WO1997016458A1 - Apolipoproteine e2 et traitement de la maladie d'alzheimer - Google Patents

Apolipoproteine e2 et traitement de la maladie d'alzheimer Download PDF

Info

Publication number
WO1997016458A1
WO1997016458A1 PCT/US1996/017629 US9617629W WO9716458A1 WO 1997016458 A1 WO1997016458 A1 WO 1997016458A1 US 9617629 W US9617629 W US 9617629W WO 9716458 A1 WO9716458 A1 WO 9716458A1
Authority
WO
WIPO (PCT)
Prior art keywords
apoe2
apolipoprotein
apoe
cell
cells
Prior art date
Application number
PCT/US1996/017629
Other languages
English (en)
Inventor
Vassilis I. Zannis
Sergie B. Aleshkov
Original Assignee
Kos Pharmaceuticals, Inc.
Trustees Of Boston University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kos Pharmaceuticals, Inc., Trustees Of Boston University filed Critical Kos Pharmaceuticals, Inc.
Priority to AU75526/96A priority Critical patent/AU720446B2/en
Priority to EP96937887A priority patent/EP0866799A4/fr
Priority to JP9517597A priority patent/JP2000500332A/ja
Priority to BR9611329-4A priority patent/BR9611329A/pt
Publication of WO1997016458A1 publication Critical patent/WO1997016458A1/fr

Links

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
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to recombinant DNA, protein purification, and Alzheimer's disease.
  • Alzheimer's disease has two major neuropathological characteristics. The first is the presence of intraneuronal neurofibrillary tangles, the major protein component of which is hyperphosphorylated tau, a microtubule-associated protein. Electron microscopy shows neurofibrillary tangles to comprise paired helical filaments ("PHF") (Selkoe (1991) Neuron 6:487-498).
  • PHF paired helical filaments
  • Amyloid deposits consist of polymers of the amyloid peptide A3, which is proteolytically derived from amyloid precursor protein ("APP").
  • APP amyloid precursor protein
  • Apolipoprotein E (“apoE”) is a 34.2 kDa protein synthesized by the liver and various peripheral tissues, including kidney, adrenal gland, astrocytes and reticuloendothelial cells.
  • ApoE is synthesized as precursor (“preapoE") with an 18-amino acid signal peptide. After the intracellular cleavage of the signal peptide, apoE is glycosylated with carbohydrate chains containing sialic acid and secreted as sialo apoE. It is subsequently desialated in plasma.
  • apoE is incorporated into lipoproteins and directs their catabolism, following recognition and binding by cell surface receptors. ApoE can be isolated from plasma and culture media of HepG-2 cells as a component of lipoprotein particles.
  • e4/E4, E3/E3, and E2/E2 There are three common alleles that encode apoE in humans.
  • the three alleles designated e4, e3, and e2 give rise to three homozygous phenotypes (i.e., E4/E4, E3/E3, and E2/E2) and three heterozygous phenotypes (i.e., E4/E3, E3/E2, and E4/E2) (Zannis and Breslow (1981)
  • apoE4 contains Arg at position 112 and Arg at position 158.
  • ApoE3 contains Cys at position 112, and Arg at position 158.
  • ApoE2 contains Cys at positions 112 and 158.
  • Electron microscopy studies showed that a combination of apoE4 with A/3 resulted in the formation of monofibrils 7 nm in diameter.
  • the matrix of monofibrils formed with apoE4 was much denser than that with apoE3 (Sanan et al. (1994) J. Clin. Invest. 94:860-869).
  • a second hypothesis is that apoE3 binds to tau, and protects it from phosphorylation in the microtubule- binding domain, while apoE4 does not bind to tau (Strittmatter et al. (1994) Exper. Neurol. 125:163-171). As a result, tau gets hyperphosphorylated and can no longer bind efficiently and stabilize microtubules, leading to the formation of neurofibrillary tangles inside neurons.
  • glycosylated apolipoprotein E2 having a conformation which is the same as, or which closely approximates, its native conformation, i.e., it is not denatured and then renatured, and exists in lipid-free as well as lipid- bound forms, binds to synthetic peptide amyloid peptide A/3 with a higher affinity than do apolipoproteins E3 and E4.
  • the invention features an isolated apolipoprotein E2 capable of specifically binding to amyloid peptide A3 with a higher affinity than apolipoproteins E3 or E4;
  • the apolipoprotein E2 can be produced by (a) transforming a eukaryotic (preferably mammalian) host cell with DNA encoding apolipoprotein E2; (b) culturing the host cell; and (c) isolating the apolipoprotein E2 under non-denaturing conditions.
  • apolipoprotein E2 refers both to the naturally-occurring amino sequence of the protein, and to variants in which there are one or more amino acid substitutions which do not substantially alter the conformation or other properties of the molecule. Examples of such mutant forms of E2 are given below in Table 1; these mutants not only retain native conformation when expressed in mammalian host cells, they exhibit superior binding properties compared to the native sequence.
  • Apolipoprotein E2 can be isolated under nondenaturing conditions by subjecting the growth medium from a culture of mammalian cells that express recombinant apolipoprotein E2 to cation exchange chromatography to produce an apolipoprotein E2-enriched cation exchange protein fraction, and then subjecting the resulting pool of apolipoprotein E2-enriched cation exchange fractions to anion exchange chromatography, thereby producing an anion exchange fraction highly enriched for apolipoprotein E2.
  • the invention also features a method for inhibiting the progression of Alzheimer's disease in a patient with early onset Alzheimer's disease, or inhibiting the onset of Alzheimer's disease in a patient genetically at risk of developing Alzheimer's disease, by administering to the brain of the patient an effective amount of apolipoprotein E2 which is glycosylated, exists in lipid-free or lipid-bound forms, and has native or close to the native conformation produced by brain cells.
  • Fig. 1 is a schematic representation of the steps leading to production of plasmids of the invention.
  • Fig. 2 is a series of gel electrophoresis and autoradiography panels of apoE2 secretion from cells of the invention.
  • Fig. 3 is a series of panels illustrating apoE2, E3, and E4 expression by various cell types using the Semliki Forest Virus expression system.
  • Fig. 4 is a pair of panels illustrating apoE expression in a bioreactor setting.
  • Fig. 5 is a set of panels showing elution profiles of apoE from ion exchange columns.
  • Fig. 6 is a set of panels showing binding to A/3 of apolipoprotein E obtained by expression in eukaryotic cells.
  • the apoE experiment shows that the ability of apolipoprotein E produced by eukaryotic cells to bind to A ⁇ follows the order: apolipoprotein E2 > apolipoprotein E3 > apolipoprotein E4.
  • Fig. 7 is a set of panels showing binding to A ⁇ of apolipoprotein E3 and apolipoprotein E4 obtained by the baculovirus expression system.
  • the ability of apoE3 and apoE4 forms thus obtained to bind to A/3 is different from that of apoE expressed by eukaryotic cells (Fig. 6) .
  • Fig. 6 is a set of panels showing binding to A/3 of apolipoprotein E obtained by expression in eukaryotic cells.
  • Fig. 8 is a set of panels showing binding to A/3 of VLDL, apoE3 and apoE4. The ability of apoE3 and apoE4 forms thus obtained to bind to A/3 is different from that of apoE expressed by eukaryotic cells (Fig. 6) .
  • Fig. 9 is a panel showing distribution of apolipoprotein E secreted by C127 cells to lipid-free and lipid-bound forms.
  • Fig. 10 is a diagram predicting binding of apolipoprotein E2 derivatives obtained from eukaryotic cells to A/3 which differ from the binding of naturally occurring apolipoproteins E3, E4 and native-sequence E2.
  • the present invention provides recombinant apolipoprotein E2 in its native conformation. Because of its native conformation, the apoE2 of the invention binds to the A ⁇ peptide with greater affinity than apoE3 or apoE4.
  • the pUC-E3 plasmid was amplified and mutagenized by PCR as described in Fig. IA, using a set of 5' and 3' amplification primers and a set of mutagenic primers.
  • the mutagenic primers were designed to replace Cys-112 with Arg, and to replace Arg- 158 with Cys.
  • the 5' external amplification primer extended from nucleotides 223 to 241 of the apoE gene (sequence numbering of Karathanassis, Haddad, Salmon and Zannis, Biochemistry and Biology of Plasma Lipoproteins 1985, pp. 475-493).
  • the 3' external amplification primer extended between nucleotides 1102-1082 in the 3' region of the cDNA.
  • codons 154-161 (nucleotides 576- 595) for alteration of Arg-158 to Cys-158 in order to produce apoE2.
  • Another set of mutagenic sense and antisense oligonucleotides covered codons 108-113 (nucleotides 438-457) for the change of Cys-112 to Arg-112 in order to produce apoE4.
  • Arg-158 ⁇ Cys For alteration of Arg-158 ⁇ Cys, two separate amplifications were utilized. The first utilized the 5' external primer and the antisense mutagenic primer covering codon 158. The second utilized the 3' external primer and the sense mutagenic primer covering codon 158. An aliquot of 4% from each reaction was mixed and amplified with the 5' and 3' external primers. The amplified fragment containing the Arg-158 ⁇ Cys mutation was used to replace the corresponding region in the pUC- E3 plasmid, as shown in Fig. IA, giving rise to pUC-E2 plasmid.
  • the amplified fragment containing the Cys-112 ⁇ Arg mutation was used to replace the corresponding region of the pUC- E3 plasmid, as shown in Fig. IA, giving rise to pUC-E4 plasmid (Fig. IA) .
  • the DNA insert obtained from the pUC-E2 derivative was initially cloned into the BamHl site of the pcDNA under the control of the CMV pcDNA promoter or into the BamHl and Bglll site of Semliki Forest Virus vector (pSFV) to generate the pcDNA-E2 derivative and the pSFV- E2 derivatives.
  • the apoE2 insert was then excised from the pUC-E2 derivative, blunt-ended with the Klenow fragment of the DNA polymerase I, and ligated to Xhol linkers digested with Xhol and cloned into the Xhol site of pBMT3X vector to generate pBMT3X-E2, expression plasmid (Fig. IB) .
  • a similar approach was utilized to generate the pBMT3X-E3 and E4 plasmids.
  • the pBMT3X is a bovine papilloma virus-based vector in which the gene of interest is transcribed under the control of the mouse metallothionein I promoter.
  • the vector also contains the human metallothionein I gene, which confers resistance to 10 ⁇ M CdCl 2 (Cladaras et al. (1987) J. Biol. Chem. 262:2310-2315).
  • the cell line was labelled with 35 S-methionine for 2 hours.
  • Media and cell lysates were immunoprecipitated with rabbit anti-apoE, mixed with 15 ⁇ g HDL containing apoE3, and analyzed with two-dimensional gel electrophoresis (PAGE) and autoradiography.
  • proteins are separated on the basis of their charge by isoelectric focusing and on the basis of their size by SDS-PAGE.
  • the gel obtained from this analysis showed the position of the marker plasma apoE3 (Fig. 2A,B,C) , and the autoradiogram showed the position of the newly synthesized apoE2 protein (Fig. 2A',B',C).
  • Superimposition of the gel on the autoradiogram established the charge and size differences between the plasma apoE3 and the newly synthesized protein (Fig. 2A",B",C") .
  • This analysis shown in Figs. 2A-C" established that the newly synthesized apoE2 is more acidic by one and two positive charges than apoE3 and apoE4, respectively.
  • the analysis also shows that the secreted protein is extensively modified and resembles the protein synthesized by hepatic and other types of cells (Zannis et al. (1986) J. Biol. Chem. 261:13415- 13421) .
  • apoE2 expression in C127 cells de ⁇ scribed above, we have also used other eukaryotic expression systems for expression of apoE2, apoE3 and apoE4 based on Semliki Forest Virus (SFV) replicon.
  • SFV Semliki Forest Virus
  • These systems consist of two plasmids - pSFVl and pSFV- helper-2.
  • the first plasmid represents the cDNA copy of Semliki Forest Virus genome with a deleted subgenomic (26S) coding region for the structural proteins of the virus, followed by a cassette of polylinker (BamHl-X al- Smal) and translational stop codons in all three reading frames.
  • the cDNA of human apoE2 was cut with Bglll and BamHl, present in the polylinker region of pSFV, and inserted via a BamHl Bglll site to create vector pSFV apoE2.
  • apoE2 cDNA expression is driven by the 26S promoter in the context of SFV genome.
  • the second plasmid is pSFV-helper 2, which retains the 5' and 3' signals needed for RNA replication, as well as the complete structural region, including its promoter, but which lacks a region required for packaging of SFV RNA into nucleocapsids.
  • the pivotal step in the development of the pSFV expression system is helper catalyzed packaging of pSFVcE2 RNA.
  • the system allows rapid transient expression of apoE2 cDNA mutants, allowing fast testing of their functions, physicochemical properties, and neuronal toxicity.
  • plasmid pSFV apoE2, or pSFV-helper 2 was transcribed in vitro under the control of an SP6 promoter. Both SFV apoE2 and SFV-helper 2 RNAs were then cotransfected by electroporation into BHK cells.
  • the SFV apoE2 RNA encodes the enzymes for RNA replication (amplification) and transcription, whereas SFV helper 2 RNA supports the production of the structural proteins via its subgenomic region. Since the helper lacks the region required for RNA packaging, RNA derived from this vector will not be packaged. Thus, transfections with recombinant and helper RNA produce only virus particles that carry recombinant RNA.
  • the viruses produced provide a one-round virus stock. After infection of cells with the recombinant virus, the cells will only express the heterologous protein (apoE2) .
  • This virus stock was utilized to infect BHK cells or primary astrocytes obtained from mice deficient in apoE.
  • confluent BHK cells or apoE deficient mouse primary astrocyte cultures were infected with recombinant SFV apoE2, virus at multiplici ⁇ ty of infection of approximately three infective units per cell for 12 hrs and grown in serum-free BHK medium for 24 h.
  • the viral titer was estimated between IO 7 and IO 8 infectious units/ml.
  • the Verax bioreactors consist of a vertical column containing microspheres in suspension (Fig. 4A) .
  • the microspheres are made of collagen and are hollow with a pore diameter which allows cell attachment and growth. Cell densities of IO 8 cells/ml are achieved.
  • the microspheres are kept in suspension by the flow of incoming culture medium.
  • a gas exchange unit provides the required 0 2 and removes excess C0 2 .
  • Temperature control keeps the medium at 37°C.
  • the medium is circulated by a pump through the bioreactor and the gas exchange unit.
  • the culture medium contain 30-50 ⁇ g apoE/ml (Fig. 4B) .
  • the medium of C127 cells expressing apoE2, apoE3 or apoE4 was purified by a scheme employing a dextran sulfate sepharose column and DEAE sepharose column frac- tionation.
  • a 20 ml dextran sulfate-sepharose column was equilibrated with 20 mM Tris-HCl, 0.2 M NaCl, pH 7.4.
  • a total of 1 liter of apoE-containing culture medium was adjusted to 0.2 M NaCl and loaded on the column at a flow rate of ⁇ 80 ml/hr.
  • the column was washed with 200 ml of 20 mM Tris-HCl, 0.2 M NaCl, pH 7.4 at the same flow rate.
  • the column was eluted with a 120 ml 0.2-1.0 M NaCl gradient in 20 mM Tris-HCl, pH 7.4 at the same flow rate; fractions of 2 ml were collected.
  • the apoE-containing samples eluted from the dextran sulfate sepharose column were adjusted to 50 mM NaCl by dilution with 20 mM Tris- HCl pH 7.4 and were loaded on a 10 ml DEAE column.
  • the column was eluted with 70 ml of 0.05-0.75 M NaCl gradient in 20 mM Tris-HCl pH 7.4. Properties of Apolipoproteins E3, E4 and E2 Produced by Mammalian Cells and Other Sources.
  • APOE3 and APOE4 Produced bv Cell Cultures to the Synthetic Amyloid Peptide A ⁇ .
  • 1 ⁇ g of apoE2 or apoE3 or apoE4 was diluted with 9 ⁇ l H 2 0 containing either 0 or 4 ⁇ M A ⁇ (40 amino acid residues in length) in 20 mM tris pH 8.0 buffer and incubated at 25°C for 12 hrs. Incubation was terminated by adding equal volume 2X Laemmli buffer (4% SDS without ⁇ -mercaptoethanol) and boiling for 5 min. The mixture was analyzed by gel electrophoresis in two gels (10% polyacrylamide - 2% SDS) and Western blotting. After blotting one of the filters (Panel A) was treated with mouse monoclonal antibody against A/3 (Fig. 6A) and the other two panels (Panels B&C) with goat polyclonal antibody against apoE.
  • FIG. 6A-C show that the ability of apoE produced by these cells to bind to A ⁇ follows the order apoE2>apoE3>apoE4. Similar results are obtained with apoE produced by apoE produced by the different expression systems described before. This binding behavior of apoE is different from that of apoE produced by the baculovirus expression system (Fig. 7) or VLDL (Fig. 8) (Strittmatter et al. (1993) Proc. Natl. Acad. Sci. USA 90:8098-8102 ⁇ .
  • Fig. 6A-C show that recombinant apoE2, apoE3 and apoE4 form stable complexes with A ⁇ . These complexes are visible in the first three lanes of Fig. 6A,B, which contain A ⁇ but not in the corresponding lanes of Fig. 6C, which does not contain A ⁇ .
  • This analysis establishes unequivocally that the binding of newly synthesized apoE secreted into the cell medium which consist mostly of modified forms of apoE with A/3 follows the order apoE2>apoE3>apoE4.
  • mice expressing natural or mutant apoE forms in an apoE deficient background (Plum et al. (1992) Cell 71:343-353) will be mated with the AD-prone mouse generated by Athena Neuroscience (Gomes et al. (1995) Nature 373:523).
  • Analysis of the offspring will provide information as to whether a specific apoE variant protects or accelerates the development of the AD phenotype in relationship to the parent AD mouse. For instance, crossing of the AD prone mouse with the EX1 or EX6 mouse will confirm that strong binding of apoE to A ⁇ is beneficial and weak binding is detrimental. If strong binding is beneficial, we will anticipate that the EX6 form will protect from AD when it is introduced into the AD-prone mouse, whereas EX1 will accelerate the development of AD.
  • the apoE2 of the invention should come in contact with neuronal cells of the brain, or with the physiological fluids in the immediate vicinity of such cells.
  • the apoE2 in that environment, binds to the A ⁇ - a yloid peptide, or to /3-amyloid precursor protein; when it binds to precursor protein; it prevents the precursor from progressing to the next stage, which is cleavage to form the fl-amyloid protein characteristic of Alzheimer's disease.
  • apoE2 binds to A/3-amyloid peptide, such binding inhibits progression of Alzheimer's disease by preventing formation of the extracellular amyloid plaques.
  • apoE2 produced by eukaryotic cells will be administered into the ventricles of the brain and will enter into the cerebrospinal fluid where it will be bound to A/3.
  • apoE2 phospholipid vesicles or triglyceride phospholipid emulsion will be administered into the ventricles of the brain.
  • the A/3 will bind to the vesicles or the emulsions and will be removed from the brain.
  • apoE2 phospholipid vesicles or the triglyceride phospholipid emulsions will be administered intravenously.
  • the complexes will cross the blood/brain barrier by interactions with the apoE receptor present in the blood/brain barrier.
  • biotinylated polyamides will be conjugated to streptavidin conjugated apoE2 according to Partridge et al. (1995) Proc. Natl. Acad. Sci. USA 92:5592-5596). Following intravenous administration, the complex will cross the blood/brain barrier by interaction with the apoE receptor present in blood brain barrier.
  • Free apoE2 or apoE2 associated with lipids can be administered to the ventricle of the brain by means of a stereotaxic instrument which is commercially available.
  • the stereotaxic coordinates of the insertion point on the skull is marked by a sterile pencil.
  • a hole of 2-3 mm is made in the skull.
  • the dura is punctured and is covered with saline soaked gel-foam using the stereotaxic holder, and a thin tube is lowered into the ventricle and is connected with the infusion pump that will deliver the - 16 - apoE2 in lipid-free or lipid-bound form into the ventricle.
  • the apoE2 solution or apoE2 phospholipid vesicles or apoE2 triglyceride phospholipid emulsions are loaded into the intraventricular pump, which has been implanted into the patient by known techniques.
  • Each apoE2 preparation is continuously infused into the ventricle of the patient's brain, at a rate sufficient to deliver 500 to 1000 ⁇ g of protein per 75 kg body weight of the patient per day.
  • Another mode of administration is to inject the protein, dissolved in a carrier liquid, into the spinal column of the patient so that it contacts the patient's cerebrospinal fluid.
  • Another mode of administration is intravenous injection of apoE2 phospholipid vesicles or apoE2 triglyceride phospholipid emulsions or conjugates of apoE2 with polyamides in physiological solutions containing 2 mg apoE2/ml. Ten mis of apoE2-containing solution will be injected twice per day.
  • the apoE2 of the invention can be administered to three classes of patients:
  • Asymptomatic patients who are over the age of 45 and who have a family history of Alzheimer's disease; one class of such patients are those carrying two copies of the apoE4 gene.
  • An alternative method of treatment is to administer DNA encoding apoE2 rather than apoE2 to protein.
  • This gene therapy method is preferably carried out using either a retroviral vector or an expression vector containing apoE2- encoding DNA, suspended in a liquid carrier; the suspension is administered either to the cerebrospinal fluid or the brain ventricle in the same manner that the protein in solution is administered.
  • Suitable plasmid and retroviral vectors are well known and publicly and commercially available.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Steroid Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne l'apolipoprotéine E2 (apoE2) de recombinaison qui se fixe au peptide amyloïde Aβ.
PCT/US1996/017629 1995-11-01 1996-10-31 Apolipoproteine e2 et traitement de la maladie d'alzheimer WO1997016458A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU75526/96A AU720446B2 (en) 1995-11-01 1996-10-31 Apolipoprotein E2 and treatment of Alzheimer's disease
EP96937887A EP0866799A4 (fr) 1995-11-01 1996-10-31 Apolipoproteine e2 et traitement de la maladie d'alzheimer
JP9517597A JP2000500332A (ja) 1995-11-01 1996-10-31 アポリポタンパク質ｅ２とアルツハイマー病の治療
BR9611329-4A BR9611329A (pt) 1995-11-01 1996-10-31 Apolipoproteìna e2 e tratamento de doença de alzheimer

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US55172295A 1995-11-01 1995-11-01
US08/551,722 1995-11-01

Publications (1)

Publication Number Publication Date
WO1997016458A1 true WO1997016458A1 (fr) 1997-05-09

Family

ID=24202413

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/017629 WO1997016458A1 (fr) 1995-11-01 1996-10-31 Apolipoproteine e2 et traitement de la maladie d'alzheimer

Country Status (7)

Country Link
EP (1) EP0866799A4 (fr)
JP (1) JP2000500332A (fr)
AU (1) AU720446B2 (fr)
BR (1) BR9611329A (fr)
CA (1) CA2236274A1 (fr)
MX (1) MX9803422A (fr)
WO (1) WO1997016458A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023587A2 (fr) * 1998-10-16 2000-04-27 Introgene B.V. Therapie genique de la maladie d'alzheimer par administration d'une apolipoproteine e codee
WO2000031548A1 (fr) * 1998-11-25 2000-06-02 Scios Inc. Prevention et traitement de troubles associes aux proteines amyloides
WO2001072802A1 (fr) * 2000-03-28 2001-10-04 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine de liaison 14 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2001079438A2 (fr) * 2000-03-29 2001-10-25 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine de liaison 9 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2005032596A1 (fr) * 2003-09-26 2005-04-14 Eli Lilly And Company Therapie de gene apoe2 lentiviral
WO2007098417A2 (fr) * 2006-02-21 2007-08-30 Oklahoma Medical Research Foundation TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE
WO2010020822A1 (fr) * 2008-08-19 2010-02-25 University Of Patras Thérapie d’hypertriglycéridémie induite par apolipoprotéine

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU587989B2 (en) * 1984-10-16 1989-09-07 Mitsubishi Chemical Corporation DMA fragments, expression vectors, proteins, hosts, and process for production of the proteins
GB9414624D0 (en) * 1994-07-20 1994-09-07 Smithkline Beecham Plc Novel method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 71, PLUMP A.S., "Severe Hypercholesterolemia and Atherosclerosis in Apolipoprotein E-Deficient Mice Created by Homologous Recombinantion in ES Cells", pages 343-353. *
JOURNAL OF BIOCHEMISTRY, 1989, Vol. 105, MAEDA H. et al., "Molecular Cloning of a Human Apolipoprotein E Variant: E5 (Glu3 - Lys3)", pages 491-493. *
JOURNAL OF LIPID RESEARCH, 1990, Vol. 31, WARDELL M.R. et al., "Apolipoprotein E2-Dunedin (288 Arg- Cys): An Apolipoprotein E2 Variant With Normal Receptor-Binding Activity", pages 535-543. *
JOURNAL OF LIPID RESEARCH, 1992, Vol. 33, HERSCOVITZ H. et al., "Expression of Human Apolipoprotein E But Not That of Apolipoprotein A-1 by Mouse C127 Cells is Associated With Increased Secretion of Lipids in the Form of Vesicles and Discs", pages 791-803. *
See also references of EP0866799A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023587A2 (fr) * 1998-10-16 2000-04-27 Introgene B.V. Therapie genique de la maladie d'alzheimer par administration d'une apolipoproteine e codee
WO2000023587A3 (fr) * 1998-10-16 2000-08-03 Introgene Bv Therapie genique de la maladie d'alzheimer par administration d'une apolipoproteine e codee
WO2000031548A1 (fr) * 1998-11-25 2000-06-02 Scios Inc. Prevention et traitement de troubles associes aux proteines amyloides
WO2001072802A1 (fr) * 2000-03-28 2001-10-04 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine de liaison 14 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2001079438A2 (fr) * 2000-03-29 2001-10-25 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine de liaison 9 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2001079438A3 (fr) * 2000-03-29 2002-02-28 Biowindow Gene Dev Inc Nouveau polypeptide, proteine humaine de liaison 9 d'une proteine precurseur de l'amyloide, et polynucleotide codant pour ce polypeptide
WO2005032596A1 (fr) * 2003-09-26 2005-04-14 Eli Lilly And Company Therapie de gene apoe2 lentiviral
WO2007098417A2 (fr) * 2006-02-21 2007-08-30 Oklahoma Medical Research Foundation TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE
WO2007098417A3 (fr) * 2006-02-21 2007-10-18 Oklahoma Med Res Found TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE
WO2010020822A1 (fr) * 2008-08-19 2010-02-25 University Of Patras Thérapie d’hypertriglycéridémie induite par apolipoprotéine

Also Published As

Publication number Publication date
EP0866799A1 (fr) 1998-09-30
MX9803422A (es) 1998-11-29
JP2000500332A (ja) 2000-01-18
AU7552696A (en) 1997-05-22
BR9611329A (pt) 1999-12-28
AU720446B2 (en) 2000-06-01
EP0866799A4 (fr) 2000-08-23
CA2236274A1 (fr) 1997-05-09

Similar Documents

Publication Publication Date Title
CALERO et al. Functional and structural properties of lipid-associated apolipoprotein J (clusterin)
EP0446017A1 (fr) Nouvelles méthodes de diagnose et de traitement par utilisation de kystique fibrosis transmembrane régulateur
US5674898A (en) Methods and therapeutic compositions for treating cystic fibrosis
US5525472A (en) Method for production and purification or recombinant Apolipoprotein E from bacteria
EP0275305A1 (fr) Sequences adn pour facteur viii:c modifie et polypeptides semblables au facteur viii:c modifie et procedes de production en grandes quantites de ces polypeptides
CA1316132C (fr) Adnc et gene de l'angiogenine humaine (facteur d'angiogenese) et methode d'expression
EP0239631A1 (fr) Apolipoproteines recombinantes et procedes
SG192693A1 (en) Lipoprotein complexes and manufacturing and uses thereof
EP0751994A1 (fr) Genes et proteines cftr tronquees en terminaison c
US5238919A (en) Peptides that inhibit von Willebrand Factor binding to the platelet SPIB receptor
Li et al. Domains of apoE required for binding to apoE receptor 2 and to phospholipids: implications for the functions of apoE in the brain
AU720446B2 (en) Apolipoprotein E2 and treatment of Alzheimer's disease
US4943527A (en) Mature apoai protein production under serum free culturing conditions
JP2009171980A (ja) 高トリグリセリド血症を誘発することなくコレステロールレベルを低下するための化合物と方法
US5622843A (en) Phospholipid transfer proteins and DNA encoding them
Zannis et al. Plasma and hepatic apoE isoproteins of nonhuman primates. Differences in apoE among humans, apes, and New and Old World monkeys.
US20060142200A1 (en) Compounds and methods for lowering cholesterol levels without inducing hypertrigylceridemia
CN1206421A (zh) 载脂蛋白e2和阿尔采默氏病的治疗
CA2042605A1 (fr) Proteines inhibitrices de la cytolyse, sequences d'adn codant pour ces proteines, et plasmides, organismes hotes et procedes permettant d'obtenir ces proteines
US20070053879A1 (en) Diagnostic and treatment methods involving the cystic fibrosis transmembrane regulator
TW202146040A (zh) 脂質代謝之動物模型及治療高脂血症或高脂血症相關疾病的方法
WO2010049103A1 (fr) Protéines apom tronquées solubles et leurs utilisations médicales
US5876974A (en) Method for producing DNA encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein in E. coli
Davis Molecular and cell biology of cystic fibrosis
EP0600875A4 (fr) Purification d'apolipoproteine e recombinante tiree de bacteries.

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 96199444.4

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA CN JP MX

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2236274

Country of ref document: CA

Ref document number: 2236274

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PA/a/1998/003422

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 1997 517597

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1996937887

Country of ref document: EP

NENP Non-entry into the national phase

Ref document number: 97517597

Country of ref document: JP

WWP Wipo information: published in national office

Ref document number: 1996937887

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1996937887

Country of ref document: EP