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EP2170929A2 - Peptides facilitant le transport retrograde et utilisations de ceux-ci - Google Patents

Peptides facilitant le transport retrograde et utilisations de ceux-ci

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Publication number
EP2170929A2
EP2170929A2 EP08772440A EP08772440A EP2170929A2 EP 2170929 A2 EP2170929 A2 EP 2170929A2 EP 08772440 A EP08772440 A EP 08772440A EP 08772440 A EP08772440 A EP 08772440A EP 2170929 A2 EP2170929 A2 EP 2170929A2
Authority
EP
European Patent Office
Prior art keywords
peptide
cargo
disease
sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP08772440A
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German (de)
English (en)
Inventor
Elaine L. Bearer
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Dart Neuroscience LLC
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Dart Neuroscience LLC
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Filing date
Publication date
Application filed by Dart Neuroscience LLC filed Critical Dart Neuroscience LLC
Publication of EP2170929A2 publication Critical patent/EP2170929A2/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention concerns peptides facilitating retrograde transport of exogenous and/ or endogenous cargo.
  • organelles move back and forth from the plasma membrane at the surface to the deeper cytoplasm and the nucleus. Movement outwards from the nucleus to the surface membrane is termed “anterograde” and inwards towards the nucleus, “retrograde”. This movement is powered by molecular motors such as kinesin, dynein and the myosins.
  • Anterograde and retrograde movements use different motors, e.g., kinesins primarily carry cargo along microtubules towards the plus end, most often oriented towards the surface membrane, and are thus thought to be anterograde motors.
  • Dynein carries cargo on microtubules towards the minus end, usually towards the nucleus, and is thus considered a retrograde motor. For directed transport to occur, organelles must recruit these motors and turn them on.
  • the surface of motile organelles should display a motor-receptor to the cytoplasm.
  • the molecular nature of such receptors was not known until the discovery that a peptide derived from amyloid precursor protein (APP) was sufficient to direct anterograde transport of exogenous cargo.
  • APP amyloid precursor protein
  • Bearer and co-workers Satpute-Krishnan P, DeGiorgis JA, Conley MP, Jang M, Bearer EL, "A peptide zipcode sufficient for anterograde transport within amyloid precursor protein," Proc. Natl. Acad. Sci.
  • the present invention is based on the identification of peptides that mediate transport of exogenous or endogenous cargo along microtubules in the retrograde direction.
  • the present invention concerns peptides facilitating retrograde transport of exogenous and/or endogenous cargo.
  • the peptides facilitating retrograde transport comprise, consist essentially of or consist of, or are at least about 95%, about 90%, about 85%, or about 80% homologous to a sequence from a trk receptor, including trkA, trkB and trkC, preferably trkB.
  • the peptides facilitating retrograde transport comprise, consist essentially of, or consist of a sequence from a trk receptor, including trkA, trkB and trkC, preferably trkB; or are at least about 95%, about 90%, about 85%, or about 80% homologous to a sequence from a trk receptor, including trkA, trkB and trkC, preferably trkB; or are less than about 20% divergent from a sequence from a trk receptor, including trkA, trkB and trkC, preferably trkB.
  • the peptides facilitating retrograde transport comprise, consist essentially of, or consist of a distal portion of the TrkB juxtamembrane region (amino acids
  • the peptides facilitating retrograde transport comprise, consist essentially of, or consist of a peptide the sequence of which is at least about 95%, about 90%, about 85%, or about 80% homologous to a sequence from a distal portion of the TrkB juxtamembrane region (comprising at least a portion of amino acids about 484 to about 513); or the peptides facilitating retrograde transport comprise, consist essentially of, or consist of a peptide the sequence of which is at least about 80% homologous, or more, to a sequence from a distal portion of the TrkA juxtamembrane region (comprising at least a portion of amino acids 484 to 513).
  • the peptides facilitating retrograde transport are about 2 to about 50 amino acids long, or about 3 to about 30 amino acids long, or about 4 to about 28 amino acids long, or about 5 to about 25 amino acids long, or about 10 to about 20 amino acids, about 12 to about 15 amino acids long; the peptide may also be about 13 amino acids long, or about 14 amino acids long, or about 15 amino acids long, or about 16 amino acids long, or about 17 amino acids long.
  • the peptides facilitating retrograde transport can be conjugated to an exogenous or an endogenous cargo.
  • the endogenous or exogenous cargo does not consist essentially of, or solely of, a sequence of a trk gene contiguous to SEQ ID NO: 1 or SEQ ID NO:2.
  • the endogenous or exogenous cargo is not a trk peptide or polypeptide, nor does it comprise a domain of a trk peptide or polypeptide.
  • the instant invention concerns a peptide facilitating retrograde transport selected from the group consisting of KlP VIE NPQ YF(J ITN (SEQ ID NO: 1 ) and KRE LGE GAF GKV FLA (SEQ ID NO: 2).
  • the invention concerns a peptide facilitating retrograde transport selected from the group consisting of KIP VIE NPQ YFG ITN (SEQ ID NO: 1 ) and KRE LGE GAF GKV FLA (SEQ ID NO: 2), which is conjugated to a cargo to yield a cargo- peptide conjugate.
  • the cargo can be an endogenous cargo or an exogenous cargo.
  • the cargo is a nucleic acid, a peptide, a polypeptide, or a non- peptide small molecule.
  • Many other embodiments of the cargo are encompassed, such as lipid vesicles, membranated vesicles, organelles, single proteins, niRNA, or packets of molecules not bound by membranes. Any cargo that is transported from cell surface to cell nucleus may be included in some embodiments of the instant invention.
  • the cargo is a neurotrophic factor.
  • the cargo-peptide conjugate is further associated with a viral or toxin-based drug delivery vehicle.
  • the invention concerns a method for facilitating retrograde neuronal transport of a cargo comprising conjugating said cargo to a peptide as hereinabove described, such as a peptide selected from the group consisting of KIP VIE NPQ YFG ITN (SEQ ID NO: 1 ) and KRE LGE GAF GKV FLA (SEQ ID NO: 2) and administering the conjugate obtained to a subject in need of treatment.
  • a peptide selected from the group consisting of KIP VIE NPQ YFG ITN (SEQ ID NO: 1 ) and KRE LGE GAF GKV FLA (SEQ ID NO: 2) and administering the conjugate obtained to a subject in need of treatment.
  • the subject in need of treatment is a human patient.
  • the subject in need of treatment is an animal; in other preferred embodiments, the subject in need of treatment is a plant.
  • the subject in need of treatment suffers from or is at risk of developing a disease or condition benefiting from facilitation of retrograde transport.
  • the subject in need of treatment suffers from or is at risk of developing a disease or condition associated with or involving neurodegeneration or nerve injury.
  • the subject suffers from or is at risk of developing a disease or condition selected from the group consisting of peripheral nerve damage caused by physical injury, diabetes, physical damage to the central nervous system, a disease of the central nervous system, brain damage associated with stroke, a neurological disorder relating to neurodegeneration, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, progressive bulbar inherited muscular atrophy, interv ertebrate disk syndromes such as a herniated, ruptured or prolapsed intervertebrate disk syndrome, cer ⁇ ical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies such as those caused by lead, dapsone, ticks, pro
  • peripheral nerve damage caused
  • the invention concerns a method for the treatment of a disease or condition benefiting from the facilitation of retrograde transport, comprising administering a subject in need an effective amount of an agent effective in the treatment of said disease or condition and further administering a peptide as hereinabove described, including a peptide selected from the group consisting of KlP VIE NPQ YFG ITN (SEQ ID NO: 1 ) and KRE LGE GAF GKV FLA (SEQ ID NO: 2).
  • the subject in need of treatment is a human patient.
  • the subject in need of treatment is an animal; in still other preferred embodiments, the subject in need of treatment is a plant.
  • the agent is administered by intramuscular injection. In yet another embodiment, the agent is administered by direct injection to the central nervous system (CNS).
  • CNS central nervous system
  • the disease or condition is selected from the group consisting of peripheral nerve damage caused by physical injury, diabetes, physical damage to the central nervous system, a disease of the central nervous system, brain damage associated with stroke, a neurological disorder relating to neurodegeneration, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, progressive bulbar inherited muscular atrophy, intervertebrate invertebrate disk syndromes such as a herniated, ruptured or prolapsed intervertebrate invertebrate disk syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies such as those caused by lead, dapsone, ticks, prophyria, systemic lupus erythematosis, Grave's diseases, Sjogren's disease, Gullain-Barre syndrome, Alzheimer'
  • the agent delivered is selected from the group consisting of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ncurotrophin-2 (NT-2), neurotrophin-3 (NT-3), ncurotiophin-4 5 (NT-4/5).
  • neurotrophin-6 NT-6
  • CNTF ciliary neurotrophic factor
  • GDNF glial cell line-derived neurotrophic factor
  • FGF's insulin-like growth factors
  • IGFs insulin-like growth factors
  • IGF-I and IGF-2 IGF-I and IGF-2
  • neurturins IGF-I and IGF-2
  • persephin persephin
  • BMP's bone mo ⁇ hogcnic proteins
  • immunophilins members of the transforming growth factor (TGF) family
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • Sonic Hedgehog Nurr- 1
  • en/ymes such as tyrosine hydroxylase and GTP-cyclohydrolase.
  • the instant invention may be used to deliver toxins or the like to a cell nucleus to cause cell death such as in cancer therapy, or for autoimmune diseases such as systemic lupus erythematosis, Grave's diseases, myasthenia gravis, or Sjogren's disease.
  • autoimmune diseases such as systemic lupus erythematosis, Grave's diseases, myasthenia gravis, or Sjogren's disease.
  • the instant invention may be used to deliv er gene therapy agents to a cell's nucleus.
  • the instant invention may be used to assist a therapy for HSV or other neurotropic viruses such as polio or West Nile Virus.
  • the instant invention may also be used, in other embodiments for delivery of therapeutics from peripheral tissue to interior nervous system, such as from the lip to the brain, from foot/leg to the spinal cord, and/or from the spinal cord to the brain.
  • the instant invention may be used to program neurons to survive and/or to maintain, grow, or remove synaptic connections.
  • Figure 1 Figure l(A) depicts individual beads tracked throughout a 100 frame video sequence.
  • Figure l (B) a different axon was injected with the same bead conjugate, the location of each bead in each frame was superimposed into the same still image.
  • Figure l (C) is a frequency histogram of distance moved between frames divided by 4 sec, giving the instantaneous velocity of the beads.
  • Figure 2 depicts an analysis of the binding of dynein to Trk-TK and Trk-SH peptide-coated magentic beads.
  • axonal transport is used herein to refer to directed transport of organelles and molecules along a nerve cell axon.
  • the "axonal transport” can be “anterograde” (outward from the cell body towards the axon) or “retrograde” (back toward the cell body (soma) and/or nucleus).
  • exogenous cargo is used herein to refer to any molecule that is not native to the organism and/or cell in which the retrograde or anterograde transport takes place.
  • anterograde transport any molecule that is not normally synthesized in the cell body from where it is transported, through axonal transport, to a target synapse is considered “exogenous;” such an exogenous cargo may be synthesized in a cell's nucleus if the gene encoding the exogenous cargo has been inserted as a transgene. This may include endogenous genes inserted for the purposes of expression as a transgene.
  • toxins and viruses utilizing retrograde transport pathways such as cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin are considered “exogenous.”
  • endogenous cargo is used herein to refer to any molecule that is native to the cell and/or the organism in which the retrograde or anterograde transport takes place. Note that in some cases an "endogenous cargo" may not be normally synthesized by the cell in question.
  • conjugate refers to any and all forms of linkage, and includes, without limitation, direct genetic or chemical fusion, and coupling though a linker or a cross-linking agent.
  • fusion is used herein to refer to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their coding nucleotide sequences, and may be referred to as a "genetic fusion".
  • fusion explicitly encompasses internal fusions, i.e., insertion of sequences of different origin within a polypeptide chain, in addition to fusion to one of its termini.
  • neurotrophin and “neurotrophic factor” and their grammatical variants are used interchangeably, and refer to a family of polypeptides comprising nerve growth factor (NGF) and sequentially related homologs: brain-derived growth factor (BDNF, a.k.a. NT-2), neurotrophin-3 (NT-3), neurotrophins-4 and -5 (NT-4/5), ncurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF).
  • NGF nerve growth factor
  • BDNF brain-derived growth factor
  • NT-3 neurotrophin-3
  • NT-4/5 neurotrophins-4 and -5
  • NT-6 ncurotrophin-6
  • CNTF ciliary neurotrophic factor
  • GDNF glial cell line-derived neurotrophic factor
  • neurotrophic factor may include native neurotrophins of any (human or non-human) animal species, and their functional derivatives, whether purified from a native source, prepared by methods of recombinant DNA technology, or chemical synthesis, or any combination of these or other methods.
  • Neurotrophic factors or neurotrophins have the amino acid sequence of a ncurotrophm occurring in nature in any human or non-human animal species, including naturally-occurring truncated and variant forms, and naturally-occurring allelic variants.
  • trk irk polypeptide
  • trk receptor and their grammatical variants are used interchangeably and refer to polypeptides of the receptor tyrosine kinase superfaniily, which are capable of binding at least one native neurotrophic factor. Specifically included within this group are trkA, trkB, and trkC, and Ltrk from the mollusk Lymnaea (Fainzilber et al., S ⁇ ence 274: 1540-1543 ( 1996)).
  • trk trk polypeptide
  • trk receptor with or w ithout an affixed capital letter (e.g., A, B or C) designating specific members within this family, specifically include “native” or “native sequence” receptors (wherein these terms are used interchangeably) from any animal species (e.g. human and other vertebrate species, including non-human higher primates and other mammals, such as mice, rats, rabbit, porcine, equine, etc.), including full length receptors, their truncated and variant forms, such as those arising by alternate splicing and/or insertion, and naturally-occurring allelic variants, as well as functional derivatives of such receptors.
  • animal species e.g. human and other vertebrate species, including non-human higher primates and other mammals, such as mice, rats, rabbit, porcine, equine, etc.
  • full length receptors truncated and variant forms, such as those arising by alternate splicing and/or
  • a “native" or “native sequence” trk polypeptide has the amino acid sequence of any form of a trk receptor as occurring in the human, including full length native human trk, truncated, tyrosine kinase (TK.) domain-deleted (spliced) forms of full length native human trk, and insertion variants of full length or truncated native human trk.
  • TK. tyrosine kinase
  • peptide As used herein, the terms “peptide,” “polypeptide” and “protein” all generally refer to a primary sequence of amino acids that are joined by covalent “peptide linkages," In general, a peptide consists of a few amino acids, typically from about 2 to about 50 amino acids, and is shorter than a protein; a peptide may consist, for example, of about 15 amino acids.
  • polypeptide may encompass either peptides or proteins.
  • protein may encompass a mult-subunit assembly incorporating multiple polypeptides and/or peptides; proteins may also have additional material attached, frequently as a post-translational modification, such as carbohydrates.
  • a polypeptide may be a fusion polypeptide in which amino acid sequences derived from two or more different polypeptides are linked in a single polypeptide chain; in some preferred embodiments, one of the two or more different polypeptides comprises the sequence set forth, e.g., in SEQ ID NO: 1 and/or SEQ ID NO:2.
  • Identity or similarity with respect to an amino acid sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with (i.e. same residue) or similar (i.e. amino acid residue from the same group based on common side-chain properties) to a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity or similarity.
  • Alignment for purposes of determining percent amino acid sequence identity or similarity can be achieved in various ways that are within the skill in the art can determine appropriate parameters for measuring alignment, including assigning algorithms needed to achieve maximal alignment over the full-length sequences being compared.
  • Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Gapped BLAST or PSI-BLAST (Altschul, et al. ( 1997) Nucleic Acids Res. 25:3389-3402), BLAST (Altschul, et al. ( 1990) J. MoI. Biol. 215:403-410), and Smith-Waterman (Smith, et al. (1981 ) J. MoI. Biol. 147: 195-197).
  • the default settings of these programs will be employed, but those skilled in the art recognize whether these settings need to be changed and know how to make the changes.
  • Other ways of optimally performing alignments for sequence comparison include the Megalign program in the Lasergene suite of software available from DNASTAR, Inc. (Madison, WI.) using default parameters. This program embodies several alignment schemes such as: Dayhoff, M.O., A Model of Evolutionary Change in Proteins - Matrices for Detecting Distant Relationships, in Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D. C. v 5 Suppl.
  • a disease or condition "benefiting from the facilitation of retrograde transport” is any disease or condition the pathology of which includes impairment in the retrograde transport or the status of which can be improved by delivery of a cargo via retrograde transport.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be pre ⁇ ented.
  • the present invention is based on the identification of peptides that mediate retrograde transport of exogenous and endogenous cargo.
  • Two peptides discovered with this activity were derived from a large domain in a trk receptor, which serves as a receptor for nerve growth factor (NGF).
  • TrkA for example, is trafficked back to the nucleus after binding to NGF.
  • the domain mediating retrograde transport comprises a distal portion of the Trk juxtamembrane region.
  • TrkA a portion of the juxtamembrane region (amino acids 484 to 513) was implicated in binding of TrkA to dynein in vitro (Yano et al., T Neurosri. 21 :RC125: l-7 (2001)).
  • Regions of 15 amino acids derived from the TrkB juxtamembrane domain were selected for testing in an in vitro motility assay as described in the Examples below.
  • the following peptides showed retrograde activity:
  • Trk-SH KIP VIE NPQ YFG ITN (SEQ ID NO: 1 ) and Trk-TK KRE LGE GAF GKV FLA (SEQ ID NO: 2).
  • peptides originating from trkA or other trk polypeptides such as trkB or trkC, including the human trk receptors and trk from other animal species can be identified by a similar approach and are expected to have similar properties.
  • the peptides of the present invention may have sequences that are not identical to any sequence present in a native trk polypeptides.
  • amino acid sequence variants of sequences present in native trk polypeptides i.e. substitution, deletion and/or insertion variants are within the scope herein.
  • Such peptides may, for example, may comprise, consist essentially of, or consist of consensus sequences obtained from two or more trk polypeptides, such as trkA, trkB and/or trkC, or from trk polypeptides of two or more animal species, including humans.
  • the peptides herein can be conjugated to an endogenous or exogenous cargo, which can be delivered to the nuclei of target cells.
  • Conjugation can be performed by any methods known in the art.
  • techniques for coupling molecules to amino acids are well known to those of skill in the art.
  • Such methodologies may be found in standard chemical text books and publications, such as, for example, U.S. Pat. No. 5,876,727; WO 99/61054; Isomura, S. ct al. LOr ⁇ Chem. 66 41 15 4121 (2001 ); Matsushita, H. et al. 57: 1006 1010. ( 1974);
  • Conjugates such as fusion proteins generated by genetic engineering may also be used to "tag" other proteins or glycoproteins with these peptides for directed delivery.
  • cholera toxin cholera toxin
  • Shiga and Shiga-like toxins Pseudomonas exotoxin A
  • ricin ricin
  • SV40 polyoma viruses
  • HSV herpes simplex viruses
  • lentiviruses utilize retrograde transport.
  • Viral vector systems suitable for retrograde transport include, without limitation, retroviral and lentiviral systems, vector systems comprising rabies G protein, HSV, adenovirus, and hybrid HSV/adenovirus vectors.
  • HSV 1 and 2 have been used as a vector for gene delivery to the nervous system and other cell types.
  • retrograde transport it is possible to get expression in both the axon terminals and the cell bodies of transduced neurons. These two parts of the cell may be located in distinct areas of the nervous system.
  • a single administration, such as injection, of a vector system may transduce many distal sites.
  • the peptides of the present invention can be used for increasing the efficacy of gene delivery using viral vectors, or to deliver toxic agents to the nuclei of cancer cells.
  • the peptides of the present invention can be also used to facilitate retrograde neuronal transport of a cargo into the central nervous system (CNS). Many CNS diseases are difficult to treat, owing in part to the difficulty of delivering therapeutic agents through the blood- brain barrier.
  • the peptides of the present invention can be used, utilizing retrograde axonal transport, to target agents into the brain. Tims, the peptides of the present invention can enhance the efficacy of toxin-based systems used for delivery of therapeutic proteins to the CNS as well as gene delivery to the CNS using viral delivery systems.
  • the peptides of the present invention can facilitate and enhance delivery of therapeutic proteins to motor neurons from the periphery following an intramuscular injection, or to enhance delivery of proteins to neurons after direct injection to the CNS, and can serv e as non-viral vectors to transport and to deliver a biological activity or gene in a neural network.
  • Genes and polypeptides the delivery of which can be effected or facilitated by using the peptides of the present invention include therapeutic molecules that find utility in the prevention or treatment of diseases or injuries associated with cellular injury or functioning, such as diseases or injuries to neurons or the nervous system.
  • therapeutic molecules may simulate the production of new cells, prevent or slow down degeneration of existing cells affected by a disease, and/or affect the metabolic functioning of cells (e.g. en/ymes).
  • diseases associated w ith or involving neurodegeneration or nerve injury include, without limitation, one or more selected from the group consisting of peripheral nerve damage caused by physical injury, diabetes, physical damage to the central nervous system, a disease of the central nervous system, brain damage associated with stroke, a neurological disorder relating to neurodegeneration, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, progressive bulbar inherited muscular atrophy, intervertebrate invertebrate disk syndromes such as a herniated, ruptured or prolapsed intervertebrate invertebrate disk syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies such as those caused by lead, dapsone, ticks, prophy
  • Polypeptides, or the encoding genes, that can be delivered with the aid of the peptides herein include therapeutic polypeptides useful in the treatment of the foregoing diseases and conditions and other diseases, including, for example, neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-2 (NT-2), ncurotropliin-3 (NT-3), ncurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factors (FGF's), insulin-like growth factors (IGF's, e.g.
  • neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-2 (NT-2), ncurotropliin-3 (NT-3), ncurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line
  • IGF- I and IGF-2 IGF- I and IGF-2
  • ncurturins IGF- I and IGF-2
  • perscphin perscphin
  • bone niorphogenic proteins BMP's
  • immunophilins members of the transforming growth factor (TGF) family
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • Sonic Hedgehog Nurr-1
  • enzymes such as tyrosine hydroxylase and GTP-cyclohydrolasc, and the like.
  • Example 1 Identification of peptide for retrograde transport in trkB receptor Regions of 15 amino acids within a large domain in the distal portion of TrkB were tested in an in vitro motility assay as described below.
  • Carboxylated microspheres (beads, 0.1 ⁇ m diameter) with red (580/605nm) and green (505/515nm) fluorescence (Invitrogen) were washed through a Low Binding Durapore filter ( 100 urn cut-off, Millipore). Uncoated beads were diluted in motility buffer (Brady, S. et ai, ( 1982) Science 218, 1 129-31 ) and injected without further treatment.
  • Peptides were cross-linked to beads via their amino terminus to allow presentation of the carboxy end of the peptide to the cytoplasm. Each peptide was cross-linked to a bead of similar spectrum as the fluorescent protein, and conjugated beads individually checked for monochromic emission by confocal microscopy. Recombinant protein was generated in PC12 cells and purified by anti-fluorescent protein affinity columns (Vector Labs). Protein concentrations were determined by Bio-Rad protein assay, and composition and purity determined by SDS-PAGE.
  • Peptides (20 ⁇ l of a 2mg/ml stock) were mixed in 300 ⁇ l of 50 niM MES buffer with 10 ⁇ l of 2% beads for 15min.
  • Cross-linker ( l -(3-dimethylaminopropyl)-3-etliylcarb ⁇ diimide hydrochloride; EDAC, Molecular Probes) was added to a concentration of 10 ⁇ g/ml and the solution adjusted to pH 6.5 with I M NaOH. After 5 hr the reaction was quenched by adding
  • Imaging Axonal Transport hv Confocal Microscopy Detection parameters were set to ensure that each color was uniquely detected in its appropriate channel by imaging beads on coverslips with the Zeiss LSM 5 H) Laser Scanning Con focal Microscope. Fluorescent bead movements in the axon were collected with K)X Plan Neolluor 0,3NA air, and 4OX ⁇ chroplan 0.8NA water correctible objectives. Green and red fluorescence and phase images were collected simultaneously using Zeiss LSM5 10 multi- tracking.
  • Rates and trajectories of APP-C beads were measured by stepping through the frames in either the Zeiss LSM browser or NlH imageJ (http://rsb.info.nih.gov/nih-image/). Only particles moving into and out of a frame were included. Rates of moving particles were statistically analyzed and graphed using Microsoft Excel. The following peptides showed retrograde activity:
  • Trk-SH KlP VIE NPQ YFG ITN SEQ ID NO: 1
  • Trk-TK KRE LGE GAF GKV FLA SEQ ID NO: 2.
  • Bearer and co-workers used the giant axon of the squid, Loligo pealei, to monitor axoplasmic transport of HSV viral particles stripped of their envelopes by detergent.
  • E. L. Bearer, X. O. Breakefield, D. Schuback, T. S. Reese, and J.H.LaVail "Retrograde axonal transport of herpes simplex virus: Evidence for a single mechanism and a role for tegument," Proc. Natl Acad. Sci. 97:8146-50 (2000).
  • the HSV particles were injected into the giant axon with the viral tegument protein, VPl 6, labeled with green fluorescent protein. Viral particles moving inside the axon were imaged by confocal microscopy.
  • Trk-TK and Trk SH peptides mediate retrograde transport of beads in axoplasm al rates consistent with dynein-mediated transport (1 -2 ⁇ m/sec).
  • Trk-TK and Trk-SH peptides bind to both dyncin and members of the kincsin family of motors with differing affinities, suggesting these peptides have differing efficiencies of retrograde delivery.
  • FIG. 2 shows that Trk-TK and Trk-SH peptide-coated magentic beads pull-dow n dynem, the retrograde motor, from rat brain extracts. Note the bands in the pellet lanes. Saturation binding occurs with 8 ⁇ l of rat brain extract of 35 mg/ml total protein concentration. Strong bands in the supernatant lanes indicate the high concentration of this dynein subunit in brain. TRk-SH binds more strongly to dynein that Trk-TK, consistent with its more robust retrograde transport capability.
  • Magnetic beads were covalently conjugated to the Trk-TK or TRK- SH peptides using EDAC (Pierce Biochemicals, and as described for fluorospheres in Satpute et al., PNAS 2006. Rat brain extract was prepared and the supernatant applied to the peptide-conjugated beads at the volumes indicated. The bead-extract suspension was brought to 50 ⁇ l with extract buffer and incubated at room temperature for 1 hr. Then the beads were collected with a magnet, the supernatant removed and the bead pellet washed, and resuspended in gel sample buffer (pellet).
  • EDAC Pulierce Biochemicals, and as described for fluorospheres in Satpute et al., PNAS 2006. Rat brain extract was prepared and the supernatant applied to the peptide-conjugated beads at the volumes indicated. The bead-extract suspension was brought to 50 ⁇ l with extract buffer and incubated at room temperature for 1 hr. Then the
  • Trk-SH is more useful for reliable retrograde targeting, while in other preferred embodiments, Trk-TK is useful when a tight association with dynein produces side-effects.
  • Nudl a soluble protein that activates dynein, enhances the capability of negative charge beads (cargo) for retrograde transport.
  • Trk-SH peptide lOpL of 1.0 ⁇ g/ml

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Abstract

L'invention concerne des peptides facilitant le transport rétrograde et des utilisations de ceux-ci. Elle concerne en particulier les peptides de KlP VIE NPQ YFG ITN (SEQ ID NO: 1) et de KRE LGE GAF GKV FLA (SEQ ID NO: 2).
EP08772440A 2007-07-05 2008-07-07 Peptides facilitant le transport retrograde et utilisations de ceux-ci Ceased EP2170929A2 (fr)

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