, Houston, Tex. 1989, incorporated herein by reference.
• Anti-VPI antibodies are crosslinked to tosylated DYNA Beads M280, and these antibody-containing beads are then contacted with colonies or plaques expressing recombinant polypeptides. Colonies or plaques expressing a VPI or VPI-related polypeptide are identified as any of those that bind the beads.
• the anti-VPI antibodies can be nonspecifically immobilized to a suitable support, such as silica or Celite® resin. This material is then used to adsorb to bacterial colonies expressing the VPI protein or VPI-related polypeptide as described herein.
• PCR amplification may be used to isolate from genomic DNA a substantially pure DNA (i.e., a DNA substantially free of contaminating nucleic acids) encoding the entire VPI or a part thereof.
• a substantially pure DNA i.e., a DNA substantially free of contaminating nucleic acids
• a DNA is at least 95% pure, more preferably at least 99% pure.
• Oligonucleotide sequences, degenerate or otherwise, that correspond to peptide sequences of VPIs disclosed herein can be used as primers.
• PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp® or AmpliTaq DNA polymerase).
• a Perkin-Elmer Cetus thermal cycler and Taq polymerase Gene Amp® or AmpliTaq DNA polymerase.
• After successful amplification of a segment of the sequence encoding a VPI that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of
• the gene encoding a VPI can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified DNA encoding a VPI of another species (e.g., mouse, human). Immunoprecipitation analysis or functional assays (e.g., aggregation ability in vitro; binding to receptor) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies that specifically recognize a VPI. A radiolabelled cDNA encoding a
• VPI can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template.
• the radiolabelled mRNA or cDNA may then be used as a probe to identify the DNA fragments encoding a VPI from among other genomic DNA fragments.
• RNA for cDNA cloning of the gene encoding a VPI can be isolated from cells that express the VPI.
• Any suitable eukaryotic cell can serve as the nucleic acid source for the molecular cloning of the gene encoding a VPI.
• the nucleic acid sequences encoding the VPI can be isolated from vertebrate, mammalian, primate, human, porcine, bovine, feline, avian, equine, canine or murine sources.
• the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell.
• Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences.
• the identified and isolated gene or cDNA can then be inserted into any suitable cloning vector.
• vector-host systems known in the art may be used.
• the vector system chosen be compatible with the host cell used.
• Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, plasmids such as PBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene) or modified viruses such as adenoviruses, adenp-associated viruses or refroviruses.
• the insertion into a cloning vector can be accomplished, for example, by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
• the ends of the DNA molecules may be enzymatically modified.
• any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
• the cleaved vector and the gene encoding a VPI may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc. s so that many copies of the gene sequence are generated.
• transformation of host cells with recombinant DNA molecules that incorporate the isolated gene encoding the VPI, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
• the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
• nucleotide sequences of the present invention include nucleotide sequences encoding amino acid sequences with substantially the same amino acid sequences as native VPIs, nucleotide sequences encoding amino acid sequences with functionally equivalent amino acids, nucleotide sequences encoding VPIs, a fragments of VPIs, VPI- related polypeptides, or fragments of VPI-related polypeptides.
• an isolated nucleic acid molecule encoding a VPI- related polypeptide can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of a VPI such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
• Standard techniques known to those of skill in the art can be used to introduce mutations, including, for example, site- directed mutagenesis and PCR-mediated mutagenesis.
• conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
• a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
• Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
• basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.
• mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
• the encoded protein can be expressed and the activity of the protein can be determined.
• the nucleotide sequence coding for a VPI, a VPI analog, a VPI-related peptide, or a fragment or other derivative of any of the foregoing can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
• the necessary transcriptional and franslational signals can also be supplied by the native gene encoding the VPI or its flanking regions, or the native gene encoding the VPI- related polypeptide or its flanking regions.
• a variety of host-vector systems may be utilized in the present invention to express the protein- coding sequence.
• mammalian cell systems infected with virus e.g., vaccinia virus, adenovirus, etc.
• insect cell systems infected with virus e.g., baculovirus
• microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
• the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
• a nucleotide sequence encoding a human gene or a nucleotide sequence encoding a functionally active portion of a human VPI
• a fragment of a VPI comprising a domain of the VPI is expressed.
• any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional and franslational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequence encoding a VPI or fragment thereof may be regulated by a second nucleic acid sequence so that the VPI or fragment is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a VPI may be controlled by any promoter or enhancer element known in the art.
• Promoters which may be used to control the expression of the gene encoding a VPI or a VPI- related polypeptide include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon Nature (1981) 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell (1980) 22:787-797), the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci.
• promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al, Cell (1984)
• mice mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, Cell (1986) 45:485-495), albumin gene control region which is active in liver (Pinkert et al, Genes andDevel. (1987) 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al, Mol. Cell. Biol. (1985) 5:1639-1648; Hammer et al, Science (1987) 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, Genes and Devel.
• beta-globin gene control region which is active in myeloid cells (Mogram et al, Nature (1 ' 985) 315:338-340; Kollias et al, Cell (1986) 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, Cell (1987) 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, Nature (1985) 314:283-286); neuronal-specific enolase (NSE) which is active in neuronal cells (Morelli et al, Gen. Virol.
• NSE neuronal-specific enolase
• BDNF brain-derived neurotrophic factor
• GFAP glial fibrillary acidic protein
• a vector in a specific embodiment, comprises a promoter operably linked to a VPI-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).
• an expression construct is made by subcloning a VPI or a VPI-related polypeptide coding sequence into the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, Gene (1988) 7:31-40). This allows for the expression of the VPI product or VPI-related polypeptide from the subclone in the correct reading frame.
• VPI coding sequence or VPI-related polypeptide coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
• Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA (1984) 81:355-359).
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
• These exogenous franslational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al, Methods in Enzymol. (1987) 153:51-544).
• Expression vectors containing inserts of a gene encoding a VPI or a VPI- related polypeptide can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
• first approach the presence of a gene encoding a VPI inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted gene encoding a VPI.
• probes comprising sequences that are homologous to an inserted gene encoding a VPI.
• the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a gene encoding a VPI in the vector.
• certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
• recombinant expression vectors can be identified by assaying the gene product (i.e., VPI) expressed by the recombinant.
• VPI gene product expressed by the recombinant.
• assays can be based, for example, on the physical or functional properties of the VPI in in vitro assay systems, e.g., binding with anti- VPI antibody.
• a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered VPI or VPI-related polypeptide may be controlled.
• different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins).
• Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system will produce an unglycosylated product and expression in yeast will produce a glycosylated product.
• Eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
• Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, neuronal cell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastomas (Sugimoto et al, J. Natl. Cancer Inst. (1984) 73: 51-57), SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704: 450-460), Daoy human cerebellar medulloblastoma (He et al, Cancer Res.
• neuronal cell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastomas (Sugimoto et al, J. Natl. Cancer Inst. (1984) 73: 51-57), SK-N-SH human neuroblastoma (Biochim. Biophy
• DBTRG-05MG glioblastoma cells (Kruse et al, In vitro Cell. Dev. Biol. (1992) 28A: 609-614), IMR-32 human neuroblastoma (Cancer Res., (1970) 30:2110-2118), 1321N1 human astrocytoma (Proc. Natl Acad. Sci. USA (1977) 74:4816), MOG-G-CCM human astrocytoma (Br. J. Cancer, (1984) 49:269), U87MG human glioblastoma-astrocytoma (Acta Patliol. Microbiol.
• cell lines which stably express the differentially expressed or pathway gene protein may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the differentially expressed or pathway gene protein.
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the differentially expressed or pathway gene protein.
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al, Cell (1977) 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA (1962) 48:2026), and adenine phosphoribosyltransferase (Lowy, et al, Cell (1980) 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al, Proc. Natl. Acad. Sci. USA (1980) 77:3567; O'Hare, et al, Proc. Natl. Acad. Sci. USA (1981) 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA (1981) 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre- Garapin, et al, J. Mol. Biol. (1981) 150:1); and hygro, which confers resistance to hygromycin (Santerre, et al, Gene (1984) 30:147) genes.
• the VPI, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence).
• the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• CHI constant domain of immunoglobulins
• CH2, CH3, or any combination thereof and portions thereof resulting in chimeric polypeptides.
• Such fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.
• Nucleic acids encoding a VPI, a fragment of a VPI, a VPI-related polypeptide, or a fragment of a VPI-related polypeptide can be fused to an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide.
• an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
• HA hemagglutinin
• a system described by Janknecht et al allows for the ready purification of non- denatured fusion proteins expressed in human cell lines (Janknecht et al, Proc. Natl. Acad. Sci. USA (1991) 88:8972-897).
• Fusion proteins can be made by ligating the appropriate nucleic 'acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in. the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
• a fusion protein may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
• cDNA and genomic sequences can be cloned and expressed.
• domains of some VPIs are known in the art and have been described in the scientific literature. Moreover, domains of a VPI can be identified using techniques known to those of skill in the art. For example, one or more domains of a VPI can be identified by using one or more of the following programs: ProDom, TMpred, and SAPS. ProDom compares the amino acid sequence of . a polypeptide to a database of compiled domains (see, e.g., http://www.toulouse.inra.fr/prodom.html; Corpet F., Gouzy J. & Kahn D., Nucleic Acids Res., (1999) 27:263-267).
• TMpred predicts membrane-spanning regions of a polypeptide and their orientation.
• This program uses an algorithm that is based on the statistical analysis of TMbase, a database of naturally occuring transmembrane proteins (see, e.g., http://www.ch.embnet.org/software/TMPRED_form.html; Hofmann & Stoffel. (1993) "TMbase - A database of membrane spanning proteins segments.” Biol. Chem. Hoppe-Seyler 347,166).
• the SAPS program analyzes polypeptides for statistically significant features like charge-clusters, repeats, hydrophobic regions, compositional domains (see, e.g., Brendel et al, Proc. Natl. Acad. Sci.
• the skilled artisan can identify domains of a VPI having enzymatic or binding activity, and further can identify nucleotide sequences encoding such domains. These nucleotide sequences can then be used for recombinant expression of a VPI fragment that retains the enzymatic or binding activity of the VPI. Based on the present description, the skilled artisan can identify domains of a VPI having enzymatic or binding activity, and further can identify nucleotide sequences encoding such domains. These nucleotide sequences can then be used for recombinant expression of
• a VPI has an amino acid sequence sufficiently similar to an identified domain of a known polypeptide.
• the term "sufficiently similar” refers to a first amino acid or nucleotide sequence which contains a sufficient number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have or encode a common structural domain or common functional activity or both.
• a VPI domain can be assessed for its function using techniques well known to those of skill in the art.
• a domain can be assessed for its kinase activity or for its ability to bind to DNA using techniques known to the skilled artisan.
• Kinase activity can be assessed, for example, by measuring the ability of a polypeptide to phosphorylate a substrate.
• DNA binding activity can be assessed, for example, by measuring the ability of a polypeptide to bind to a DNA binding element in a electromobility shift assay.
• the function of a domain of a VPI is determined using an assay described in one or more of the references identified in Table XI, infra.
• a VPI, VPI analog, VPI-related protein or a fragment or derivative of any of the foregoing may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
• immunogens can be isolated by any convenient means, including the methods described above.
• Antibodies of the invention include, but are not limited to polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
• the immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA ) or subclass of immunoglobulin molecule.
• antibodies that recognize gene products of genes encoding VPIs are publicly available.
• antibodies that recognize these VPIs and/or their isoforms include antibodies recognizing: VPI-2, VPI-6, VPI-7, VPI-8, VPI-9, VPI- 10, Wil l, VPI-14, VPI-15, VPI-17, VPI-23, VPI-24, VPI-25, VPI-27, VPI-31, VPI-43, VPI-48, VPI- 49, VPI-50, VPI-56, VPI-57, VPI-58, VPI-61, VPI-62, VPI-63, VPI-64, VPI-65, VPI-66, VPI- 67, VPI-70, VPI-77, VPI-79, VPI-80, VPI-81, VPI-83, VPI-84, VPI-87, VPI-88 * , VPI-90, VPI- 91, VPI-92, VPI-93, VPI-94, VPI-97, VPI-98, VPI-99, VPI-109, VPI-110, V
• methods known to those skilled in the art are used to produce antibodies that recognize a VPI, a VPI analog, a VPI-related polypeptide, or a derivative or fragment of any of the foregoing.
• antibodies to a specific domain of a VPI are produced.
• hydrophilic fragments of a VPI are used as immunogens for antibody production.
• screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
• ELISA enzyme-linked immunosorbent assay
• an antibody that specifically binds a first VPI homolog but which does not specifically bind to (or binds less avidly to) a second VPI homolog one can select on the basis of positive binding to the first VPI homolog and a lack of binding to (or reduced binding to) the second VPI homolog.
• the present invention provides an antibody (preferably a monoclonal antibody) that binds with greater affinity (preferably at least 2-fold, more preferably at least 5-fold still more preferably at least 10- fold greater affinity) to a VPI than to a different isoform or isoforms (e.g., glycoforms) of the VPI.
• Polyclonal antibodies that may be used in the methods of the invention are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Unfractionated immune serum can also be used. Various procedures known in the art may be used for the production of polyclonal antibodies to a VPI, a fragment of a VPI, a VPI-related polypeptide, or a fragment of a VPI-related polypeptide. In a particular embodiment, rabbit polyclonal antibodies to an epitope of a VPI or a VPI- related polypeptide can be obtained.
• VPI native or a synthetic (e.g., recombinant) version of a VPI, a fragment of a VPI, a VPI-related polypeptide, or a fragment of a VPI-related polypeptide, including but not limited to rabbits, mice, rats, etc.
• the Preferred Technology described herein provides isolated VPIs suitable for such immunization. If the VPI is purified by gel electrophoresis, the VPI can be used for immunization with or without prior extraction from the polyacrylamide gel.
• adjuvants may be used to enhance the immunological response, depending on the host species, including, but not limited to, complete or incomplete Freund's adjuvant, a mineral gel such as aluminum hydroxide, surface active substance such as lysolecithin, pluronic polyol, a polyanion, a peptide, an oil emulsion, keyhole limpet hemocyanin, dinitrophenol, and an adjuvant such as BCG (bacille Calmette-Guerin) or corynebacterium parvum. Additional adjuvants are also well known in the art.
• any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
• the hybridoma technique originally developed by Kohler and Milstein (Nature (1975) 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.
• Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
• the hybridoma producing the mAbs of the invention may be cultivated in vitro or in vivo.
• monoclonal antibodies can be produced in germ-free animals utilizing known technology (PCT/US90/02545, incorporated herein by reference).
• the monoclonal antibodies include but are not limited to human monoclonal antibodies and chimeric monoclonal antibodies (e.g., human-mouse chimeras).
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those .having a human immunoglobulin constant region and a variable region derived from a murine mAb. (See, e.g., Cabilly et al, U.S. Patent No. 4,816,567; and Boss ' et al, U.S. Patent No.
• Humanized antibodies are antibody molecules from non- human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al, 1988, Science 240:1041-1043; Liu et al, Proc. Natl. Acad. Sci. USA (1987) 84:3439-3443; Liu et al, J. Immunol. (1987) 139:3521-3526; Sun et al, Proc.
• Fully human antibodies are particularly desirable for therapeutic treatment of human subjects.
• Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
• the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a VPI of the invention.
• Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
• the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
• companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
• Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
• a selected non-human monoclonal antibody e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, Bio/technology (1994) 12:899- 903).
• the antibodies of the present invention can also be generated using various phage display methods known in the art.
• phage display methods functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them.
• phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
• Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labelled antigen or antigen bound or captured to a solid surface or bead.
• Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
• Phage display methods that, can be used to make the antibodies of the present invention include those disclosed in Brinkman et al, J. Immunol. Methods (1995) 182:41-50 ; Ames et al, J. Immunol. Methods (1995) 184:177-186 ; Kettleborough et al, Eur. J. Immunol.
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
• techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in
• the invention further provides for the use of bispecific antibodies, which can be made by methods known in the art.
• Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, Nature (1983) 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, EMBO J. (1991) 10:3655-3659 .
• antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
• the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions.
• DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
• the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
• the invention provides functionally active fragments, derivatives or analogs of the anti-VPI immunoglobulin molecules.
• Functionally active means that the fragment, derivative or analog is able, to elicit anti-anti-idiotype antibodies (i.e., tertiary antibodies) that recognize the same antigen that Is recognized by the antibody from which the fragment, derivative or analog is derived.
• antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen.
• synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.
• the present invention provides antibody fragments such as, but not limited to, F(ab')2 fragments and Fab fragments. Antibody fragments which recognize specific epitopes may be generated by known techniques.
• F(ab')2 fragments consist of the variable region, the light chain constant region and the CHI domain of the heavy chain and are generated by pepsin digestion of the antibody molecule.
• Fab fragments are generated by reducing the disulfide bridges of the F(ab')2 fragments.
• the invention also provides heavy chain and light chain dimers of the antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent 4,946,778; Bird, Science (1988) 242:423-42; Huston et al, Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al, Nature (1989) 334:544-54), or any other molecule with the same specificity as the antibody of the invention.
• Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E.
• the invention provides fusion proteins of the immunoglobulins of the invention (or functionally active fragments thereof), for example in which the immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the immunoglobulin.
• a covalent bond e.g., a peptide bond
• the immunoglobulin, or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.
• fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.
• the immunoglobulins of the invention include analogs and derivatives that are either modified, i.e, by the covalent attachment of any type of molecule as long as such covalent attachment that does not impair immunospecific binding.
• the derivatives and analogs of the immunoglobulins include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the analog or derivative may contain one or more non-classical amino acids.
• the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the VPIs of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
• the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
• a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, BioTechniques (1994) 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• the nucleic acid encoding the antibody may be obtained by cloning the antibody. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe " specific for the particular gene sequence.
• a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody
• antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies.
• a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et al, Science (1989) 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al, Nature (1991) 352:624; Hane et al, Proc. Natl. Acad. Sci. USA (1997) 94:4937).
• nucleic acid encoding at least the variable domain of the antibody molecule may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464).
• Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available.
• the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group.
• Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson et al, J. Biol. Chem. (1978) 253:6551), PCT based methods, etc.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human antibody constant region, e.g., humanized antibodies.
• the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
• methods for preparing the protein of the invention by expressing nucleic acid containing the antibody molecule sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing an antibody molecule coding sequences and appropriate transcriptional and franslational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al, (1990,
• the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
• the host cells used to express a recombinant antibody of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, Gene (1986) 45:101; Cockett et al, Bio/Technology ( 1990) 8 :2).
• host-expression vector systems may be utilized to express an antibody molecule of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculoviras) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
• vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. (1983) 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
• the virus grows in Spodoptera frugiperda cells.
• the antibody coding sequence may be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under confrol of an AcNPV promoter (for example the polyhedrin promoter).
• a number of viral-based expression systems e.g., an adenovirus expression system
• a host cell strain may be chosen which modulates the expression of the inserted sequences, or. modifies and processes the gene product in the specific fashion desired.
• Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
• stable expression is preferred.
• cells lines that stably express an antibody of interest can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable (e.g., neomycin or hygromycin), and selecting for expression of the selectable marker.
• a selectable e.g., neomycin or hygromycin
• the expression levels of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• a marker in the vector system expressing antibody is amplifiable
• increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, 1983, Mol. Cell. Biol. 3:257).
• the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature (1986) 322:52; Kohler, Proc. Natl. Acad. Sci. USA (1980) 77:2197).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• the antibody molecule of the invention may be purified by any method known in the art for purification of an antibody molecule, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• chromatography e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography
• centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed.
• a system described by Janknecht et al allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al, Proc. Natl. Acad. Sci. USA (1991) 88:8972-897).
• the gene of interest' is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
• the tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid- agarose columns and histidine-tagged proteins are selectively eluted with imidazole- containing buffers.
• anti-VPI antibodies or fragments thereof are conjugated to a diagnostic or therapeutic moiety.
• the antibodies can be used for diagnosis or to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Patent No.
• Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 1251, 1311, ll lln and 99Tc.
• Anti-VPI antibodies or fragments thereof can be conjugated to a therapeutic agent or drug moiety to modify a given biological response.
• the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G- CSF), nerve growth factor (NGF) or other growth factor.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumor necrosis factor,
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
• An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with cytotoxic factor(s) and/or cytokine(s).
• test samples of cerebrospinal fluid (CSF), serum, plasma or urine obtained from a subject suspected of having or known to have Vascular Dementia can be used for diagnosis or monitoring.
• CSF cerebrospinal fluid
• a decreased abundance of one or more VFs or VPIs (or any combination of them) in a test sample relative to a control sample (from a subject or subjects free from Vascular Dementia) or a previously determined reference range indicates the presence of Vascular Dementia; VFs and VPIs suitable for this purpose are identified in Tables I and IV, respectively, as described in detail above.
• an increased abundance of one or more VFs or VPIs (or any combination of them) in a test sample compared to a control sample or a previously determined reference range indicates the presence of Vascular Dementia; VFs and VPIs suitable for this purpose are identified in Tables II and V, respectively, as described in detail above.
• the relative abundance of one or more VFs or VPIs (or any combination of them) in a test sample compared to a control sample or a previously determined reference range indicates.
• a subtype of Vascular Dementia e.g., familial or sporadic Vascular Dementia).
• the relative abundance of one or more VFs or VPIs (or any combination of them) in a test sample relative to a control sample or a previously determined reference range indicates the degree or severity of Vascular Dementia.
• detection of one or more VPIs described herein may optionally be combined with detection of one or more additional biomarkers for
• Vascular Dementia Any suitable method in the art can be employed to measure the level of
• VFs and VPIs including but not limited to the Preferred Technology described herein, kinase assays, immunoassays to detect and/or visualize the VPI (e.g., Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc.).
• an assay for that function may be used to measure VPI expression.
• a decreased abundance of mRNA encoding one or more VPIs identified in Table IV (or any combination of them) in a test sample relative to a control sample or a previously determined reference range indicates the presence of Vascular Dementia.
• an increased abundance of mRNA encoding one or more VPIs identified in Table V (or any combination of them) in a test sample relative to a control sample or previously determined reference range indicates the presence of Vascular Dementia.
• Any suitable hybridization assay can be used to detect VPI expression by detecting and/or visualizing mRNA encoding the VPI (e.g., Northern assays, dot blots, in situ hybridization, etc.).
• labelled antibodies, derivatives and analogs thereof, which specifically bind to a VPI can be used for diagnostic purposes to detect, diagnose, or monitor Vascular Dementia.
• Vascular Dementia is detected in an animal, more preferably in a mammal and most preferably in a human.
• the invention provides methods for identifying agents (e.g., candidate compounds or test compounds) that bind to a VPI or have a stimulatory or inhibitory effect on the expression or activity of a VPI.
• the invention also provides methods of identifying agents, candidate compounds or test compounds that bind to a VPI-related polypeptide or a VPI fusion protein or have a stimulatory or inhibitory effect on the expression or activity of a VPI-related polypeptide or a VPI fusion protein.
• agents, candidate compounds or test compounds include, but are not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs.
• Agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one- compound” library method; and synthetic library methods using affinity chromatography selection.
• biological libraries are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam,
• Libraries of compounds may be presented, e.g., presented in solution (e.g., Houghten, Bio/Techniques (1992) 13:412-421), or on beads (Lam, Nature (1991) 354:82-84), chips (Fodor, Nature (1993) 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci.
• agents that interact with (i.e., bind to) a VPI, a VPI fragment (e.g. a functionally active fragment), a VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein are identified in a cell-based assay system.
• a VPI fragment e.g. a functionally active fragment
• a VPI-related polypeptide e.g. a functionally active fragment
• a VPI-related polypeptide e.g. a fragment of a VPI-related polypeptide, or a VPI fusion protein
• cells expressing a VPI, a fragment of a VPI, a VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein are contacted with a candidate compound or a control compound and the ability of the candidate compound to interact with the VPI is determined.
• this assay may be used to screen a plurality (e
• the cell for example, can be of prokaryotic origin (e.g., E. coli) or eukaryotic origin (e.g., yeast or mammalian). Further, the cells can express the VPI, fragment of the VPI, VPI-related polypeptide, a fragment of the VPI-related polypeptide, or a VPI fusion protein endogenously or be genetically engineered to express the VPI, fragment of the VPI, VPI-related polypeptide, a fragment of the VPI- related polypeptide, or a VPI fusion protein.
• prokaryotic origin e.g., E. coli
• eukaryotic origin e.g., yeast or mammalian.
• the cells can express the VPI, fragment of the VPI, VPI-related polypeptide, a fragment of the VPI-related polypeptide, or a VPI fusion protein endogenously or be genetically engineered to express the VPI, fragment of the VPI, VPI-related polypeptide, a
• the VPI, fragment of the VPI, VPI-related polypeptide, a fragment of the VPI-related polypeptide, or a VPI fusion protein or the candidate compound is labelled, for example with a radioactive label (such as P, S or I) or a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine) to enable detection of an interaction between a VPI and a candidate compound.
• a radioactive label such as P, S or I
• a fluorescent label such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine
• the ability of the candidate compound to interact directly or indirectly with a VPI, a fragment of a VPI, a VPI-related polypeptide, a fragment of a VPI- related polypeptide, or a VPI fusion protein can be determined by methods known to those of skill in the art.
• the interaction between a candidate compound and a VPI, a fragment of a VPI, a VPI-related polypeptide, a fragment of a VPI- related polypeptide, or a VPI fusion protein can be determined by flow cytometry, a scintillation assay, immunoprecipitation or western blot analysis.
• agents that interact with (i.e., bind to) a VPI, a VPI fragment (e.g., a functionally active fragment) a VPI-related polypeptide, a fragment of a VPI-related polypeptide, .or a VPI fusion protein are identified in a cell-free assay system.
• a native or recombinant VPI or fragment thereof, or a native or recombinant VPI-related polypeptide or fragment thereof, or a VPI-fusion protein or fragment thereof is contacted with a candidate compound or a control compound and the ability of the candidate compound to interact with the VPI or VPI-related polypeptide, or VPI fusion protein is determined.
• this assay may be used to screen a plurality (e.g. a library) of candidate compounds.
• the VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI-fusion protein is first immobilized, by, for example, contacting the VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI- related polypeptide, or a VPI fusion protein with an immobilized antibody which specifically recognizes and binds it, or by contacting a purified preparation of the VPI, VPI fragment, VPI- related polypeptide, fragment of a VPI-related polypeptide, or a VPI fusion protein with a surface designed to bind proteins.
• VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein may be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate. Further, the VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide may be a fusion protein comprising the VPI or a biologically active portion thereof, or VPI-related polypeptide and a domain such as glutathionine- S-fransferase.
• VPI, VPI fragment, VPI-related polypeptide, fragment of a VPI-related polypeptide or VPI fusion protein can be biotinylated using techniques well known to those of skill in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL).
• biotinylation kit Pierce Chemicals; Rockford, IL.
• the ability of the candidate compound to interact with a VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein can be determined by methods known to those of skill in the art.
• a cell-based assay system is used to identify agents that bind to or modulate the activity of a protein, such as an enzyme, or a biologically active portion thereof, which is responsible for the production or degradation of a VPI or is responsible for the post- franslational modification of a VPI.
• a protein such as an enzyme, or a biologically active portion thereof, which is responsible for the production or degradation of a VPI or is responsible for the post- franslational modification of a VPI.
• a plurality e.g., a library
• cells that naturally or recombinantly express: (i) a VPI, an isoform of a VPI, a VPI homolog a VPI-related polypeptide, a VPI fusion protein, or a biologically active fragment of any of the foregoing; and (ii) a protein that is responsible for processing of the VPI, VPI isoform, VPI homolog, VPI-related polypeptide, VPI fusion protein, or fragment in order to identify compounds that modulate the production, degradation, or post-translational modification of the VPI, VPI isoform, VPI homolog, VPI-related polypeptide, VPI fusion protein or fragment.
• compounds identified in the primary screen can then be assayed in a secondary screen against cells naturally or recombinantly expressing the specific VPI of interest.
• the ability of the candidate compound to modulate the production, degradation or post-translational modification of a VPI, isoform, homolog, VPI- related polypeptide, or VPI fusion protein can be determined by methods known to those of skill in the art, including without limitation, flow cytometry, a scintillation assay, imrnimoprecipitation and western blot analysis.
• agents that competitively interact with (i.e., bind to) a VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein are identified in a competitive binding assay.
• cells expressing a VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein are contacted with a candidate compound and a compound known to interact with the VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide or a VPI fusion protein; the ability of the candidate compound to competitively interact with the VPI, VPI fragment, VPI-related polypeptide, fragment of a VPI-related polypeptide, or a VPI fusion protein is then determined.
• agents that competitively interact with (i.e., bind to) a VPI, VPI fragment, VPI- related polypeptide or fragment of a VPI-related polypeptide are identified in a cell-free assay system by contacting a VPI, VPI fragment, VPI-related polypeptide, fragment of a VPI-related polypeptide, or a VPI fusion protein with a candidate compound and a compound known to interact with the VPI, VPI-related polypeptide or VPI fusion protein.
• the ability of the candidate compound to interact with a VPI, VPI fragment, VPI-related polypeptide, a fragment of a VPI-related polypeptide, or a VPI fusion protein can be determined by methods known to those of skill in the art. These assays, whether cell-based or cell- free, can be used to screen a plurality (e.g., a library) of candidate compounds.
• agents that modulate i.e., upregulate or downregulate the expression of a VPI, or a VPI-related polypeptide are identified by contacting cells (e.g., cells of prokaryotic origin or eukaryotic origin) expressing the VPI, or VPI- related polypeptide with a candidate compound or a control compound (e.g., phosphate buffered saline (PBS)) and determining the expression of the VPI, VPI-related polypeptide, or VPI fusion protein, mRNA encoding the VPI, or mRNA encoding the VPI-related polypeptide.
• a candidate compound or a control compound e.g., phosphate buffered saline (PBS)
• the level of expression of a selected VPI, VPI-related polypeptide, mRNA encoding the VPI, or mRNA encoding the VPI-related polypeptide ' in the presence of the candidate compound is compared to the level of expression of the VPI, VPI-related polypeptide, mRNA encoding the VPI, or mRNA encoding the VPI-related polypeptide in the absence of the candidate compound (e.g., in the presence of a control compound).
• the candidate compound can then be identified as a modulator of the expression of the VPI, or a VPI-related polypeptide based on this comparison.
• the candidate compound when expression of the VPI or mRNA is significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of expression of the VPI or mRNA.
• the candidate compound when expression of the VPI or mRNA is significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the expression of the VPI or mRNA.
• the level of expression of a VPI or the mRNA that encodes it can be determined by methods known to those of skill in the art. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR,. and protein levels can be assessed by western blot analysis.
• agents that modulate the activity of a VPI, or a VPI- related polypeptide are identified by contacting a preparation containing the VPI or VPI-related polypeptide, or cells (e.g., prokaryotic or eukaryotic cells) expressing the VPI or VPI-related polypeptide with a test compound or a control compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the VPI or VPI-related polypeptide.
• the activity of a VPI or a VPI-related polypeptide can be assessed by detecting induction of a cellular signal transduction pathway of the VPI or VPI-related polypeptide (e.g.
• telomeres intracellular Ca2+, diacylglycerol, IP3, etc.
• detecting catalytic or enzymatic activity of the target on a suitable substrate detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a VPI or a VPI-related polypeptide and is operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
• a reporter gene e.g., a regulatory element that is responsive to a VPI or a VPI-related polypeptide and is operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
• a cellular response for example, cellular differentiation, or cell proliferation.
• the candidate compound can then be identified as a modulator of the activity of a VPI or VPI-related polypeptide by comparing the effects of the candidate compound to the control compound.
• Suitable control compounds include phosphate buffered saline (PBS) and normal saline (NS).
• agents that modulate i.e., upregulate or downregulate) the expression, activity or both the expression and activity of a VPI or VPI-related polypeptide are identified in an animal model.
• suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats.
• the animal used represents a model of Vascular Dementia (e.g., animal models of cerebral beta-amyloid angiopathy (Walker, Brain Res. Rev. (1997) 25:70-84), animal models of vascular dementia with emphasis on stroke-prone spontaneously hypertensive rats (Saito et al., Clin. Exp. Pharmacol. Physiol.
• test compound or a control compound is administered (e.g., orally, rectally or parenterally such as intraperitoneally or intravenously) to a suitable animal and the effect on the expression, activity or both expression and activity of the VPI or VPI-related polypeptide is determined. Changes in the expression of a VPI or VPI-related polypeptide can be assessed by the methods outlined above.
• a VPI or VPI-related polypeptide is used as a "bait protein" in a two-hybrid assay or three hybrid assay to identify other proteins that bind to or interact with a VPI or VPI-related polypeptide
• a VPI or VPI-related polypeptide see, e.g., U.S. Patent No. 5,283,317; Zervos et al, Cell (1993) 72:223-232; Madura et al, J. Biol. Chem. (1993) 268:12046-12054; Bartel et al, Bio/Techniques (1993) 14:920-924; Iwabuchi et al, Oncogene (1993) 8:1693-1696; and PCT Publication No.
• binding proteins are also likely to be involved in the propagation of signals by the VPIs of the invention as, for example, upstream or downstream elements of a signaling pathway involving the VPIs of the invention.
• Table XI enumerates scientific publications describing suitable assays for detecting or quantifying enzymatic or binding activity of a VPI, a VPI analog, a VPI- related polypeptide, or a fragment of any of the foregoing. Each such reference is hereby incorporated in its entirety.
• as assay referenced in Table XI is used in the screens and assays described herein, for example to screen for or identify a compound that modulates the activity of (or that modulates both the expression and activity of) a VPI, VPI analog, or VPI- related polypeptide, a fragment of any of the foregoing.
• This invention further provides novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
• VPIs The invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound.
• Such compounds include but are not limited to: VPIs, VPI analogs, VPI-related polypeptides and derivatives (including fragments) thereof; antibodies to the foregoing; nucleic acids encoding VPIs, VPI analogs, VPI-related polypeptides and fragments thereof; antisense nucleic acids to a gene encoding a VPI or VPI- related polypeptide; and modulator (e.g., agonists and antagonists) of a gene encoding a VPI or VPI-related polypeptide.
• An important feature of the present invention is the identification of genes encoding VPIs involved in Vascular Dementia. Vascular Dementia can be treated
• a therapeutic compound that promotes function or expression of one or more VPIs that are decreased in the CSF of Vascular Dementia subjects having Vascular Dementia or prevented by administration of a therapeutic compound that reduces function or expression of one or more VPIs that are increased in the CSF of subjects having Vascular Dementia.
• one or more antibodies each specifically binding to a VPI are administered alone or in combination with one or more additional therapeutic compounds or treatments.
• additional therapeutic compounds or treatments include, but are not limited to, antithrombic therapies such as Danaparoid, Nadroparin and Tinzaparin, thrombolytic and defibrinogenating agents such as Pro-urokinase, sfreptokinase, tissue plasmoinogen activator and urokinase, antiplatelet agents such as aspirin, Buflomedil (Cucinotta et al. J Int Med Res (1992) 20:136-49), neuroprotective agents such as Propentofylline (Rother et al.
• cytoprotective agents currently under clinical evaluation such as the calcium antagonists Nimodipine andNicadipine, NMDA antagonists such as Selfotel, Dextrorphan, Cerestat, Eliprodil, Lamortigine, GABA agonists, Kappa- selective opiod antagonists, Lubeluzole, Free radicalscavengers, anti-ICAM antibodies and GM-1 ganglioside, Abbokinase®, Activase®, Aggrenox®, Anti-ICAM- 1 antibody, Anti-beta- 2-integrin antibody, Arvin®, Atacand®, CerAxon®, Cerebyx®, Ceresine®, Cerestat®, Cervene®, Coumadin®, Fiblast®,Fraxiparine®, Freedox®, Innohep®, Kabikinase®, Klerval®, LeukArrest®, Lipitor®, Lovenox®, Neurogard
• a biological product such as an antibody is allogeneic to the subject to which it is administered.
• a human VPI or a human VPI- related polypeptide, a nucleotide sequence encoding a human VPI or a human VPI- related polypeptide, or an antibody to a human VPI or a human VPI-related polypeptide is administered to a human subject for therapy (e.g. to ameliorate symptoms or to retard onset or progression) or prophylaxis.
• Vascular Dementia is treated or prevented by administration to a subject suspected of having or known to have Vascular Dementia or to be at risk of developing Vascular Dementia of a compound that modulates (i.e., increases or decreases) the level or activity (i.e., function) of one or more VPIs or the level of one or more VFs that are differentially present in the CSF of subjects having Vascular Dementia compared with CSF of subjects free from Vascular
• Vascular Dementia is treated or prevented by administering to a subject suspected of having or known to have Vascular Dementia or to be at risk of developing Vascular Dementia a compound that upregulates (i.e., increases) the level or activity (i.e., function) of one or more VPIs or the level of one or more VFs that are decreased in the CSF of subjects having Vascular Dementia.
• a compound is administered that downregulates the level or activity (i.e., function) of one or more VPIs or the level of one or more VFs that are increased in the CSF of subjects having Vascular Dementia.
• Examples of such a compound include but are not limited to: VPIs, VPI fragments and VPI- related polypeptides; nucleic acids encoding a VPI, a VPI fragment and a VPI-related polypeptide (e.g., for use in gene therapy); and, for those VPIs or VPI-related polypeptides with enzymatic activity, compounds or molecules known to modulate that enzymatic activity.
• VPI agonists can be identified using in vitro assays.
• Vascular Dementia is also treated or prevented by administration to a subject suspected of having or known to have Vascular Dementia or to be at risk of developing Vascular Dementia of a compound that downregulates the level or activity of one or more VPIs or the level of one or more VFs that are increased in the CSF of subjects having Vascular Dementia.
• a compound is administered that upregulates the level or activity of one or more VPIs or the level of one or more VFs that are decreased in the CSF of subjects having Vascular Dementia.
• VPI antisense oligonucleotides examples include, but are not limited to, VPI antisense oligonucleotides, ribozymes, antibodies directed against VPIs, and compounds that inhibit the enzymatic activity of a VPI.
• VPI antagonists and small molecule VPI antagonists can be identified using in vitro assays.
• therapy or prophylaxis is tailored to the needs of an individual subject.
• compounds that promote the level or function of one or more VPIs, or the level of one or more VFs are therapeutically or prophylactically administered to a subject suspected of having or known to have Vascular Dementia, in whom the levels or functions of said one or more VPIs, or levels of said one or more VFs, are absent or are decreased relative to a confrol or normal reference range.
• compounds that promote the level or function of one or more VPIs, or the level of one or more VFs are therapeutically or prophylactically administered to a subject suspected of having or known to have Vascular Dementia in whom the levels or functions of said one or more VPIs, or levels of said one or more VFs, are increased relative to a confrol or to a reference range.
• compounds that decrease the level or function of one or more VPIs, or the level of one or more VFs are therapeutically or prophylactically administered to a subject suspected of having or known to have Vascular Dementia in whom the levels or functions of said one or more VPIs, or levels of said one or more VFs, are increased relative to a control or to a reference range.
• compounds that decrease the level or function of one or more VPIs, or the level of one or more VFs are therapeutically or prophylactically administered to a subject suspected of having or known to have Vascular Dementia in whom the levels or functions of said one or more VPIs, or levels of said one or more VFs, are decreased relative to a control or to a reference range.
• VPI function or level, or VF level due to the administration of such compounds can be readily detected, e.g., by obtaining a sample (e.g., a sample of CSF, blood or urine or a tissue sample such as biopsy tissue) and assaying in vitro the levels of said VFs or the levels or activities of said VPIs, or the levels of mRNAs encoding said VPIs. or any combination of the foregoing.
• a sample e.g., a sample of CSF, blood or urine or a tissue sample such as biopsy tissue
• assays can be performed before and after the administration of the compound as described herein.
• the compounds of the invention include but are not limited to any compound, e.g., a small organic molecule, protein, peptide, antibody, nucleic acid, etc.
• antithrombic therapies such as Danaparoid, Nadroparin and Tinzaparin
• thrombolytic and defibrinogenating agents such as Pro-urokinase, streptokinase, tissue plasmoinogen activator and urokinase
• Alzheimer Dis Assoc Disord (1998) 12 Suppl 2:S29-35, Rother et al. Dement Geriatr Cogn Disord (1998) 9 Suppl 1:36-43), cholinesterase inhibitors such as rivastigmine, galantamine (Kumar et al.
• cytoprotective agents currently under clinical evaluation such as the calcium antagonists Nimodipine andNicadipine, NMDA antagonists such as Selfotel, Dextrorphan, Cerestat, Eliprodil, Lamortigine, GABA agonists, Kappa-selective opiod antagonists, Lubeluzole, Free radicalscavengers, anti-ICAM antibodies and GM-1 ganglioside, Abbokinase®, Activase®, Aggrenox®, Anti-ICAM- 1 antibody, Anti-beta-2-integrin antibody, Arvin®, Atacand®, CerAxon®, Cerebyx®, Ceresine®, Cerestat®, Cervene®, Coumadin®, Fiblast®,Fraxiparine®, Freedox®, Innohep®, Kabikinase®, Klerval®, LeukArrest®, Lipitor®, Lovenox®
• nucleic acids comprising a sequence encoding a VPI, a VPI fragment, VPI-related polypeptide or fragment of a VPI-related polypeptide, are administered to promote VPI function by way of gene therapy.
• Gene therapy refers to administration to a subject of an expressed or expressible nucleic acid.
• the nucleic acid produces its encoded polypeptide that mediates a therapeutic effect by promoting VPI function.
• the compound comprises a nucleic acid encoding a VPI or fragment or chimeric protein thereof, said nucleic acid being part of an expression vector that expresses a VPI or fragment or chimeric protein thereof in a suitable host.
• a nucleic acid has a promoter operably linked to the VPI coding region, said promoter being inducible or constitutive (and, optionally, tissue-specific).
• a nucleic acid molecule is used in which the VPI coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the VPI nucleic acid (Roller and Smithies, Proc. Natl. Acad. Sci. USA (1989) 86:8932-8935; Zijlstra et al, Nature (1989) 342:435-438).
• Delivery of the nucleic acid into a subject may be direct, in which case the subject Hs directly exposed to the nucleic acid or nucleic acid-carrying vector; this approach is known as in vivo gene therapy.
• delivery of the nucleic acid into the subject may be indirect, in which case cells are first transformed with the nucleic acid in vitro and then transplanted into the subject; this approach is known as ex vivo gene therapy.
• the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
• Biolistic, Dupont by coating with lipids, cell-surface receptors or transfecting agents; by encapsulation in liposomes, microparticles or microcapsules; by administering it in linkage to a peptide which is known to enter the nucleus; or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. (1987) 262:4429- 4432). which can be used to target cell types specifically expressing the receptors.
• a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al,); WO 92/22635 dated December 23, 1992 (Wilson et al,); WO92/20316 dated November 26, 1992 (Findeis et al,); W093/14188 dated July 22, 1993 (Clarke et al,), WO 93/20221 dated October 14, 1993 (Young)).
• the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Roller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, Nature (1989) 342:435-438).
• a viral vector that contains a nucleic acid encoding a VPI is used.
• a refroviral vector can be used (see Miller et al, Meth. Enzymol. (1993) 217:581-599). These refroviral vectors have been modified to delete refroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
• the nucleic acid encoding the VPI to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a subject.
• refroviral vectors More detail about refroviral vectors can be found in Boesen et al, Biotherapy (1994) 6:291-302, which describes the use of a refroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illusfrating the use of refroviral vectors in gene therapy are: Clowes et al, J. Clin. Invest. (1994) 93:644-651; Kiem et al, Blood (1994) 83:1467-1473; Salmons and Gunzberg, Human Gene Therapy (1993) 4:129-141; and Grossman and Wilson, Curr. Opin. in Genetics andDevel. (1993) 3:110-114.
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. Biol. Med. (1993) 204:289-300; U.S. Patent No. 5,436,146).
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
• the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a subject.
• the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
• introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
• Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. (1993) 217:599-618; Cohen et al, Mefh.
• the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• the resulting recombinant cells can be delivered to a subject by various methods known in the art.
• epithelial cells are injected, e.g., subcutaneously.
• recombinant skin cells may be applied as a skin graft onto the subject.
• Recombinant blood cells e.g., hematopoietic stem or progenitor cells
• the amount of cells envisioned for use depends on the desired effect, the condition of the subject, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to neuronal cells, glial cells (e.g., oligodendrocytes or astrocytes), epithelial cells,, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood or fetal liver.
• glial cells e.g., oligodendrocytes or astrocytes
• epithelial cells e.g., endothelial cells
• keratinocytes keratin
• the cell used for gene therapy is autologous to the subject that is treated.
• a nucleic acid encoding a VPI is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem or progenitor cells which can be isolated and maintained in vitro can be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald,. Meth. Cell Bio. (1980) 21A:229; and Pittelkow and Scott, Mayo Clinic Proc. (1986) 61:771).
• the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
• Direct injection of a DNA coding for a VPI may also be performed according to, for example, the techniques described in United States Patent No. 5,589,466. These techniques involve the injection of "naked DNA", i.e., isolated DNA molecules in the absence of liposomes, cells, or any other material besides a suitable carrier.
• naked DNA comprising (a) DNA encoding a VPI and (b) a promoter are injected into a subject to elicit an immune response to the VPI.
• Vascular Dementia is treated or prevented by administration of a compound that antagonizes (inhibits) the level(s) and/or function(s) of one or more VPIs which are elevated in the CSF of subjects having Vascular Dementia as compared with CSF of subjects free from Vascular Dementia.
• Compounds useful for this purpose include but are not limited to anti-VPI antibodies (and fragments and derivatives containing the binding region thereof), VPI antisense or ribozyme nucleic acids, and nucleic acids encoding dysfunctional VPIs that are used to "knockout" endogenous VPI function by homologous recombination (see, e.g., Capecchi, Science (1989) 244:1288-1292).
• Other compounds that inhibit VPI function can be identified by use of known in vitro assays, e.g., assays for the ability of a test compound to inhibit binding of a VPI to another protein or a binding partner, or to inhibit a known VPI function.
• Such inhibition is assayed in vitro or in cell culture, but genetic assays may also be employed.
• the Preferred Technology can also be used to detect levels of the VPI before and after the administration of the compound.
• suitable in vitro or in vivo assays are utilized to determine the effect of a specific compound and whether its administration is indicated for treatment of the affected tissue, as described in more detail below.
• a compound that inhibits a VPI function is administered therapeutically or prophylactically to a subject in whom an increased CSF level or functional activity of the VPI (e.g., greater than the normal level or desired level) is detected as compared with CSF of subjects free from Vascular Dementia or a predetermined reference range.
• an increased CSF level or functional activity of the VPI e.g., greater than the normal level or desired level
• Methods standard in the art can be employed to measure the increase in a VPI level or function, as outlined above.
• Preferred VPI inhibitor compositions include small molecules, i.e., molecules of 1000 daltons or less. Such small molecules can be identified by the screening methods described herein.
• VPI expression is inhibited by use of VPI antisense nucleic acids.
• the present invention provides the therapeutic or prophylactic use of nucleic acids comprising at least six nucleotides that are antisense to a gene or cDNA encoding a VPI or a portion thereof.
• a VPI "antisense" nucleic acid refers to a nucleic acid capable of hybridizing by virtue of some sequence complementarity to a portion of an RNA (preferably mRNA) encoding a VPI.
• the antisense nucleic acid may be complementary to a coding and or noncoding region of an mRNA encoding a VPI.
• Such antisense nucleic acids have utility as compounds that inhibit VPI expression, and can be used in the treatment or prevention of Vascular Dementia.
• the antisense nucleic acids of the invention are double-stranded or single- stranded oligonucleotides, RNA or DNA or a modification or derivative thereof, and can be directly administered to a cell or produced infracellularly by transcription of exogenous, introduced sequences.
• the invention further provides pharmaceutical compositions comprising an effective amount of the VPI antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra.
• the invention provides methods for inhibiting the expression of a VPI nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing the cell with an effective amount of a composition comprising a VPI antisense nucleic acid of the invention.
• VPI antisense nucleic acids and their uses are described in detail below.
• the VPI antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides ranging from 6 to about 50 oligonucleotides.
• the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
• the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof and can be single- stranded or double-stranded.
• the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
• the oligonucleotide may include other appended groups such as peptides; agents that facilitate transport across the cell membrane (see, e.g., Letsinger et al, Proc. Natl. Acad. Sci. USA (1989) 86:6553-6556; Lemaitre et al, Proc. Natl. Acad. Sci. USA (1987) 84:648-652; PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, e.g., PCT Publication No.
• VPI antisense oligonucleotide is provided, preferably of single-stranded DNA.
• the oligonucleotide may be modified at any position on its structure with substituents generally known in the art.
• the VPI antisense oligonucleotide may comprise at least one of the following modified base moieties: 5-fTuorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5 -carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine,
• modified base moieties 5-fTuorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5 -car
• the oligonucleotide comprises at least one modified sugar moiety, e.g., one. of the following sugar moieties: arabinose, 2-fluoroarabinose, xylulose, and hexose.
• the oligonucleotide comprises at least one of the following modified phosphate backbones: a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, a formacetal, or an analog of formacetal.
• the oligonucleotide is an ⁇ -anomeric oligonucleotide.
• An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, 1987, Nucl. Acids Res. 15:6625-6641).
• the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent.
• Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
• an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
• phosphorothioate oligonucleotides may be synthesized by the method of Stein et al, (Nucl. Acids Res. (1988) 16:3209), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. N ⁇ tl. Ac ⁇ d. Sci. USA (1988) 85:7448-7451).
• the VPI antisense nucleic acid of the invention is produced infracellularly by transcription from an exogenous sequence.
• a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
• RNA antisense nucleic acid
• Such a vector would contain a sequence encoding the VPI antisense nucleic acid.
• Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Such vectors can be constructed by recombinant DNA technology standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the
• VPI antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Examples of such promoters are outlined above.
• the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene encoding a VPI, preferably a human gene encoding a VPI. However, absolute complementarity, although preferred, is not required.
• a sequence "complementary to at least a portion of an RNA,” as referred to herein, means a sequence having sufficient complementarity to be able to hybridize under stringent conditions (e.g., highly stringent conditions comprising hybridization in 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C and washing in O.lxSSC/0.1% SDS at 68 °C, or moderately stringent conditions comprising washing in 0.2xSSC/0.1% SDS at 42 °C ) with the RNA, forming a stable duplex; in the case of double-stranded VPI antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
• stringent conditions e.g., highly stringent conditions comprising hybridization in 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C and washing in O.lxSSC/0.1
• the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA encoding a VPI it may contain and still form a stable duplex (or triplex, as the case may be).
• One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
• the VPI antisense nucleic acids can be used to treat or prevent Vascular Dementia when the target VPI is overexpressed in the CSF of subjects suspected of having or suffering from Vascular Dementia.
• a single-stranded DNA antisense VPI oligonucleotide is used.
• RNA types which express or overexpress RNA encoding a VPI can be identified by various methods known in the art. Such cell types include but are not limited to leukocytes (e.g., neutrophils, macrophages, monocytes) and resident cells (e.g., astrocytes, glial cells, neuronal cells, and ependymal cells). Such methods include, but are not limited to, hybridization with a VPI-specific nucleic acid (e.g., by Northern hybridization, dot blot hybridization, in situ hybridization), observing the ability of RNA from the cell type to be translated in vitro into a VPI, immunoassay, etc. In a preferred aspect, primary tissue from a subject can be assayed for VPI expression prior to freatment, e.g., by immunocytochemistry or in situ hybridization.
• leukocytes e.g., neutrophils, macrophages, monocytes
• resident cells e.g., astrocytes,
• compositions of the invention comprising an effective amount of a VPI antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a subject having Vascular Dementia.
• Vascular Dementia can be determined by standard clinical techniques.
• compositions comprising one or more VPI antisense nucleic acids are administered via liposomes, microparticles, or microcapsules.
• such compositions may be used to achieve sustained release of the VPI antisense nucleic acids.
• symptoms of Vascular Dementia may be ameliorated by decreasing the level of a VPI or VPI activity by using gene sequences encoding the VPI in conjunction with well-known gene "knock-out,” ribozyme or triple helix methods to decrease gene expression of a VPI.
• ribozyme or triple helix molecules are used to modulate the activity, expression or synthesis of the gene encoding the VPI, and thus to ameliorate the symptoms of Vascular Dementia.
• Such molecules may be designed to reduce or inhibit expression of a mutant or non-mutant target gene. Techniques for the production and use of such molecules are well known to those of skill in the art.
• Ribozyme molecules designed to catalytically cleave gene mRNA transcripts encoding a VPI can be used to prevent translation of target gene mRNA and, therefore, expression of the gene product.
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi, Current Biology (1994) 4:469-471). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event.
• composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety.
• ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs encoding a VPI
• the use of hammerhead ribozymes is preferred.
• Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'- UG-3'.
• the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the mRNA encoding the VPI, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
• the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al, Science, (1984) 224:574-578; Zaug and Cech, Science, (1986) 231, 470-475; Zaug, et al, Nature, (1986) 324, 429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, Cell, (1986) 47:207-216).
• Cech-type ribozymes such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and that has been extensively described by Thomas Cech and collaborators (Zaug, et al, Science, (1984) 224:
• the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
• the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in the gene encoding the VPI.
• the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that express the VPI in vivo.
• a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that fransfected cells will produce sufficient quantities of the ribozyme to destroy endogenous mRNA encoding the VPI and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficacy.
• Endogenous VPI expression can also be reduced by inactivating or "knocking out” the gene encoding the VPI, or the promoter of such a gene, using targeted homologous recombination (e.g., see Smithies, et al, Nature (1985) 317:230-234; Thomas and Capecchi,
• a mutant gene encoding a non-functional VPI (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous gene (either the coding regions or regulatory regions of the gene encoding the VPI) can be used, with or without a selectable marker and/or a negative selectable marker, to fransfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene.
• ES embryonic stem
• inactive target gene e.g., see Thomas and Capecchi, 1987 and Thompson, 1989, supra.
• this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.
• the endogenous expression of a gene encoding a VPI can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the gene (i.e., the gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene encoding the VPI in target cells in the body.
• Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and ' composed of deoxynucleotides.
• the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
• Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC+ triplets across the three associated strands of the resulting triple helix.
• the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
• nucleic acid molecules may be chosen that are purine- rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
• the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule.
• Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
• the technique may so efficiently reduce or inhibit the transcription (triple helix) or translation (antisense, ribozyme) of mRNA produced by normal gene alleles of a VPI that the situation may arise wherein the concentration of VPI present may be lower than is necessary for a normal phenotype.
• gene therapy may be used to introduce into cells nucleic acid molecules that encode and express the VPI that exhibit normal gene activity and that do not contain sequences susceptible to whatever antisense, ribozyme, or triple helix treatments are being utilized.
• normal VPI can be co- administered in order to maintain the requisite level of VPI activity.
• Antisense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
• RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
• antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
• the present invention also provides assays for use in drug discovery in order to identify or verify the efficacy of compounds for treatment or prevention of Vascular Dementia.
• Test compounds can be assayed for their ability to restore VF or VPI levels in a . subject having Vascular Dementia towards levels found in subjects free from Vascular Dementia or to produce similar changes in experimental animal models of Vascular Dementia.
• Compounds able to restore VF or VPI levels in a subject having Vascular Dementia towards levels found in subjects free from Vascular Dementia or to produce similar changes in experimental animal models of Vascular Dementia can be used as lead compounds for further drug discovery, or used therapeutically.
• VF and VPI expression can be assayed by the Preferred Technology, immunoassays, gel electrophoresis followed by visualization, detection of VPI activity, or any other method taught herein or known to those skilled in the art.
• Such assays can be used to screen candidate drugs, in clinical monitoring or in drug development, where abundance of a VF or VPI can serve as a surrogate marker for clinical disease.
• in vitro assays can be carried out with cells representative of cell types involved in a subject's disorder, to determine if a compound has a desired effect upon such cell types.
• Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
• suitable animal model systems prior to administration to humans, any animal model system known in the art may be used.
• animal models of Vascular Dementia include, but are not limited to, animal models of cerebral beta-amyloid angiopathy (Walker, Brain Res. Rev. (1997) 25:70-84), animal models of vascular dementia with emphasis on stroke-prone spontaneously hypertensive rats (Saito et al., Clin. Exp. Pharmacol. Physiol.
• transgenic animals can be produced with "knock-out" mutations of the gene or genes encoding one or more VPIs.
• a "knock-out" mutation of a gene is a mutation that causes the mutated gene to not be expressed, or expressed in an aberrant form or at a low level, such that the activity associated with the gene product is nearly or entirely absent.
• the transgenic animal is a mammal, more preferably, the transgenic animal is a mouse.
• test compounds that modulate the expression of a VPI are identified in non-human animals (e.g, mice, rats, monkeys, rabbits, and guinea pigs), preferably non-human animal models for Vascular Dementia, expressing the VPI.
• a test compound or a confrol compound is administered to the animals, and the effect of the test compound on expression of one or more VPIs is determined.
• a test compound that alters the expression of a VPI can be identified by comparing the level of the selected VPI or VPIs (or mRNA(s) encoding the same) in an animal or group of animals treated with a test compound with the level of the
• VPI(s) or mRNA(s) in an animal or group of animals treated with a control compound can be used to determine the mRNA and protein levels, for example, in situ hybridization.
• the animals may or may not be sacrificed to assay the effects of a test compound.
• test compounds that modulate the activity of a VPI or a biologically active portion thereof are identified in non-human animals (e.g., mice, rats, monkeys, rabbits, and guinea pigs), preferably non-human animal models for Vascular Dementia, expressing the VPI.
• a test compound or a confrol compound is administered to the animals, and the effect of a test compound on the activity of a VPI is determined.
• a test compound that alters the activity of a VPI can be identified by assaying animals treated with a control compound and animals treated with the test compound.
• the activity of the VPI can be assessed by detecting induction of a cellular second messenger of the VPI (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic or enzymatic activity of the VPI or binding partner thereof, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a VPI of the invention operably linked to a nucleic acid encoding a detectable marker, such as luciferase or green fluorescent protein), or detecting a cellular response (e.g., cellular differentiation or cell proliferation).
• a reporter gene e.g., a regulatory element that is responsive to a VPI of the invention operably linked to a nucleic acid encoding a detectable marker, such as luciferase or green fluorescent protein
• a cellular response e.g., cellular differentiation or cell proliferation.

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