EP2170929A2

EP2170929A2 - Peptides facilitating retrograde transport and their uses

Info

Publication number: EP2170929A2
Application number: EP08772440A
Authority: EP
Inventors: Elaine L. Bearer
Original assignee: Dart Neuroscience LLC
Current assignee: Dart Neuroscience LLC
Priority date: 2007-07-05
Filing date: 2008-07-07
Publication date: 2010-04-07
Also published as: AU2008272810A1; WO2009006638A3; WO2009006638A2; CA2692603A1

Abstract

The present invention concerns peptides facilitating retrograde transport and their uses. In particular, the invention concerns the peptides of KlP VIE NPQ YFG ITN (SEQ ID NO: 1) and KRE LGE GAF GKV FLA (SEQ ID NO: 2).

Description

PEPTIDES FACILITATING RETROGRADE TRANSPORT AND THEIR USES

FIELD QF THE INVENTION

The present invention concerns peptides facilitating retrograde transport of exogenous and/ or endogenous cargo. BACKGROUND OF THE INVENTION

Inside cells, 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.

Thus, 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. In particular, 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. USA 103: 16532-37 (2006)) identified a short peptide sequence ( 15 amino acids) from the cytoplasmic C terminus of amyloid precursor protein (APP-C) sufficient to mediate the anterograde transport of peptide- conjugated beads in the squid giant axon. The APP-C domain is conserved (13 out of 15 amino acids aa) from squid to human, and peptides from either squid or human APP behave similarly. Thus, the authors Bearer and co-workers identified a conserved peptide zipcode sufficient to direct anterograde transport of exogenous cargo and suggested that one of APP's roles may be to recruit and activate axonal machinery for endogenous cargo transport. For further details see, also U.S. Application No. 60/852,896, filed on October 18, 2006 and/or publication WO 2008/060811 (PCT/US2007/081854), the entire disclosures of which are hereby expressly incorporated by reference.

SUMMARY OF THE INVENTION

The present invention is based on the identification of peptides that mediate transport of exogenous or endogenous cargo along microtubules in the retrograde direction. In general, the present invention concerns peptides facilitating retrograde transport of exogenous and/or endogenous cargo.

In some embodiments, 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.

In other embodiments, 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.

In other embodiments, the peptides facilitating retrograde transport comprise, consist essentially of, or consist of a distal portion of the TrkB juxtamembrane region (amino acids

484 to 513); or 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).

In some embodiments, 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.

In all embodiments, the peptides facilitating retrograde transport can be conjugated to an exogenous or an endogenous cargo. In preferred embodiments, 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. In many preferred embodiments, the endogenous or exogenous cargo is not a trk peptide or polypeptide, nor does it comprise a domain of a trk peptide or polypeptide. In a further aspect, 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).

In another aspect, 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.

In various embodiments, the cargo can be an endogenous cargo or an exogenous cargo. In some embodiments, 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. In a further embodiment, the cargo is a neurotrophic factor. In other embodiments, the cargo-peptide conjugate is further associated with a viral or toxin-based drug delivery vehicle.

In another aspect, 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.

In a preferred embodiment, the subject in need of treatment is a human patient. In some preferred embodiments, the subject in need of treatment is an animal; in other preferred embodiments, the subject in need of treatment is a plant. In another embodiment, the subject in need of treatment suffers from or is at risk of developing a disease or condition benefiting from facilitation of retrograde transport.

In another embodiment, 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. In yet another embodiment, 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, prophyria, systemic lupus erythematosis, (jraxc's diseases, Sjogren's disease, Gullain-Barre syndrome, Al/heimer's disease, Huntington's Disease, amyotrophic lateral sclerosis, stroke, epilepsy, mental retardation, and Parkinson's disease.

In a further aspect, 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). In a preferred embodiment, the subject in need of treatment is a human patient. In other preferred embodiments, the subject in need of treatment is an animal; in still other preferred embodiments, the subject in need of treatment is a plant.

In another embodiment, the agent is administered by intramuscular injection. In yet another embodiment, the agent is administered by direct injection to the central nervous system (CNS).

In a further embodiment, 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's disease, Huntington's Disease, amyotrophic lateral sclerosis, stroke, epilepsy, mental retardation, and Parkinson's disease.

In a still further embodiment, 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), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factors (FGF's), insulin-like growth factors (IGFs, e.g. IGF-I and IGF-2), neurturins, persephin, bone moφhogcnic proteins (BMP's), immunophilins, members of the transforming growth factor (TGF) family, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), Sonic Hedgehog, Nurr- 1 , en/ymes such as tyrosine hydroxylase and GTP-cyclohydrolase.

In further embodiments, 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.

In other embodiments, the instant invention may be used to deliv er gene therapy agents to a cell's nucleus.

In still other embodiments, 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.

In further embodiments, the instant invention may be used to program neurons to survive and/or to maintain, grow, or remove synaptic connections.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Figure l(A) depicts individual beads tracked throughout a 100 frame video sequence. In 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: Figure 2 depicts an analysis of the binding of dynein to Trk-TK and Trk-SH peptide-coated magentic beads.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et til., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For puφoses of the present invention, the following terms are defined below.

The term "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).

The term "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. Thus, for example, in the case of 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. For retrograde transport, for example, toxins and viruses utilizing retrograde transport pathways, such as cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A and ricin are considered "exogenous." The related term "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.

The term "conjugate" or "conjugated" 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.

The term "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". The term "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.

The terms "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). The terms "ncurolrophin" and "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.

"Native" or "native sequence" 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.

The terms "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., Sαence 274: 1540-1543 ( 1996)). The terms "trk", "trk polypeptide" and "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.

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. For sequences of trk polypeptides from human and other animal species see, for example, U.S. Patent No. 5,844,092; 5,877,016; 5,910,574; 6,027,927; 6,153,189; 5,348,856; and 5,688,91 1 , the entire disclosures of which are expressly incorporated by reference herein. For publications relating to trk, see Coulier F., et al., "Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain," MoI Cell Biol. 1990 Aug: 10(8):4202-lϋ; Martin-Zanca D. et al., "A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences," Nature. 1986 Feb 27-Mar 5;319(6056):743-8; and Klein, R. et al., EMBO J. 8, 3701 -3709 ( 1989). 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 The term "polypeptide" may encompass either peptides or proteins. The term "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. In some embodiments, 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). Preferably, 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. 3 pp 345- 58, 1978; Hein J., Unified Approach to Alignment and Phylogenes pp. 626-45 Methods in En/ymology v. 183, Academic Press, Inc., San Diego, CA, 1990; Higgiπs, D. G. and Sharp, P.M., CABIOS 5: 151 -53, 1989; Myers, E.W. and Muller, W, CABlOS 4: 1 1 - 17, 1988; Robinson, E.D. Comb. Theor 1 1 : 105, 1971 ; Santou, N, Ncs, M., MoI. Biol, 12vol. 4:406-25, 1987; Sneath, P. H. A. and Sokal, R. R. Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Ca., 1973; and Wilbur, WJ. and Lipman, DJ. , Proc. Nat'l. Acad. Sci. 80:726-30, 1983.

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. In the case of 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).

Other 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.

In addition, the peptides of the present invention may have sequences that are not identical to any sequence present in a native trk polypeptides. Thus, 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. Thus, 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);

Langone, J. L. and Van Vunakis, H., Methods Enzymol. 84:628 640 ( 1982); Wong, Chemistry of Protein Conjugation and Cross-Linking. CRC Press, Inc., Boca Raton, FIa. ( 1991 ). 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.

The ability of the peptides of the present invention to carry cargo to the nuclei of cells is a major tool both in clinical and laboratory practice. It is known that retrograde transport pathways are utilized by certain viruses and toxins for delivery from the cell surface to the endoplasmic reticulum (ER). Thus, for example, cholera toxin, Shiga and Shiga-like toxins, Pseudomonas exotoxin A, ricin. SV40, polyoma viruses, herpes simplex viruses (HSV), and 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. By 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. As a result, 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.

In addition, 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. In general, such 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).

Particular diseases that may be treated (including prevention) with the aid of the peptides of the present invention include, without limitation diseases associated w ith or involving neurodegeneration or nerve injury. Such diseases 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, prophyria, systemic lupus erythematosis. Grave's diseases, Sjogren's disease, Gullain-Barre syndrome, Alzheimer's disease, Huntington's Disease, amyotrophic lateral sclerosis, stroke, epilepsy, mental retardation, and Parkinson's disease.

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. IGF- I and IGF-2), ncurturins, perscphin, bone niorphogenic proteins (BMP's), immunophilins, members of the transforming growth factor (TGF) family, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), Sonic Hedgehog, Nurr-1 , enzymes such as tyrosine hydroxylase and GTP-cyclohydrolasc, and the like.

Further details of the invention are illustrated by the following non-limiting examples. 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.

Peptide Conjugation to Fluorescent Microspheres

Carboxylated microspheres (beads, 0.1 μm 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 μl of a 2mg/ml stock) were mixed in 300 μl of 50 niM MES buffer with 10 μl of 2% beads for 15min. Cross-linker ( l -(3-dimethylaminopropyl)-3-etliylcarbυdiimide hydrochloride; EDAC, Molecular Probes) was added to a concentration of 10 μg/ml and the solution adjusted to pH 6.5 with I M NaOH. After 5 hr the reaction was quenched by adding

2M glycine for a final of 0.1 M. Conjugation was confirmed by analyzing peptide concentrations before and after by protein assay. Peptide:bead ratio was adjusted that equivalent molar amounts of each protein would be conjugated to the beads. Efficiency of conjugation was relatively similar for all peptides. Binding sites on the beads ( 10⁶) were saturated by using 300-1000 fold excess peptide.

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 Λchroplan 0.8NA water correctible objectives. Green and red fluorescence and phase images were collected simultaneously using Zeiss LSM5 10 multi- tracking.

Analysis of Transport

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 ) and Trk-TK KRE LGE GAF GKV FLA (SEQ ID NO: 2). Exaniple. 2: Observation of retrograde transport using detergent-stripped HSV particles

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.

Bearer et al. concluded that (i) HSV recruits the squid retrograde transport machinery; and (ii) viral tegument and capsid but not envelope are sufficient for this recruitment. Example 3: Observation of retrograde transport in axoplasm

Trk-TK and Trk SH peptides mediate retrograde transport of beads in axoplasm al rates consistent with dynein-mediated transport (1 -2 μm/sec).

In Figure l (A) individual beads were tracked throughout a 100 frame video sequence using the "Track point" function in Metamorph and the step size between frames automatically loaded into an Excel spreadsheet. In (B), a different axon injected with the same bead conjugate, the location of each bead in each frame is superimposed into the same still image. Note the beaded strings emanating from the central mass of beads at the injection site (right side of the image) and leading towards the synapse, towards the left, retrograde side of the image. Frequency histogram of distance moved between frames divided by 4 sec, gives the instantaneous velocity of the beads, peaking at 1 -2 μm/sec (C).

Method: Peptides were conjugated to beads as described (Satpute et al. PNAS 2006).

In this case, 100 nm red Fluorospheres conjguated to Trk-SH peptide were injected into the squid axon and imaged by confocal laser scanning microscope on a Zeiss 510. Images were captured at q4sec intervals (A and B, above). Analysis was performed with NIH share-ware

ImageJ and Mctamorph software.

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.

Figure 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 μl 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.

Method: Magnetic beads (Dynal) 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 μl 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). The supernatant was precipitated with 10% Trichloroacetatc, pelleted at 15,000xg for 15 min and the pellet resuspended in the same volume as the bead pellet. Parallel lanes were loaded equivalent volumes and run through a 10% SDS-PAGE and transferred to nictrocellulose membranes according to established protocols. Blots were probed for dynein (Abeam).

Both Trk-derived peptides bound to dynein, and Trk-SH bound more dynein from the same extract than Trk-TK. Both types of beads bound a small amount of the anterograde motor, kinesin 3, and both moved in the anterograde direction as well.

In some preferred embodiments, 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. K?yMll2kJlJifl∞J_JlLNudl

Nudl, a soluble protein that activates dynein, enhances the capability of negative charge beads (cargo) for retrograde transport.

Normally COOH -beads are transported uniquely anterograde. With injection of K) pL of 7 ng/ml NdI-GST, negative charge beads are radically re-directed and transported retrograde.

Many tested peptides do not mediate any transport, and some mediate only anterograde and not retrograde transport. Table 1 lists many of the peptides tested.

Note that injection of soluble Trk-SH peptide ( lOpL of 1.0 μg/ml) inhibits retrograde but not anterograde transport of Trk-SH beads.

The following peptides have been tested for the ability to mediate anterograde and retrograde transport in axons using methods as described herein and as described in Satpute et al., 2006, U.S. Application No. 60/852,896, filed on October 18, 2006 and/or publication WO 2008/06081 1 (PCT/US2007/081854).

Table I a

Table I b

Table I c

Table I d

Name Sequence Oriirin Source

Other peptides tested with anterograde but not retrograde activity in the squid axon

The terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalent of the invention shown or portion thereof, but it is rccogni/cd that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the inventions embodied herein disclosed can be readily made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling w ithin the generic disclosure also form the part of these inv entions. This includes within the generic description of each of the inventions a proviso or negative limitation that will allow removing any subject matter from the genus, regardless or whether or not the material to be removed was specifically recited.

In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recogni/e that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. The steps depicted and/or used in methods herein may be performed in a different order than as depicted and/or stated. The steps are merely exemplary of the order these steps may occur. The steps may occur in any order that is desired such that it still performs the goals of the claimed invention.

From the description of the invention herein, it is manifest that various equivalents can be used to implement the concepts of the present invention without departing from its scope. Moreover, while the invention has been described with specific reference to certain embodiments, a person of ordinary skill in the art would recognize that changes can be made in form and detail without departing from the spirit and the scope of the invention. The described embodiments are considered in all respects as illustrative and not restrictive. It should also be understood that the invention is not limited to the particular embodiments described herein, but is capable of many equivalents, rearrangements, modifications, and substitutions without departing from the scope of the invention. Thus, additional embodiments are within the scope of the invention and within the following claims. All references cited throughout the disclosure are hereby expressly incorporated by reference, including any drawings, figures and tables.

Claims

WHAT IS CLAIMED IS:

1. A peptide facilitating retrograde transport of an exogenous and/ or endogenous cargo,

2. The peptide of claim 1 comprising a sequence from a native trk receptor.

3. The peptide of claim 1 consisting essentially of a sequence from a native trk receptor.

4. The peptide of claim 1 consisting of a sequence from a native trk receptor.

5. The peptide of any one or claims 2 to 4 wherein said native trk receptor is of human origin.

6. The peptide of any one or claims 1 to 4 wherein said native trk receptor is selected from the group consisting of trkA, trk B and trkC receptors.

7. The peptide of claim 1 comprising a sequence having at least about 95% sequence identity to a native trk receptor.

8. The peptide of claim 1 comprising a sequence having at least about 98 % sequence identity to a native trk receptor. 9. The peptide of claim 1 comprising a sequence having at least about 99"« sequence identity to a native trk receptor.

K). The peptide of claim 1 consisting essentially of a sequence having at least about 95% to 99% sequence identity to a native trk receptor.

1 1. The peptide of claim 1 consisting of a sequence having at least about 95% to 99% sequence identity to a domain of a native trk receptor.

12. The peptide of claim 1 comprising a sequence having at least about 96% to 99°i) sequence identity to a peptide from amino acids 484 to 513 of human trkA, wherein said peptide is about 10 to about 20 amino acids in length.

13. 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).

14. The peptide of claim 13 which is KIP VIE NPQ YFG ITN (SEQ ID NO:

I ⁾.

15. The peptide of claim 13 which is KRE LGE GAF GKV FLA (SEQ ID NO: 2). 16. The peptide of claim 13, claim 14, or claim 15 conjugated to a cargo to prepare a cargo-peptide conjugate.

17. The peptide of 16 wherein said cargo is an endogenous cargo.

18. The peptide of claim 16 wherein said cargo is an exogenous cargo.

19. The peptide of claim 19 wherein said exogenous cargo is a nucleic acid

20. The peptide of claim 18 said exogenous cargo is a peptide, a polypeptide, or a protein.

21. The peptide of claim 18 wherein said exogenous cargo is a non-peptide 5 small molecule.

22. The peptide of claim 18 wherein said exogenous cargo is a neurotrophic factor.

23. The peptide of claim 16 wherein said cargo-peptide conjugate is further associated with a viral or toxin-based delivery vehicle.

K) 24. A method for facilitating retrograde neuronal transport of a cargo comprising conjugating said cargo to 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) and administering the conjugate obtained to a subject in need of treatment.

25. The method of claim 24 wherein the subject in need of treatment is a 1 5 human patient

2(). The method of claim 25 wherein said cargo is an endogenous cargo.

27. The method of claim 25 wherein said cargo is an exogenous cargo.

28. The method of claim 27 wherein said exogenous cargo is a nucleic acid.

29. The method of claim 27 wherein said exogenous cargo is a peptide a 0 polypeptide, or a protein.

30. The method of claim 27 wherein said exogenous cargo is non-peptide small molecule.

31 . The method of claim 27 wherein said exogenous cargo is a neurotrophic factor. 5 32. The method of claim 25 wherein said conjugate is further associated with a viral or toxin-based delivery vehicle.

33. The method of claim 25 wherein said human patient suffers from or is at risk of developing a disease or condition benefiting from facilitation of the retrograde transport. 0 34. The method of claim 25 wherein said patient suffers from or is at risk of developing a disease associated with or involving neurodegeneration or nerve injury.

35. The method of claim 25 wherein said patient 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 neurodegencration, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, progressive bulbar inherited muscular atrophy, intervertebrate disk syndromes such as a herniated, ruptured or prolapsed interv ertebrate 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's disease, Huntington's Disease, amyotrophic lateral sclerosis, stroke, epilepsy, mental retardation, and Parkinson's disease.

36. 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 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).

37. The method of claim 36 wherein said agent is administered by intramuscular injection.

38. The method of claim 36 wherein said agent is administered by direct injection to the CNS. 39. The method of claim 36 wherein said disease or condition is selected from the group consisting of a disease or condition such as 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's disease, Huntington's Disease, amyotrophic lateral sclerosis, stroke, epilepsy, mental retardation, and Parkinson's disease.

40. The method of claim 36 wherein said agent 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 ( N F-3), neurotrophin-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 (IGPs, e.g. IGF-] and IGF-2), neurturins, persephin, bone moφhogenic proteins (BMP's), immunophilins, members of the transforming growth factor (TGF) family, epidermal growth factor (EGF), plateled-derived growth factor (PDGF), Sonic Hedgehog, Nurr-1 , enzymes such as tyrosine hydroxylase and GTP- cyclohydrolase.