EP1446014A2 - Introduction du gene wlds pour la prevention de la degenerescence axonale dans les maladies neurologiques - Google Patents

Introduction du gene wlds pour la prevention de la degenerescence axonale dans les maladies neurologiques

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Publication number
EP1446014A2
EP1446014A2 EP02800391A EP02800391A EP1446014A2 EP 1446014 A2 EP1446014 A2 EP 1446014A2 EP 02800391 A EP02800391 A EP 02800391A EP 02800391 A EP02800391 A EP 02800391A EP 1446014 A2 EP1446014 A2 EP 1446014A2
Authority
EP
European Patent Office
Prior art keywords
seq
amino acid
polynucleotide sequence
set forth
polynucleotide
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.)
Withdrawn
Application number
EP02800391A
Other languages
German (de)
English (en)
Other versions
EP1446014A4 (fr
Inventor
Jonathan D. Glass
Mark M. Rich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Emory University
Original Assignee
Emory University
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Filing date
Publication date
Application filed by Emory University filed Critical Emory University
Publication of EP1446014A2 publication Critical patent/EP1446014A2/fr
Publication of EP1446014A4 publication Critical patent/EP1446014A4/fr
Withdrawn legal-status Critical Current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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention is generally related to polynucleotides and polypepetides and, more particularly, is related to polynucleotides and polypepetides relating to the prevention and/or treatment of axonal degeneration.
  • Axonal degeneration is a pathological substrate leading to loss of neurological
  • CNS central nervous system
  • PNS peripheral nervous system
  • HMSN-1 hereditary motor sensor neuropathies
  • axonal degeneration is the pathological finding most highly correlated with severity of
  • Wallerian degeneration is the simplest and most thoroughly studied model of
  • axonal cytoskeleton in axotomized nerve fibers is a calcium-dependent process.
  • reduction of calcium to below a critical threshold of 200 ⁇ M delays the onset of axotomy-induced axonal degeneration.
  • Calpains are ubiquitous calcium-dependent cysteine proteases involved in both physiological and pathological cellulai functions.
  • protecting against axotomy-induced axonal degeneration may be protective in
  • peripheral neuropathy an in vitro model of toxin-induced axonal degeneration using
  • Vincristine is a chemotherapeutic agent used to treat leukemias and other types
  • vincristine causes
  • the slow Wallerian degeneration (Wld s ) mouse is a spontaneously occurring mutant strain of mouse that demonstrates the remarkable phenotype of prolonged
  • the Wld s mutation is created by the splicing of
  • embodiments of the present invention include
  • polynucleotides and polypeptides that can be used to treat axonal degeneration and related diseases.
  • the present invention provides for probes, expression
  • a representative embodiment of the present invention includes a method of preventing axonal degeneration in a host having a nervous system dysfunction.
  • composition that includes a polynucleotide selected from: a polynucleotide sequence set forth in SEQ ID NO:l, or a degenerate variant of the SEQ ID NO:l; a polynucleotide selected from: a polynucleotide sequence set forth in SEQ ID NO:l, or a degenerate variant of the SEQ ID NO:l; a
  • polynucleotide sequence at least 90% identical to the polynucleotide sequence set
  • polynucleotide sequence set forth in SEQ ID NO: 1 and a polynucleotide sequence at
  • the present invention provides for a method of
  • composition includes a polypeptide selected from: an amino acid sequence set
  • the present invention provides for a method of
  • the composition includes a polynucleotide selected from: a polynucleotide sequence
  • the present invention provides for a method of protecting axons from axon degeneration by exposing the axons to a composition.
  • the composition includes a polypeptide selected from: an amino acid sequence set forth in SEQ ID NO:2, or conservatively modified variants thereof; an amino acid sequence that is at least 90% identical to SEQ ID NO:2; an amino acid sequence that
  • the present invention provides for a method of treating a condition comprising administering to a host in need of treatment an
  • the polypeptide is selected from: an amino acid sequence set forth in SEQ ID NO:2, or conservatively modified variants thereof; an amino acid sequence that is at least 90% identical to SEQ ID NO:2; an amino acid sequence that is at least 75% identical to SEQ ID NO:2; and an amino acid sequence
  • the present invention provides for a method of
  • treating a condition comprising administering to a host in need of treatment an
  • the polynucleotide is selected from: a
  • polynucleotide sequence set forth in SEQ ID NO: 1 or a degenerate variant of the SEQ
  • polynucleotide sequence at least 90% identical to the polynucleotide sequence set forth in SEQ ID NO:l; a polynucleotide sequence at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:l ; and a polynucleotide
  • the present invention provides for a
  • composition comprising a polypeptide in combination with a pharmaceutically acceptable carrier.
  • the polypeptide is selected from: an amino acid
  • the present invention provides for a
  • composition comprising a polynucleotide in combination with a
  • the polynucleotide is selected from: a
  • Figure 1 is a serial montage phase-contrast photomicrographs of the same
  • Figure 2 is a graphic representation of changes in DRG area (mm ) and neurite
  • Figure 3 is a direct comparison of mo ⁇ hology on day 3 of cultures exposed to vincristine alone (A), or with addition of AK295 (B) or EGTA (C).
  • DRGs are stained with MAP-5 antibody.
  • Figure 4 is a graph of a quantitative measure of axonal survival with EGTA
  • Figures 5A-F is a comparison of protective effects of AK295 in vincristine
  • FIGs. 5A and 5D are unmanipulated controls (11 days in culture), 5B and 5E are untreated cultures exposed
  • Figs. 5C and 5F are the same respective injuries in media containing 50 ⁇ M AK 295.
  • Figure 6 is representative photo-montages of DRG cultures comparing
  • Figure 7 is dose-dependent response of cultured Wld s and C57BL/6 axons to vincristine. Comparisons of individual groups are shown on the graph: * p ⁇ 0.01,
  • Figure 8 are comparisons of normalized data for halo area and axon length at each dose of vincristine. Data are graphed as mean ⁇ SEM. * p ⁇ 0.05; # p ⁇ 0.01 ; $ p
  • Figures 9A and 9B are comparisons of sensory neurite growth (length and area) in C57BL/6 (solid lines) and Wld s axons (dotted lines). For Figure 9A, there are
  • axis percent of day 0, +/- SEM.
  • X-axis days after exposure to vincristine.
  • FIGS. 10A, A', B and B' are representative photomicrographs demonstrating axonal growth in a vincristine-exposed Wld s culture. The same DRG is shown at day 10 (Figs. 10A and A') and day 20 (Figs. B and B'). The arrows depict the same point
  • Figures 11 A, B, and C are Western blots using the polyclonal Wld s antibody demonstrating adenoviral expression of the Wld protein in HEK 293 A cells (Fig.
  • overlay panel demonstrates that the protein expression is in axons.
  • Figure 12 depicts the protective effect of expression of Wld in rat DRG
  • adenovirus expressing only lacZ (dotted line) rapidly die when exposed to 0.01 ⁇ M vincristine. Cultures infected with the Wld expressing adenovirus (dashed line) show
  • Figures 13 A, B and C are photomicrographs of representative cultures, demonstrating the pathological effects of vincristine exposure after 10 days in
  • Fig. 13C is a higher power view demonstrating the continuity of axons. Cultures are stained with MAP-5 for
  • polynucleotides that can be used to prevent and/or treat a number of neurological
  • affinity tag is used herein to denote a polypeptide segment that can
  • Affinity tags include a poly-histidine
  • streptavidin binding peptide or other antigenic epitope or binding
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified ribonucleic acid (RNA) or
  • DNA deoxyribonucleic acid
  • Polynucleotides include,
  • polynucleotide refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide also includes DNAs or RNAs containing one or more modified
  • Modified bases include, for example, tritylated bases and unusual bases such as
  • polynucleotide embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide” also embraces
  • oligonucleotides relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids
  • Polypeptide refers to both short chains, commonly referred to as
  • Polypeptides may contain amino acids other than the 20 gene-encoded
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques, which are well known in the art. Such modifications are described in
  • Modifications may occur anywhere in a polypeptide, including the peptide
  • a given polypeptide may contain
  • Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translation natural processes or
  • Modifications include acetylation, acylation,
  • heme moiety covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,
  • Variant refers to a polynucleotide or polypeptide that differs from a
  • variant of a polynucleotide differs in nucleotide sequence from another, reference
  • Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination.
  • a substituted amino acid sequence may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • identity also means the degree of sequence
  • Preferred methods to determine identity are designed to give the largest match
  • sequences can be determined by using analysis software (i.e., Sequence Analysis
  • polynucleotide sequence of the present invention may be
  • nucleotide alterations may include up to a certain integer number of nucleotide alterations as compared to
  • nucleotide deletion substitution, including transition and transversion, or
  • alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleotide alterations is determined by multiplying the total number of nucleotides in
  • polypeptide may alter the polypeptide encoded by the polynucleotide following such
  • polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of
  • a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the
  • degenerate nucleotide sequence denotes a sequence of nucleotides
  • expression vector is used to denote a DNA molecule, linear or circular, which includes a segment encoding a polypeptide of interest operably linked
  • promoter and terminator sequences include promoter and terminator sequences, and may also include one or more origins
  • Expression vectors are generally derived from plasmid or viral DNA, or may
  • isolated when applied to a polynucleotide, denotes that the
  • polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • cDNA are those that are separated from their natural environment and include cDNA and
  • isolated polynucleotide molecules of the present invention are free of
  • isolated polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal
  • the isolated polypeptide is substantially free of other amino acids
  • polypeptides particularly other polypeptides of animal origin. It is preferred to have a wide range of polypeptides of animal origin. It is preferred to have a wide range of polypeptides of animal origin. It is preferred to have a wide range of polypeptides of animal origin. It is preferred to have a wide range of polypeptides of animal origin. It is preferred to have a wide range of polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more
  • operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended pu ⁇ oses
  • transcription initiates in the promoter and proceeds through the coding segment
  • promoter is used herein for its art-recognized meaning to denote a
  • Promoter sequences are commonly, but not
  • secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide
  • the larger polypeptide is commonly cleaved to remove the secretory
  • condition and “conditions” denote a state of health that can be
  • axonal degeneration and nervous system disorder processes that involve the axonal degeneration and nervous system disorder.
  • the processes that involve axonal degeneration and nervous system disorder are discussed below, and are to be included as condition(s) that can be treated by
  • the term "host” includes both humans, mammals (e.g., cats, dogs, horses,
  • predisposed to condition(s) can be defined as hosts that do not exhibit overt
  • beneficial or desired clinical results include, but are not limited to, alleviation of
  • treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • modulate and “modulation” denote adjustment or regulation of the activity of a compound or the interaction between one or more compounds.
  • phenotype means a property of an organism that can be detected, which is usually produced by interaction of an organism's genotype and environment.
  • open reading frame means the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • Codon means a specific triplet of mononucleotides in the DNA chain. Codons correspond to specific amino acids or to start and stop of translation by
  • wild-type means that the nucleic acid fragment does not comprise
  • wild-type protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.
  • chimeric protein means that the protein comprises regions which
  • the protein comprises wild-type regions from one protein and wild-type regions from another
  • mutation means a change in the sequence of a wild-type nucleic acid sequence or a change in the sequence of a peptide.
  • Such mutation may be a point mutation such as a transition or a transversion.
  • the mutation may be a deletion, an
  • terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Similarly, unless specified
  • the lefthand end of single-stranded polynucleotide sequences is the 5' end; the lefthand direction of double-stranded polynucleotide sequences is referred to as
  • agent is used herein to denote a chemical compound, a mixture of
  • a biological macromolecule a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly
  • Wallerian degeneration is model of axonal degeneration.
  • polypeptides and polynucleotides e.g., Wallerian degeneration (Wld s ) gene and the corresponding protein
  • the polypeptides and polynucleotides is inserted into a vector or otherwise introduced to
  • the host which is used to infect an host's tissue so that the gene expresses the Wld s
  • polypeptides and polynucleotide of the present invention can be any polypeptide that is significantly less than that occurring in organisms without the Wld s gene.
  • the polypeptides and polynucleotide of the present invention can be any polypeptide that is significantly less than that occurring in organisms without the Wld s gene.
  • the polypeptides and polynucleotide of the present invention can be any polypeptide that is significantly less than that occurring in organisms without the Wld s gene.
  • disorders such as, but not limited to, degenerative, heritable, and
  • process and polypeptides and polynucleotide of the present invention include but are
  • peripheral and specific neuropathies not limited to, peripheral and specific neuropathies, direct axonal injury, trauma and
  • ischemia stroke, Alzheimer's disease, Charcot-Marie-Tooth, chronic spinocerebellar degeneration and primary demyelinating diseases, such as for example multiple
  • embodiments of the present invention include
  • polypeptide are designated "WLDS polypeptides", while embodiments of the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids having the amino acids
  • WLDS polynucleotides are designated "WLDS polynucleotides.”
  • embodiments of the present invention provide WLDS polynucleotides, including DNA and RNA molecules that encode the WLDS polypeptides.
  • SEQ ID NO:l is a degenerate polynucleotide
  • nucleic acid sequence that encompasses polynucleotide that encodes the WLDS polypeptide of SEQ ID NO:2.
  • the degeneracy of nucleic acid is well known in the art and as such
  • amino acids as well as possible codons that can be associated with the amino acids.
  • degenerate codon for example, the degenerate codon for
  • WSN serine
  • AGR arginine
  • polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant amino acid sequences.
  • variant sequences by reference to the amino acid sequence of SEQ ID NO:2.
  • variant WLDS polynucleotides that encode polypeptides that can treat a
  • WLDS polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Table 1.
  • those of skill in the art can use standard software to devise WLDS variants (i.e.,
  • polynucleotides and polypeptides based upon the polynucleotide and amino acid
  • polynucleotides of the present invention can include DNA and RNA molecules.
  • RNA is isolated from a tissue or cell that produces WLDS RNA. Such tissues and cells can be
  • RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient
  • Polynucleotides encoding WLDS polypeptides are then identified and isolated by hybridization or PCR, for example.
  • WLDS polynucleotides can also be synthesized using techniques widely
  • polypeptide of SEQ ID NO:2 The term “substantially homologous” is used herein to denote polypeptides having about 50%, about 75%, about 85%, and preferably about 90% sequence identity to the sequence shown in SEQ ID NO:2. Percent sequence identity is determined by conventional methods as discussed above. In addition,
  • homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, and/or additions. These changes are preferably of
  • homologous polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the homologous
  • polypeptide and the affinity tag are polypeptide and the affinity tag.
  • embodiments of the present invention include polynucleotides that
  • Conservative amino acid substitutions can be based upon the chemical properties of the amino acids. That is,
  • variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO:2., in which an alkyl amino acid is substituted for an alkyl amino acid in a WLDS
  • an aromatic amino acid is substituted for an aromatic amino acid in a
  • WLDS polypeptide a sulfur-containing amino acid is substituted for a sulfur-
  • a hydroxy-containing amino acid is
  • amino acid is substituted for an acidic amino acid in a WLDS polypeptide, a basic
  • amino acid is substituted for a basic amino acid in a WLDS polypeptide, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in a WLDS polypeptide.
  • common amino acids for example, a "conservative amino acid
  • substitution is illustrated by a substitution among amino acids within each of the
  • “conservative amino acid” variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase
  • variant WLDS polypeptides can be identified by the ability to
  • WLDS polypeptides having conservative amino acid variants can also comprise non-naturally occurring amino acid residues.
  • amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline,
  • methylproline 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenyl-alanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
  • nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out
  • Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated
  • E. coli cells are cultured in the absence of a natural amino acid that is to
  • amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine.
  • amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.
  • Chemical modification can be combined with site-directed mutagenesis to further
  • Essential amino acids in the polypeptides of the present invention can be any amino acids residues.
  • Essential amino acids in the polypeptides of the present invention can be any amino acids residues.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as
  • Variants of the disclosed WLDS polypeptide can be generated through DNA
  • variant polypeptides are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by
  • mutagenesis and assay provides for rapid "evolution" of sequences by selecting for
  • Mutagenesis methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays. Mutagenized DNA molecules that encode active polypeptides
  • Such polypeptides may also include additional polypeptide segments as generally disclosed herein.
  • a fusion protein consists essentially of a first portion and a
  • the first portion includes a polypeptide comprising a sequence of amino acid residues that is at least about 50%, about 75%, about 85%, and preferably about 90% identical in amino acid sequence to
  • SEQ ID NO:2 and the second portion is any other heterologous non WLDS
  • polypeptide The other polypeptide may be polypeptides that do not inhibit the
  • WLDS polypeptide such as a signal peptide to facilitate secretion of
  • the fusion protein or an affinity tag.
  • WLDS polypeptides of the present invention including full-length
  • polypeptides can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous
  • DNA and grown in culture include bacteria, fungal cells, and cultured higher
  • Eukaryotic cells particularly cultured cells of multicellular
  • WLDS polynucleotides sequence encoding WLDS polypeptides are operably linked to other genetic elements required for its expression, generally
  • vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within
  • selectable markers may be provided on separate vectors, and
  • replication of the exogenous DNA may be provided by integration into the host cell genome.
  • Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art.
  • a WLDS polypeptide To direct a WLDS polypeptide into the secretory pathway of a host cell, a
  • secretory signal sequence also known as a leader sequence, signal sequence, prepro sequence or pre sequence
  • the secretory signal sequence is provided in the expression vector.
  • sequence may be derived from another secreted protein or synthesized de novo.
  • secretory signal sequence is operably linked to the WLDS polynucleotide sequence, (i.e., the two sequences are joined in the correct reading frame and positioned to direct
  • Secretory signal sequences are commonly positioned 5' to the polynucleotide
  • sequences may be positioned elsewhere in the polynucleotide sequence of interest
  • a purified polypeptide is substantially free of other polypeptides, particularly other
  • Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid
  • Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE
  • chromatographic media include those
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads,
  • cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used.
  • These supports may be modified with reactive groups
  • chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation,
  • the WLDS polypeptides of the present invention can be isolated by
  • IMAC adso ⁇ tion
  • Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution,
  • glycosylated proteins include purification of glycosylated proteins by lectin affinity chromatography and ion
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., Glu-Glu tag
  • WLDS polypeptides or fragments thereof may also be prepared through
  • WLDS polypeptides may be prepared as
  • viruses for this pu ⁇ ose include adenovirus, he ⁇ esvirus, vaccinia
  • Adenovirus a double-stranded DNA virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus
  • adenovirus system offers several advantages: adenovirus
  • adenoviruses can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • the WLDS polypeptide can be inserted into portions of the adenovirus by
  • the WLDS polypeptide may be
  • the essential El gene has been
  • adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells.
  • the host's tissue e.g., liver
  • the host's tissue will express and process (and, if a secretory
  • liver, and effects on the condition to be treated can be determined.
  • WLDS polypeptides can also be used to prepare antibodies that may inhibit axonal degeneration.
  • the WLDS polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • Suitable antigens would be the WLDS polypeptide encoded by SEQ ID NO:2, for example.
  • Antibodies generated from this immune response can be isolated and purified as
  • the immunogenicity of a WLDS polypeptide may be increased
  • an adjuvant such as alum (aluminum hydroxide) or Freund's
  • Polypeptides useful for immunization also include
  • fusion polypeptides such as fusions of WLDS or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a
  • macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum
  • BSA albumin
  • tetanus toxoid tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-binding
  • polyclonal antibodies polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments.
  • Genetically engineered intact antibodies or fragments such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant
  • half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • antibody display libraries in phage or similar vectors for instance, through use of
  • WLDS polypeptides binding domains can be obtained by screening random
  • peptide libraries displayed on phage phage display
  • bacteria such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a
  • WLDS polynucleotide probes or primers can be RNA
  • DNA can be either cDNA or genomic DNA.
  • DNA can be either cDNA or genomic DNA.
  • polynucleotide polynucleotide
  • probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, between about 17 and 25 nucleotides, and between about 25 and 36 nucleotides. Probes and primers are generally synthetic
  • oligonucleotides may be generated from cloned cDNA or genomic sequences or its complements.
  • Analytical probes will generally be about 20 nucleotides in length
  • nucleotides in length preferably 15 or more nucleotides, more preferably 20-
  • Probes can be labeled to provide a detectable signal, such as with an enzyme,
  • biotin a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes,
  • WLDS polypeptides may be used within diagnostic systems to detect axonal
  • Altered levels of WLDS polypeptides may be indicative of pathological conditions, as defined above.
  • RNA In a basic assay, a single-stranded probe molecule is incubated with RNA,
  • Nucleic acid probes can be any nucleic acid probes.
  • radioisotopes such as P or S.
  • WLDS WLDS
  • polynucleotide can be detected with a nonradioactive hybridization method (Isaac
  • nonradioactive detection is achieved by enzymatic conversion
  • Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • WLDS polynucleotide probes are also useful for in vivo diagnosis. As an
  • 18 F-labeled WLDS polynucleotides can be administered to a subject and
  • PCR reverse transcriptase kinase kinase kinase
  • Standard techniques for performing PCR are well-known (Mathew (Ed.), Protocols in Human Molecular Genetics, (Humana Press, Inc. 1991), White (Ed.), PCR Protocols: Current Methods
  • PCR amplification products can be detected using a variety of approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be
  • the WLDS polypeptides and the pharmaceutically acceptable salts and solvates thereof can be prepared in a physiologically acceptable formulation, such as
  • the WLDS polypeptide can be combined with a
  • the WLDS polynucleotide for the WLDS polypeptide can delivered in a vector for continuous administration using gene therapy techniques.
  • the vector may be administered in a vehicle having specificity for a target site, such
  • WLDS compositions may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular,
  • WLDS compositions may conveniently be presented in unit dosage form and
  • Such techniques include
  • WLDS compositions suitable for oral administration may be presented as
  • WLDS compositions suitable for topical administration in the mouth include
  • lozenges having the ingredients in a flavored basis, usually sucrose and
  • acacia or tragacanth pastilles, having a WLDS polypeptide in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes, having one or more of
  • compositions of the present invention administered in a suitable liquid carrier.
  • WLDS compositions suitable for topical administration to the skin may be
  • WLDS compositions for rectal administration may be presented as a
  • suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • solid include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is taken, (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose).
  • the carrier is a liquid (for example, a nasal spray or as nasal
  • WLDS polypeptides can be admixed in an aqueous or oily solution, and
  • WLDS compositions suitable for vaginal administration may be presented as
  • WLDS compositions suitable for parenteral administration include aqueous
  • non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may
  • WLDS compositions may be
  • vials may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately
  • enteral or parenteral administration can be used to fabricate the compositions.
  • Gelatin lactose, starch, magnesium stearate, talc, vegetable and animal fats and oils,
  • gum gum, polyalkylene glycol, water, or other known carriers may all be suitable as carrier
  • WLDS compositions may be used as the active ingredient in combination with
  • one or more pharmaceutically acceptable carrier mediums and/or excipients As used herein, one or more pharmaceutically acceptable carrier mediums and/or excipients. As used herein, one or more pharmaceutically acceptable carrier mediums and/or excipients. As used herein, one or more pharmaceutically acceptable carrier mediums and/or excipients. As used herein, one or more pharmaceutically acceptable carrier mediums and/or excipients. As used herein, one or more pharmaceutically acceptable carrier mediums and/or excipients.
  • pharmaceutically acceptable carrier medium includes any and all carriers, solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid
  • binders lubricants, adjuvants, vehicles, delivery systems, disintegrants, absorbents,
  • preservatives surfactants, colorants, flavorants, or sweeteners and the like, as suited to
  • WLDS compositions may be combined with pharmaceutically
  • compositions comprising, and, optionally, sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • sustained-release matrices such as biodegradable polymers
  • acceptable excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • a therapeutically effective amount of WLDS compositions When used in the above or other treatments, a therapeutically effective amount of WLDS compositions may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt.
  • a “therapeutically effective amount" of a WLDS By a "therapeutically effective amount" of a WLDS
  • polypeptide it is meant a sufficient amount of one or more of the components to treat a
  • compositions will be decided by the attending physician within the scope of sound
  • the specific therapeutically effective dose level for any particular host will depend upon a variety of factors, including for example, the disorder being treated and the severity of the disorder; activity of the specific composition employed;
  • the specific composition employed the age, body weight, general health, sex and diet of the patient; the time of administration; route of administration; rate of excretion of
  • WLDS compositions are preferably formulated in dosage unit form for ease of
  • Dosage unit form refers to
  • WLDS composition a physically discrete unit of WLDS composition appropriate for the host to be treated.
  • Each dosage should contain the quantity of WLDS compositions calculated to produce
  • the desired therapeutic affect either as such, or in association with the selected
  • preclinical testing is usually quite conservative.
  • a standard measure of toxicity of a drug in preclinical testing is the percentage of animals (rodents) that die because of treatment.
  • the dose at which 10% of the animals die is known as the LDj 0 , which
  • the adjustment for body surface area includes host
  • a therapeutically effective dose level will depend on many variables
  • compositions at relatively low levels and increase the dosage until the desired effect
  • WLDS compositions may be used in combination with other WLDS compositions, medicines and/or procedures for the treatment of the conditions
  • WLDS compositions may be used with a sustained-release matrix.
  • a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution.
  • the matrix is acted upon by enzymes and body fluids.
  • sustained-release matrix desirably is chosen from biocompatible materials such as
  • polylactides polylactic acid
  • polyglycolide polymer of glycolic acid
  • polylactide co-glycolide copolymers of lactic acid and glycolic acid
  • polyanhydrides polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides,
  • poly(ortho)esters polypeptides, hyaluronic acid, collagen, chondroitin sulfate,
  • carboxcylic acids fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
  • biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or
  • WLDS compositions may also be administered in the
  • liposomes are generally derived from
  • Liposomes are formed by mono- or multi- lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non- toxic, physiologically-acceptable and metabolizable lipid capable of forming
  • liposomes can be used.
  • the liposome can contain, in addition to WLDS compositions, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are described below.
  • toxin-induced axonal degeneration is a calcium-dependent, calpain mediated process, and that pharmacological inhibition of calpains is protective.
  • Tissue culture dishes for example, BD FalconTM tissue culture dishes, manufactured by BD Biosciences Discovery Labware in Bedford, Massachusetts, United States, and commercially available from American Scientific and Industrial Supplies of Radnor, Pennsylvania, United States
  • Tissue culture dishes for example, BD FalconTM tissue culture dishes, manufactured by BD Biosciences Discovery Labware in Bedford, Massachusetts, United States, and commercially available from American Scientific and Industrial Supplies of Radnor, Pennsylvania, United States
  • rat tail collagen Type 1 , available from Becton Dickinson
  • DMEM formerly GIBCO brand, now manufactured commercially available from Invitrogen Co ⁇ oration, Carlsbad, California, United States
  • the dishes are washed twice with PBS buffer (pH 7.4), filled with 550 ⁇ l medium and pre- incubated at 37 C for at least 2 hours.
  • Standard media can be, for example, MEM (GIBCO, free calcium 1.8 mM), supplemented with 1 % N2 supplement (GIBCO), 7S NGF (manufactured by Alomone Labs, Jerusalem, Israel) 100 ng/ml, and 1.4 mM L- glutamine (manufactured by and commercially availabe from manufactured by and commercially available from Sigma- Aldrich Company, St. Louis, Missouri, United States). Calcium-free medium is prepared in the same manner, replacing MEM with S-MEM (GIBCO).
  • DAB diaminobenzidine
  • Vincristine Neuropathy DRGs are allowed to mature for 5 days (with a media change on day 3) creating a lush halo of neurites. This method of allowing neuritic extension to proceed before addition of a neuro toxin tests the effect of the toxin on established neurites as opposed to the effect on primary neuritic outgrowth.
  • the in vitro paradigm is partially comparable to the clinical situation in that an "established" peripheral nervous system is exposed to a toxic agent.
  • DRGs After 6 days of treatment (11 days in culture) immunostained DRGs are quantitated for degree of axonal degeneration. Images of the DRGs and neurites are captured onto disk using a computerized video imaging system, and are analyzed using NIH Image version 1.61. DRG areas are calculated by tracing the outside circumference of the remaining culture halo. The length of the longest neurite of each DRG is measured from the center of the DRG to the distal end of the neurite, so that cultures without remaining neurites still have positive values. These quantitative data are subjected to ANOVA, with post-test correction for multiple comparisons. 1.1.3. Wallerian Degeneration
  • Vincristine induced axonal degeneration To determine the sensitivity of cultured DRG neurites to vincristine, drug concentrations of 0.01 to 4 ⁇ M are added to 5 day-old cultures. Axonal degeneration occurs very quickly (within 1-3 days) at concentrations 0.05 ⁇ M. At 0.01 ⁇ M, the process of axonal degeneration is relatively slow, allowing for discrimination of changes between treated and untreated cultures over time. Signs of degeneration including axonal beading and segmentation are identified as early as twelve hours after exposure to vincristine. At 24 to 48 hours of exposure, the distal portions of axons show significant pathological changes.
  • Degeneration proceeds in a distal to proximal pattern along neuritic bundles until fibers are completely replaced by axonal debris at 5 to 6 days ( Figure 1).
  • the neuronal cell bodies also show changes, with the DRG becoming smaller over the 6 day time course. These changes are not quantified herein.
  • Vincristine exposure produces a progressive reduction in the area of the DRG neuritic halo (Table 4). At day 0, the DRG area is 28.98 ⁇ 1.76 mm 2 and after three days exposure to vincristine is reduced by 76.6% to 6.78 ⁇ 1.06 mm 2 (P ⁇ 0.01).
  • the DRG area is 2.45 ⁇ 0.74 mm 2 , and at 6 days 0.16 ⁇ 0.61 mm 2 , representing respectively a 91.5% and a 97.9% reduction in area.
  • axons continued to grow during the experimental period, increasing to 33.62 ⁇ 2.98 mm 2 on day 3, 41.94 ⁇ 1.64 mm 2 on day 4, and 45.14 ⁇ 4.35 mm 2 on day 6.
  • the area of DRG halo is 155.8 % larger than at treatment day 0.
  • the neuroprotective effects of a low calcium environment lead us to test whether calpains are also important in the pathogenesis of vincristine neuropathy.
  • AK295 additive of the experimental calpain inhibitor AK295 is effective in preventing axonal degeneration at doses of either 50 ⁇ M (Table 4) or 10 ⁇ M (not shown), and is ineffective at 1 ⁇ M (not shown).
  • the neuroprotective effect of AK295 is equal to that of either 2 mM EGTA or calcium- free media.
  • the low calcium environment or treatment with AK295 does not provide complete protection against axonal degeneration.
  • a graded effect is revealed. Neurites exposed to vincristine but maintained in the neuroprotective media stop growing (Figure 2), and show mo ⁇ hologic changes typical of axonal degeneration (Figure 3). These changes are, however, both qualitatively and quantitatively less severe than those seen in cultures treated with vincristine alone.
  • Wallerian Degeneration Axotomized neurites are completely degenerated by 72 hours after transection. Addition of EGTA at the time of axotomy provides significant protection against Wallerian degeneration (Figure 4). Addition of the calpain inhibitor AK295 is as protective as EGTA. A direct comparison of the protective effects of AK295 in vincristine neuropathy and Wallerian degeneration is demonstrated in Figure 5. The preservation of axons with AK295 is not a subtle finding, and is seen easily in these low power photomicrographs.
  • the pathogenesis of vincristine neuropathy is thought to be a consequence of its primary antineoplastic function as a mitotic spindle inhibitor.
  • Experimental data suggest that vincristine alters the structure of axonal microtubules, leading to abnormalities in fast axonal transport.
  • Dysfunctional axonal transport is a major theory for the pathogenesis of a variety of toxic neuropathies, and is considered a causative factor in the case of vincristine.
  • Intoxication of laboratory animals, or direct exposure of nerves to vincristine has been reported to cause alterations in the structure and shape of axonal microtubules, shortening of microtubule lengths, or changes in microtubule distribution within the axon.
  • Calpain activation has also been implicated in the pathogenesis of a wide variety of neurologic disorders such as stroke, head and spinal cord trauma, and even Alzheimer disease. It is believed that elevated intracellular calcium in injured cells leads to pathologic activation of calpains, and results in neuronal/ axonal degeneration. In the case of vincristine neuropathy, altered axonal transport may lead to elevations of intracellular calcium and activation of calpains through metabolic compromise, alteration of calcium conductance, or loss of membrane integrity. The therapeutic potential of calpain inhibitors is based on their effectiveness in preventing axonal degeneration, but may be somewhat limited by their inherent cellular toxicity.
  • Calpains are ubiquitous cytosolic enzymes that are putatively involved in a number of normal cellular functions. Certainly, chronic calpain inhibition has the potential for interfering with these functions. Previous uses of AK295 have been in acute neurologic injuries, where drug exposure has been limited to single or short term dosing. In our DRG cultures we find that chronic calpain inhibition is neither toxic to neurites, nor does it affect their normal growth.
  • Calpain inhibition may be a reasonable strategy for preventing axonal degeneration and preserving neurologic function in acute injuries such as for example, but not limited to, stroke and trauma, or in chronic disorders such as for example, but not limited to, diabetes mellitus, hereditary neuropathies, or multiple sclerosis.
  • concentrations are: Vin 0.01 ⁇ M, AK295 50 ⁇ M, EGTA 2 mM. Numbers in parentheses are the number of experiments performed.
  • DRG Dorsal root ganglion
  • Tissue culture dishes (BD FalconTM) of 35x10mm were pre-coated with rat tail
  • DRGs were dissected from newborn mice (C57BL/6, Charles River and WLD S , breeding colony maintained at the Emory
  • MEM MEM
  • N2 GABA
  • 7S NGF 100 ng/ml
  • Vincristine sulfate salt (Sigma) was dissolved in culture medium, aliquoted
  • DRGs were allowed to mature for 5 days to create a lush halo of neurites. This method of allowing neuritic extension to proceed before addition of
  • each DRG served as its own control by normalizing data at days 4, 8, and 10 to the
  • Table 5 shows the comparisons of axonal lengths and halo areas between C57BL/6 and Wld s . Note that there were no differences between the cultures at the time of vincristine exposure (day 0). Except for the lowest dose (0.01 ⁇ M) at the earliest evaluation time (4 days),
  • Figure 3 compares graphically the normalized data from C57BL6 and Wld
  • Wld s mutation that slows Wallerian degeneration after axotomy also provides
  • Vincristine neurotoxicity provides a good model for non-traumatic neuropathy because it is clinically relevant (peripheral
  • the C57BL/6 is the parent strain for the Wld s , and is so closely genetically related that tissue grafts between C57BL/6 and
  • neurotrophin deprivation involved activation of caspases, whereas axonal
  • anti-apoptotic Bcl-2 protein protects neuronal cell bodies from degeneration but not
  • axonal death likely exists that is distinct from those involved in neuronal death. Since axonal degeneration is such an important feature of neurologic disease and
  • Wallerian degeneration are that axonal neurofilaments from the Wld are relatively resistant to calcium-mediated degradation and specifically to calpain.
  • neuropathy like other neuropathic disorders, is a slowly evolving process, making it amenable to early detection, treatment, and prevention. Unlike axotomy-induced
  • axonal degeneration where rapid degradation and removal of the distal nerve stump may be preferable for initiation of the regenerative process, axonal degeneration in
  • Wld gene may provide a novel approach to prevention or treatment of axonal
  • mice C57BL/6 (wild- type) mice and Sprague Dawley rats were obtained from Jackson
  • Adenovirus was generated as previously described. Briefly, the modified transfer vector was linearized with Nhel for preparation of the recombinant
  • adenovirus adenovirus
  • pAdLink.l CMV/Wld /IRES/GFP linearized transfer DNA
  • Recombinant adenovirus/Wld s /IRES/GFP was selected by checking
  • Adenovirus titers were determined by TCID-50 (Tissue
  • the Wld s gene was introduced into rat DRG cells by replacement of standard media with media containing recombinant adenovirus (10 9 particles/cc) expressing c either the lacZ gene (control) or the Wld gene. Cultures had extended neurites for 6
  • the Wld s polyclonal antibody was produced in a New Zealand rabbit using
  • the immunogen was a peptide sequence (YLVPDLVQEYTEK) unique to the Wld s mutant protein conjugated to thyroglobulin. Rabbit serum was
  • Positive controls were HEK293 A or DRG cells infected with adenovirus expressing the Wld protein.
  • Negative controls were cells infected with
  • DRG Dorsal root ganglia
  • Standard media was MEM (GIBCO), supplemented with 1% N2 supplement
  • adenovirus cultures were grown for either 5 (mouse) or 6 (rat) days to allow for a rich
  • Wld s were compared to all succeeding days using ANOVA for repeated measures.
  • Rat DRG cultures were used to test whether expression of the Wld s transgene
  • mice showed growth arrest for the first 10 days, and then resumed growth to reach 125% and 150% of control values for length and area, respectively (Figure 9B; Figure
  • adenovirus similarly resulted in expression of the Wld s protein as demonstrated by
  • the Wld s protein could be shown to colocalize
  • Figure 13 depicts the relative resistance to axonal degeneration of cultures expressing the Wld gene.
  • ubiquitin degradation pathway that has been shown to be important in several models of cell death and, specifically, neurodegeneration.
  • chemotherapeutic drugs including vincristine, paclitaxel, cisplatin, and suramin
  • the Wld s gene may provide a beneficial effect for patients
  • lacZ are cultures exposed to adenoviruses expressing the Wld and lacZ genes
  • Tables 7 A and 7B that follow depict values for DRG axonal length and area in vincristine treated cultures. Row “a” shows are uninfected cultures treated with
  • Rows “b” and “c” are infected with adenoviruses expressing the Wld and lacZ genes, respectively, and treated with vincristine. All adenoviruses expressed

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Abstract

L'invention concerne des polypeptides qui peuvent protéger les axones de la dégénérescence, ainsi que des méthodes d'utilisation de ces polypeptides. Elle concerne aussi des polynucléotides qui peuvent protéger les axones de la dégénérescence, ainsi que des méthodes d'utilisation de ces polynucléotides. Elle concerne encore des polynucléotides codant pour les polypeptides de l'invention. Elle concerne enfin des compositions pharmaceutiques destinées à traiter ces troubles.
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Title
CONFORTI L ET AL: "A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 97, no. 21, 10 October 2000 (2000-10-10), pages 11377-11382, XP002953257 ISSN: 0027-8424 & DATABASE GENBANK 2 October 2000 (2000-10-02), CONFORTI; ET AL: "Mus musculus UFD2/D4COLE1E fusion protein mRNA" XP002953260 retrieved from GENBANK Database accession no. AF260924 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 2001, COLEMAN M P ET AL: "Protecting axons: The molecular basis of slow Wallerian degeneration" XP002299951 Database accession no. PREV200200038753 & SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 27, no. 2, 2001, page 2623, 31ST ANNUAL MEETING OF THE SOCIETY FOR NEUROSCIENCE; SAN DIEGO, CALIFORNIA, USA; NOVEMBER 10-15, 2001 ISSN: 0190-5295 *
FERNANDO S ET AL: "Characterization of the human homologue of a candidate gene for slow Wallerian degeneration" AMERICAN JOURNAL OF HUMAN GENETICS, AMERICAN SOCIETY OF HUMAN GENETICS, CHICAGO, IL, US, vol. 67, no. SUPPLEMENT 2, October 2000 (2000-10), page 178, XP002953261 ISSN: 0002-9297 *
See also references of WO03029414A2 *
WANG M-S ET AL: "Pathogenesis of axonal degeneration: parallels between Wallerian degeneration and vincristine neuropathy" JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY, NEW YORK, NY, US, vol. 59, no. 7, July 2000 (2000-07), pages 599-606, XP002953258 ISSN: 0022-3069 *
WANG M-S ET AL: "The gene for low Wallerian degeneration (WldS) is also protective against vincristine neuropathy" NEUROBIOLOGY OF DISEASE, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 8, 20 December 2000 (2000-12-20), pages 155-161, XP002953259 ISSN: 0969-9961 *

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