WO2017060510A1 - Méthodes et compositions pharmaceutiques pour le traitement de la maladie d'alzheimer - Google Patents

Méthodes et compositions pharmaceutiques pour le traitement de la maladie d'alzheimer Download PDF

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WO2017060510A1
WO2017060510A1 PCT/EP2016/074133 EP2016074133W WO2017060510A1 WO 2017060510 A1 WO2017060510 A1 WO 2017060510A1 EP 2016074133 W EP2016074133 W EP 2016074133W WO 2017060510 A1 WO2017060510 A1 WO 2017060510A1
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mice
app
interleukin
ps1ae9
disease
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PCT/EP2016/074133
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English (en)
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Nathalie Cartier
David Klatzmann
Sandro ALVES
Guillaume CHURLAUD
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Paris-Sud
Commissariat à l'énergie atomique et aux énergies alternatives
Université Pierre Et Marie Curie (Paris 6)
Assistance Publique-Hôpitaux De Paris (Aphp)
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Publication of WO2017060510A1 publication Critical patent/WO2017060510A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease.
  • AD Alzheimer's disease
  • AD Amyloid- ⁇ peptide
  • APP ⁇ -amyloid precursor protein
  • Interleukin-2 IL-2
  • BBB blood-brain-barrier
  • IL-2 has numerous effects on hippocampal neurons where its receptors are enriched, modifying cognitive performances in rodents (Hanisch et al., 1997; Lacosta et al., 1999).
  • IL-2 shifts cellular and molecular substrates of learning and memory, like long-term potentiation (LTP) (Tancredi et al., 1990) and release of acetylcholine (Seto et al., 1997), the later playing a key role in cognition. Furthermore, IL-2 can afford trophic support to both neurons and glia (Awatsuji et al., 1993), affecting the morphology of neurite branching of rat hippocampal cultures (Sarder et al., 1996), enhancing dendritic development and spinogenesis (Shen et al., 2010), thus playing a role in neuronal development (Sarder et al., 1993). Importantly, IL-2 knockout (KO) mice display cytoarchitectural hippocampal modifications (Beck et al., 2005), impaired learning and memory ability and altered hippocampal development (Petitto et al., 1999).
  • LTP long-term pot
  • Tregs cytokine controlling regulatory T cells
  • Tregs represent a subset of T cells which main role is to control inflammation and (likewise) autoimmunity.
  • the anti-inflammatory effects of Tregs have been observed in various models of inflammatory diseases in mice. Little is known about Tregs and neuroinflammation.
  • peripheral blood Tregs numbers have been described as the best biomarkers predicting prognostic in ALS, with fewer Tregs correlating with worse clinical outcome.
  • Recent clinical trials of IL-2 at low dose showed that it is safe and improves autoimmune and alloimmune inflammatory conditions in human.
  • the present invention relates to methods and pharmaceutical compositions for the treatment of Alzheimer's disease.
  • the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:
  • Interleukin-2 (IL-2) knockout mice have impaired learning and memory ability. Furthermore, IL-2 at low dose stimulates regulatory T cells (Tregs) which main role is to control inflammation. As neuroinflammation contributes to neurodegeneration in Alzheimer's disease (AD), the inventors investigated IL-2 in AD. They first showed that IL-2 is decreased in hippocampal biopsies from of AD patients. The inventors then treated with IL-2, APP/PS1AE9 mice having established AD. IL-2 induced systemic Treg expansion and activation, all along the 5-months follow-up.
  • Tregs regulatory T cells
  • IL-2 induced astrocytic activation and recruitment around amyloid plaques, a decrease of amyloid plaques load and of the ⁇ (42/40) ratio, and a restoration of the N-methyl-D-aspartate receptor subunit NR2A. Noteworthy, this tissue remodeling was associated with the recovery of memory deficits. Thus, IL-2 can alleviate AD in APP/PS1AE9 AD mice and this should prompt the investigation of low-dose IL-2 in AD.
  • an object of the present invention relates to a method of treating Alzheimer's disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated viral (AAV) vector comprising a polynucleotide encoding for an interleukin 2 (IL-2) polypeptide.
  • AAV adeno-associated viral
  • the term "subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the invention is a human.
  • a "subject in need thereof denotes a subject, preferably a human suffering from Alzheimer's disease.
  • Alzheimer's disease denotes chronic neurodegenerative disease that usually starts slowly and gets worse over time.
  • AD Alzheimer's disease
  • amyloid deposits intracellular neurofibrillary tangles
  • neuronal loss and a decline in cognitive function (Hardy and Allsop, 1991, Selkoe, 2001).
  • the most common early symptom is difficulty in remembering recent events (short-term memory loss).
  • symptoms can include: problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, not managing self-care, and behavioral issues.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the method of the present invention is particularly suitable for activating astrocytes, improving clearance of clearance of ⁇ from the brain, improving amyloid pathology, and/or improving subject's memory learning and/or cognition.
  • IL-2 has its general meaning in the art and refers to the interleukin-2.
  • the term "IL-2" polypeptide designates any source of IL-2, including mammalian sources such as e.g., human, mouse, rat, primate, and pig...
  • IL-2 may be or comprise the native polypeptide sequence, or can be an active variant of the native IL-2 polypeptide.
  • the IL-2 polypeptide is derived from a human source.
  • the IL-2 polypeptide of the present invention comprises a amino acid sequence having at least 90% of identity with SEQ ID NO: l.
  • a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences.
  • Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.
  • the alignment tools ALIGN Myers and Miller, CABIOS 4: 11-17, 1989
  • LFASTA Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996
  • ALIGN compares entire sequences against one another
  • LFASTA compares regions of local similarity.
  • these alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance.
  • the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • the BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266: 131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.
  • polynucleotide encoding for a IL-2 polypeptide refers to any nucleic acid molecule encoding for the IL-2 polypeptide as defined above.
  • nucleic acid molecule has its general meaning in the art and refers to a DNA molecule.
  • the term captures sequences that include any of the known base analogues of DNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5- bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 - methylpseudouracil, 1 -methyl guanine, 1- methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-
  • the polynucleotide of the present invention comprises a sequence having at least 80% (i.e. 80; 81, 82, 83, 84, 85, 86, 87, 88, 89 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100%) of identity with SEQ ID NO:2.
  • SEQ ID NO:2 (IL-2, Homo Sapiens):
  • AAV has its general meaning in the art and is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all serotypes and variants both naturally occurring and engineered forms.
  • AAV refers to AAV type 1 (AAV-1), AAV type 2 (AAV- 2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV- 6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8) and AAV type 9 (AAV9).
  • rAAV vector refers to an AAV vector comprising the polynucleotide of interest (i.e the polynucleotide encoding for the IL-2 polypeptide).
  • the rAAV vectors contain 5' and 3' adeno- associated virus inverted terminal repeats (ITRs), and the polynucleotide of interest operatively linked to sequences, which regulate its expression in a target cell.
  • the AAV vector of the present invention typically comprises regulatory sequences allowing expression and, secretion of the encoded polypeptide (i.e. the IL-2 polypeptide), such as e.g., a promoter, enhancer, polyadenylation signal, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTD), and the like.
  • the vector comprises a promoter region, operably linked to the polynucleotide of interest, to cause or improve expression of the protein in infected cells.
  • a promoter may be ubiquitous, tissue- specific, strong, weak, regulated, chimeric, inducible, etc., to allow efficient and suitable production of the protein in the infected tissue.
  • the promoter may be homologous to the encoded protein, or heterologous, including cellular, viral, fungal, plant or synthetic promoters.
  • regulated promoters include, without limitation, Tet on/off element- containing promoters, rapamycin-inducible promoters and metallothionein promoters.
  • ubiquitous promoters include viral promoters, particularly the CMV promoter, CAG promoter (chicken beta actin promoter with CMV enhancer), the RSV promoter, the SV40 promoter, etc. and cellular promoters such as the PGK (phosphoglycerate kinase) promoter.
  • the promoters may also be neurospecific promoters such as the Synapsin or the NSE (Neuron Specific Enolase) promoters (or NRSE (Neuron restrictive silencer element) sequences placed upstream from the ubiquitous PGK promoter), or promoters specific for RPE cell types such as the RPE65, the BEST1, the Rhodopsin or the cone arrestin promoters.
  • the vector may also comprise target sequences for miRNAs achieving suppression of transgene expression in non- desired cells.
  • the vector comprises a leader sequence allowing secretion of the encoded protein.
  • Fusion of the polynucleotide of interest with a sequence encoding a secretion signal peptide will allow the production of the therapeutic protein in a form that can be secreted from the transduced cells.
  • signal peptides include the albumin, the ⁇ -glucuronidase, the alkaline protease or the fibronectin secretory signal peptides.
  • the recombinant AAV vector of the present invention is produced using methods well known in the art.
  • the methods generally involve (a) the introduction of the rAAV vector into a host cell, (b) the introduction of an AAV helper construct into the host cell, wherein the helper construct comprises the viral functions missing from the rAAV vector and (c) introducing a helper virus into the host cell. All functions for rAAV virion replication and packaging need to be present, to achieve replication and packaging of the rAAV vector into rAAV virions.
  • the introduction into the host cell can be carried out using standard virological techniques simultaneously or sequentially.
  • the host cells are cultured to produce rAAV virions and are purified using standard techniques such as CsCl gradients. Residual helper virus activity can be inactivated using known methods, such as for example heat inactivation.
  • the purified rAAV vector is then ready for use in the method of the present invention.
  • a “therapeutically effective amount” of AAV vector as above described is meant a sufficient amount of the AAV vector for the treatment of Alzheimer's disease. It will be understood, however, that the total dosage of the AAV vector of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the compound it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • 10 8 to 10 10 viral genomes (vg) are administered per dose in mice.
  • the doses of AAV vectors to be administered in humans may range from 10 10 to 10 12 vg.
  • Administering the recombinant AAV vector of the present invention vector to the subject is preferably performed by sublingually, subcutaneously, intramuscularly, intravenously, transderaially delivery.
  • the recombinant AAV vector of the present invention is not administered to the subject by intraventricular or intracerebral injection.
  • the recombinant AAV vector of the present invention is administered to the subject by the intravenous injection.
  • the recombinant AAV vector of the present invention is typically formulated into pharmaceutical compositions.
  • the AAV vector of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • the term "pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the AAV vector of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifusoluble agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed, will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the invention will be further illustrated by the following figures and examples.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Interleukin-2 protein levels are decreased in hippocampal biopsies from
  • FIG. 1 Tregs are expanded and activated in blood and brain during Interleukin- 2 treatment.
  • Training phase consisted of daily sessions with three trials/day during 5 consecutive days. Four hours after the last training trial (day 5), the platform was removed and memory retention was assessed during a probe test.
  • Figure 4 Interleukin-2 expression rescues structural and functional synaptic deficits in APP/PS1AE9 mice.
  • LTP Long-term potentiation
  • TBS theta-burst stimulation
  • (B) spine number was reduced in APP/PS1AE9 (1.12+ 0.03) compared to littermate controls injected with the control vector (1.42+ 0.05) or rAAV-IL-2 (1.35+ 0.04), a phenotype which could be rescued by injection with rAAV-IL-2 (1.29+ 0.04).
  • N 3 animals per genotype, 8-10 dendrites per animal. Values represent means+SEM.
  • FIG. 5 AAV-mediated Interleukin-2 administration decreases amyloid patology in the hippocampus of APP/PS1AE9 mice.
  • For optical densitometry quantification (A and B) signal intensities were normalized to GAPDH used as a loading control.
  • Figure 7 Western blot analysis showing increased levels of IL-2 in hippocampal biopsies from APP/PS1AE9 mice injected with rAAV8-IL-2, relatively to APP/PS1AE9 mice injected with rAAV8-LUC or littermates injected with both vectors.
  • For quantification signal intensities were normalized to GAPDH, used as a loading control. Experiments were performed using 6-7 mice per group. Values represent means+SEM.
  • Figure 8 Ex vivo bioluminescence imaging in brain and liver at 2 weeks after intraperitoneal injection of 10E10 vg rAAV8-CAG-LUC vector in 8 months old APP/PS 1 ⁇ 9 transgenic mice.
  • Figure 9 (A, B and C) ELISA quantification (MSD immunoassay) showing increased levels of ⁇ peptides (38, 40 and 42) and ⁇ -CTF (D) in hippocampal samples from 8 months aged APP/PS 1 ⁇ 9 mice compared to age-matched wild- type littermates.
  • Figure 10 Western-blots showing no major differences in the levels of the microglial markers Arginase-1 (A), TGF- ⁇ (B), TREM-2 (C) and Insulin degrading enzyme (IDE) (D) in hippocampal biopsies of APP/PS 1 ⁇ 9 mice injected with rAAV8-IL2 or the control vector rAAV8-LUC.
  • A Arginase-1
  • B TGF- ⁇
  • TREM-2 C
  • IDE Insulin degrading enzyme
  • APP/PS1AE9 mice overexpress the mutated human APP (Swedish mutation, K595N/M596L) gene as well as the Human PS1 gene deleted from its ex on 9 (Jankowsky et al., 2004).
  • APP/PS1AE9 mice and wild-type littermates were bred and maintained in our animal facility under specific pathogen-free conditions. Mice were housed in a temperature-controlled room and maintained on a 12 h light/dark cycle. Food and water were available ad libitum.
  • Recombinant AAV8 vectors were generated by triple transfection of human embryonic kidney 293T cells, as described previously (Churlaud et al., 2014).
  • Transgenes used were luciferase (LUC) and murine Interleukin-2 (IL-2) driven by the hybrid cytomegalovirus enhancer/chicken beta-actin constitutive promoter (CAG).
  • LOC luciferase
  • IL-2 murine Interleukin-2 driven by the hybrid cytomegalovirus enhancer/chicken beta-actin constitutive promoter
  • PBS phosphate-buffered saline
  • Sera were collected, frozen and kept at -80 °C until use.
  • Levels of Interleukin-2 were measured using a mouse Interleukin-2 ELISA (eBioscience) according to the manufacturer's recommendations.
  • Tregs were defined as CD25+ Foxp3+ cells among CD4+ cells, and activated effector T cells as CD25+ cells among CD4+ Foxp3- cells.
  • FACS fluorescence-activated cell sorting
  • APP/PS1AE9 mice and wild-type littermates were sacrificed 5 months post-injection (13 months old).
  • the animals given an overdose of sodium pentobarbital, were perfused transcardially with ice-cold PBS 0,1M before brain extraction.
  • freshly perfused brain was dissociated and digested in collagenase/DNase solution in RPMI medium (Churlaud et al., 2014).
  • a Percoll (Sigma-Aldrich) gradient was used to isolate brain- infiltrating lymphocytes. Cells were then stained as described earlier for blood.
  • the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde (PFA) in 0.1M PBS for 1 week. Brains were cryoprotected by incubation in a 30% sucrose/0.1 M PBS solution. Coronal brain sections (40 ⁇ ) were cut on a freezing microtome (Leica, Wetzlar, Germany), collected serially, and stored at -20°C until additional analysis. The right hemisphere was dissected to extract the hippocampus, used biochemistry analysis. Samples were then homogenized in a lysis buffer (TBS, NaCl 150mM and Triton 1%) containing phosphatase (Pierce) and protease (Roche) inhibitors.
  • Total protein concentrations were determined using the BCA kit (Pierce). Equal amounts of total protein extract (30 ⁇ g) were electrophoretically separated using SDS-PAGE in 4-12% Bis-Tris gels (NuPAGE® Novex Bis-tris midi gel 15 or 26 wells, Life Technologies, Carlsbad, USA) and transferred to nitrocellulose membranes. Blocked membranes (5% non-fat dry milk in TBS-0.1% Tween-20) were incubated with primary antibodies overnight at 4°C, and washed three times with TBS-0.1% Tween-20 (T-BST) for 10 min. Membranes were then labeled with secondary IgG-HRP antibodies raised against each corresponding primary antibody.
  • ECL chemiluminescent reagent (Clarity Western ECL substrate; GE Healthcare, Little Chalfont, UK) according to the instructions of the supplier. Peroxydase activity was detected with camera system Fusion TX7 (Fisher Scientific). Normalization was done by densitometry analysis with the Quantity One ID image analysis software (version 4.4; Biorad, Hercules, CA, USA). The optical densities were normalized with respect to a "standard protein" (GAPDH). A partition ratio was calculated and normalized with respect to the sample with the highest value defined as 1.
  • GFAP Protein (GFAP) Dako 1:5000 1:4000 rat monoclonal anti-mTREM-2B R&D systems 1:2000 - rabbit anti-ionized calcium
  • binding adapter molecule 1 (Ibal) Wako 1:500 1:3000 rabbit anti-Insulin degrading
  • ⁇ -38, ⁇ -40 and ⁇ -42 were measured using the MSD Human ⁇ 42 V-PLEX Kit and the triplex ⁇ Peptide Panel 1 (6E10) V-PLEX Kit (Mesoscale Discovery, Rockville, MD, USA).
  • ⁇ -CTF was measured using the Human APP ⁇ -CTF Assay Kit (IBL, Hamburg, Germany).
  • Interleukin-2 was measured using the MSD proinflammatory panel 1 (Mesoscale Discovery, Rockville, MD, USA).
  • ELISA assays were performed following supplier instructions.
  • the immunohistochemical procedure was initiated, by incubating slices in 88% formic acid solution for 15 min (antigen retrieval) and then by quenching endogenous peroxidase by incubating free-floating sections in hydrogen peroxide for 30 min at room temperature (RT). After three washes, slices were blocked in PBS/0.1% Triton X-100 containing 10% Normal Goat Serum (NGS, Gibco) for lh at RT. The sections were then incubated with the primary antibody (4G8), overnight at 4°C.
  • the sections were incubated with the corresponding biotinylated secondary antibody (1:250; Vector Laboratories Inc., CA, USA) diluted in PBS/0.1% Triton X-100 and 10% NGS for 2 h at RT. After three washes, bound antibodies were visualized by the ABC amplification system (Vectastain ABC kit, Vector Laboratories, West Grove, USA) and 3,3' -diaminobenzidine tetrahydrochloride (peroxidase substrate kit, DAB, Vector Laboratories, CA, USA) as the substrate.
  • the sections were mounted, dehydrated by passing twice through ethanol and toluol solutions, and coverslipped with Eukitt® (O. Kindler GmbH & CO, Freiburg, Germany).
  • Plaques, GFAP and Ibal immunoreactivity were quantified using Image J (NIH, Bethesda, USA) or Icy (Institut Pasteur, Paris, France). Laserpower, numeric gain and magnification were kept constant between animals to avoid potential technical artefacts. Images were first converted to 8-bit gray scale and binary thresholded to highlight a positive staining. At least 3 sections per mouse (between -1.7 mm to -2.3 mm caudal to bregma) were quantified. The average value per structure was calculated for each mouse. For quantification of Ibal and GFAP immunoreactivity around plaques, a region of interest (ROI) was drawn around the center of the plaque. The diameter of the circular ROI was set as 3 times the diameter of the plaque. Mean fluorescence intensity values were measured for either Ibal or GFAP immunoreactivity and were processed via Icy software (Institut Pasteur, Paris, France). Analysis of data was blind with respect to treatments and genotypes.
  • ROI region of interest
  • the second hemisphere was transferred into cold (4 °C) artificial cerebrospinal fluid (ACSF), containing the following (in mM): 124 NaCl, 4.9 KC1, 1.2 KH2P04, 2.0 MgS04, 2.0 CaC12, 24.6 NaHC03, 10 D-glucose, equilibrated with 95% 02 and 5% C02.
  • the hippocampus was dissected from the second hemisphere and transverse hippocampal slices of 400 ⁇ were cut using tissue chopper. Hippocampal slices were incubated at 32 °C in an interface chamber with the constant flow of carbogenated (95% 02 and 5% C02) ACSF for 2 hours prior recording.
  • fEPSPs Field excitatory postsynaptic potentials
  • Golgi staining was performed using the Golgi Staining Kit (FD NeuroTechnologies, Columbia, USA) according to the manufacturer's instructions. All procedures were performed under dark conditions. Brains hemispheres used for Golgi cox staining were immersed in 2 ml mixtures of equal parts of kit solutions A and B and stored at RT for 2 weeks. Then, brain tissues were stored in solution C at 4°C for at least 48h and up to 7 days before sectioning. Solutions A, B and C were renewed within the first 24h. Coronal sections of 200 ⁇ were cut with a vibrating microtome (Leica, VT1200S) while embedded in 2% Agar in 0.1M PBS.
  • a vibrating microtome Leica, VT1200S
  • Imaging and analysis of spine density in Golgi- Cox stained slices Imaging of dendritic branches of hippocampal pyramidal neurons was done with an Axioplan 2 imaging microscope (Zeiss) using a 63x oil objective (NA 1.3) and a z-stack thickness of 0.5 ⁇ under reflected light. The number of spines was determined per micrometer of dendritic length (in total 100 ⁇ ) at apical compartments using ImageJ (1.48v, National Instruments of Health, USA). Three animals per genotype and 8-10 neurons per animal were analyzed blinded to genotype and injected AAV. Data were analyzed using Graphpad Prism (Version 5.01) software. Spine density is expressed as mean + SEM. Differences between genotypes were detected with one-way ANOVA followed by Bonferroni's post hoc test using IBM SPSS Statistics 21.
  • Interleukin-2 expression is decreased in the hippocampus of AD patients
  • Peripheral Interleukin-2 delivery induces increased Interleukin-2 and Tregs levels in the brain of APP/PS1AE9 mice As Alzheimer's disease is a slow developing disease, we anticipated that long term Tregs stimulation could be necessary for obtaining therapeutic benefit. As Interleukin-2 has a short half-life in mice, maintaining an effect on Tregs would require frequent sub-cutaneous (sc) injections that could have an effect on mice behavior.
  • sc sub-cutaneous
  • Interleukin-2 by intraperitoneal (ip) injection of a recombinant adeno-associated virus (AAV) coding for murine Interleukin-2, which allows sustained and stable release of Interleukin-2 for at least 20 weeks (Churlaud et al., 2014; Wang et al., 2005).
  • AAV adeno-associated virus
  • serum Interleukin-2 was undetectable in mice receiving rAAV8-LUC, and was of 25.2 + 5.3 or 29.5 + 4.8 pg/mL (mean + SEM) in Interleukin-2 treated littermates and APP/PS1AE9, respectively (Fig.2A).
  • serum Interleukin-2 levels are those necessary for expanding and activating Tregs without effects on effector T cells, as previously described (Churlaud et al., 2014).
  • peripheral Interleukin-2 production expanded blood Tregs, significantly more in the APP/PS1AE9 mice than wild type littermates (Fig 2C).
  • Tregs from Interleukin-2 treated mice were also more activated as assessed by increased CD25 cell surface expression (Rosenzwajg et al., 2015) (Fig 2E). Mice were sacrificed at 5.5 months post- injection (13.5 months of age). In hippocampal biopsies, there was a significant increase of Interleukin-2 levels only in Interleukin-2 treated APP/PS1AE9 mice (Figure 2B). These levels were approximately doubled compare to Interleukin-2 treated wild-type littermates and rAAV8-LUC controls, as detected both by western blot ( Figure 7) and ELISA ( Figure 2B).
  • luciferase expression could be detected in most of peripheral organs (liver, heart, kidney and spleen). In accordance with the known tropism of AAV8, most of the expression was detected in the liver. In contrast, no expression could be detected in the brain ( Figure 8A).
  • ITR2 inverted terminal repeat
  • mice were tested in the Morris water- maze place navigation task (Fig. 3).
  • Treated APP/PS1AE9 mice or wild-type littermates were tested 5 months post-injection. All mice learned platform position across time during learning session, as demonstrated by decreased latency (data not shown) or path length (data not shown) to reach the platform over the 5 days of training. Noteworthy, an overall significantly improved learning was detected in rAAV-IL- 2 treated littermates (p ⁇ 0.05). Average swimming speed was comparable in all groups ruling out potential motor abilities differences (data not shown).
  • Interleukin-2 rescues impaired synaptic plasticity and restores decreased spine density in APP/PS1AE9 mice
  • Paired-pulse facilitation in APP/PS1AE9 mice administered with rAAV-LUC was also significantly (p ⁇ 0.05, ANOVA) higher compared with littermates. Paired-pulse facilitation was not different between APP/PS1AE9 mice administered with rAAV-IL-2 or rAAV-LUC (data not shown).
  • Spine density was analyzed as a correlate of excitatory synapses in the same animals used for electrophysiological recordings.
  • Spine density of mid- apical dendritic segments (between 100 and 400 ⁇ form soma) of the hippocampal CAl pyramidal layer was analyzed by the Golgi cox method (data not shown).
  • Interleukin-2 peripheral administration alleviates hippocampal amyloid pathology in APP/PS1AE9 mice
  • APP/PS 1 ⁇ 9 mice show highly abundant plaques from 6 months (Jankowsky et al., 2004). Interleukin-2 treatment was started at 8 months of age, a time at which we already evidence increased levels of ⁇ peptides ( ⁇ 38, ⁇ 40 and ⁇ 42) and ⁇ -CTF in hippocampus from APP/PS 1 ⁇ 9 mice (Figure 9). Mice were sacrificed at 13.5 months of age. Hippocampal APP levels were slightly increased in mice receiving rAAV8-IL-2 compared to rAAV8-LUC injected mice (Fig. 5A-B).
  • Interleukin-2 administration induces widespread recruitment of astrocytes in the vicinity of amyloid plaques
  • Amyloid plaques in APP/PS1AE9 mice were surrounded by microglia and astrocytes at the time of the treatment (8 months) (data not shown).
  • microglial Ibal The protein levels of microglial Ibal were not statistically different in the hippocampus of IL-2 treated APP/PS1AE9 mice compared to APP/PS1AE9 mice receiving rAAV8-LUC (Fig. 6A); no differences were detected in littermates receiving rAAV8-LUC or rAAV8-IL-2. No differences were observed in the expression of microglia markers, cytokine transforming growth factor-beta (TGF- ⁇ ), Arginase-1 and Triggering Receptor Expressed on Myeloid cells (TREM2) or insulin-degrading enzyme (IDE), which contributes to ⁇ clearance (Leissring et al., 2003) ( Figure 10). No major difference in Ibal immunoreactivity around hippocampal amyloid plaques was observed (Fig. 6C).
  • TGF- ⁇ cytokine transforming growth factor-beta
  • TREM2 Arginase-1
  • IDE insulin-degrading enzyme
  • Interleukin-2 administration activates the JAK/STAT3 pathway in the hippocampus of APP/PS1AE9 mice
  • the hippocampal AAV-mediated overexpression of the anti-inflammatory cytokines Interleukin-10 or Interleukin-4 in AD mice enhanced neurogenesis and improved spatial learning and ⁇ deposition in APP/PSl mice (Kiyota et al., 2010; Kiyota et al., 2012; Latta et al., 2015).
  • two recent studies support a detrimental impact of Interleukin-10 in Alzheimer's disease pathology (Chakrabarty et al., 2015; Guillot-Sestier et al., 2015).
  • IL- ⁇ in Alzheimer's disease mice did not result in the expected exacerbation of the amyloid plaque deposition, but instead in plaque improvement (Shaftel et al., 2007).
  • AAV-mediated expression of Interleukin-6 (Chakrabarty et al., 2010) and TNF-a (Chakrabarty et al., 2011) induced massive gliosis that suppressed ⁇ deposition.
  • immune checkpoint blockade directed against the programmed death- 1 (PD-1) pathway evoked an interferon (IFN)-y-dependent systemic immune response leading to clearance of plaques and improved cognitive performance (Baruch et al., 2016).
  • IFN interferon
  • Interleukin-2 prompts Treg expansion and activation in the brain of APP/PS1AE9 mice and improves Alzheimer's disease pathology. Increased Interleukin-2 concentrations were observed in the hippocampus of Interleukin-2 treated APP/PS1AE9 mice but not in Interleukin-2 treated littermates. Since AAV vectors do not transduce brain cells and Interleukin-2 is produced in the periphery, we may speculate that CNS penetration of peripherally produced Interleukin-2 could be favored in APP/PS1AE9 mice due to BBB leakage. Indeed, the BBB is relatively impermeable in healthy subjects, however compromised in Alzheimer's disease given disruption of the tightly packed endothelial cells that support brain vasculature.
  • BBB permeability has been reported in Alzheimer' s disease mouse models (Tanifum et al., 2014) and was shown to increase before plaque formation in Alzheimer's disease mice (Ujiie et al., 2003). This has important implications for the route of administration of therapeutic molecules such as Interleukin-2 (Waguespack et al., 1994).
  • Tregs have a controversial role in controlling or worsening Alzheimer's disease. Treg depletion was reported to improve Alzheimer's disease in 5XFAD mice (Baruch et al., 2015) or to accelerate the onset of cognitive deficits in APPPS1 mice (Dansokho et al., 2016). In any case, how transient Treg ablation (observed in the periphery but not asserted in the brain) can impact a disease that develops along months is unexplained.
  • Tregs recruitment to cerebral sites of Alzheimer's disease pathology may have led to reduction of gliosis and ⁇ plaque, with improvement of cognitive functions (Baruch et al., 2015).
  • Treg depletion might not be an optimal method to assess Treg role in Alzheimer's disease.
  • Other investigators used an anti-CD25 monoclonal antibody to deplete Tregs in vivo in APPPS1 mice.
  • Treg depletion is only transient and CD25 is also expressed by other immune cells such as NK cells, activated B cells, activated effector T cells, myeloid cells, which could induce non expected effects by depleting these populations (Baeyens et al., 2013).
  • NK cells activated B cells
  • activated effector T cells activated effector T cells
  • myeloid cells which could induce non expected effects by depleting these populations.
  • the astrocytic phenotypic activation was correlated with stimulation of the JAK/STAT3 pathway that was shown to activate astrocytes in models of acute brain injury and is involved in cell growth, neuronal survival and differentiation (Bareyre et al., 2011; Lang et al., 2013).
  • Astrocytes have been described as a potential source of brain Interleukin-2 (Eizenberg et al., 1995). Astrocytes make part of the BBB and may well be the primary brain parenchymal cell type encountered by peripheral Tregs. Indeed, previous reports have described astrocyte/T cells interactions (Barcia et al., 2013).
  • astrocytes may influence Alzheimer's disease-like pathogenesis through invasion of plaques as an attempt to clear ⁇ and limit its extracellular deposition.
  • mouse astrocytes were reported to degrade amyloid-beta in vitro and in situ (Wyss-Coray et al., 2003), and exogenous astrocytes transplanted into the brain of plaque -bearing Alzheimer' s disease mice, were shown to migrate towards ⁇ deposits, internalizing them (Pihlaja et al., 2008). Furthermore, it was shown that attenuating astrocyte activation accelerates plaque deposition in Alzheimer's disease mice (Kraft et al., 2013).
  • Interleukin-2 treatment and consequent astrocytic activation were accompanied by a reduction of amyloid plaques and a decrease in the ⁇ 42/ ⁇ 40 ratio (Murray et al., 2012).
  • the strong ⁇ 40 increase in Interleukin-2 treated APP/PS1AE9 mice is in line with in vitro and in vivo data showing that ⁇ 40 protects neurons from ⁇ 42 induced damage in culture and in rat brain (Zou et al., 2003) and inhibits amyloid deposition in Alzheimer's disease mice, protecting them from premature death (Kim et al., 2007).
  • Interleukin-2 is an approved drug used for the stimulation of effector cells for the treatment of metastatic melanoma and renal cell carcinoma. In these indications it is given at very high doses (up to 160 MIU per day) and actually poorly used because of severe side effects (Klatzmann and Abbas, 2015). The demonstration that low-dose Interleukin-2 is safe and selectively activates and expands Tregs without activating effector T cells in humans has changed the paradigm for Interleukin-2 therapeutic use. Interleukin-2 is now intensively developed as a stimulant of Tregs at daily dose around 1 to 3 MIU.
  • Interleukin-2 is well tolerated in humans with autoimmune diseases (Castela et al., 2014; Hartemann et al., 2013; He et al., 2016; Saadoun et al., 2011).
  • these doses lead to increased serum concentration of Interleukin-2 that are in the range of the long-term elevated concentrations observed during pregnancy (Curry et al., 2008).
  • a pre-clinical study of the long-term effects of Interleukin-2 in mice showed that a yearlong treatment is well tolerated (Churlaud et al., 2014).
  • Long-term treatment with low-dose Interleukin-2 of Alzheimer's disease patients can be envisioned.
  • IL-2 deficiency results in altered septal and hippocampal cyto architecture: relation to development and neurotrophins. J Neuroimmunol. 160, 146-53.
  • Transplanted astrocytes internalize deposited beta-amyloid peptides in a transgenic mouse model of Alzheimer's disease. Glia. 56, 154-63.
  • Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain. Brain Res. 625, 347-50.
  • Interleukin-2 enhances dendritic development and spinogenesis in cultured hippocampal neurons.
  • Anat Rec Hoboken
  • Interleukin-2 does not cross the blood-brain barrier by a saturable transport system. Brain Res Bull. 34, 103-9.
  • Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol. 23, 321-8.
  • Amyloid beta-protein (Abeta)l-40 protects neurons from damage induced by Abetal-42 in culture and in rat brain. J Neurochem. 87, 609-19.

Abstract

La présente invention concerne des méthodes et des compositions pharmaceutiques pour le traitement de la maladie d'Alzheimer. En particulier, la présente invention concerne une méthode de traitement de la maladie d'Alzheimer chez un sujet en ayant besoin, comprenant l'administration au sujet d'une quantité thérapeutiquement efficace d'un vecteur viral adéno-associé recombinant comprenant un polynucléotide codant pour un polypeptide d'interleukine 2 (IL -2).
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109999179A (zh) * 2019-05-08 2019-07-12 山东省分析测试中心 低剂量白介素2用于制备治疗阿尔茨海默病药物中的应用
GB201912863D0 (en) 2019-09-06 2019-10-23 Babraham Inst Novel method
WO2022189797A1 (fr) * 2021-03-10 2022-09-15 Babraham Institute Nouveau procédé
WO2023111560A1 (fr) * 2021-12-14 2023-06-22 Babraham Institute Nouvelles méthode et composition comprenant de l'il-2 et une fraction de ciblage spécifique d'un tissu ou d'un organe

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094605A2 (fr) * 2000-06-09 2001-12-13 University Of Florida Research Foundation, Inc. Compositions a base de vecteur viral associe a l'adenovirus et leurs utilisations therapeutiques
WO2011133890A1 (fr) * 2010-04-23 2011-10-27 University Of Massachusetts Vecteurs aav ciblant le système nerveux central et leurs procédés d'utilisation
WO2014206899A1 (fr) * 2013-06-25 2014-12-31 Icm (Institut Du Cerveau Et De La Moelle Épinière) Stimulation de cellules treg pour le traitement de la maladie d'alzheimer et de troubles apparentés
WO2015042521A1 (fr) * 2013-09-20 2015-03-26 University Of Virginia Patent Foundation Compositions et méthodes destinées à traiter les maladies et les troubles auto-immuns et inflammatoires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001094605A2 (fr) * 2000-06-09 2001-12-13 University Of Florida Research Foundation, Inc. Compositions a base de vecteur viral associe a l'adenovirus et leurs utilisations therapeutiques
WO2011133890A1 (fr) * 2010-04-23 2011-10-27 University Of Massachusetts Vecteurs aav ciblant le système nerveux central et leurs procédés d'utilisation
WO2014206899A1 (fr) * 2013-06-25 2014-12-31 Icm (Institut Du Cerveau Et De La Moelle Épinière) Stimulation de cellules treg pour le traitement de la maladie d'alzheimer et de troubles apparentés
WO2015042521A1 (fr) * 2013-09-20 2015-03-26 University Of Virginia Patent Foundation Compositions et méthodes destinées à traiter les maladies et les troubles auto-immuns et inflammatoires

Non-Patent Citations (86)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NAT. GENET., vol. 6, 1994, pages 119 - 129
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
AWATSUJI, H. ET AL.: "Interleukin-2 as a neurotrophic factor for supporting the survival of neurons cultured from various regions of fetal rat brain", J NEUROSCI RES., vol. 35, 1993, pages 305 - 11
BAEYENS, A. ET AL.: "Limitations of IL-2 and rapamycin in immunotherapy of type 1 diabetes", DIABETES, vol. 62, 2013, pages 3120 - 31
BARCIA, C., SR. ET AL.: "Imaging the microanatomy of astrocyte-T-cell interactions in immune-mediated inflammation", FRONT CELL NEUROSCI., vol. 7, 2013, pages 58
BAREYRE, F.M. ET AL.: "In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration", PROC NATL ACAD SCI USA., vol. 108, 2011, pages 6282 - 7
BARUCH, K. ET AL.: "Breaking immune tolerance by targeting Foxp3(+) regulatory T cells mitigates Alzheimer's disease pathology", NAT COMMUN., vol. 6, 2015, pages 7967
BARUCH, K. ET AL.: "PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease", NAT MED., 2016
BECK, R.D., JR. ET AL.: "IL-2 deficiency results in altered septal and hippocampal cytoarchitecture: relation to development and neurotrophins", J NEUROIMMUNOL., vol. 160, 2005, pages 146 - 53
BELOOSESKY, Y. ET AL.: "Cytokine levels and phagocytic activity in patients with Alzheimer's disease", GERONTOLOGY, vol. 48, 2002, pages 128 - 32
BIRCH, A.M.; KATSOURI, L.; SASTRE, M.: "Modulation of inflammation in transgenic models of Alzheimer's disease", J NEUROINFLAMMATION., vol. 11, 2014, pages 25
CASTELA, E. ET AL.: "Effects of low-dose recombinant interleukin 2 to promote T-regulatory cells in alopecia areata", JAMA DERMATOL., vol. 150, 2014, pages 748 - 51
CHAKRABARTY, P. ET AL.: "Hippocampal expression of murine TNFalpha results in attenuation of amyloid deposition in vivo", MOL NEURODEGENER, vol. 6, 2011, pages 16
CHAKRABARTY, P. ET AL.: "IL-10 alters immunoproteostasis in APP mice, increasing plaque burden and worsening cognitive behavior", NEURON, vol. 85, 2015, pages 519 - 33
CHAKRABARTY, P. ET AL.: "Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition", FASEB J., vol. 24, 2010, pages 548 - 59
CHIBA, T. ET AL.: "Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons", MOL PSYCHIATRY, vol. 14, 2009, pages 206 - 22
CHURLAUD GUILLAUME ET AL: "Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer", CLINICAL IMMUNOLOGY, vol. 151, no. 2, 2014, pages 114 - 126, XP028838230, ISSN: 1521-6616, DOI: 10.1016/J.CLIM.2014.02.003 *
CHURLAUD, G. ET AL.: "Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer", CLIN IMMUNOL., vol. 151, 2014, pages 114 - 26
CLARE H LATTA ET AL: "Determining the role of IL-4 induced neuroinflammation in microglial activity and amyloid-? using BV2 microglial cells and APP/PS1 transgenic mice", JOURNAL OF NEUROINFLAMMATION, BIOMED CENTRAL LTD., LONDON, GB, vol. 12, no. 1, 4 March 2015 (2015-03-04), pages 41, XP021218142, ISSN: 1742-2094, DOI: 10.1186/S12974-015-0243-6 *
CORPET ET AL., NUC. ACIDS RES., vol. 16, 1988, pages 10881 - 10890
CURRY, A.E. ET AL.: "Maternal plasma cytokines in early- and mid-gestation of normal human pregnancy and their association with maternal factors", J REPROD IMMUNOL., vol. 77, 2008, pages 152 - 60
DANSOKHO, C. ET AL.: "Regulatory T cells delay disease progression in Alzheimer-like pathology", BRAIN, 2016
EIZENBERG, O. ET AL.: "Interleukin-2 transcripts in human and rodent brains: possible expression by astrocytes", J NEUROCHEM., vol. 64, 1995, pages 1928 - 36
FARFARA D.; V. LIFSHITZ; D. FRENKEL: "Neuroprotective and neurotoxic properties of glial cells in the pathogenesis of Alzheimer's disease", JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, vol. 12, 2008, pages 762 - 780
FERRARI, R. ET AL.: "Frontotemporal dementia and its subtypes: a genome-wide association study", LANCET NEUROL., vol. 13, 2014, pages 686 - 99
FONTENOT, J.D.; GAVIN, M.A.; RUDENSKY, A.Y.: "Foxp3 programs the development and function of CD4+CD25+ regulatory T cells", NAT IMMUNOL., vol. 4, 2003, pages 330 - 6
GALIMBERTI D.; E. SCARPINI: "Progress in Alzheimer's disease", JOURNAL OF NEUROLOGY, vol. 259, 2012, pages 201 - 211
GISH.; STATES, NATURE GENET., vol. 3, 1993, pages 266 - 272
GUILLOT-SESTIER, M.V. ET AL.: "1110 deficiency rebalances innate immunity to mitigate Alzheimer-like pathology", NEURON, vol. 85, 2015, pages 534 - 48
HANISCH, U.K. ET AL.: "Neurotoxic consequences of central long-term administration of interleukin-2 in rats", NEUROSCIENCE, vol. 79, 1997, pages 799 - 818
HARTEMANN, A. ET AL.: "Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial", LANCET DIABETES ENDOCRINOL., vol. 1, 2013, pages 295 - 305
HE, J. ET AL.: "Low-dose interleukin-2 treatment selectively modulates CD4(+) T cell subsets in patients with systemic lupus erythematosus", NAT MED., vol. 22, 2016, pages 991 - 3
HEIM, M.H.; 1999: "The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus", J RECEPT SIGNAL TRANSDUCT RES., vol. 19, pages 75 - 120
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151 - 153
HIGGINS; SHARP, GENE, vol. 73, 1988, pages 237 - 244
HUANG ET AL., COMP. APPLS BIOSCI., vol. 8, 1992, pages 155 - 165
JANKOWSKY, J.L. ET AL.: "Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase", HUM MOL GENET., vol. 13, 2004, pages 159 - 70
KIM, J. ET AL.: "Abeta40 inhibits amyloid deposition in vivo", J NEUROSCI., vol. 27, 2007, pages 627 - 33
KIYOTA, T. ET AL.: "AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice", GENE THER., vol. 19, 2012, pages 724 - 33
KIYOTA, T. ET AL.: "CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PS1 bigenic mice", FASEB J., vol. 24, 2010, pages 3093 - 102
KLATZMANN, D.; ABBAS, A.K.: "The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases", NAT REV IMMUNOL., vol. 15, 2015, pages 283 - 94
KRAFT, A.W. ET AL.: "Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice", FASEB J., vol. 27, 2013, pages 187 - 98
LACOSTA, S.; MERALI, Z.; ANISMAN, H.: "Influence of acute and repeated interleukin-2 administration on spatial learning, locomotor activity, exploratory behaviors, and anxiety", BEHAV NEUROSCI., vol. 113, 1999, pages 1030 - 41
LANG, C. ET AL.: "STAT3 promotes corticospinal remodelling and functional recovery after spinal cord injury", EMBO REP., vol. 14, 2013, pages 931 - 7
LATTA, C.H. ET AL.: "Determining the role of IL-4 induced neuroinflammation in microglial activity and amyloid-beta using BV2 microglial cells and APP/PS 1 transgenic mice", J NEUROINFLAMMATION, vol. 12, 2015, pages 41
LEISSRING, M.A. ET AL.: "Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death", NEURON, vol. 40, 2003, pages 1087 - 93
LORITA DUDUS ET AL: "Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV", VISION RESEARCH., vol. 39, no. 15, 1 July 1999 (1999-07-01), GB, pages 2545 - 2553, XP055239319, ISSN: 0042-6989, DOI: 10.1016/S0042-6989(98)00308-3 *
M. MIZUI ET AL: "IL-2 Protects Lupus-Prone Mice from Multiple End-Organ Damage by Limiting CD4-CD8- IL-17-Producing T Cells", THE JOURNAL OF IMMUNOLOGY, vol. 193, no. 5, 25 July 2014 (2014-07-25), US, pages 2168 - 2177, XP055239184, ISSN: 0022-1767, DOI: 10.4049/jimmunol.1400977 *
MADDEN ET AL., METH. ENZYMOL., vol. 266, 1996, pages 131 - 141
MONJE M. L.; H. TODA; T. D. PALMER: "Inflammatory blockade restores adult hippocampal neurogenesis", SCIENCE, vol. 302, 2003, pages 1760 - 1765
MURRAY, P.S. ET AL.: "beta-Amyloid 42/40 ratio and kalirin expression in Alzheimer disease with psychosis", NEUROBIOL AGING, vol. 33, 2012, pages 2807 - 16
MYERS; MILLER, CABIOS, vol. 4, 1989, pages 11 - 17
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
PEARSON ET AL., METH. MOL. BIOL., vol. 24, 1994, pages 307 - 31
PEARSON; LIPMAN, PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 2444
PETITTO, J.M. ET AL.: "Impaired learning and memory and altered hippocampal neurodevelopment resulting from interleukin-2 gene deletion", J NEUROSCI RES., vol. 56, 1999, pages 441 - 6
PIHLAJA, R.; 2008 ET AL.: "Transplanted astrocytes internalize deposited beta-amyloid peptides in a transgenic mouse model of Alzheimer's disease", GLIA, vol. 56, pages 154 - 63
ROGERS J.; J. LUBER-NAROD; S. D. STYREN; W. H. CIVIN: "Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease", NEUROBIOLOGY OF AGING, vol. 9, 1988, pages 339 - 349
ROSENZWAJG, M. ET AL.: "Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients", J AUTOIMMUN., vol. 58, 2015, pages 48 - 58
SAADOUN, D. ET AL.: "Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis", N ENGL J MED., vol. 365, 2011, pages 2067 - 77
SAMBAMURTI K.; N. H. GREIG; T. UTSUKI; E. L. BARNWELL; E. SHARMA; C. MAZELL; N. R. BHAT; M. S. KINDY; D. K. LAHIRI; M. A. PAPPOLLA: "Targets for AD treatment: conflicting messages from gamma-secretase inhibitors", JOURNAL OF NEUROCHEMISTRY, vol. 117, 2011, pages 359 - 374
SARDER, M. ET AL.: "Comparative effect of IL-2 and IL-6 on morphology of cultured hippocampal neurons from fetal rat brain", BRAIN RES., vol. 715, 1996, pages 9 - 16
SARDER, M.; SAITO, H.; ABE, K.: "Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain", BRAIN RES., vol. 625, 1993, pages 347 - 50
SCHWAB, C.; MCGEER, P.L.: "Inflammatory aspects of Alzheimer disease and other neurodegenerative disorders", J ALZHEIMERS DIS., vol. 13, 2008, pages 359 - 69
SELKOE D. J.: "The molecular pathology of Alzheimer's disease", NEURON, vol. 6, 1991, pages 487 - 498
SETO, S D..; KAR, R.: "Quirion, Evidence for direct and indirect mechanisms in the potent modulatory action of interleukin-2 on the release of acetylcholine in rat hippocampal slices", BRITISH JOURNAL OF PHARMACOLOGY, vol. 120, 1997, pages 1151 - 1157
SHAFTEL, S.S. ET AL.: "Chronic interleukin-lbeta expression in mouse brain leads to leukocyte infiltration and neutrophil-independent blood brain barrier permeability without overt neurodegeneration", J NEUROSCI., vol. 27, 2007, pages 9301 - 9
SHEN, Y. ET AL.: "Interleukin-2 enhances dendritic development and spinogenesis in cultured hippocampal neurons", ANAT REC (HOBOKEN, vol. 293, 2010, pages 1017 - 23
SKAPER, S.D.: "The brain as a target for inflammatory processes and neuroprotective strategies", ANN N Y ACAD SCI., vol. 1122, 2007, pages 23 - 34
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482
SNYDER, E.M. ET AL.: "Regulation of NMDA receptor trafficking by amyloid-beta", NAT NEUROSCI., vol. 8, 2005, pages 1051 - 8
STREIT W. J.: "Microglial response to brain injury: a brief synopsis", TOXICOLOGIC PATHOLOGY, vol. 28, 2000, pages 28 - 30
T KIYOTA ET AL: "AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice", GENE THERAPY, vol. 19, no. 7, 15 September 2011 (2011-09-15), GB, pages 724 - 733, XP055239170, ISSN: 0969-7128, DOI: 10.1038/gt.2011.126 *
T. KIYOTA ET AL: "CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PS1 bigenic mice", THE FASEB JOURNAL, vol. 24, no. 8, 1 August 2010 (2010-08-01), BETHESDA; US, pages 3093 - 3102, XP055239344, ISSN: 0892-6638, DOI: 10.1096/fj.10-155317 *
TANCREDI V.; C. ZONA; F. VELOTTI; F. EUSEBI; A. SANTONI: "Interleukin-2 suppresses established long-term potentiation and inhibits its induction in the rat hippocampus", BRAIN RESEARCH, vol. 525, 1990, pages 149 - 151
TANIFUM, E.A. ET AL.: "Cerebral vascular leak in a mouse model of amyloid neuropathology", J CEREB BLOOD FLOW METAB., vol. 34, 2014, pages 1646 - 54
UJIIE, M. ET AL.: "Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model", MICROCIRCULATION, vol. 10, 2003, pages 463 - 70
VOM BERG, J. ET AL.: "Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline", NAT MED., vol. 18, 2012, pages 1812 - 9
WAGUESPACK, P.J.; BANKS, W.A.; KASTIN, A.J.: "Interleukin-2 does not cross the blood-brain barrier by a saturable transport system", BRAIN RES BULL., vol. 34, 1994, pages 103 - 9
WANG, Z. ET AL.: "Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart", NAT BIOTECHNOL., vol. 23, 2005, pages 321 - 8
WEGIEL, J. ET AL.: "The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APP(SW) mice", NEUROBIOL AGING., vol. 22, 2001, pages 49 - 61
WYSS-CORAY, T. ET AL.: "Adult mouse astrocytes degrade amyloid-beta in vitro and in situ", NAT MED., vol. 9, 2003, pages 453 - 7
XIE, L. ET AL.: "Cerebral regulatory T cells restrain microglia/macrophage-mediated inflammatory responses via IL-10", EUR J IMMUNOL., vol. 45, 2015, pages 180 - 91
ZHANG; MADDEN, GENOME RES., vol. 7, 1997, pages 649 - 656
ZOU, K. ET AL.: "Amyloid beta-protein (Abeta) 1-40 protects neurons from damage induced by Abetal-42 in culture and in rat brain", J NEUROCHEM., vol. 87, 2003, pages 609 - 19

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