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WO1999008533A1 - Regeneration d'axones dans le systeme nerveux central - Google Patents

Regeneration d'axones dans le systeme nerveux central Download PDF

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WO1999008533A1
WO1999008533A1 PCT/US1998/016794 US9816794W WO9908533A1 WO 1999008533 A1 WO1999008533 A1 WO 1999008533A1 US 9816794 W US9816794 W US 9816794W WO 9908533 A1 WO9908533 A1 WO 9908533A1
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rho
collapsin
adeno
exoenzyme
racl
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PCT/US1998/016794
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Stephen M. Strittmatter
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Yale University
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Priority to EP98943195A priority Critical patent/EP1011330A4/fr
Priority to AU91042/98A priority patent/AU735607B2/en
Priority to JP2000509293A priority patent/JP2001515018A/ja
Priority to CA002300878A priority patent/CA2300878A1/fr
Publication of WO1999008533A1 publication Critical patent/WO1999008533A1/fr

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • 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
    • C12N2799/028—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a herpesvirus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions

  • This invention relates to therapies for promoting central nervous system axon growth, including adenoviral-mediated gene therapy that results in a modification of growth cone signal transduction protein function.
  • the treatment methods are particularly directed to recovery from acute or chronic spinal cord injury, traumatic brain injury, and white matter stroke.
  • NI-35 35 kDa
  • 205 kDa into liposomes after SDS-PAGE 35 kDa
  • NI-35 inhibits axonal extension and induces growth cone collapse (id. , and Bandtlow, et al., 1993).
  • An antibody to NI-35 promotes some axonal regeneration after spinal cord transection, demonstrating the physiological relevance of this inhibition (Schnell, et al., 1994).
  • Transplantation of olfactory ensheathing cells at the site of spinal cord injury can also promote a degree of axonal regeneration, presumably by substituting for the oligodendrocytes which normally produce inhibitory compounds (Li, et al., 1997; Imaizumi, et al., 1998; Mukhopadhyay, et al. , 1994).
  • CNS myelin inhibition of neurite growth is also mediated in part by myelin associated glycoprotein (MAG; Mukhopadhyay, et al. , 1994; McKerracher, et al., 1994).
  • MAG myelin associated glycoprotein
  • MAG may or may not contribute to myelin inhibition of axonal regeneration (Bartsch, et al., 1995; Schafer, et al. , 1996). If the inhibitory effects of CNS myelin on axon outgrowth can be prevented in vivo, then increased recovery from spinal cord trauma and other instances of CNS axonal injury is likely to occur.
  • Neuronal growth cones possess the sensory apparatus to distinguish amongst innumerable potential pathways and targets during nervous system development and regeneration (for a review, see Strittmatter, 1995).
  • Extracellular signals induce changes in the actin-based cytoskeleton of the growth cone and hence its morphology and motility. The molecular mechanisms whereby extracel- lular clues are transduced to cytoskeletal rearrangements are defined poorly.
  • the semaphorin/collapsin family of proteins has been recognized as one important negative regulator of axon outgrowth and terminal arborization (Luo, et al., 1993; Kolodkin, et al. , 1992, 1993).
  • Chick collapsin-1 induces growth cone collapse and a cessation of neurite outgrowth from at least a subset of DRG neurons (Raper and Kapfhammer, 1990; Luo, et a , 1993).
  • Insect semaphorins have a demonstrated in vivo role during axonal pathfinding and synaptic terminal branching (Kolodkin, et al., 1992; Matthes, et al., 1995).
  • rho subfamily of monomeric ras-related GTP- binding proteins have prominent effects on the actin-based cytoskeleton and on cell shape (Hall, 1990; 1994).
  • rho activation has been linked to stress fiber form ation and focal adhesions, racl activation with membrane ruffling and lamelipodia, and cdc42 activation with filopodial formation (Nobes and Hall, 1995).
  • Single amino acid substitutions have been identified which produce constitutively active or dominant negative forms of each of these proteins.
  • the C3 transferase from C. botulinum ADP-ribosylates rho specifically and inactivates the G protein.
  • a downstream target of activated rho has been identified as myosin light chain phosphorylase (Kimura, et al., 1996), and an inhibitor of myosin light chain kinase, KT5926, also blocks LPA-induced neurite retraction (Jalink, et al. , 1994).
  • Rho protein inhibitors may be introduced mechanically to the axons or their non-neuronal support tissue, or introduced by administering replication-deficient adeno, adeno-associated, or herpes viruses that express inhibitors.
  • the inhibitor is C. botulinum C3 exoenzyme; in another it is a chimeric C. botulinum C2/C3 inhibitor.
  • the invention correspondingly provides pharmaceutical compositions containing rho protein inhibitors for the treatment of central nervous system injuries using the methods disclosed herein. Also provided are screens that can be used to detect axon regenerative activity in panels of compounds by assaying for rho inhibitory activity.
  • Figure 1 shows line graphs illustrating that collapsin-1 -induced growth cone collapse is attenuated by KT5926 and PTX.
  • A Two hours prior to the assay, the indicated concentrations of KT5926 were added to the DRG explant culture medium. Low concentrations of KT5926 shifted the collapsin dose response curve to the right by a factor of 5. KT5926 had no direct effect on growth cone collapse in the absence of collapsin- 1. The means from 4-6 separate experiments are shown. For each point, the SEM was less than 10% of the value shown.
  • Figure 2 shows growth cone collapse and neurite outgrowth in DRG neurons triturated with rho subfamily proteins.
  • A The protein preparations used for trituration were separated by SDS-PAGE and stained with Coomassie Blue. The migration of 45, 36, 25 and 21 kDa Mr standards is shown at the right.
  • B DRG neurons were triturated with the indicated proteins at 5 mg/ml for rho family proteins and 0.1 mg/ml for C3 transferase. After 4 hours of culture, growth cone collapse was assessed with (gray bars) or without (solid bars) a 20 min exposure to 200 pM collapsin-His 6 . The data are averages + SEM for 3-9 separate experi- ments.
  • DRG neurons were triturated with the indicated proteins and exposed to collapsin- 1 as described in B. Actin was visualized by staining formalin-fixed cells with TRITC-phalloidin. Magnification, 500 X.
  • DRG neurons were triturated with the indicated proteins at 5 mg/ml for rho family proteins and 0.1 mg/ml for C3 transferase.
  • Figure 3 shows racl in collapsin- 1 regulation of growth cone motility.
  • DRG neurons were triturated with buffer or various concentrations of the indicated G proteins.
  • Growth cone collapse with or without a 20 minute exposure to collapsin-His 6 was determined as in Figure 2. The data are averages + SEM for 2-4 separate experiments.
  • B DRG neurons were triturated with 0 or 2.5 mg/ml N17rac and 0 or 5 mg/ml of the following constitutively active G proteins: B is N17rac, C is N17rac+V14rho, D is N17rac+V12rac, and E is N17rac+V12cdc42; A is buffer. Growth cone collapse was determined in the absence (solid bars) or the presence (gray bars) of 200 pM collapsin-1. Note that V12 rac partially reverses the N17rac inhibition of collapsin-induced growth cone collapse.
  • C After trituration with buffer (•), constitutively active V12rac (O) or dominant negative N17rac ( ⁇ ), growth cone collapse was quantitated for DRG neurons exposed to the indicated concentrations of collapsin.
  • FIG. 4 shows C3 transferase action on DRG neurons.
  • DRG neurons were triturated and cultured as described in Figure 2. The data are averages + SEM for 2-4 separate experiments.
  • A The indicated concentrations of C3 transferase were present during the trituration of DRG neurons. Growth cone collapse in the presence and absence of 200 pM collapsin- 1 was determined as in Figure 2.
  • B After trituration with buffer, 4 ⁇ g/ml C3 transferase, 5 mg/ml V14rho, or both proteins, neurons were exposed to 0 (gray bars) or 200 pM (solid bars) collapsin-His 6 and growth cone collapse was quantitated.
  • B (and C), A is buffer, B is C3, C is V14rho, D is C3+V14rho, E is C3+V12- rac, and E is C3+V12cdc42.
  • C Average total neurite outgrowth per cell triturated as in B was determined after plating with (gray bars) or without (solid bars) the presence of 200 pM collapsin-His 6 .
  • Figure 5 shows the effects of C3 transferase are not blocked by N17rac.
  • DRG neurons were triturated with buffer, 5 mg/ml for N17rac, 0.1 mg/ml for C3 transferase or both proteins. The data are averages + SEM for 3-5 separate experiments.
  • Figure 6 shows that growth cone collapse by myelin or LPA is not blocked by N17rac. DRG neurons were triturated with the indicated proteins as in Figure 2. The data are averages + SEM for 3 separate experiments.
  • A Neurons were cultured for 4 hours and growth cone collapse was assessed after a 30 minute exposure to buffer (solid bars), or CNS myelin extract (5 ⁇ g protein/ml, gray bars).
  • Figure 7 is a model drawing for rho/rac regulation of DRG growth cone function. Three states for DRG growth cones are classified by morphologic appearance, neurite outgrowth rate, rho activation state and racl activation.
  • Figure 8 schematically illustrates an adenovirus transfer vector map illustrating the major elements for expression of C3 exoenzyme or racl together with tau-EGFP.
  • a polycistronic message is encoded: a Kozak translation initiation site and the coding sequence of C3 exoenzyme or of racl ending in a stop sequence is followed by a ribosomal reentry site and a second Kozak translation initiation site and the sequence for a marker protein.
  • the marker consists of a fragment of tau protein for axonal targeting followed by an enhanced fluorescence variant of GFP.
  • Figure 9 is an immunoblot of adenovirus-directed expression of racl mutants.
  • COS-7 cells were infected with recombinant adenoviruses expressing wild type racl (lane 1), V 12 racl (lane 2), N17 racl (lane 3), or no racl protein (lane 4). Analysis of cells 24 hours after infection indicates that the low endoge- nous level of racl is greatly increased by recombinant adeno virus infection.
  • FIG. 10 histologically shows adenovirus-directed expression of C3 exoenzyme.
  • COS-7 cells were infected with recombinant adenovirus expressing GFP (control, top panel) or C3 plus GFP (bottom panel).
  • GFP control, top panel
  • C3 plus GFP bottom panel
  • actin filaments were visualized by rhodamine-phalloidin staining.
  • the altered structure of the C3-expressing cells can be seen. Over 95% of cells were infected in the cultures.
  • Figure 11 shows that recombinant adenovirus expressing C3 prevents myelin-induced inhibition of neurite outgrowth.
  • DRG neuronal cultures were infected with the C3/GFP adenovirus and then cultured for 4 days. Fluorescence microscopy demonstrates expression of the marker protein in cells with a neuronal phenotype (top panel). The cells were trypsinized and replaced without additions, with collapsin- 1, or with extracts of CNS myelin. Note that neurite outgrowth is not decreased by the addition of these inhibitory factors (bottom panel) . In control cultures, collapsin and CNS myelin decreased outgrowth by about 60% .
  • Figure 12 shows expression from the C3 recombinant adenovirus in rat cerebral cortex.
  • the C3/EGFP adenovirus was injected into the cerebral cortex of 8 week old rats. Seven days later, the animals were sacrificed and the brains were examined by fluorescence microscopy. Note the intense cellular EGFP fluorescence at the injection site in the cerebral cortex. Similar results have been obtained with survival times up to 4 weeks. Similar expression is also obtained in DRG after local injection.
  • This invention is based upon the finding that rho protein inhibition promotes axonal regeneration after central nervous system injury by blocking the action of molecules in the injured spinal cord or brain which otherwise stymie functional recovery.
  • axon regeneration is enhanced and growth promoted by administering an effective amount of at least one rho protein inhibitor to a patient in need of such treatment, i.e. , suffering from acute or chronic spinal cord injury, traumatic brain injury, white matter stroke, or other central nervous system injury that damaged axons and disrupted axonal tracts.
  • rho protein inhibitor is meant any inhibitor of rho protein function, analogues that bind to receptors, antibodies to the proteins or protein fragments, and the like. Mixtures of inhibitors can also be employed, as well as inhibitors of rho protein synthesis or stability.
  • Rho protein inhibitors include any inhibitor of rho, rac, cdc42 or other protein in the GTP-binding subfamily.
  • patients include both animals and human beings; the invention has utility in both medical and veterinary applications.
  • Systemic administration can be via any method known in the art such as, for example, oral administration of losenges, tablets, capsules, granules, or other edible compositions; subcutaneous, intravenous, intramuscular, or intradermal administration, e.g., by sterile injections; parenteral administration of fluids and the like.
  • Typical systemic administrations involve the use of the inhibitor dispersed or solubilized in a pharmaceutically acceptable carrier.
  • At least one inhibitor is typically introduced into the axons or their non-neuronal support tissue.
  • Local administration of inhibitors includes, but is not limited to, mechanical introduction of the inhibitor using any means such as injections, by perfusion or injection of the tissue with a composition containing the inhibitor in a pharmaceutically acceptable carrier, often in connection with ingredients that enhance penetration and uptake and/or the inhibitory activity, and by injection of recombinant viruses expressing inhibitors.
  • C. botulinum C3 inhibitor which inhibits rho proteins, is introduced intraneuronally to a patient using a replication-deficient adeno, adeno-associated, or herpes virus that express the C3.
  • Recombinant adenoviruses for example, have been utilized to direct neuronal expression of foreign genes over weeks to months with limited immunologic reaction in the CNS (Choi-Lumdberg, et al. , 1997).
  • Adeno-associated viruses are employed in some embodiments because of their lower toxicity and long-term protein expression.
  • C3 inhibitor An alternate to the C3 inhibitor is a recombinant binary delivery system for the C3 exoenzyme, recently developed using the cell surface and binding components from the C. botulinum C2 toxin (Barth, et al., 1998). The actin ADP-ribosylation activity was deleted from the C2 toxin and the C3 enzyme activity was substituted.
  • This C3 chimeric protein is reported to enter non- neuronal cells at least 100-fold more efficiently than C3 exoenzyme itself. Use of this embodiment can involve direct injection of the molecule into the nervous system and achieve rho inhibition without the potential non-specific effects of viral injection.
  • compositions or formulations of the invention may also contain other carriers, adjuvants, stabilizers, preservatives, dispersing agents, and other agents conventional in the art having regard to the type of formulation in question.
  • the invention provides not only methods for stimulating axon regeneration and corresponding treatments for a variety of central nervous system injuries and pharmaceutical compositions used in the various therapies, but it also provides for screens that can be used to assay for rho protein inhibitory activity.
  • panels of natural or synthetic compounds, including a variety of biological materials are screened for potential in axon regenerative therapy using a rho protein inhibition assay such as racl inhibition. Screening tests may be quantitative or qualitative. Typical methods involve comparing inhibition observed by a panel of test compounds with control inhibition observed, for example, with C. botulinum C3 exoenzyme. The presence of inhibition indicates a potential agent for the stimulation of axon regeneration. Inhibitors identified by the screen can then be further tested, particularly for efficacy in either local and/or systemic administration.
  • This example provides evidence that racl mediates collapsin- 1 -induced growth cone collapse.
  • collapsin- 1/semaphorin III(D) inhibits axonal out- growth by collapsing the neuronal growth cone lamelipodial and filopodial structures.
  • growth cone collapse is associated with actin depolymerization, the small GTP-binding proteins of the rho subfamily was studied for its participation in collapsin-1 signal transduction. Recombinant rho, racl and cdc42 proteins were triturated into embryonic chick DRG neurons.
  • Constitutively active racl increases the proportion of collapsed growth cones, and dominant negative racl inhibits collapsin- 1 -induced growth cone collapse and collapsin- 1 inhibition of neurite outgrowth.
  • DRG neurons treated with dominant negative racl remain sensitive to myelin-induced growth cone collapse. Similar mutants of cdc42 do not alter growth cone structure, neurite elongation or collapsin sensitivity.
  • activated rho has no effect
  • inhibition of rho with botulinum C3 transferase stimulates the outgrowth of DRG neurites.
  • C3-treated growth cones exhibit little or no lamelipodial spreading and are minimally responsive to collapsin- 1 and myelin.
  • G proteins G proteins, collapsin, myelin.
  • Monomeric human G proteins and C. botulinum C3 transferase were produced in bacteria as GST fusion proteins and then treated with thrombin to remove the GST moiety (Nobes and Hall, 1995). Thrombin was removed from the samples by absorption to p-aminobenzamidine-agarose.
  • the following derivatives were produced: wild type rho A (rho), a constitutively active form of rho A with gly at position 14 mutated to val (V14 rho), wild type racl (rac), a constitutively active form of racl with Gly at position 12 mutated to Val (V12 rac), a dominant negative form of racl with thr at position 17 mutated to Asn (N17rac), wild type cdc42 (cdc42), a constitutively active form of cdc42 with Gly at position 12 mutated to Val (V12 cdc42), a dominant negative form of cdc42 with Thr at position 17 mutated to Asn (N17cdc42), and the C3 exoenzyme from C. botulinum (C3).
  • the rho and V14rho proteins contain a substitution of Asn at position 25 for Phe to enhance stability in E. coli.
  • Collapsin-His 6 was prepared as previously described (Goshima, et al. , 1995). Myelin fractions were prepared from bovine brain, and proteins extracted with 2% octylglucoside were tested in growth cone collapse after removal of detergent by dialysis (Igarashi, et al., 1992).
  • neurons were plated in 25 volumes of F12 medium with 10% FBS and 50 ng/ml 7S-NGF on a glass surface precoated sequentially with 100 ⁇ /ml poly-L-lysine and 20 ⁇ /ml laminin.
  • triturated neurons were transferred to serum-free medium (F12 medium with 1 % fatty acid-free BSA and 50 ng/ml 7S-NGF) for 3 hours prior to the growth cone collapse assay. Neurite outgrowth and growth cone collapse. For outgrowth assays, triturated cells were plated for 1.5-2 hours and then agents to be tested were added to the medium.
  • a number of other agents had little or no effect on collapsin- 1 action including tyrosine kinase inhibitors, protein kinase A inhibitors, voltage-sensitive Ca channel blockers and depolarization with KC1.
  • LPA and thrombin are mediated by receptors linked to heterotrimeric G proteins (Jalink, et al. , 1994). Whether recombinant collapsin-1 action also involves trimeric G protein activation was considered.
  • Pertussis toxin (PTX) ADP-ribosylates the a subunit of heterotrimeric G proteins of the Go/i class and blocks their activation by receptors. Growth cone collapse by crude whole brain membrane extracts (BME, which contains collapsin- 1) is blocked by PTX (Igarashi, et al , 1992), but this is due to the cell surface binding properties of PTX rather than its modification of G proteins (Kindt and Lander, 1995).
  • the isolated oligomer B fraction of PTX contains the cell surface binding domain but does not block purified recombinant collapsin- 1 -induced growth cone collapse ( Figure IB).
  • Figure IB The decrease in collapsin- 1 potency by intact PTX suggests that collapsin- 1 action involves heterotrimeric G protein action, strengthening the similarity with LPA and thrombin action.
  • the failure of PTX blockade at higher collapsin- 1 concentrations may be attributable to either PTX-insensitive G proteins or to non-G protein-dependent mechanisms.
  • Oligomer B blockade of BME action may reflect the inhibition of collapsing agents other than collapsin- 1 in the crude extract.
  • Collapsin-1 sensitivity in DRG neurons containing rho subfamily proteins Neurons triturated with rho family members were exposed to collapsin- 1, and then growth cone morphology and neurite extension were examined. In control cultures, exposure to collapsin- 1 for 30 minutes increases the percentage of collapsed growth cones from 15% to 70% (Figure 2B,C). Exposure to collapsin- 1 during the interval from 2-5 hours after plating decreases the extent of outgrowth by 50% (Figure 2D). Collapsin- 1 -induced changes in growth cone collapse and neurite outgrowth are markedly attenuated in neurons treated with dominant negative N17rac ( Figure 2B-D).
  • the collapsin- 1 dose response curve for DRG growth cone collapse is shifted to the right by a factor of 15 (EC50 from 60 pM to 2 nM, Figure 3C).
  • the residual weak effect of collapsin- 1 as a growth cone collapse factor in N17rac-triturated cells may be due to incomplete racl blockade achieved by the trituration method, or to non-racl- dependent collapsin- 1 -induced growth cone collapse mechanisms.
  • trituration with constitutively active V12rac induces collapse of 20% of growth cones ( Figure 2B).
  • the dose response curve for collapsin- 1-induced growth cone collapse is shifted to the left by a factor of 2 following trituration with constitutively active V12rac (EC50 from 60 pM to 30 pM, Figure 3C).
  • racl is an endogenous modulator of collapsin- 1 -induced growth cone collapse, it must be present in the growth cone. Histologic staining for racl demonstrates that the protein is found in growth cones and is present in filopodial structures at the very tip of the growth cone. Thus, the protein is in a position to mediate collapsin- 1 action.
  • Dominant negative racl does not block the effects of rho inactivation.
  • the decrease in growth cone area caused by C3 transferase treatment is associated with increased neurite extension, whereas that caused by collapsin- 1 is associated with decreased extension. It was considered whether dominant negative racl could block the effects of rho inhibition by C3 transferase, as it blocks collapsin- 1 action.
  • C3 transferase and N17rac are cotriturated, DRG neurites resemble C3-triturated neurites ( Figure 5).
  • Rho may act in separate pathway (s) and/or function downstream of racl to regulate growth cone morphology and neurite extension.
  • Inhibitory effects of myelin are not mediated by rho family members.
  • Components of CNS myelin have inhibitory influences on neurite regeneration and alter cultured DRG neuron morphology in a fashion similar to collapsin- 1 (Bandtlow, et al., 1993).
  • Growth cone collapse after exposure to CNS myelin extract is not alt ered by trituration with N17rac ( Figure 6A,B). This indicates that the Ca +2 i-dependent pathway utilized by inhibitory components of myelin (Bandtlow, et al., 1993) is distinct from the racl -dependent pathway utilized by collapsin-1.
  • Rho subfamily members do not have these effects.
  • the presence of racl in the growth cone is consistent with a role in collapsin- 1 signaling. Constitutively active V12rac weakly mimics collapsin- 1 action.
  • V12rac action may be due to (1) the contribution of non-racl dependent mechanisms in collapsin- 1 -induced collapse, (2) the inefficiency of the trituration method or (3) desensitizing mechanisms occurring during the 3-5 hours after trimration.
  • collapsin- 1 action is inhibited by N17rac, the effect of other extracellular proteins which induce the same morphologic changes is not blocked by trituration with N17rac. This indicates that racl is specifically involved in collapsin- 1 action and that the Ca +2 -mediated growth cone collapse induced by components of CNS myelin does not utilize this monomeric G protein.
  • the myosin light chain kinase inhibitor, KT5926 may counteract myosin 1 ight chain phosphorylase regulation by rho (Kimura, et al. , 1996). In so doing, this compound partially reproduces the C3 transferase effect and decreases collapsin- 1 sensitivity.
  • Rhol is capable of reorganizing the actin-based cytoskeleton in non-neuronal cells and of activating a number of protein kinases (Nobes and Hall, 1995; Hall, 1994; Cosco, et al., 1995; Minden, et al. , 1995). Collapsin- 1 -induced changes in cell shape may be mediated by protein kinases such as PAK (Manser, et al., 1994). After activation by racl, such kinases are hypothesized to modulate cytoskeletal function.
  • PAK Manser, et al., 1994
  • This example reports expression and biological activity of recombinant C3 adenovirus used for rho protein inhibition, and the in vivo modulation of neuronal rho protein activity.
  • the C3 exoenzyme from C. botulinum ADP- ribosylates rho specifically and inactivates this G protein.
  • the contribution of this class of G proteins to the regulation of neuronal growth cone motility has only recently come under investigation.
  • lysophosphatidic acid induces rapid neurite retraction through a GPCR (Jalink, et al. , 1994).
  • Recombinant rho, racl and cdc42 proteins were triturated into embryonic chick DRG neurons in Example 1.
  • the response of axons to collapsin- 1 (sema- phorin D/III), a prototypic diffusible axon repellent was examined.
  • Constitatively active racl increases the proportion of collapsed growth cones, and dominant negative racl blocks collapsin-induced growth cone collapse and collapsin inhibition of neurite outgrowth.
  • DRG neurons treated with dominant negative racl remain sensitive to myelin-induced growth cone collapse.
  • Similar mutants of cdc42 do not alter growth cone structure, neurite elongation or collapsin sensitivity. Whereas the addition of activated rho has no effect, inhibition of rho with botulinum C3 exoenzyme stimulates the outgrowth of DRG neurites.
  • C3-treated growth cones exhibit little or no lamelipodial spreading and are insensitive to collapsin or LPA. While CNS myelin extracts reduce outgrowth from control neurons by 50%, this inhibitory extract does not reduce outgrowth from C3-treated cultures.
  • purified protein is loaded into neurons by mechanical means. It does not enter neurons or ADP-ribosylate rho without trituration of individual cells.
  • adeno- and herpes viruses that express the C3 protein were derived. These vectors express C3 together with an enhanced fluorescent version of green fluorescent protein (EGFP, Clontech).
  • EGFP green fluorescent protein
  • Such vectors have allowed expression of other foreign proteins in neurons for 2 weeks (HSV, Carlezon, et al. , 1997) to 2 months (adeno, Choi-Lumbdberg, et al., 1997) without toxic effects.
  • the adeno- viruses are El and E3 deleted, so that they are replication defective (He, et al. , 1998).
  • the herpes virus preparations utilize the amplicon system; C3 and EGFP sequences were inserted into a plasmid containing the immediate early promotor 4/5 of HSV and an HSV packaging site. Recombinant virus preparations are obtained from a packaging cell line after sequential transfection with the amplicon plasmid and infection with a immediate early gene 2 deletion mutant of HSV (Neve, et al. , 1997).
  • Expression cassettes for the protins of interest were constructed in a transfer vector, pQBI-AdBM5, with expression driven from the major late promot- er of adenovirus ( Figure 8; Massie, et al., 1995).
  • the linear transfer vector was co-transfected with the long arm of Clal-cut E1/E3 -deleted viral DNA into HEK 293 cells.
  • the viruses are replication-defective, viral particles can be amplified in these cells because they are stably transfected to express the El protein element which is missing from replication-defective viruses.
  • Viral stocks were plaque-purified twice, enriched by cesium chloride equilibrium centrifuga- tion, and titered.
  • Such viral stocks were utilized to infect COS-7 kidney cells. Within 24 hours of infection, greater than 95% of the cells express the GFP marker protein as judged by the bright green fluorescence of living cells. The expression of the racl proteins was verified by immunoblot analysis ( Figure 9). The expression of the C3 exoenzyme was documented indirectly by observing the change in actin filament staining in the virus-infected cells ( Figure 10). The C3-expressing COS cells exhibit extensive protrusions without the lamelipodial spreading seen in control cultures. The C3 virus was used to modulate rho function in DRG sensory neurons in culture.
  • the C3-expressing virus was injected into the cerebral cortex of 8-week- old male rats, with the goal of infecting cortico-spinal pyramidal neurons.
  • One week after injection large number of cells express the GFP marker (Figure 12).
  • the results show that the C3 viruses do infect sensory neurons in cultare, direct expression of EGFP and render the neurons insensitive to semD and CNS myelin. It is clear that injection of the adenovirus into adult rat cerebral cortex or DRG allows expression of the EGFP marker for at least 3 weeks.
  • Nissl stained preparations there is no major cellular toxicity associated with viral injection.
  • Strittmatter SM (1995) The Neuroscientist 1: 255-258. Strittmatter SM (1996) The Neuroscientist 2: 83-86.

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Abstract

Des thérapies permettant de traiter diverses lésions du système nerveux central y compris les lésions médulaires aigues ou chroniques, les lésions chromatiques du cerveau et les accidents vasculaires atteignant la substance blanche implique l'administration d'inhibiteurs de protéine rho pour activer la régénération des axones. Dans des formes de réalisations classiques, on utilise l'administration locale, cette dernière pouvant comprendre l'injection d'un virus de recombinaison qui exprime un inhibiteur. Dans une forme de réalisation, l'inhibiteur est la l'exoensyme C3 C.botulinium ou une structure C2/C3 C.botulinum chimérique exprimée dans un virus adéno déficient au niveau de la réplication, un virus adéno associé ou un virus associé.
PCT/US1998/016794 1997-08-13 1998-08-12 Regeneration d'axones dans le systeme nerveux central WO1999008533A1 (fr)

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EP98943195A EP1011330A4 (fr) 1997-08-13 1998-08-12 Regeneration d'axones dans le systeme nerveux central
AU91042/98A AU735607B2 (en) 1997-08-13 1998-08-12 Central nervous system axon regeneration
JP2000509293A JP2001515018A (ja) 1997-08-13 1998-08-12 中枢神経軸索再生
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Cited By (12)

* Cited by examiner, † Cited by third party
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WO1999023113A3 (fr) * 1997-10-31 1999-08-26 Lisa Mckerracher Antagonistes de la famille rho et leur utilisation pour bloquer l'inhibition de l'excroissance des neurites
WO2002051429A3 (fr) * 2000-12-22 2003-06-19 Migragen Ag Utilisation d'une composition pour stimuler la croissance neuronale, inhiber la formation de tissu cicatriciel, reduire un dommage secondaire et/ou l'accumulation de macrophages
EP1334729A1 (fr) * 2002-02-07 2003-08-13 Botulinum Toxin Research Associates, Inc. Utilisation thérapeutique de non-neurotoxic botulinum clostridium toxine type C2
WO2004006947A1 (fr) * 2002-07-12 2004-01-22 Yihai Cao Methode permettant d'inhiber la permeabilite vasculaire et l'oedeme tissulaire
WO2004009126A1 (fr) * 2002-07-19 2004-01-29 Health Protection Agency Agents cibles de regeneration des nerfs
US6855688B2 (en) 2001-04-12 2005-02-15 Bioaxone Thérapeutique Inc. ADP-ribosyl transferase fusion proteins, pharmaceutical compositions, and methods of use
US7169783B2 (en) 1998-11-02 2007-01-30 Universite De Montreal (+)-Trans-4-(1-aminoethyl)-1-(4-pyridycarbamoyl)-cyclohexane and method for promoting neural growth in the central nervous system and in a patient at a site of neuronal lesion
WO2007106991A1 (fr) * 2006-03-17 2007-09-27 Mcgill University Identification de la protéine crmp4 en tant que régulateur convergent de l'inhibition de la croissance des axones
US7749496B2 (en) 2001-10-12 2010-07-06 Case Western Reserve University Neuronal regeneration
WO2014113539A1 (fr) * 2013-01-16 2014-07-24 Bal Ram Singh Compositions chimères de botulinum pour thérapie de régénération axonale pendant une lésion de la moelle épinière
WO2017058819A1 (fr) * 2015-10-02 2017-04-06 The Regents Of The University Of California Cellules progénitrices enrichies en cellules gliales dérivées de cellules souches pluripotentes induites pour le traitement d'un avc affectant la substance blanche
US11008388B2 (en) 2015-04-28 2021-05-18 Mitsubishi Tanabe Pharma Corporation RGMa binding protein and use thereof

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CA2325842C (fr) 2000-11-02 2007-08-07 Lisa Mckerracher Methodes de production et d'administration de preparations combinant un antagoniste de rho et un adhesif tissulaire aux systemes nerveux central et peripherique blesses de mammiferes et utilisations de ces preparations
US7442686B2 (en) 2001-04-12 2008-10-28 Bioaxone Therapeutique Inc. Treatment of macular degeneration with ADP-ribosyl transferase fusion protein therapeutic compositions
US7795218B2 (en) 2001-04-12 2010-09-14 Bioaxone Therapeutique Inc. ADP-ribosyl transferase fusion variant proteins

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LUO L., ET AL.: "DISTINCT MORPHOGENETIC FUNCTIONS OF SIMILAR SMALL GTPASES: DROSOPHILA DRAC1 IS INVOLVED IN AXONAL OUTGROWTH AND MYOBLAST FUSION.", GENES AND DEVELOPMENT., COLD SPRING HARBOR LABORATORY PRESS, PLAINVIEW, NY., US, vol. 08., 1 January 1994 (1994-01-01), US, pages 1787 - 1802., XP002915255, ISSN: 0890-9369 *
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NIKOLIC M., ET AL.: "THE P35/CDK5 KINASE IS A NEURON-SPECIFIC RAC EFFECTOR THAT INHIBITS PAK1 ACTIVITY.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 395., 10 September 1998 (1998-09-10), United Kingdom, pages 194 - 198., XP002915253, ISSN: 0028-0836, DOI: 10.1038/26034 *
NISHIKI T., ET AL.: "ADP-RIBOSYLATION OF THE RHO/RAC PROTEINS INDUCES GROWTH INHIBITION, NEURITE OUTGROWTH AND ACETYLCHOLINE ESTERASE IN CULTURED PC-12 CELLS.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 167., no. 01., 28 February 1990 (1990-02-28), US, pages 265 - 272., XP002915251, ISSN: 0006-291X, DOI: 10.1016/0006-291X(90)91760-P *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999023113A3 (fr) * 1997-10-31 1999-08-26 Lisa Mckerracher Antagonistes de la famille rho et leur utilisation pour bloquer l'inhibition de l'excroissance des neurites
US7169783B2 (en) 1998-11-02 2007-01-30 Universite De Montreal (+)-Trans-4-(1-aminoethyl)-1-(4-pyridycarbamoyl)-cyclohexane and method for promoting neural growth in the central nervous system and in a patient at a site of neuronal lesion
WO2002051429A3 (fr) * 2000-12-22 2003-06-19 Migragen Ag Utilisation d'une composition pour stimuler la croissance neuronale, inhiber la formation de tissu cicatriciel, reduire un dommage secondaire et/ou l'accumulation de macrophages
US6855688B2 (en) 2001-04-12 2005-02-15 Bioaxone Thérapeutique Inc. ADP-ribosyl transferase fusion proteins, pharmaceutical compositions, and methods of use
US7749496B2 (en) 2001-10-12 2010-07-06 Case Western Reserve University Neuronal regeneration
EP1334729A1 (fr) * 2002-02-07 2003-08-13 Botulinum Toxin Research Associates, Inc. Utilisation thérapeutique de non-neurotoxic botulinum clostridium toxine type C2
WO2004006947A1 (fr) * 2002-07-12 2004-01-22 Yihai Cao Methode permettant d'inhiber la permeabilite vasculaire et l'oedeme tissulaire
WO2004009126A1 (fr) * 2002-07-19 2004-01-29 Health Protection Agency Agents cibles de regeneration des nerfs
WO2007106991A1 (fr) * 2006-03-17 2007-09-27 Mcgill University Identification de la protéine crmp4 en tant que régulateur convergent de l'inhibition de la croissance des axones
WO2014113539A1 (fr) * 2013-01-16 2014-07-24 Bal Ram Singh Compositions chimères de botulinum pour thérapie de régénération axonale pendant une lésion de la moelle épinière
US11008388B2 (en) 2015-04-28 2021-05-18 Mitsubishi Tanabe Pharma Corporation RGMa binding protein and use thereof
WO2017058819A1 (fr) * 2015-10-02 2017-04-06 The Regents Of The University Of California Cellules progénitrices enrichies en cellules gliales dérivées de cellules souches pluripotentes induites pour le traitement d'un avc affectant la substance blanche

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JP2001515018A (ja) 2001-09-18
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CA2300878A1 (fr) 1999-02-25
EP1011330A1 (fr) 2000-06-28
EP1011330A4 (fr) 2001-05-16

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