WO2010088729A1 - Compositions et leurs utilisations - Google Patents

Compositions et leurs utilisations Download PDF

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Publication number
WO2010088729A1
WO2010088729A1 PCT/AU2010/000113 AU2010000113W WO2010088729A1 WO 2010088729 A1 WO2010088729 A1 WO 2010088729A1 AU 2010000113 W AU2010000113 W AU 2010000113W WO 2010088729 A1 WO2010088729 A1 WO 2010088729A1
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WIPO (PCT)
Prior art keywords
rap
polypeptide
cell
binding
composition
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PCT/AU2010/000113
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English (en)
Inventor
Megan Kerr
David Small
Alfons Lawen
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University Of Tasmania Through The Menzies Research Institute
Monash University
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Priority claimed from AU2009900408A external-priority patent/AU2009900408A0/en
Application filed by University Of Tasmania Through The Menzies Research Institute, Monash University filed Critical University Of Tasmania Through The Menzies Research Institute
Publication of WO2010088729A1 publication Critical patent/WO2010088729A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • compositions and methods for treating or preventing symptoms of amyloidoses, Alzheimer's disease or related conditions characterised by amyloid deposits in the brain, memory loss and dementia relate generally to compositions and methods for treating or preventing symptoms of amyloidoses, Alzheimer's disease or related conditions characterised by amyloid deposits in the brain, memory loss and dementia.
  • the specification considers compositions that reduce the activity or pathogenesis of A ⁇ comprising agents capable of binding to ⁇ -amyloid peptide (A ⁇ ).
  • proteopathies comprise a group of clinically diverse disorders characterised by the damaging accumulation of aggregated proteins in cells and tissues of the body.
  • proteopathies include, inter alia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses.
  • Proteins normally fold into preferred, 'native' conformations in which they can carry out their customary functions in the cell. However, in proteopathies a protein assumes an atypical, three-dimensional conformation, which often is enriched in ⁇ -sheet structure. 13
  • Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with similar protein molecules.
  • Each proteopathy has a characteristic signature that includes the accumulation of a particular protein as extracellular deposits and/or intracellular inclusions or aggregations in certain organs. Such deposits and inclusions are considered central to the pathology of proteopathies and attempts to develop effective therapies for the proteopathies have been directed inter alia toward reducing the production of the proteins, blocking their aggregation, or augmenting their removal.
  • Alzheimer's disease is a common and debilitating neurodegenerative proteopathy resulting in progressive loss of memory and cognitive ability that eventually lead to dementia and death.
  • Related conditions include conditions that are characterised by amyloid deposits in the brain and memory loss, such as found in Lewy body dementia, in muscles such as in inclusion body mycositis, or in cerebral blood vessels such as in cerebral amyloid angiopathy.
  • Alzheimer's disease is characterized by accumulation of ⁇ -amyloid protein (A ⁇ ) in the brain, extracellularly as amyloid plaques and cerebral amyloid angiopathy, and intracellularly as neurofibrillary tangles (NFTs).
  • a ⁇ ⁇ -amyloid protein
  • a ⁇ is neurotoxic and that oligomeric forms of A ⁇ are the most potent neurotoxin (Lambert et al, Proc Natl Acad Sci USA 25:6448-6453, 1998; Small et al, Nat Rev Neurosci 2:595-598, 2001; Walsh et al, Nature 416:535-539, 2002; Gong et al, Proc Natl Acad Sci U S A 700:10417-10422, 2003; Cleary et al, Nat Neurosci 5:79-84, 2005; Lacor et al, J Neurosci 27:796-807, 2007).
  • a number of A ⁇ receptors have been proposed including LRPl (Deane et ah, Neuron 43:333-344, 2004), the ⁇ 7-nicotinic acetylcholine receptor (ct7nAChR) (Wang et ah, J. Biol. Chern. 275: 5626-5632, 2000), the ⁇ 75 neurotrophin receptor (Yaar et ah, J. Clin. Invest. 100: 2333-2340, 1997) and the receptor for advanced glycation endproducts (RAGE) (Yan et ah, Proc. Natl. Acad. Set USA 94: 5296-5301, 1997).
  • LRPl Deane et ah, Neuron 43:333-344, 2004
  • ct7nAChR ⁇ 7-nicotinic acetylcholine receptor
  • RAGE receptor for advanced glycation endproducts
  • Chem. 283: 34554-34562, 2008 have shown that A ⁇ does not bind directly to LRPl in endothelial cells and our own studies (Small et ah, 2007 (supra)) have shown that A ⁇ does not bind directly to the ⁇ 7nAChR. Instead, our studies (Subasinghe et ah, 2003 (supra); Small et ah, 2007 (supra)) and the work of others (Simakova and Arispe, J. Neurosci. 27: 13719-13729, 2007; Davis and Berkowitz, Biophys. J.
  • RAP The 39 kDa receptor-associated protein
  • Bu The 39 kDa receptor-associated protein (RAP) is a major ligand of many low-density lipoprotein receptor family members (Bu, Int. Rev. Cytol. 209: 79-116, 2001).
  • RAP is of interest, as a carrier, for the therapy of brain diseases as the protein is actively transported across the blood-brain barrier (Pan et ah, J Cell Sci. 117: 5071-5078, 2004) and has been proposed as a vehicle for drug delivery of active agents to the brain (Prince et ah, J Biol
  • a cell includes a single cell, as well as two or more cells; reference to “an agent” includes one agent, as well as two or more agents; and so forth.
  • the present invention is based upon the finding by the inventors that receptor associated protein (RAP) binds to ⁇ -amyloid protein (A ⁇ ) and directly modulates various activities of A ⁇ in vitro or in vivo.
  • RAP receptor associated protein
  • a ⁇ ⁇ -amyloid protein
  • LRPl lipoprotein receptors
  • RAP is found herein to bind to extracellular A ⁇ in a lipoprotein receptor independent manner and to enhance binding of A ⁇ to neuronal cells.
  • RAP polypeptide binds strongly to A ⁇ and enhances binding and/or uptake of A ⁇ to/by neuronal cells.
  • RAP down modulates A ⁇ aggregation.
  • RAP reduced the neurotoxic ability of A ⁇ to induce memory loss or cognitive impairment in an animal model of AD.
  • RAP-A ⁇ interaction is clearly demonstrated by the co-immunoprecipitation of both A ⁇ and RAP using an anti-RAP antibody. This procedure also revealed that some RAP and A ⁇ remained associated during SDS-PAGE suggesting that the interaction is particularly stable. It is likely that the 46 kDa RAP-A ⁇ complex contains one molecule of A ⁇ and one molecule of RAP, based on the apparent molecular mass of the complex during SDS-PAGE. However, the possibility that higher or lower molecular weight forms of A ⁇ may bind to RAP cannot be excluded at this stage and will be determined experimentally as described herein. The co-localization of RAP and A ⁇ at the cell membrane suggests that the two proteins can remain associated while bound to the cell surface.
  • the present invention provides a composition comprising a molecule having the activity of RAP including the herein disclosed ability to bind A ⁇ and reduce A ⁇ oligomerisation.
  • the molecule is a RAP polypeptide or a variant thereof including a functional part (fragment or portion of RAP), or an analog or agonist thereof which is capable of binding to A ⁇ or a precursor thereof.
  • the subject molecule or agent is for use in the treatment or prophylaxis of symptoms of AD or a related condition in a subject.
  • the symptoms of AD include memory loss associated with AD in a subject.
  • the RAP polypeptide is capable of binding to a lipoprotein receptor under physiological conditions. In other embodiments, the RAP polypeptide is incapable of binding to a lipoprotein receptor under physiological conditions.
  • composition comprising a RAP polypeptide or variant thereof as defined herein as the active ingredient, active in preventing the pathogenesis associated with A ⁇ is provided.
  • the RAP polypeptide is RAP or a fragment of RAP.
  • the RAP polypeptide is a variant of RAP.
  • the RAP polypeptide is a RAP peptidomimetic or other peptide such as a stapled peptide.
  • the RAP polypeptide is a small molecule analog or agonist of RAP.
  • the RAP polypeptide is human or mammalian.
  • the analog or agonist is a small molecule, an antibody, a nucleic acid or a peptide.
  • the molecules of the present invention are conveniently provided in a medicament form such as a pharmaceutical composition.
  • the present invention provides a method of identifying a candidate agent that modulates A ⁇ activity.
  • the methods are suitable for identifying agents that modulate amyloid activity in related conditions other than AD.
  • the activity of A ⁇ is selected from the group consisting of A ⁇ aggregation or oligomerisation, A ⁇ fibril formation, A ⁇ induced increase in intracellular calcium (Ca 2+ ),
  • the candidate agent also a RAP polypeptide or agonist
  • said method comprises:
  • the method comprises:
  • the system comprises an in vitro cell, such as a brain cell or a neural cell.
  • the cell is a neuroblastoma cell.
  • the system comprises an animal model of AD.
  • the system further comprises a RAP polypeptide and step ii) comprises instead determining the ability of the agent to reduce the binding between the RAP polypeptide and A ⁇ .
  • the present invention contemplates a method of generating a RAP polypeptide (RAP fragment, mutant, agonist, mimetic) capable of agonising RAP activity by binding to A ⁇ or other amyloid peptide and down-regulating the activity of A ⁇ .
  • the method comprises the steps of:
  • mutating one or more residues of an A ⁇ binding domain of RAP ii) contacting the mutated A ⁇ -binding domain with A ⁇ ; iii) detecting the presence of binding between the mutated RAP and A ⁇ thereby identifying amino acid residues associated with a binding interaction between the A ⁇ -binding domain of RAP and A ⁇ or other amyloid peptide; and iv) generating a RAP polypeptide agonist which mimics the wild-type RAP polypeptide at the residues essential for binding to occur between RAP polypeptide and A ⁇ and which inhibits the activity of A ⁇ .
  • the present invention contemplates a method of generating a RAP polypeptide (RAP fragment, mutant (variant), agonist, mimetic) capable of agonising or mimicking the functional activity of RAP by binding to an A ⁇ peptide and down- regulating the activity of A ⁇ .
  • the method comprises the steps of:
  • mutating one or more residues of an A ⁇ binding domain of RAP ii) contacting the mutated A ⁇ -binding domain with A ⁇ ; iii) detecting the presence of binding between the mutated RAP and A ⁇ thereby identifying amino acid residues essential for a binding interaction between the A ⁇ -binding domain of RAP and A ⁇ ; and iv) generating a RAP polypeptide or a RAP polypeptide analog which mimics a RAP polypeptide at the residues essential for binding to occur between RAP polypeptide and A ⁇ .
  • the present invention contemplates a method of generating a RAP polypeptide (a RAP fragment, mutant or variant, agonist, mimetic or analog) capable of agonising or mimicking RAP activity by binding to A ⁇ and down-regulating the activity of A ⁇ .
  • the method comprises the steps of:
  • mutating one or more residues of a RAP polypeptide or a fragment thereof ii) contacting the mutated RAP with A ⁇ ; iii) detecting the presence of binding between the mutated RAP and A ⁇ thereby identifying amino acid residues associated with a binding interaction between RAP and A ⁇ ; and iv) generating a RAP polypeptide or analogs which mimics the wild-type RAP polypeptide at the residues essential for binding to occur between RAP polypeptide and A ⁇ and which inhibits the activity of A ⁇ .
  • the RAP polypeptide is a fragment of RAP comprising all or part of Domain 1 (amino acids 1 to 112), Domain 2 (amino acids 113 to 215), or Domain 3 (amino acids 216 to 323) of the mature protein.
  • the amino acid sequence of these fragments are set out in SEQ ID NOs: 2, 3 and 4.
  • the amino acid sequence of the RAP precursor protein is set out in SEQ ID NO: 1.
  • the RAP polypeptide has the amino acid sequence set out in SEQ ID NO: 5, lacking the first 34 amino acid residues set out in SEQ ID NO: 1.
  • the RAP polypeptide has the ER retention signal C-terminal (HDEL) removed. Analogs and variants of all fragments are contemplated as a matter of routine.
  • the RAP polypeptide is also tested for its ability to cross biological membranes.
  • an indicator of the activit y of the complex between the RAP polypeptide or RAP polypeptide analog and A ⁇ is the memory performance of an animal model of AD.
  • the candidate agent (RAP polypeptide) is generated by methods such as, but not limited to, in silico screening, high throughput chemical screening, function based assay or structure-activity relationships.
  • the agents may be a proteinaceous or non- proteinaceous molecule derived from natural, synthetic or recombinant sources. Useful sources include screening libraries such as natural product libraries, chemical molecule libraries, peptide libraries, pharmaceutical product libraries, combinatorial libraries, phage display libraries and in vitro translation libraries, as known in the art.
  • RAP and/or A ⁇ are endogenously produced within a cell. In other instances, these agents are supplied exogenously.
  • the cell is a brain cell or a neural cell as referred to hereinabove as a component of the system.
  • the invention provides a composition comprising a complex comprising an isolated RAP polypeptide and A ⁇ .
  • RAP is covalently bound to A ⁇ .
  • kits comprising the complex or a specific-binding agent thereto for the diagnosis or prognosis of AD and related conditions are contemplated.
  • the present invention provides for the use of RAP or a complex comprising RAP and A ⁇ in the manufacture of a medicament in the treatment of AD or a related condition in a subject.
  • Reference herein to manufacture includes selection or design of a medicament.
  • the invention provides a method for the treatment or prophylaxis of a subject with AD or a related condition or who is at risk of developing same said method comprising administering to the subject an amount of a RAP polypeptide or an agent capable of producing same or a RAP analog or agonist capable of binding to A ⁇ (or an A ⁇ precursor for a time and under conditions effective to reduce A ⁇ aggregation, A ⁇ oligomerisation, A ⁇ fibril formation, amyloid neuropathy and/or enhance memory performance.
  • the subject is a human.
  • the invention contemplates a combination therapeutic protocol for the treatment or prophylaxis of symptoms of a condition characterised by aggregation of A ⁇ in brain tissue, said protocol comprising the administration of a composition as defined above or herein and one or more other treatments,
  • the other treatment is administration of a neurotrophin or other agent to enhance nerve cell regeneration, growth or development.
  • RAP polypeptide includes all biologically active naturally occurring forms of RAP as well as biologically active portions or fragments thereof.
  • variants including mutants, analogs and mimetics
  • derivatives of a RAP polypeptide that bind A ⁇ as disclosed herein refers to the ability of RAP in accordance with the present invention to bind to A ⁇ , or modify the activity of A ⁇ and/or reduce one or more symptoms of AD, such as memory loss. Mutants, analogs and mimetics are therefore selected for their ability to target A ⁇ at the structural and/or functional level.
  • the invention provides for and includes methods of screening for functional variants of RAP wherein the functional variant inter alia retains the ability to bind to A ⁇ and/or prevent or ameliorate the development of symptoms of proteopathies including neurodegenerative changes in a subject.
  • RAP binding to A ⁇ and internalisation by neural cells is independent of binding to lipoprotein receptors such as LRPl
  • RAP polypeptides are tested for their ability to bind A ⁇ .
  • an ability to bind LRPl is not required.
  • the invention provides a method for modulating aggregation of A ⁇ .
  • Oligomeric forms of A ⁇ rather than the monomeric or fibrillar forms are the most potent neurotoxins, and accordingly, reducing the formation of oligomers
  • the methods are useful in vitro.
  • the method comprises contacting A ⁇ or cells producing A ⁇ with a RAP polypeptide.
  • a ⁇ includes without limitation A ⁇ 1-40 and A ⁇ 1-42 peptides.
  • the invention provides a method for modulating cellular uptake of A ⁇ .
  • full length RAP polypeptide enhances the cell surface association and uptake of A ⁇ by cells.
  • the cell is a neuronal call.
  • the method comprises contacting A ⁇ with a RAP polypeptide
  • the present invention provides a method for preventing or reducing symptoms of AD, said method comprising administering to a subject an effective amount of a RAP polypeptide.
  • the subject may be administered in vivo or ex vivo.
  • the method comprises administering RAP polypeptide or an agent from which RAP polypeptide is producible.
  • the subject is tested for AD, cognitive impairment and/or loss of memory before or after administration of RAP polypeptide.
  • the RAP polypeptide is a portion or fragment of a full length RAP. In other embodiments, the RAP polypeptide is a variant or derivative of RAP comprising conservative amino acid changes as described herein and known in the art. In some embodiments, basic residues are conserved. In some embodiments, the RAP polypeptide comprises Domain 1 (Dl) 5 Domain 2 (D2) or Domain 3 (D3), of any combination of one or more of these domains of RAP. In other embodiments, the RAP polypeptide variant has at least 80%, 90% or 95% amino acid sequence identity to a naturally occurring RAP polypeptide over a reference region of at least 40, 50, 100, 150, 200 or 250 contiguous amino acids.
  • the variant RAP is encoded by a sequence of nucleotides that hybridise in conditions of medium or high stringency to the complement of the nucleotide sequences encoding a RAP polypeptide, such as described herein and including a fragment thereof.
  • RAP polypeptide is a variant, analog or mimetic of RAP that may comprise, for example, a protein, peptide, nucleic acid, small or large molecule or aptamer or antibody.
  • the invention provides a genetically modified cell genetically modified to express or over express a RAP polypeptide.
  • the invention provides an antibody that recognises (binds to) RAP or A ⁇ when RAP and A ⁇ are bound to each other but not when unbound.
  • the invention provides methods of diagnosis and diagnostic markers for subjects at risk for or exhibiting one or more symptoms of AD.
  • labelled RAP is provided as a marker for A ⁇ ; alternatively labelled A ⁇ is provided as a marker for RAP.
  • antibodies to RAP or a RAP binding agent is provided suitable for detection of a RAP molecule in biological samples.
  • extracellular RAP is to be detected. In other embodiments, intracellular RAP is detected.
  • Figure 1 is a photographic representation depicting binding and internalisation of FluoA ⁇ !- 42 by SH-SY5Y cells.
  • Cells were incubated with FluoA ⁇ 1-4 2 (1 ⁇ M) for 1 hours (h) (upper panel), 4 h (middle panel) or 24 h (lower panel), then the cell membrane was labeled by incubation with ice-cold Alexa-555-CTX B subunit (Alexa555-CTX) (250 ng/ml). Fluorescence images were captured by confocal microscopy. Scale bar, 20 ⁇ m.
  • Figure 2 is a representation of data illustrating that cell-bound A ⁇ colocalizes with exogenous RAP but not endogenous LRPl.
  • Panel A Effect of RAP and anti-LRPl antibody on association of FluoA ⁇ i -42 with SH-S Y5 Y cells.
  • Cells were treated with freshly prepared FluoA ⁇ 1-42 (1 ⁇ M) in the presence of RAP (5 ⁇ g/ml) (grey bars) or in the absence of RAP (black bars). Incubations were also performed in the absence (Control) or presence of anti-LRP antibody R2629 (10 ⁇ g/ml). AU incubations were for 4 h. Cells were analyzed for fluorescence by flow cytometry. Data represent means of three independent experiments ⁇ SEM.
  • Panels C and D Localization of FluoA ⁇ i. 42 RAP and LRPl on SH-SY5Y cells. Cells were incubated with freshly prepared FluoA ⁇ 1-42 (1 ⁇ M) in the presence of RAP (5 ⁇ g/ml) for 6 h, then rinsed, fixed and permeabilized. In panel C 5 LRPl was detected using antibody R2629 (1 ⁇ g/ml). Arrows indicate cell surface-bound FluoA ⁇ 1-42 and arrow heads show LRPl immunoreactivity. Scale bar, 10 ⁇ m. Panel D shows co-localization of A ⁇ and RAP at the cell surface.
  • the cells were fixed, permeabilized, and stained for RAP with anti-RAP monoclonal antibody 7Fl. Fluorescence was visualized by confocal microscopy. Asterisks show regions of co- localization. Blue channel shows AlexaFluor-555-CTX fluorescence. Scale bar, 20 ⁇ m.
  • Figure 3 is a photographic representation depicting A ⁇ and RAP co-immunoprecipitation.
  • a ⁇ 1-40 or A ⁇ 1-42 (1 ⁇ M) and/or RAP (5 ⁇ g/ml) were incubated in NaCl/P i5 pH 7.4 at 37°C for 6 h. Incubation mixtures were then further incubated with monoclonal antibody 7Fl- or an unrelated transferrin receptor antibody H68.4-bound protein G agarose. Beads were then washed thoroughly and then bound proteins denatured by heating in SDS sample buffer. Proteins were separated on a 12% Tris-glycine gel, transferred to nitrocellulose membrane, and immunoblotted using anti-A ⁇ monoclonal antibody 6E10 (left panel). The membrane was then stripped and re-probed using anti-RAP monoclonal antibody 7Fl (right panel), f Under these conditions of electrophoresis, A ⁇ migrated with the solvent front at -4-6 kDa.
  • Figure 4 is a photographic representation of SDS-PAGE analysis showing that RAP alters A ⁇ 1-4 o oligomerization and induces the formation of an SDS-stable RAP-A ⁇ complex.
  • a ⁇ 1-4 o (upper panel) or A ⁇ 1-42 (lower panel) (1 ⁇ M) and/or RAP (5 ⁇ g/ml) were incubated in NaCl/Pj, pH 7.4 at 37°C for the times indicated (A) or for 96 h (B).
  • proteins (20 ng A$ per lane) were separated by 16.5% Tris-tricine (A) or 15% Tris-glycine (JS) SDS-PAGE, transferred to nitrocellulose and immunoblotted with 6E10.
  • B The membrane was then stripped and immunoblotted with 7Fl (right panel).
  • FIG. 5 is a photographic representation illustrating that RAP inhibits A ⁇ 1-42 aggregation.
  • a ⁇ 1-42 (1 ⁇ M) and/or RAP (5 ⁇ g/ml) were incubated in NaCl/Pj, pH 7.4 at 37°C for the times indicated. Proteins were then deposited onto a surface of freshly cleaved highly oriented pyrolytic graphite, washed, dried and visualized by AFM. Left panels show images of A ⁇ 1-42 incubated alone. Middle panels show incubations with A ⁇ 1-4 2 incubated in the presence of RAP. Right panel shows RAP alone. Scale bar, 100 nm.
  • Figure 6 are a graphical representations showing the effect of RAP on the A ⁇ 1-42 -induced increase in intracellular Ca 2+ .
  • Freshly prepared A ⁇ 1-42 was first dissolved in HFIP prior to dilution into calcium imaging buffer. Cells were loaded with fluo-4 and treated with Ap 1- 42 in the absence or presence of RAP, with or without R2629 pre-treatment. Changes in intracellular Ca 2+ were detected as a change in fluorescence over time.
  • A Graph shows the change in fluorescence from baseline (AF/F) with time. Values are means of AF/F calculated for 4 wells + SEM.
  • B Effect of RAP and an anti-LRPl antibody (R2629) on A ⁇ -induced Ca2+ increase.
  • Figure 7 is a graphical representation of data showing that RAP co-injection blocks the amnestic effect of A ⁇ 1-42 in day-old chicks.
  • Physiological saline containing A ⁇ 1-42 (10 pmo I/hemisphere) with or without RAP (50 pg/hemisphere) was injected into the avian cortical region intermediate medial mesopallium of each hemisphere 45 min before bead discrimination training.
  • bead discrimination was tested at 120 min after training, providing a discrimination ratio (DR) score for each chick.
  • DR discrimination ratio
  • For complete memory retention a DR of 1.0 is obtained, whereas for complete loss of memory, a DR of 0.5 is obtained.
  • Figure 8 is a photographic and graphical representation illustrating decreased expression of RAP in AD brain.
  • A&B Representative micrographs of anti-RAP immunhistochemistry in the CAl of a control (A) and sporadic AD (B) case. Scale bar in A is equivalent for B.
  • the majority of neurons in control CAl are RAP immunoreactive (A) whereas many neurons in AD cases were RAP-negative (B).
  • Graph shows means ⁇ SEM. BRIEF DESCRIPTION OF THE TABLES
  • Table 1 provides a description of the SEQ ID NOs provided herein.
  • Table 3 provides an amino acid sub-classification.
  • Table 4 provides exemplary amino acid substitutions.
  • Table 5 provides a list of non-natural amino acids contemplated in the present invention.
  • Table 6 provides control and AD cases used for RAP immunohistochemical analysis.
  • the term "about” refers to a quantity, level, value, percentage, dimension, size, or amount that varies by as much as 30%, 20% or 10% to a reference quantity, level, value, percentage, dimension, size, or amount.
  • RAP polypeptide includes compounds that induce the desired pharmacological and/or physiological effect of RAP as disclosed herein.
  • the term also encompasses pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, derivatives, analogs and the like.
  • pharmaceutically acceptable and pharmacologically active ingredients include but not limited to salts, esters, amides, prodrugs, active metabolites, derivatives, analogs and the like.
  • this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
  • the phrase is not to be construed narrowly but extends to proteinaceous molecules including all forms of peptide, polypeptide and protein as well as mimetics and chemical analogs thereof as well as cellular agents.
  • agent in the phrase “agent from which RAP polypeptide is producible” includes a cell which is capable of producing and secreting RAP polypeptide as well as a polynucleotide comprising a nucleotide sequence that encodes a RAP polypeptide.
  • the RAP-encoding nucleotide sequence is operably connected to a regulatory element in a nucleic acid construct.
  • agent extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells.
  • Analogs contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.
  • biologically active portion is meant a portion part or fragment of a RAP polypeptide such as for example a RAP polypeptide whose amino acid sequence is set out in SEQ ID NO: 1 or SEQ ID NO: 5. In accordance with the present invention, the portion retains at least one of the herein described activities of a RAP polypeptide including binding to A ⁇ .
  • biologically active portion includes peptides, for example, of at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350 contiguous amino acids (and every integer in between) which comprise an activity of a reference RAP polypeptide including binding to A ⁇ .
  • a biologically active portion is a RAP polypeptide without a signal sequence or ER retention signal.
  • a fragment is a contiguous sequence of amino acids that comprises or consists of one or more of domain 1 (SEQ ID NO: 2), domain 2 (SEQ ID NO: 3), or domain 3 (SEQ ID NO: 4) of RAP. Portions of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques.
  • peptides can be produced by digestion of a peptide or polypeptide of the invention with proteinases such as endoLys-C, endoArg- C, endoGlu-C and staphylococcus V8-protease.
  • the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Recombinant nucleic acid or synthetic techniques can also be used to produce such portions.
  • furin cleavage is preferred.
  • cell is meant any prokaryotic or eukaryotic cell.
  • the cell is a neural cell.
  • a syngeneic cell is preferred which is genetically identical to the subject or is genetically compatible to minimize any immune response.
  • co-administered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • the subject composition may be administered together with another agent in order to enhance its activity.
  • simultaneous administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
  • the oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.
  • derivative is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art.
  • derivative also includes within its scope alterations that have been made to a RAP polypeptide including additions, or deletions that provide for functionally equivalent molecules.
  • a functional derivative of a polynucleotide encoding a RAP polypeptide comprises a sequence of nucleotides having at least 80% or 90% or 95% similarity identity to the polynucleotide over a reference window of comparison.
  • a "part” or “portion” of a polynucleotide is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides.
  • This definition includes all sizes in the range of 10-35 nucleotides including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600 nucleotides or nucleic acid molecules having any number of nucleotides within these values.
  • an effective amount in the context of treating AD is meant the administration of that amount of active to a subject, either in a single dose or as part of a series or slow release system, that is effective for treatment typically in a statistically significant number of subjects.
  • the effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention.
  • expression vector any autonomous genetic element capable of directing the transcription of a polynucleotide contained within the vector and suitably the synthesis of a peptide or polypeptide encoded by the polynucleotide.
  • expression vectors are known to practitioners in the art.
  • the term "gene” as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions.
  • the gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the DNA sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • Hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base-pairing rules.
  • match and mismatch refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • hydrogen bonding which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • nucleobases nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances as known to those of skill in the art.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g. total cellular) DNA or RNA.
  • an "isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • an "isolated polynucleotide”, as used herein, refers to a polynucleotide, isolated from the sequences which flank it in a naturally-occurring state, e.g. a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • an "isolated peptide” or an “isolated polypeptide” and the like, as used herein refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.
  • an isolated composition, complex, polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means.
  • modulation or “modulator” in relation to a particular target is meant directly or indirectly up-regulating or down-regulating the level, effects or activity of the target.
  • the effects of A ⁇ may be down-regulated by binding to RAP polypeptide.
  • mutants includes the substitution or deletion of one or more amino acids within one or more domains of RAP.
  • Insertional amino acids sequence mutants are those in which one or more amino acid residues are introduced into a predetermined site in a protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional mutants include the removal of one or more amino acids.
  • Substitutional mutants contain at least one residue that have been inserted in place of the wild-type (parent or naturally) occurring residue. Substitutions are either conservative or non-conservative.
  • Neurotrophins include without limitation nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin 4/5 and neurotrophin 6.
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • Neurotrophins are a group of small structurally and chemically related proteins which support the survival and development of neurones and maintain neuronal phenotypes.
  • operably connected means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation of the gene.
  • polynucleotide means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation of the gene.
  • polynucleotide means placing a structural gene under the regulatory control of a promoter, which then controls the transcription and optionally translation of the gene.
  • polynucleotide include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. ⁇ -anomeric nucleic acids, etc.).
  • uncharged linkages e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.
  • charged linkages e.g. phosphorothioates, phosphorodithioates, etc.
  • pendent moieties
  • RNA forms of the genetic molecules of the present invention are generally niRNA or iRNA including siRNAs.
  • the genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules.
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polynucleotide variant and “variant” also include naturally-occurring allelic variants.
  • polypeptide proteinaceous molecule
  • peptide protein
  • protein protein
  • polypeptide protein
  • protein protein
  • polymer polymer of amino acid residues and to variants and synthetic analogues of the same.
  • amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • Reference to peptides includes a foldamer, peptido- including cyclic peptidomimetic, constrained or stapled peptides. These terms do not exclude modifications, for example, glycosylations, aceylations, phosphorylations and the like.
  • Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.
  • polypeptide variant refers to polypeptides which are distinguished from a reference polypeptide by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, one or more amino acid residues of a reference polypeptide are replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g.
  • sequence identity will be understood to mean the "match percentage" calculated by an appropriate method.
  • sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP 5 BESTFIT, FASTA 5 and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP 5 BESTFIT Garnier et al
  • FASTA 5 and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA
  • the best alignment i.e. resulting in the highest percentage homology over the comparison window
  • small molecule refers to a non-peptide molecule that has a molecular mass of up to about 1500 Daltons, such as from about 200 or 400 to 1000 Daltons, or 600 to about 1200 Daltons, or from about 500 to 1500 Daltons.
  • Stringency refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the observed degree of complementarity between sequences.
  • Stringent conditions refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize.
  • the stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used.
  • stringent conditions are selected to be about 10°C to 2O 0 C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.
  • a preferred polynucleotide will hybridize to a RAP sequence or its complement under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions.
  • Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C.
  • Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 niM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA 3 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • 1 niM EDTA 1 niM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C
  • 2xSSC 0.1% SDS
  • BSA 3 1 mM EDTA
  • 40 mM NaHPO4 pH 7.2
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42 0 C, and at
  • Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA 5 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at 42 0 C.
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65°C.
  • Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Current Protocols in Molecular Biology (supra) at pages 2.10.1 to 2.10.16 and Sambrook et at, eds. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, 1989 at sections 1.101 to 1.104.
  • subject refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the medical protocols of the present invention.
  • a subject regardless of whether a human or non-human animal or embryo may be referred to as an individual, subject, animal, patient, host or recipient.
  • the present invention has both human and veterinary applications.
  • an "animal” specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as companion animals. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry.
  • treatment or “therapy” are used interchangeably in their broadest context and include any measurable or statistically significant change in one or more symptoms or frequency of one or more assessable indications of AD.
  • vector is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast, virus, mammal, avian, reptile or fish into which a polynucleotide can be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e.
  • a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication e.g. a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art.
  • the present invention provides, inter alia methods for treating symptoms of AD in a subject, as disclosed in the Summary, including the administration to a subject of an effective amount of RAP polypeptide or an agent from which a RAP polypeptide is producible.
  • RAP species homologs sharing more that about 60% amino acid sequence similarity have been identified in man, mice, rat, chicken, zebrafish, pig, invertebrates such as worms, mosquitoes and fruit flies.
  • a RAP polypeptide encompasses any naturally-occurring RAP polypeptide from any animal species as well as their biologically active portions and variants or derivatives of these, as defined herein.
  • RAP polypeptides may be prepared by any suitable procedure known to those of skill in the art.
  • the polypeptides may be prepared by a procedure including the steps of: (a) preparing a construct comprising a polynucleotide sequence that encodes RAP polypeptide and that is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the RAP polypeptide; and (d) isolating the RAP polypeptide from the host cell.
  • the nucleotide sequence encodes at least a biologically active portion of the sequence set forth in SEQ ID NO: 1, or a variant thereof.
  • Recombinant RAP polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook et al, 1989 (supra), in particular Sections 16 and 17; Ausubel et al, 1994 (supra), in particular Chapters 10 and 16; and Coligan et al, Current Protocols in Protein Science John Wiley & Sons, Inc. 1995-1997, in particular Chapters 1, 5 and 6.
  • the RAP polypeptides may be synthesized by chemical synthesis, e.g. using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et al, Science 269:202, 1995.
  • the RAP polypeptide may be produced by any convenient method such as by purifying the polypeptide from naturally- occurring reservoirs including blood or serum. Methods of purification include lectin (e.g. wheat genu agglutinin) affinity chromatography or separation. The identity and purity of derived RAP is determined for example by SDS-polyacrylamide electrophoresis or chromatographically such as by high performance liquid chromatography (HPLC).
  • lectin e.g. wheat genu agglutinin affinity chromatography or separation.
  • HPLC high performance liquid chromatography
  • the RAP polypeptide of the present invention includes all biologically active naturally occurring forms of RAP as well as biologically active portions (fragments) thereof, and variants or derivatives of these.
  • Biological activity as used herein refers to the ability of RAP polypeptide to modulate an activity of A ⁇ or reduce or otherwise ameliorate a symptom of AD or a related proteopathy.
  • Biologically active portions of RAP polypeptide include parts of the amino acid sequence set out in SEQ ID NO: 1 or SEQ ID NO: 5.
  • a biologically active portion of a full-length RAP polypeptide may comprise, for example, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120 or 150, or even at least about 200, 220, 240, 260, 280, 300, 310, 320, or 330 contiguous amino acid residues, or almost up to the total number of amino acids present in a full-length RAP polypeptide.
  • the portion is a "biologically- active portion" having no less than about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 99% of the activity of the full-length RAP polypeptide from which it is derived.
  • Suitable biologically active portions include soluble forms of the polypeptide without a leader or signal peptide.
  • RAP polypeptides include "variant" polypeptides that are distinguished from a naturally- occurring RAP polypeptide or from a biologically active portion thereof by the addition, deletion and/or substitution of at least one amino acid residue.
  • variants include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native RAP polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more or 99% or more sequence similarity or identity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a native RAP polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more or 99% or more sequence similarity or identity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a native RAP polypeptide will have
  • RAP polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • a naturally occurring isolated RAP polypeptide or its encoding sequences may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of a RAP polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad.
  • Variant RAP polypeptides containing conservative amino acid substitutions at one or various locations along their sequence, as compared to the parent RAP amino acid sequence are encompassed.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Acidic The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having an acidic side chain include glutamic acid and aspartic acid.
  • the residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g. histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having a basic side chain include arginine, lysine and histidine.
  • the residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e. glutamic acid, aspartic acid, arginine, lysine and histidine).
  • amino acids having acidic or basic side chains i.e. glutamic acid, aspartic acid, arginine, lysine and histidine.
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
  • Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
  • proline This description also characterizes certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity.
  • "small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not.
  • Amino acids having a small side chain include glycine, serine, alanine and threonine.
  • the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains.
  • the structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the ⁇ - amino group, as well as the ⁇ -carbon.
  • amino acid similarity matrices e.g. PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al, 1978 ⁇ supra
  • PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al, 1978 ⁇ supra
  • Gonnet et al., Science 256(5062): 11443-11445, 1992 include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.
  • the degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behavior.
  • Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large.
  • the residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not.
  • Small residues are, of course, always nonaromatic.
  • amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 3.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
  • similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
  • the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
  • the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
  • the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers, 1993.
  • a predicted non-essential amino acid residue in a RAP polypeptide is typically replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a RAP polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.
  • the present invention also contemplates variants of the naturally-occurring RAP polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues.
  • variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to a parent RAP polypeptide sequence as, for example, set forth in SEQ ID NO: 1.
  • variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to a parent RAP polypeptide sequence as, for example, set forth in SEQ ID NO: 1 or the mature polypeptides lacking residues 1 to 34 of SES ID NO: 1.
  • sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60 ,70, 80, 90, 100 or more amino acids but which retain the properties of the parent RAP polypeptide are contemplated.
  • RAP polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to RAP polynucleotide sequences, or the non-coding strand thereof.
  • Illustrative RAP polynucleotide sequences encode the polypeptide described in SEQ ID NO: 1 , 2, 3 or 4.
  • variant polypeptides differ from a RAP sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s).
  • variant polypeptides differ from the corresponding sequence in any one of SEQ ID NO: 1, 2, 3 or 4 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.
  • Naturally-occurring RAP polypeptides contain a significant number of structural characteristics in common with each other. An alignment shows positions that are amenable to conservative substitution and others that accommodate non- conservative substitutions.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities.
  • the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type.
  • amino acid residues that are absolutely conserved between the RAP polypeptides of human, mice, and zebrafish may be unamenable to alteration.
  • a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a RAP polypeptide as, for example, set forth in any one of SEQ ID NO: 1, 2 or 4 and has the activity of a RAP polypeptide as described herein.
  • RAP polypeptides Another useful group of compounds that function as RAP polypeptides are functional derivatives, analogs and mimics (mimetics) of RAP.
  • these molecules retain the ability to ameliorate symptoms of AD or related proteopathies, or to bind to A ⁇ and ameliorate its effects, enhance its uptake for degradation or transport away from neurons.
  • Analogs and may also possess additional characteristics which improve their efficacy, such as exhibiting a longer half life in vivo or alternatively which are, for example, readily synthesized or readily taken up across the blood-brain barrier or by neurons or other cells.
  • a peptide mimetic or mimic has some chemical similarity to the parent molecule, e.g. RAP, but agonizes its activity.
  • a peptide mimic may be a peptide-containing molecule which mimics elements of protein secondary structure (as described for example in Johnson et at, "Peptide Turn Mimetics” in Biotechnology and Pharmacy, Pezzuto et a!., Eds., Chapman and Hall, New York, 1993).
  • Non-peptide "small molecules” are often preferred for many in vivo pharmaceutical applications and accordingly mimetics may be designed for pharmaceutical use. Mimetic design, synthesis and testing is available to avoid randomly screening large numbers of molecules for a particular property, particularly where a lead compound has already been identified.
  • residues critical for binding are identified and this framework used as a pharmacophore.
  • the structure may then be modeled using computational and other analyses. Alternatively, the three dimensional structure of RAP and A ⁇ are know and RAP polypeptides may be designed in silico along the same lines.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson, Bio/Technology, P:19-21, 1991, Henchy et al, Curr Opin Chem Biol 12(6):692-7 ' , 2008 and reference referred to therein).
  • Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins.
  • An example of rational drug design is the development of HIV protease inhibitors (Erickson et al, Science, 249:527-533, 1990).
  • target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol, 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. This term also does not exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS);
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino- 3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenyl glycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • Suitable ⁇ -amino acids include, but are not limited to, L- ⁇ -homoalanine, L- ⁇ - homoarginine, L- ⁇ -homoasparagine, L- ⁇ -homoaspartic acid, L- ⁇ -homoglutamic acid, L- ⁇ - homoglutamine, L- ⁇ -homoisoleucine, L- ⁇ -homoleucine, L- ⁇ -homolysine, L- ⁇ - homomethionine, L- ⁇ -homophenylalanine, L- ⁇ -homoproline, L- ⁇ -homoserine, L- ⁇ - homothreonine, L- ⁇ -homotryptophan, L- ⁇ -homotyrosine, L- ⁇ -homovaline, 3-amino- phenylpropionic acid, 3-amino-chlorophenylbutyric acid, 3-amino-fluorophenylbutyric acid, 3-amino-bromopheynyl butyric
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid contemplated herein is shown in Table 5.
  • Sugar amino acids are sugar moieties containing at least one amino group as well as at least one carboxyl group.
  • Sugar amino acids may be based on pyranose sugars or furanose sugars. Suitable sugar amino acids may have the amino and carboxylic acid groups attached to the same carbon atom, ⁇ -sugar amino acids, or attached to adjacent carbon atoms, ⁇ -sugar amino acids. Suitable sugar amino acids include but are not limited to
  • Sugar amino acids may be synthesized starting from commercially available monosaccharides, for example, glucose, glucosamine and galactose.
  • the amino group may be introduced as an azide, cyanide or nitromethane group with subsequent reduction.
  • the carboxylic acid group may be introduced directly as CO 2 , by Wittig reaction with subsequent oxidation or by selective oxidation of a primary alcohol.
  • the RAP analog or agonist is a small chemical molecule.
  • New chemical entities, natural products, combinatorial synthetic organic or inorganic compounds, peptide/polypeptide/protein, nucleic acid molecules and libraries or phage or other display technology comprising these are all available to screen or test for suitable agents.
  • Natural products include those from coral, soil, plant, or the ocean or Antarctic environments. Libraries of small organic molecules can be generated and screened using high-throughput technologies known to those of skill in this art. See for example United States Patent No. 5,763,623 and United States Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesized having different substitutions of a common or subset of parent structures. Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function, for example, varying in chain length, ring size or number or substitutions. Virtual libraries are also contemplated and these may be constructed and compounds tested in silico (see for example, US Publication No.
  • agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997; United States Patent No. 5,738,996; and United States Patent No. 5,807,683).
  • Libraries of compounds may be presented, for example, in solution (e.g.
  • the proteins necessary for high capacity assays may be produced in bacteria.
  • One useful assay suitable for high throughput is Amplified Luminescent Proximity Homogenous Assay (ALPHA) technology described in Glickman et al, J Biomol Screen. 7(l):3-10, 2002.
  • APHA Amplified Luminescent Proximity Homogenous Assay
  • RAP and A ⁇ have been determined and this facilitates the design of binding agents that modulate A ⁇ activity.
  • Peptide or non-peptide mimetics are anticipated to mimic elements of protein secondary structure and permit molecular interactions similar to the natural molecule. Leads selected may require some or considerable modification to enhance their biological, biochemical and pharmacological properties. Lead compounds identified in screening process can be optimised by molecular modelling in silico.
  • Three-dimensional representations of the structure of one or more binding sites of A ⁇ and/or RAP or a variant, derivative or analog of either of these molecules are used to identify interacting molecules that, as a result of their shape, reactivity, charge potential etc. favourably interacts or associate.
  • the skilled person can screen three-dimensional structure databases of compounds to identify those compounds having functional groups that will fit into one or more of the binding sites.
  • Combinational chemical libraries can be generated around such structures to identify those with high affinity binding to appropriate binding sites.
  • Agents identified from screening compound databases or libraries are then fitted to three-dimensional representations of RAP or A ⁇ binding sites in fitting operations using, for example docking software programs.
  • a potential modulator may be evaluated "in silico" for its ability to bind to a A ⁇ binding site prior to its actual synthesis and testing.
  • the quality of the fit of such entities to binding sites may be assessed by, for example, shape complementarity by estimating the energy of the interaction (Meng et al, J. Comp. Chem., i3:505-524, 1992).
  • the design of chemical entities that associate with A ⁇ or other amyloid proteins will involve consideration of two factors.
  • the compound must be capable of physically and structurally associating with A ⁇ .
  • Non-covalent molecular interactions important in the association of a compound with its interacting partners include hydrogen bonding, van der Waal's and hydrophobic interactions.
  • the compound must be able to assume a conformation that allows it to associate with A ⁇ . Although certain portions of the compound will not directly participate in this association with A ⁇ , those portions may still influence the overall conformation of the molecule.
  • Such conformation requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the active site, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with amyloid peptides.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e. the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should of course be understood that components known in the art to alter conformation should be avoided.
  • Putative binding agents may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the one or more binding sites. Selected fragments or chemical entities may then be positioned in a variety of orientations, or "docked,” to target binding sites. Docking may be accomplished using software, such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM or AMBER. Specialised computer programs may be of use for selecting interesting fragments or chemical entities. These programs include, e.g.
  • Useful programs to aid the skilled addressee in connecting chemical entities or fragments include CAVEAT (University of California, USA), 3D database systems and HOOK (Molecular Simulations, USA)
  • De-novo ligand design methods include those described in LUDI (Molecular Simulations, USA), LEGEND (Molecular Simulations, USA), LeapFrog (Tripos Inc.,) SPROUT (University of Leeds, UK) and the like.
  • the medicament is an autologous cell derived from the subject to be treated or a syngeneic cell.
  • the cell is genetically modified in order to secrete a RAP polypeptide. Other cells, such as neurones secrete RAP polypeptide naturally.
  • the cell is a genetically modified neuronal cell capable of producing RAP polypeptide.
  • the cell is a stem cell or progenitor cell for a neuronal cell.
  • the stem cell is an neuronal cell progenitor cell. Genetically modified neuronal stem cells are conveniently used in order to treat proteopathies.
  • a polynucleotide encoding a RAP polypeptide is engineered within an expression construct or shuttle vector and operably linked to a regulatory element (e.g. a promoter) that is operable in the cell in which it is desired to express the polynucleotide.
  • a regulatory element e.g. a promoter
  • the nucleic acid construct that is delivered remains episomal and induces an endogenous and otherwise silent gene.
  • a selective marker gene such as an antibiotic resistance marker gene is employed to facilitate selection of appropriately modified cells.
  • the polynucleotide (cDNA) is selected (amplified) or modified by removal of sequences encoding signal sequences to facilitate secretion of a soluble or mature RAP polypeptide.
  • Mammalian expression vectors capable of expression in mammalian epidermal cells are, for example, routinely available. Construction of recombinant DNAs comprising RAP polynucleotides and a mammalian vector capable of expressing inserted DNAs in cultured human or animal cells, can be carried out by standard gene expression technology using methods well known in the art for expression of such a relatively simple polypeptide. Promoters for selective expression in a range of cells have also been identified and documented.
  • viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses, adenovirus, vaccinia virus, adeno- associated virus, herpesviruses including HSV and EBV, Antiviruses, Sindbis and Semliki Forest virus and retroviruses of avian and murine origin. Viral and non-viral methods of polynucleotide delivery are available. Various viral vectors are routinely used to transform a range of different cell types with adequate efficiency.
  • the RAP polynucleotide comprises a nucleotide sequence that encodes wholly or partially the amino acid sequence set forth in SEQ ID NO: 1, or a sequence having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO: 1.
  • the RAP polynucleotide comprises all or part of the nucleotide sequence encoding the polypeptide described in SEQ ID NO: 1, or a sequence having at least 80% sequence identity to SEQ ID NO: 1, or a sequence that hybridizes to the nucleotide sequence encoding the sequence set out in SEQ ID NO: 1 or to a complementary form thereof under at least medium stringency conditions.
  • the present invention provides contemplates an antibody or antigen-binding fragment thereof which mimics the ability of RAP to bind to A ⁇ and modulate its functional activity.
  • compounds which have the potential to act as modulators include small chemical molecules which can penetrate a cell membrane or via an ion channel or other pore and an antigen binding agent which has the capacity for intracellular transmission such as cartilage fish-derived antibodies (e.g. shark antibodies, see for example, Lui et al., BMC Biotechnol. 7:78, 2007).
  • an antigen binding agent, or functionally active fragment thereof, which has the capacity for intracellular transmission also includes antibodies such as camelids and llama antibodies, scFv antibodies, intrabodies or nanobodies, e.g. scFv intrabodies and VH H intrabodies.
  • Such antigen binding agents can be made as described by Harmsen & De Haard, Appl. Microbiol. Biotechnol. 77(l):l3-22, 2007; Tibary et al, Soc. Reprod. Fertil. Suppl. 64:291-313, 2007; Muyldermans, J Biotechnol. 74:211-302, 2001; and references cited therein.
  • such agents may comprise a cell-penetrating peptide sequence or nuclear-localizing peptide sequence such as those disclosed in Constantini et al., Cancer Biotherm. Radiopharm. 25(7 ⁇ :3-24, 2008.
  • Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al, Biochem J. 390( ⁇ t2):407-418, 2005 and Meyer-Losic et al., J Med Chem. 49(23 ⁇ :6908-6916, 2006.
  • the invention provides the therapeutic use of fusion proteins of the agents (or functionally active fragments thereof), for example but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C-terminus, to a cell-penetrating peptide or nuclear-localizing peptide sequence.
  • a covalent bond e.g. a peptide bond
  • the present invention provides a composition for use in therapy.
  • the composition comprises a pharmaceutically acceptable carrier, diluent and/or excipient.
  • proteopathies Diseases associated with the accumulation of insoluble protein deposits (amyloid) are known as "proteopathies” or “proteinopathies amyloidoses” or “conformational diseases” and comprise clinically and pathologically diverse disorders.
  • proteopathies are AA amyloidosis, AH (heavy chain) amyloidosis, AL (light chain) amyloidosis, Alexander disease, Alzheimer's disease, amyotrophic lateral sclerosis, aortic medial amyloidosis, apoAl amyloidosis, apoA2 amyloidosis, apoA4 amyloidosis, CADASIL, cardiac atrial amyloidosis, cataract, cerebral amyloid angiopathy, corneal lactoferrin amyloidosis, Creutzfeld-Jacob disease, mad cow disease, critical illness myopathy, cutaneous lichen amyloidosis, dialysis amyloidosis, Familial amyloidotic neuropathy, familial
  • references herein to the phrase "related conditions" in the context of AD in some embodiments includes conditions such as those listed supra which are characterised by amyloid deposits in the brain, memory loss and dementia. In other embodiments, the term extends to amyloidoses generally, including those listed supra.
  • the composition of the present invention in some embodiments, comprises a cellular agent.
  • the cell is a genetically modified syngeneic cell that produces a RAP polypeptide.
  • the cell is a viral cell capable of transforming cells and causing them to produce RAP polypeptide.
  • the cell naturally produces RAP polypeptide such as a cell of the neural lineage.
  • the present invention provides a use of a RAP polypeptide or an agent from which a RAP polypeptide is producible or a RAP analog or mimetic in the manufacture of a medicament for the treatment of AD.
  • the medicament is suitable for local or systemic administration by any route, such as without limitation by patch, cellular transfer, implant, orally, intravenously, intravesicaly, intracerebrally, intradermally, intramuscularly, intraperitoneally, intrathecally, subcutaneously, sublingually, rectally, vaginally, iiitraocularly, nasally, respiratorialy, nasopharyngeal, subcutaneously, cutaneously, topically and transdermally.
  • any route such as without limitation by patch, cellular transfer, implant, orally, intravenously, intravesicaly, intracerebrally, intradermally, intramuscularly, intraperitoneally, intrathecally, subcutaneously, sublingually, rectally, vaginally, iiitraocularly, nasally, respiratorialy, nasopharyngeal, subcutaneously, cutaneously, topically and transdermally.
  • compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Company, Easton, PA, U.S.A., 1990).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract. See for example, International Patent Publication No. WO 96/11698.
  • the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the actual amount of active agent administered and the rate and time-course of administration will depend on the nature and severity of the burn injury. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes into account the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences ⁇ supra).
  • the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the agent chosen. A broad range of doses may be applicable.
  • a patient for example, from about 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7 ng, 0.8 ng. 0.9 ng, or 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg. 0.9 mg to about 1 to 10 mg or from 5 to 50 mg of RAP polypeptide oragent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the agents may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules).
  • the agent or composition comprising the agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application).
  • acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
  • the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
  • a binder such as tragacanth, corn starch or gelatin
  • a disintegrating agent such as alginic acid
  • a lubricant such as magnesium stearate.
  • Mouse monoclonal antibodies were purchased as follows: anti- RAP (clone 7Fl) from Merck-Calbiochem (Kilsyth, VIC 5 Australia), anti-A ⁇ (clone 6E10) from Sigma- Aldrich (Castle Hill, NSW, Australia), anti-transferrin receptor (clone H68.4) from Invitrogen-Zymed (Mt Waverley, VIC, Australia). Heparin from porcine intestinal mucosa and methyl anthranilate were purchased from Sigma-Aldrich.
  • Hoechst 33342, fiuo-4 acetoxymethyl (AM) ester, and all AlexaFluor-conjugated secondary antibodies and choleratoxin B subunits (CTX) were purchased from Invitrogen-Molecular Probes (Mt Waverley, VIC, Australia). Protein G agarose was purchased from Roche (Castle Hill, NSW, Australia). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG was purchased from GE Lifesciences-Amersham (Rydalmere, NSW, Australia).
  • HRP horseradish peroxidase
  • a ⁇ peptide preparation and aggregation Synthetic A ⁇ 1-42 N-terminally labeled with fluorescein (FluoA ⁇ 1-42 ) and recombinant A ⁇ 1-40 were purchased from rPeptides (Athens, GA, USA). A ⁇ 1-42 was purchased from Keck Laboratories (New Haven, CT, USA). All peptides were of >95% purity as assessed by high performance liquid chromatography and mass spectrometry. A ⁇ peptides were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and stored at -8O 0 C.
  • DMSO dimethyl sulfoxide
  • a ⁇ 1-42 (2.5 mg/ml) was dissolved in hexafluoroisopropanol, diluted in distilled water at 250 ⁇ g/ml, then centrifuged at 14,000 x g at room temperature for 20 min prior to addition to cells. "Aging" of A ⁇ peptides was performed by incubating the peptide (1 ⁇ M) in the absence or presence of RAP (5 ⁇ g/ml) in 50 mM NaH 2 PO 4 containing 100 mM NaCl (NaCl/Pi), pH 7.4 at 37 0 C.
  • SH-SY5Y neuroblastoma cell culture SH-SY5Y cells were obtained from the American Type Culture Collection (CRL-2266; Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen-Gibco, Mt Waverley, VIC, Australia) containing 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine and 10% (v/v) fetal bovine serum (FBS) 5 in an atmosphere of 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • F12 fetal bovine serum
  • Cells were passaged by rinsing with warm phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 6.5 mM Na 2 HPO 4 , 1.76 mM KH 2 PO 4 ), pH 7.4, then dissociating with trypsin.
  • Serum-free medium was DMEM/F12 containing penicillin, streptomycin and 10 mM HEPES, pH 7.4 at 37°C.
  • confocal microscopy cells were plated on 13 mm autoclaved coverslips in 24-well plates and grown to 50% confluence before treatment.
  • cells were plated in 48-well plates and 96-well plates, respectively, and grown to 80-90% confluence before treatment.
  • cells were plated in black, clear-bottomed 96-well plates (Coming, Lindfield, NSW, Australia) and grown to 50% confluence before treatment. Cells were rinsed once in serum-free medium, then treated with FluoA ⁇ 1-42 (1 ⁇ M), or A ⁇ 1-42 (1 ⁇ M), with or without RAP (5 ⁇ g/ml), in serum-free medium at 37 0 C.
  • Immunolabeled cells on coverslips were rinsed, incubated with Hoechst 33342 (1 ⁇ g/ml) in PBS for 5 min, then rinsed and mounted on microscopy slides in Fluoromount G (Southern Biotech, Birmingham, AL, USA). Images were captured on an Olympus FVlOOO confocal microscope, using Kalman integration of two scans and sequential scanning of channels to minimize bleed-through. Images were processed using the ImageJ software (Abramoff et at, Biophotlnt 11:36-42, 2004). Flow cytometry.
  • SH-S Y5 Y cells were incubated with FluoA ⁇ 1-42 for 4 h then placed on ice, rinsed with ice-cold PBS and allowed to lift in PBS containing 1 mM EDTA and 1% (v/v) FBS. After 30 min, cells were transferred to polystyrene tubes and triturated. Twenty min prior to analysis, propidium iodide (PI; 0.1 ⁇ g/ml) was added to each tube. Ten thousand cells were read from each well at a rate of 100-300 cells per second in a FC500 cytometer (Beckman Coulter, Gladesville, NSW, Australia). Each incubation was performed in triplicate and experiments were performed at least twice. Pi-negative single cells were selected for inclusion in data analysis using WinMDI software (v2.9).
  • Protein G agarose beads were incubated in lysis buffer containing 1 ⁇ g of either 7Fl or H68.4 for 2 h at 4 0 C. Solutions containing A ⁇ and/or RAP in NaCl/Pj were diluted 1:1 in lysis buffer, and then 500 ⁇ l of this solution was incubated with 20 ⁇ l of antibody-coated beads for 3 h at 4 0 C.
  • the beads were then rinsed thrice in lysis buffer and twice in 50 mM Tris-HCl, pH 7.5 containing 0.1% (v/v) Nonidet P40 and 0.05% (v/v) sodium deoxycholate, then boiled in an equal volume of 2* SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% [v/v] SDS, 20% [v/v] glycerol, 0.01% [w/v] bromophenol blue) and ⁇ -mercaptoethanol ( ⁇ -ME) to 5% (v/v).
  • SDS sample buffer 125 mM Tris-HCl, pH 6.8, 4% [v/v] SDS, 20% [v/v] glycerol, 0.01% [w/v] bromophenol blue
  • ⁇ -ME ⁇ -mercaptoethanol
  • Incubations with primary (6E10, 1:1000; 7Fl, 1:4000) and secondary (anti-mouse HRP, 1:4000) antibodies were carried out in blocking buffer for 1.5 h and 1 h, respectively, and detection was achieved via enhanced chemiluminescence (GE Lifesciences-Amersham). When required, antibodies were stripped from the membrane by incubating in 62.5 mM Tris-HCl, pH 6.7, containing 2% (v/v) SDS and 100 mM ⁇ -ME at 50°C for 30 min before reprobing.
  • Atomic force microscopy AFM was performed essentially as previously described (Hou et al, J Neurochem 100:446-457, 2007). Samples were applied to a substrate of highly oriented pyrolytic graphite, which was then briefly rinsed with distilled deionized water and dried under a constant flow of nitrogen. Imaging was performed by tapping mode in air using NSCl 5 silicon probes (Mikromasch, Tallinn, Estonia) on a Nanoscope IV Multimode scanning probe microscope (Veeco Corp., Santa Barbara, CA, USA). AFM images were analysed using the WSxM 4.0 software (Horcas et al, Rev Sci lustrum 75:013705, 2007).
  • Intracellular Ca 2+ measurements were performed in DMEM/F12 without phenol red (Invitrogen). Cells were loaded with fluo-4 AM (2 ⁇ M) for 7-10 min at 37 0 C. Following removal of fluo-4 AM, cells were rinsed and incubated for a further 30 min in 100 ⁇ l medium at 37 0 C to allow complete de-esterification. Fluorescence was measured in a microplate reader (FluoStar Optima, BMG Labtechnologies, Offenburg, Germany) equipped with fluorescence optics (excitation 485 nm, emission 520 nm). Fluorescence measurements were made every 8 s.
  • Baseline was determined for 56 s before addition of A ⁇ 1-42 (10 ⁇ M; final concentration of 1 ⁇ M) with or without RAP (50 ⁇ g/ml; final concentration of 5 ⁇ g/ml), and the response was monitored for a further 200 s. Quantitation of intracellular Ca 2+ response was achieved by subtracting the average background response to vehicle treatments from A ⁇ treatments, then calculating AF/F values where AF represents the fluorescence change of cells compared to 13
  • Chick discriminative avoidance memory task The ability of RAP to influence the functional effects of A ⁇ was tested in a recently established in vivo model of A ⁇ -induced inhibition of memory consolidation using a discriminative avoidance task (Gibbs et ah, Neurobiol Aging, [Available online, accessed July 15, 2008] doi:10.1016/j.neurobiolaging.2008.05.018 5 2008).
  • a ⁇ 1-42 was diluted from DMSO in cold physiological saline (0.9% [w/v] NaCl) to yield a final peptide concentration of 2 ⁇ M.
  • RAP (10 ⁇ g/ml) was immediately added and samples were stored at 4 0 C for no more than 1 h prior to injecting into day-old chicks.
  • Brain tissue Brain tissue from 7 AD and 8 clinical and neuropathological controls were available for immunohistochemical analysis (Table 6). Diagnosis of AD (and no other neurodegenerative condition) or control (without neurological or neuropathological disease) was based on longitudinal clinical and systematic neuropathological assessments, as previously described (Shepherd et al, Neurobiol. Dis. 9: 249-257, 2002). AU cases were matched for sex, age and post-mortem delay. The post-mortem delay was 24 + 6 h for control tissue and 22 + 8 h for AD tissue (mean + SEM). Immunohisto chemistry of brain tissue.
  • the primary antibody was incubated overnight at 4 0 C at a concentration of l ⁇ g/ml. Sections were then sequentially incubated with biotinylated secondary antibodies (Vector biotinylated secondary immunoglobulin G antibodies) for 2 hr at room temperature, streptavidin- conjugated horseradish peroxidase (Vector Elite ABC) for 30 minutes at 25°C, and with 3,3'-diaminobenzidine in H 2 O 2 until the reaction products were visualised (5-10 min).
  • biotinylated secondary antibodies Vector biotinylated secondary immunoglobulin G antibodies
  • streptavidin- conjugated horseradish peroxidase Vector Elite ABC
  • RAP-positive neurons Quantification of RAP -positive neurons. Quantification was carried out using an 11x11 eye piece grid at 20Ox magnification on a Zeiss microscope in the CAl of the hippocampus. Neurons were identified by the presence of a clear nucleolus and defined cytoplasm. Both total (cresyl violet positive) and RAP-imrnunoreactive neurons were counted in the eye piece grid and the percentage of RAP-immunoreactive neurons calculated. Ten repeated measurements on all slides from one case at different time intervals did not vary by more than 5%. Counts on the same slides from four cases by different investigators varied on average by 7 %.
  • Extracellular RAP polypeptide binds strongly to A ⁇ and increases A ⁇ binding to neuronal cells independent of presence of LDL receptor
  • Fluorescein-labeled A ⁇ 1-42 was incubated with SH-SY5Y cells and its cell association was evaluated by confocal microscopy ( Figure 1). A brief incubation on ice with Alexa 555- conjugated CTX allowed visualization of the plasma membrane. Following 1 hour (h) of incubation, little, if any, FluoA ⁇ 1-42 was observed in association with the cells. However, after 4 h of incubation, cell-associated FluoA ⁇ i- 42 was observed. Most of the FluoA ⁇ 1-42 was intracellular, distributed in a punctate fashion. However, some of the FluoA ⁇ i. 42 bound to the cell surface. After a longer (24 h) incubation, almost all of the FluoA ⁇ 1-42 fluorescence was intracellular, with little fluorescence detected at the plasma membrane.
  • a ⁇ 1-42 aggregated to form higher molecular weight structures that failed to enter the polyacrylamide gel (data not shown). Some oligomeric species were observed ( Figure 4B), but overall, the pattern of aggregation was not easily analyzed using gel electrophoresis. Therefore, we examined A ⁇ i -42 aggregation by AFM, a technique that gives a qualitative assessment of the morphology of A ⁇ aggregation. A ⁇ 1-42 was diluted in the absence or presence of RAP then either examined immediately or aged for 6 h prior to analysis ( Figure 5).
  • AFM studies showed that RAP inhibited formation of A ⁇ 1-42 protofibrils.
  • the freshly prepared A ⁇ 1-42 formed globular structures with an apparent diameter of 20 nm that displayed a strong tendency to align along the graphite surface, as previously reported (Losic et al. % 2006).
  • fresh A ⁇ 1-42 in the presence of RAP generally did not form these aligned patterns, but instead consisted of smaller structures with an apparent diameter ranging from 15 nm through to 35 nm.
  • RAP alone formed discrete structures of approximately 25 or 40 nm in diameter; it was not clear which structures in the sample containing both RAP and A ⁇ 1-4 2 represented A ⁇ i.42, RAP, or a complex of the two proteins.
  • RAP polypeptide blocks A ⁇ -induced neurotoxic effects in vitro and in vivo
  • a ⁇ neurotoxicity is highly dependent on its aggregation state (Walsh et al, 2002 (supra)). Therefore, it was hypothesized that RAP, which inhibited A ⁇ aggregation, would influence A ⁇ neurotoxicity.
  • RAP which inhibited A ⁇ aggregation
  • One response of cells to extracellular A ⁇ is an increase in intracellular Ca 2+ (Small et al, J Alzheimer s Dis 16: 225-233, 2008).
  • neurons and neuron-like cells display immediate intracellular Ca 2+ changes in response to oligomeric, but not monomeric or fibrillar, A ⁇ (Demuro et al, 2005 (supra); Kelly and Ferreira, J Biol Chem 281:28079-28089, 2006).
  • Freshly prepared A ⁇ 1-42 was used, which was previously shown to contain toxic oligomeric species (Gibbs et al, 2008 (supra)) and to potently inhibit memory consolidation in the chick (Gibbs et al, 2008 (supra)).
  • a ⁇ 1-42 was freshly diluted from stocks in the absence or presence of RAP, and then injected into the brains of chicks 45 min prior to their training for discriminative avoidance of red versus blue beads. Consistent with our previous report (Gibbs et al, 2008 (supra)), chicks injected with A ⁇ 1-42 alone did not avoid the red beads upon testing 120 min after training, thus their discrimination ratio (DR) score was close to that of chance alone (0.5) ( Figure 9).
  • DR discrimination ratio
  • chicks injected with an A ⁇ 1-42 solution that also contained RAP exhibited less amnesia as they avoided the red beads, giving a DR of close to 1.0.
  • the behavior of chicks injected with A ⁇ 1-42 and RAP together was indistinguishable from that of chicks injected with vehicle alone.
  • the co-injection of RAP prevented the amnestic effect of A ⁇ 1-42 in chicks.
  • Tissue from a transgenic mouse model of AD (such as Tg2576) taken at different ages is analysed for RAP by western blotting and immunohistochemical staining.
  • the distribution of RAP is compared with that of age-matched background strain control mice.
  • the tissue distribution of staining is compared with that of various markers of A ⁇ deposition and toxicity such as A ⁇ , tau and ubiquitin.
  • Tissue from AD brain is analysed for RAP by western blotting and immunohistochemical staining.
  • the distribution of RAP is compared with that of age-matched controls or other neurological diseases. Wherever possible, tissue are matched for sex, age and post-mortem interval.
  • the tissue distribution of staining are compared with that of various markers of A ⁇ deposition and toxicity such as A ⁇ , tau and ubiquitin.
  • RAP 5 as it occurs naturally in the body or as administered in its naturally occurring form, is actively taken up across the blood-brain barrier via a receptor-mediated mechanism (Pan et al., 2004 (supra)) and is relatively stable in blood. Therefore, peripheral intravenous administration of RAP results in rapid incorporation of the brain into the brain parenchyma.
  • RAP polypeptide is administered intravenously to Tg2576 mice.
  • RAP polypeptide on amyloid load (as measured with Thioflavin S and A ⁇ immunohistochemistry), A ⁇ levels (ELISA and western blotting) and various markers of amyloid neuropathology (such as tau and ubiquitin) are determined.
  • Treated Tg2576 mice are also be tested for memory using a Morris Water Maze and the effects of RAP polypeptide examined.
  • Non-conventional Code Non-conventional Code amino acid amino acid ⁇ -aminobutyric acid Abu L-N-methylalamne Nmala ⁇ -amino- ⁇ -methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
  • D-N-methyllysine Dnmlys N-methyl- ⁇ -aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-niethylmethionine Dnmmet

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Abstract

L'invention porte entre autres sur l'utilisation d'un polypeptide de protéine associé à un récepteur (RAP) ou d'un analogue fonctionnel de celui-ci dans le but de réduire l'oligomérisation et le dépôt d'amyloïde et de réduire les caractéristiques neuropathologiques associées à Aβ. Ainsi, l'invention fournit une composition comprenant un RAP ou un analogue de celui-ci qui se lie au peptide β-amyloïde (Aβ) pour utilisation dans le traitement ou la prophylaxie d'un symptôme de la maladie d'Alzheimer (AD) ou d'un état apparenté chez un sujet. Un symptôme illustratif est la perte ou la consolidation de la mémoire. Sous un autre aspect, l'invention porte sur des procédés de criblage pour des variantes fonctionnelles du polypeptide RAP ou analogues par le test de la capacité d'un agent à entrer en compétition avec succès avec le RAP pour se lier à Aβ.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2919798A1 (fr) * 2012-11-14 2015-09-23 Sagetis Biotech, SL Polypeptides pour le transport à travers la barrière hémato-céphalique
EP3668891B1 (fr) * 2017-08-16 2023-07-26 Lgv1 S.R.L. Isoform vtft d'une proteine bpifb4 destinee aux maladies neuronales et aux blessures

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2007035716A2 (fr) * 2005-09-16 2007-03-29 Raptor Pharmaceutical Inc. Compositions de proteines rap specifiques des proteines contenant du cr, et leurs utilisations

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2007035716A2 (fr) * 2005-09-16 2007-03-29 Raptor Pharmaceutical Inc. Compositions de proteines rap specifiques des proteines contenant du cr, et leurs utilisations

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KANEKIYO, T. ET AL.: "Receptor-Associated Protein Interacts with Amyloid-P Peptide and Promotes Its Cellular Uptake", JOURNAL OF BIOLOGICAL CHEMISHY, vol. 284, no. 48, 2009, pages 33352 - 33359 *
WILHELTUS, M. M. M. ET AL.: "Lipoprotein Receptor-Mediated Protein- Mediates Amyloid-beta- Mediated Cell Death of Cerebrovascular Cells", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 171, no. 6, 2007, pages 1989 - 1999 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2919798A1 (fr) * 2012-11-14 2015-09-23 Sagetis Biotech, SL Polypeptides pour le transport à travers la barrière hémato-céphalique
EP3668891B1 (fr) * 2017-08-16 2023-07-26 Lgv1 S.R.L. Isoform vtft d'une proteine bpifb4 destinee aux maladies neuronales et aux blessures

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