WO1999053945A1 - Molecules inhibant la croissance neuronale ou leurs derives utilises pour immuniser des mammiferes et ainsi favoriser la regeneration de l'axone - Google Patents

Molecules inhibant la croissance neuronale ou leurs derives utilises pour immuniser des mammiferes et ainsi favoriser la regeneration de l'axone Download PDF

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WO1999053945A1
WO1999053945A1 PCT/CA1999/000304 CA9900304W WO9953945A1 WO 1999053945 A1 WO1999053945 A1 WO 1999053945A1 CA 9900304 W CA9900304 W CA 9900304W WO 9953945 A1 WO9953945 A1 WO 9953945A1
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growth
myelin
spinal cord
regeneration
immunogenic
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PCT/CA1999/000304
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WO1999053945A8 (fr
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Samuel David
Lisa Joan Mckerracher
Peter Erich Braun
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Samuel David
Lisa Joan Mckerracher
Peter Erich Braun
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Publication of WO1999053945A8 publication Critical patent/WO1999053945A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K2121/00—Preparations for use in therapy

Definitions

  • This invention pertains to the field of nerve regeneration.
  • Axon growth inhibitory activity is an important cause of the failure of axon regeneration in the central nervous system (CNS).
  • CNS central nervous system
  • Axon growth inhibitors are predominantly associated with CNS myelin and constitute an important barrier to regeneration.
  • axon growth inhibitory activity is present in CNS myelin and the plasma membrane of oligodendrocytes, which synthesize myelin in the CNS (see Schwab et al., (1993) Ann. Rev. Neurosci. 16:565-595 for review).
  • the growth follows specific pathways that are delineated by cells and extracellular matrix that are located along the way. The guidance of the growth depends on various classes of adhesion molecules, on intercellular signals, as well as on factors that repel and inhibit growth cones.
  • the growth cone located at the end of a rapidly growing neurite grows about 1 mm per day.
  • the cone consists of a broad and flat expansion, with numerous long microspikes or filopodia that extend like fingers. These filopodia are continually active. Some filopodia retract back into the growth cone. Other filopodia continue to elongate and wave around, touch and adhere to the substratum.
  • the webs or veils between the filopodia form lamellipodia.
  • the growth cone can explore the area that is ahead of it and on either side with its lamellipodia and filopodia. When such an elongation comes in contact with a surface that is unfavorable, it withdraws. When such an elongation comes in contacts with a surface that is favorable, it persists longer and can steer the growth cone moving it in that direction. Hence, the growth cone can be guided by subtle variation in surface properties of the substrata.
  • N-CAM immunoglobulin superfamily
  • Ca2+-dependent cadherin family eg. N-cadherin
  • Both families are usually present on the surface of growth cones, axons, and other cell types that interact with such cones. These include glial cells found in the CNS as well as muscle cells in the periphery of the body. Growth cones can also move over various components of the extracellular matrix (eg. laminin), which they bind to via cell-surface matrix receptors found in the integrin family.
  • laminin extracellular matrix
  • Various combinations of adhesion molecules allow for greater variety in the surface properties of growth cones, thereby allowing for subtle and complex pathway selection according to the combinations of molecules on the surfaces of cells.
  • the myelin associated inhibitors appear to be a major contributor to the failure of axon regeneration in the CNS in vivo.
  • Other non-myelin associated axon growth inhibitors in the CNS may play a lessor role.
  • inhibitors that are not derived from myelin and are associated with the scar at the site of a CNS lesion may also play a role in blocking some of the axon regeneration after injuries
  • MAG Myelin-Associated Glycoprotein
  • MAG myelin-associated glycoprotein
  • MAG axonal but not dendritic growth cones
  • CHO cells expressing MAG on their surface are an inhibitory substrate for neurite growth from a variety of primary neurons from the CNS and PNS as compared to mock transfected cells (Mukhopadhyay et ai, (1994); DeBellard et ai, (1996)).
  • arretin Another growth inhibitory protein has been identified, termed arretin (Xio et al, (1994) Soc. NeuroScience Abstract, vol. 23).
  • the apparent molecular weight of arretin is approximately 70 kDa, and it has potent growth inhibitory activity when plated as a substrate.
  • Arretin causes growth cone collapse, and recent work has shown that the 70 kDa component consists of at least two separate inhibitors. Monoclonal antibodies raised against these inhibitors are able to block neurite growth on myelin and extracts of myelin.
  • tenascin-C Four members of the tenascin family have been identified and characterized: tenascin-C, tenascin-R, tenascin-X and tenascin-Y (Bristow etai, Cell Biol., 122, 265-278, 1993; Erickson, H P., J. Cell Biol., 120, 1079-1081, 1993).
  • Tenascin-X and tenascin-Y are not prominent in the nervous system. Studies have indicated that both tenascin R and tenascin C are minor inhibitory components of octlyglucoside extracts of myelin. These data suggest that growth inhibitory proteins from the CNS matrix may become associated with isolated myelin fragments.
  • Tenascin-C is important in the development of the nervous system and it is the best characterized member of this protein family. It is generated by alternative splicing (Weller et al., J. Cell Biol., 1 12, 355-362, 1991 ; Sriramarao and Bourdon, Nucl. Acids Res., 21, 347-362, 1993) and the variants are expressed both in the nervous system and in several non-neural tissues. Tenascin-C has been suggested to be involved in neuron-glia adhesive and migratory events and to promote axon outgrowth after injury of peripheral nerves.
  • Tenascin-R has a modular structure similar to TN-C, previously designated Jl-160/180 and janusin in rodents, or restriction in chicken (Pesheva et al, J. Cell Biol., 109, 1765-1778, 1989; Fusset ⁇ /., J. Neurosci. Res., 29, 299-307, 1991, and J. CellBiol., 120, 1237-1249, 1993).
  • Tenascin-R is predominantly expressed by oligodendrocytes during the onset and early phases of myelin formation and remains detectable in myelin-forming oligodendrocytes in the adult, and is also expressed by neurons (Pesheva etai, 1989; Fuss etai, 1993). Tenascin-R has been shown to be involved in promotion of neurite outgrowth and morphological polarization of differentiating neurons when presented as a uniform substrate (Lochter and Schachner, J. Neurosci., 13, 3986-4000, 1993; Lochter et al, Eur. J. Neurosci., 6, 597-606, 1994).
  • tenascin-R When offered as a sharp substrate boundary with a neurite outgrowth conducive molecule, tenascin-R is repellent for growth cone advance (Taylor etai, J. Neurosci. Res., 35, 347-362, 1993; Pesheva et ai, 1993).
  • CSPGs Chondroitin Sulfate Proteoglycans
  • GAGs Glycosaminoglycans
  • GAGs are polymers of disaccharide repeats, which are mostly highly sulphated and negatively charged.
  • the main glycosaminoglycans in PGs are chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate.
  • CSPGs Chondroitin sulfate proteoglycans
  • the NG2 proteoglycan also inhibits neurite growth after digestion with chondroitinase ABC, indicating that the inhibitory activity is a property of the core protein and not the covalently attached chondroitin sulfate glycosaminoglycan chains (Dou and Levine, J. Neurosci., 14, 7616-7628, 1994), but for many other types of CSPGs the inhibitory activity resides in the glycosaminoglycan.
  • This highly sulfated proteoglycan which is a potent inhibitor of neurite growth in vitro has been shown to be involved in the differentiation of developing retinal ganglion cells, and by acting as an inhibitory substrate serves to appropriately guide ganglion cell axons toward the optic disc (Brittis and Silver, Proc. Nat. Acad. Sci. USA., 19, 7539-7542, 1992).
  • McKeon et al, (1991) J. Neurosci. 1 1 :3398-411 have reported that astrocytes harvested from the site of cerebral cortical lesions express increased amounts of CSPG, which reduces neurite growth on these cells in vitro.
  • proteoglycans are a very heterogenous class of proteins with diverse biological activities it is essential that individual, identified proteins be considered.
  • Phosphacan is a proteoglycan in brain recognized by the 3F8 antibody (Maurel et al, Proc. Nat. Acad. Sci. USA, 91, 2512-2516, 1994), and by the 6B4 antibody (Maeda et al, Neurosci., 67, 23-35, 1995).
  • Phosphacan is a splice variant of a receptor-type protein tyrosine phosphatase, although phosphacan itself lacks the phosphatase domains. It is a protein with an apparent molecular weight of approximately 500 kDa, having a core glycoprotein of approximately 400 kDa.
  • the HNK-1 monoclonal antibody recognizes a 3-sulphated carbohydrate epitope, and this epitope is strongly represented in phosphacan from 7-day brain, but not in adult brain (Rauch et al, J. Biol Chem., 266, 14785-14801, 1991).
  • phosphacan is immunostained on radial glia and on neurons (Maeda et al, 1995) and generally it is expressed in both white matter and grey matter regions (Meyer-Puttlitz, et al, j. Comp. Neurol. 366, 44-54, 1996). and therefore, unlike the myelin inhibitors, it is not localized only to white matter areas. It appears to be synthesized only by astroglia (Engel et al, 1996).
  • Versican a CSPG originally isolated from fibroblasts, also called PG-M, has an apparent molecular weight of approximately 900 kDa, with a core protein of approximately300 to 400 kDa (Braunewell et al, Eur. J. Neurosci., 7, 792-804, 1995; Naso et al, 1994).
  • Versican belongs to a family of aggregating CSPGs; other members of the family include the cartilage-derived aggrecan, and two PGs expressed in the nervous system, neurocan and brevican (Dours-Zimmermann and Zimmerermann, J. Biol. Chem., 269, 32992-32998, 1994).
  • Versican is widely distributed in adult human tissues, associated with connective tissue of various organs, in certain muscle tissues, epithelia, and in central and peripheral nervous tissues.
  • versican isoforms are known (Vo, VI, V2, V3), derived by alternative splicing. They vary in calculated mass from approximately 370 kDa (Vo) to approximately 72 kDa (V3). It has been suggested that the association of versican expression with cell migration and proliferation in vivo and its adhesion inhibitory properties in vitro point to pathological processes such as tumorigenesis and metastasis (Bode-Lesniewska et al, Histol. & Cyto., 44, 303-312, 1996; Naso et al,. J. Biol Chem., 269, 32999-33008, 1994).
  • CSPGs related to versican are brevican (Mr approximately 145 kDa) and neurocan (Mr > 300 kDa). Neither of these is known to be expressed by oligodendrocytes and are therefore not expected to be present in CNS myelin (Engel et al. , J. Comp. Neurol. 366, 34-43 , 1996; Yamada et al. . Biol. Chem., 269, 101 19-10126, 1994).
  • CNS myelin The growth inhibitory properties of CNS myelin have been demonstrated in a number of different laboratories by a wide variety of techniques, including plating neurons on myelin substrates or cryostat sections of white matter, and observations of axon contact with mature oligodendrocytes (Schwab et ai, (1993) Annu. Rev. Neurosci. 16:565-595 ).
  • Schwab et al. generated a monoclonal antibody to an inhibitor which recognizes two proteins on Western blots with molecular weights of 35 and 250 kDa. In vitro assays have demonstrated that this antibody was able to block some of the neurite growth inhibitory activity of CNS myelin.
  • This monoclonal antibody was introduced by either being infused directly via osmotic pumps or by transplanting the hybridoma cells into young adult rats was shown to stimulate the growth of a small number of corticospinal tract axons after lesions of the spinal cord (Schnell & Schwab (1990) Nature 343 :269-272).
  • a further strategy is to use monoclonal antibodies to block specific myelin-associated inhibitors of axon growth (Schnell & Schwab (1990) Nature 343:269-272; Bregman et al, (1995) Nature 378: 498-501.
  • SCH94.03 A monoclonal polyreactive antibody called SCH94.03 has been isolated following immunization of mice with spinal cord homogenate. This antibody augments repair of myelin after intracerebral inoculation of genetically susceptible mice with TMEV, a pathogenic virus that produces immune-related CNS demyelination (Miller et al, (1994)). These investigators have also shown that this antibody can pass through the blood-brain barrier and enter the CNS tissue. Furthermore, polyreactive antibodies generated following immunization with spinal cord homogenate tend to be polyreactive against proteins expressed in myelin (see Asakura et al, (1995)), likely due to the highly charged nature of many of these molecules.
  • the present invention relates to regeneration of central nervous system (CNS) axons in mammals.
  • CNS central nervous system
  • the present invention relates to compositions comprising immunogenic agents derived from neuron growth inhibitory molecules and the method of administering this composition a mammal to cause polyreactive antibodies to be generated in vivo, that bind to axon growth inhibitors, thereby promoting axon regeneration.
  • the immunogenic agents are derived from mammalian central nervous system tissue homogenate.
  • the central nervous system tissue may comprises one or more components of the group comprising spinal cord, brain, brainstem, and optic nerves.
  • the immunogenic agents are derived from mammalian CNS tissue that is largely myelin.
  • the immunogenic agents are derived from mammalian peripheral nervous system tissue homogenate.
  • the immunogenic agents are purified or recombinant proteins, or fragments or derivatives thereof. Examples of specific immunogenic agents that may be used include MAG, arretin, and proteoglycans. These proteins may be used alone or in combination.
  • the immunogenic agents are provided by gene therapy.
  • the immunogenic agents may be derived from any mammal, but are preferably human.
  • kits comprising the immunogenic compositions and instructions for use in immunization and neuron regrowth.
  • the present invention describes methods of promoting axon repair and/or regeneration in a subject by blocking axon growth inhibitors, comprising immunizing said subject with therapeutically effective amounts of an immunogenic composition.
  • This immunization may be carried out in conjunction with other known therapies, including cell or tissue transplants, surgical resections to remove scar tissue, and the administration of neurotrophin or growth factors to stimulate the growth state of neurons.
  • These methods may be used to promote repair following injuries, whether acute or chronic, such as spinal cord injuries, optic nerve injuries, and injuries to other white matter tracts.
  • These methods may also be used when a subject requires repair and/or regeneration due to disease, such as strokes, multiple sclerosis, diseases of the optic nerve, and other conditions in which damage to CNS axons occur.
  • the present invention describes the use of immunogenic compositions as prophylactic vaccines.
  • FIG. 1 Regeneration of corticospinal tract fibers in the injured adult mouse spinal cord, (a.) Micrograph of the lesioned corticospinal tract in a mouse immunized with spinal cord homogenate in LFA. WGA-HRP labels the tract rostral (toward the left) and caudal (toward the right) to the lesion (arrow). Many regenerated axons can be seen caudally. (b.) Higher magnification of an adjacent section showing the scar at the site of lesion. WGA-HRP-labeled axons can be seen coursing through the scar (arrow; same area shown at higher magnification in insert).
  • Figure 2 Percentage of double-labeled neurons retrogradely labeled with Fluorogold and Fluororuby. This is an index of the proportion of corticospinal tract neurons that regenerated their axons.
  • FIG. 5 Assessment of myelin-reactive antibodies in immunized mice.
  • a. Western blot of spinal cord proteins showing an example of the binding of the sera from an immunized mouse (left lane) and a control mouse (right lane) both of which were sacrificed 3 weeks after spinal cord hemisection.
  • the antibodies in the sera of immunized mice bound variably to multiple bands (not yet identified) as compared to controls.
  • MW markers (arrowheads): 103, 76, and 49 kDa.
  • Figure 6 The technique used to study axon regeneration in the optic nerve of rats after immunization, (a) The retinal ganglion cell axons in the optic nerve are transected by constriction with 10.0 sutures, (b) Other treatments, such as the application of neurotrophins, C3 enzyme or other agents that help stimulate regeneration can be added to the site of injury to act synergistically with the immunization approach, (c) Regenerating axons are detected by anterograde labeling after injection of tracer into the eye.
  • FIG. 7 Immunization controls.
  • the top panel shows longitudinal section from an animal that received injection of adjuvant alone. The site of the crush is detected where anterogradely labeled axons stop abruptly. The few axons that extend past the crush are found only along the peripheral region of the optic nerve.
  • the bottom panel shows longidudinal section from a different control animal from that shown above. Bar, 100 ⁇ m
  • Figure 8 The region of the crush in an immunized rat.
  • the top panel shows immunoreactivity for glial fibrillary acid protein showing the region of the crush.
  • the bottom panel shows an adjacent section showing some anterogradely labeled axons that extend past the crush in the central region of the optic nerve. Bar, 50 ⁇ m.
  • Figure 9 Longitudinal sections from an immunized rat.
  • the top, middle and bottom panels show three different sections from the same optic nerve from an animal treated with the imunization procedure. Anterogradely labeled axons that extend past the crush in the central region of the optic nerve can be seen in all three sections. Bar, lOO ⁇ m.
  • Figure 10 Longitudinal sections from an immunized rat. This section is from a different animal than that shown in Figures 8 and 9. In this animal, axons were observed to extend farther than 1 mm from the site of the crush. Bar, 100 ⁇ m.
  • the present invention resides in the discovery that the administration of compositions, comprising immunogenic molecules derived from neuron growth inhibitors, to a mammal cause polyreactive antibodies to be generated that bind to neuron growth inhibitors at the site of neuron damage or disease, thereby promoting axon regeneration.
  • this type of immunization approach can be used to promote axon regeneration in an injured or diseased human CNS.
  • the compositions can also be used as prophylactic vaccines.
  • compositions of the present invention comprise immunogenic agents that cause polyreactive antibodies to be generated in a subject, which bind to the subject ' s own neuron growth inhibitory molecules and block their effect, thereby allowing for axon regeneration.
  • Immunogens correspond to a class of molecules that elicit an immune response through classical immunologic pathways, as in the non-limiting example of the incorporation in an MHC molecule of an antigen processing cell where the immunogens can potentially interact with antigen specific T cell receptors.
  • immunogens can bind to antigen specific binding regions of immunoglobulins which may lead to stimulating the B lymphocytes(if on the surface of B lymphocytes), but alternatively could elicit an immune response through other means, e.g., by the activation of complement, or the modulation of Fc receptors.
  • the individual doses of individual immunogens may by themselves be subimmunogenic, provided that in aggregate, when administered according to the schedule, an immunogenic effect is achieved.
  • compositions of the present invention create an immunogenic response by causing polyreactive antibodies to be generated in a subject.
  • Polyreactive antibodies are natural autoantibodies that bind to a large variety of structurally unrelated antigens. They are generally of the IgM class. These natural autoantibodies, which are present in the sera of healthy humans and rodents, are encoded by germline genes with no or few mutations. Generally, they recognize long stretches of acidic amino acids and thus have broad antigen binding capabilities. The physiological function of natural autoantibodies is not known, but they may participate in general homeostasis. (Asakura et /., (1995) Molec. Brain. Res. 34:283-293)
  • Any individual proteins shown to have neuron growth inhibitory properties in vitro are suitable immunogenic agents of the compositions of the present invention.
  • the immunogenic agents of the composition are inhibitors of axonal growth, including both myelin-associated inhibitors, such as NI 35/250, MAG, arretin, and the tenascins, and non-myelin- associated inhibitors, including inhibitors present in scar tissue, such as the proteoglycans CSPGs, phosphacan, and versican.
  • myelin-associated inhibitors such as NI 35/250, MAG, arretin, and the tenascins
  • non-myelin- associated inhibitors including inhibitors present in scar tissue, such as the proteoglycans CSPGs, phosphacan, and versican.
  • These immunogenic agents may be delivered alone or in combination to stimulate an appropriate polyreactive antibody response to promote axon regeneration.
  • immunogenic agents may be mammalian or submammalian, but preferably are human.
  • Any method of providing a composition of immunogenic agents to a subject may be used.
  • Strategies include the administration of purified growth inhibitor proteins, or fragments thereof, the administration of recombinant growth inhibitor proteins, or fragments thereof, and gene therapy.
  • Immunogenic agents may be obtained from a natural source; for example, immunogenic agents may be derived from CNS tissue (including spinal cord, brain, brainstem, and optic nerves). In particular, immunogenic agents may be obtained from spinal cord extracts. Immunogenic agents may also be derived from the peripheral nervous system.
  • immunogenic agents may be associated with any component of the CNS tissue, they associate preferentially with myelin components (Mckerracher et al, (1994) Neuron 13 : 229-246 and 805-811). Accordingly, myelin may be purified to develop a more refined composition of immunogenic agents.
  • the spinal cord tissue is homogenized in a physiological buffer and subjected to a standard protocol to separate the myelin.
  • the spinal cord is used here because it has a high content of myelin. This procedure, however, can also be done with any other parts of the CNS (such as the brain, brainstem, optic nerves, etc.).
  • the biochemical separation of myelin from the spinal cord homogenate will yield two components - (1) the purified myelin, and (2) the non-myelinated components.
  • the purified myelin can be subjected to further biochemical separations, such as detergent extractions, followed by various types of separations techniques, including anion or cation exchange chromatography, but not limited exclusively to these forms of separations.
  • the various partially purified myelin components can be used to immunize mice, and the effectiveness of these components to promote axon regeneration assessed.
  • An immunogenic agent mixture may comprise either whole myelin or partially purified components of myelin.
  • purified myelin-associated axon growth inhibitors such as NI 35/250, MAG, arretin, or the tenascins, could be used alone or in combination to stimulate an appropriate polyreactive response to promote axon regeneration.
  • growth inhibitors not primarily associated with myelin could also be used in the composition.
  • growth inhibitors associated with scar tissue such as proteoglycans, may be used.
  • the growth inhibitor proteins, or fragments thereof may be made by recombinant means utilizing available sequence data. These recombinant proteins could be combined to create a well-defined immunogen composition.
  • Purified or recombinant proteins could be used alone or in combination in the composition.
  • the composition does not have to contain only previously identified molecules, but could also contain unidentified growth inhibitors.
  • composition may be prepared in pharmaceutically acceptable form according to known method steps, based on the teaching and guidance presented herein.
  • Recombinant technology and monoclonal antibodies and fragments thereof may also be used (See, e.g., Ausubel et al, eds, (1987, 1993) Current Protocols in Molecular Biology (Greene Publishing Assoc. and Wiley Interscience, N.Y., N.Y.); Coligan et al, eds., (1992, 1993) Current Protocols in Immunology (Greene Publishing Assoc. and Wiley Interscience, N.Y., N.Y.); Sanbrook et al, infra; and Harlow, infra).
  • Purification of immunogenic agents helps minimize the presence of impurities in a immunization composition. Purification may be performed by any method that produces a highly pure end product as in the non-limiting examples of: chromatography, electrokinetic processes, membrane processes, centrifugation, and extraction (Biotechnology 5:789-793). Extensive reviews of separation techniques are included in several different texts including the book by Juan A. Asenjo, Separation Process in Biotechnology, published Marcel Dekker Inc., New York (1990) (see especially Chapters 19, 20).
  • the composition should preferably contain as few impurities as possible, as well as maintain a very high consistency of the amounts and ratio of an immunization composition components including immunogens and adjuvants in each dose with little or no lot-to-lot variation.
  • An immunization composition immunogens should preferably be characterized by their unique physical properties to allow their composition in an immunization composition to be measured. The movement of an immunogen or its immunogenic components through a gel as in the non-limiting examples of chromatography or electrophoresis will allow detection of bands of impurities in the immunization composition.
  • the total amount of impurities comprising molecules unrelated to the desired an immunization composition components should be less than 0.5% by weight and even more preferably less than 0.1%.
  • the desired an immunization composition antigens should preferably be defined by molecular weight and molecules should preferably differ by less than 5% of this number or be considered separate immunogen.
  • the variation in the amount of a particular defined immunogenic composition component, such as an immunogen or adjuvant, should preferably be less than 2% by weight.
  • immunization compositions eg. vaccines
  • a non-limiting example of how immunization compositions (eg. vaccines) are currently tested for purity is described in a paper on manufacturing of Neisseria meningitis vaccines (Avshalom Mizrahi, Bacterial Vaccines, Alan R. Liss Inc. (1990), pages 123-145) and differences to the current inventions are clear.
  • Gene therapy can also be used to provide immunogenic agents to a subject.
  • Gene therapy methods include the use of recombinant viral vectors encoding growth inhibitors.
  • viruses for this invention include adenoviruses, adeno-associated virus, herpes simplex viruses, the ALDS virus, and retroviruses well known to those skilled in the art.
  • the viral vector employed may, in one embodiment, be an adenoviral vector that includes essentially the complete adenoviral genome (Shenk et al. , ( 1984) Curr. Topics Microbiol Immun. 111 (3 ) : 1 -39) .
  • the viral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.
  • the viruses used in the construction of viral vectors are rendered replication-defective to remove the effects of viral replication on the target cells.
  • any mammalian growth inhibitor can be employed in the present invention.
  • the growth inhibitor is human.
  • the DNA sequences can be either cDNA or genomic DNA. DNA encoding the entire growth inhibitor, or any portion thereof, may be used. Due to the degeneracy of the genetic code, other DNA sequences that encode substantially the same growth inhibitor or a functional equivalent can also be used. Multiple gene copies may also be used.
  • the DNA sequences encoding the growth inhibitor are under the control of a suitable promoter.
  • the viral vectors of the present invention will be by procedures well established in the pharmaceutical arts, e.g. by direct delivery to the target organ, tissue or site, intranasally, intravenously, intramuscularly, subcutaneously, intradermally and through oral administration, either alone or in combination.
  • the viral vectors are administered by injecting vector suspension into various locations of the nervous system, or by injection into nerves, or injection into peripheral tissues such as skin or muscles, which are innervated by neurons.
  • the vector enters the neurons via the axons or axon terminals, and the vector genome is transported retrogradely in the axon to the nucleus.
  • the dosages administered will vary from subject to subject and will be determined by the level of enhancement of neurofilament function balanced against any risk or deleterious side effects. Monitoring levels of transduction, neurofilament expression and/or the presence or levels of normal neurofilament will assist in selecting and adjusting the dosages administered.
  • compositions are Compositions:
  • the pharmaceutical composition may also contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Such carriers may include depot adjuvants that release an immunogen in vivo over a prolonged period as compared to administration of an unbound immunogen.
  • the depot adjuvant comprises an aluminum, calcium or salts thereof, such as aluminum sulfate (alum), aluminum phosphate, calcium phosphate or aluminum hydroxide, see, e.g., Gregoriades, G. et al., Immunological Adjuvants and Vaccines, Plenum Press, New York, 1989.
  • Immunogenic compositions of the present invention may also include suitable solutions for administration, intramuscularly, intravenously, subcutaneously, dermally, orally, mucosally, or rectally or by any other injection, and contain from about 0.001 to 99.999 percent, preferably from about 20 to 75 percent of active component (i.e. the immunogen) together with the excipient.
  • Compositions which can be administered rectally include suppositories.
  • compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients, such as suitable adjuvants, which are known in the art.
  • Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods. See, e.g., Berker, supra, Goodman, supra, and Avery, supra, which are entirely incorporated herein by reference, included all references cited therein.
  • the immunogenic compositions of the present invention may optionally include immunomodulators other than antigens. Such immunomodulators may also be administered separately as a part of the program.
  • the immunogenic compositions of the present invention may also include pharmaceuticals whose primary activity is non-immunological.
  • Said non vaccine pharmaceutical agent may be of any class of pharmaceutical agents including the non limiting examples of agents to ameliorate the following diseases: infectious, cardiovascular, gastrointestinal, endocrine/hormonal, renal, neurological, psychiatric, muscular, skeletal/orthopedic, hematological, hepatic, pancreatic, metabolic, neoplastic, inflammatory/rheumatic, reproductive, dietary/nutritional, ophthalmologic, otologic, pulmonary/respiratory, dermatologic, allergic, and surgical as in anesthetics. See Physicians Desk Reference, Medical Economics Data Production, Montvale, N.J.
  • these pharmaceuticals are ones which, when screened as taught herein, are shown to help reduce the incidence or severity of a chronic immune-mediated disorder, or at least do not worsen the disorder.
  • the immunogenic compositions of the present invention could be used to treat patients with acute or chronic spinal cord injuries.
  • immunizations with an appropriate antigen will be started and may be combined with other forms of therapy such as neurotrophin or growth factors to stimulate the growth state of the neurons, and/or surgical resections to remove the scar tissue at the site of CNS lesions, with or without transplantations of suitable materials (such as embryonic tissue transplants, purified non-neuronal cell transplants, inert materials, etc.), to serve as a suitable tissue bridge to span the area that has undergone the resection.
  • suitable materials such as embryonic tissue transplants, purified non-neuronal cell transplants, inert materials, etc.
  • This immunization approach can also be used to treat patients with strokes (particularly those with lesions in the axon tracts, e.g., the internal capsule) to promote regeneration of axons.
  • This immunization approach can also be used in patients with Multiple Sclerosis, particularly in patients who display signs of axonal damage such as of the optic nerve, or other long axon tracts in the spinal cord or brain.
  • a modification of this immunization approach can be used as a prophylactic vaccine in healthy individuals. This would prime the immune system such that later immunizations given to an individual who has sustaied CNS injury or onset of CNS disease (such as MS or stroke) would provoke a more rapid and strong antibody production to stimulate regeneration of axons.
  • the following immunization protocol is used to preferentially result in the production of a polyreactive autoantibody response. Variations of this protocol that results in the production of polyreactive antibodies to axon growth inhibitors to be used as a therapy in humans would also be included in this invention. While it is possible that the purposes of the present invention can be served with a single administration, especially when the immunogen is a strong one, and in that case a single dosing schedule is within the compass of the present invention, it is desirable to administer two or more dosings for greater surety. Preferably, the number of dosings is at least three, more preferably at least four, and still more preferably, at least five. There is no set maximum number of vaccinations, however it is good clinical practice not to immunize more often than necessary to achieve the desired effect.
  • In vivo assays include the non-limiting examples of antibody responses and delayed type hypersensitivity responses.
  • the antibody responses primarily measures B-cell function as well as B-cell T-cell interactions while the delayed type hypersensitivity responses measure T-cell immunity.
  • Phenotypic cell assays can also be performed to determine the frequency of certain cell types.
  • Peripheral blood cell counts may be performed to determine the number of lymphocytes or macrophages in the blood.
  • Antibodies may be used to screen peripheral blood lymphocytes to determine the percent of cells expressing a certain antigen as in the non-limiting example of determining CD4 cell counts and CD4/CD8 ratios.
  • a pharmaceutically acceptable dose is a dose where the clinical benefits of said product outweighs the toxicity at said dose.
  • Non-limiting examples of said toxicity include acute or subacute reactions like fever, shock or seizures, which may lead to permanent sequela and chronic toxicity like cancer, as is known and recognized in the relevant parts.
  • a pharmaceutically acceptable dose according to this definition can vary according to the severity of the illness being modulated by the immunogenic agent. It is logical that a high dose of an agent which causes significant toxicity may be pharmaceutically acceptable in certain situations.
  • a pharmaceutically acceptable dose will depend on the structure of the particular agent and/or the condition of the recipient. Some reagents may be more toxic than others while some may be more immunogenic than others. In a like manner, some individuals may be more responsive to a given dose while others may be more sensitive to the toxic effects at that dose. There is thus an individual variation within the definition of pharmaceutically acceptable dose as well as species, racial, age, and population variation, all of which should be taken into account when dosing an individual. Such consideration has, of course, been given to other prophylactic agents.
  • One animal model that can be used to assess the effectivenes of immunogenic agents is spinal cord injury in the adult mouse.
  • the protein levels of immunogenic agents are monitored by observing optical density (OD) measurements.
  • the dorsal portion of the spinal cord in the lower thoracic region is lesioned with microscissors (hemisection) as described by Li et al, (1996) J. Neurosci. Res. 46:404-414, and the immunization continued for another 3 weeks post-lesion period in the same way as above. After this 3 week period, Wheatgerm conjugated horseradish peroxidase (WGA-HRP) is injected into the sensory-motor cortex. After 24 hours, blood is collected for testing and the animals are perfused with fixatives for HRP histochemistry. The protocol used for the HRP histochemistry in vivo assay of regeneration will be similar to that described (Li et al, (1996) J. Neurosci. Res. 46:404-414).
  • retrograde neuronal tracers can be used to assess regeneration. This is done by applying a fluoresecent dye such as Fluorogold to the site of the spinal cord lesion at the time of lesioning. After 3 weeks post-lesion, during which the animals receive immunizations, the animals will receive a 1 ⁇ l injection of a second fluorescent dye such as Fluororuby at a point between 5-6 mm caudal to the site of the spinal cord lesion. Three days later the animals are sacrified by perfusion with a fixative such as 4% paraformaldehye and the brain cut of a cryostat through the regions containing the sensory-motor cortex.
  • a fluoresecent dye such as Fluorogold
  • the antibodies likely enter into the degenerating fiber pathways in the white matter as the blood-brain barrier is compromised due to disease, injury, or trauma.
  • myelin-reactive antibodies which are produced by our immunization procedure are likely to enter into the degenerating fiber pathways in the white matter as the blood-brain barrier is opened by the trauma and the action of activated microglia. These antibodies then bind to inhibitors associated with myelin and thus stimulate axon regeneration over long distances.
  • This approach to stimulate regeneration in the CNS is useful therapy to be applied after spinal cord injuries, other types of CNS traumas, stokes, multiple sclerosis, optic nerve damage, and other neurological conditions in which axon regeneration is desired.
  • This approach is particularly useful because one could start immunizing a person at the scene of an accident involving spinal cord injury, or very soon thereafter;, thus, there would be an immediate production of polyreactive autoantibodies and the corresponding stimulation of axon regeneration.
  • This treatment could be combined with other types of therapies such as growth factor or neurotrophin therapies, or methylprednisolone treatments.
  • compositions ofthe present invention could also be used as prophylactic vaccines to be given to healthy individuals. Commencement of immunization after the onset of CNS lesion or disease (such as a stroke) would then lead to more rapid and stronger antibody production.
  • mice 4-8 weeks old are deeply anaesthetized with chloral hydrate, and perfused via the heart with 0. IM phosphate buffer. Brains and spinal cords are removed and collected separately in buffer containing protease inhibitors.
  • Purified myelin is prepared by the procedure of Norton and Poduslo (1973), with the inclusion of protease inhibitors at every step. Briefly, this consists of homogenizing the tissue in 0.32 M sucrose containing protease inhibitors, with a motor-driven Dounce homogenizer. The homogenate is then filtered through a cheese cloth and then overlaid on 0.85 M sucrose and centrifuged for 30 min at 75,000g. The material at the interface is collected and resuspended in ice cold water and centrifuged for 25 min at 25,000 g. The pellet is again resuspended in the same way and centrifuged for 15 min at 10,000 g. This step is repeated.
  • the pellet is then resuspended in 0.32 M sucrose, overlaid on 0.85 M sucrose, and centrifuged for 1 h at 75,000 g. Purified myelin, which separates at the interface, is collected and washed twice with ice cold water.
  • Purified myelin is extracted for 2 hr at 20EC with 1% octylglucoside (1ml per milligram of protein) in 0.2 M phosphate buffer (pH 6.8) containing 0.1 M Na 2 SO 4 , lmM EDTA, ImM dithiothreitol, and a composition of protease inhibitors.
  • the extracts are centrifuged at 400,000 g.min and applied to a DEAE-Sepharose column (Pharmacia; 1 cm x 1 cm).
  • EXAMPLE H In Vitro Assay for Testing the Efficacy of the Immunogenic Composition to Stimulate Neurite Growth on a CNS Inhibitory Substrate
  • the efficacy ofthe immunogenic composition ofthe present invention was assessed by measuring neurite growth in vitro, using both NGl 08- 15 cells and primary neurons. Neurite growth was assessed using a previously-described assay (Mckerracher et al, (1994) Neuron 13 : 229-246 and 805-811).
  • Purified myelin (8 ⁇ g/ well) or the appropriate antigen (e.g., purified inhibitory components used for immunization) was plated onto poly-L-lysine (PLL)-coated 96 well plates and left overnight. The wells were then washed with buffer. Cyclic AMP primed NGl 08- 15 cells were first labeled with Dil ( 1.1 'dioctadecyl-3 , 3 , 3 ', 3 '-tetramethylindocarbocyanine perchlorate, 3 ⁇ g/ml) in serum-free tissue culture medium, then rinsed and maintained in culture for 24 h prior to use in the assay.
  • Dil 1.1 'dioctadecyl-3 , 3 , 3 ', 3 '-tetramethylindocarbocyanine perchlorate, 3 ⁇ g/ml
  • DMEM Dulbecco's minimal essential medium
  • the unlabeled NGl 08- 15 cells were plated and used in the neurite growth assay as noted above. At the end ofthe experiment, however, these cells were fixed as described above and stained with Coomassie blue for 5 min.
  • Neurite growth was assessed by estimating the percentage of cells extending neurites greater than 1 cell body diameter in length using an inverted microscope equipped with fluorescence optics. Counts were made from duplicate wells in each experiment.
  • Embryonic day 19 rat hippocampal neurons were prepared as described previously (Banker and Cowan, 1977).
  • Neonatal rat cerebellar granule cells were purified by Percoll density gradient centrifugation as described by Hatten (1985). These neurons were then plated onto round glass coverslips coated with the immunogenic composition. After 18-20 hrs, the cultures were fixed and labeled with a monoclonal antibody against GAP -43 and visualized with a goat anti-mouse Ig conjugated to rhodamine.
  • the following example illustrates that immunization with the immunogenic composition stimulated axon regeneration in a mammalian subject following spinal cord injury.
  • mice were immunized subcutaneously twice weekly with an immunogenic composition.
  • the composition consisted of 50 ⁇ g of either adult mouse spinal cord or purified CNS myelin homogenized in phosphate buffered saline and mixed with an equal volume of incomplete Freund ' s adjuvant.
  • the animals were anesthetized.
  • the mid to lower thoracic spinal cord was exposed and a dorsal hemisection lesion done with a pair of microscissors.
  • the lesions were carried out in such as way as to cut both corticospinal tracts that run from the motor cortex to various spinal cord levels.
  • the area was closed by sutures, and the animals were allowed to survive for another three weeks.
  • the animals continued to receive twice weekly immunizations during this 3 week period.
  • mice were injected into a site about 5-6 mm caudal to the lesion.
  • Animals were sacrificed one week later and cryostat sections ofthe motor cortex examined for the presence of double-labeled neurons.
  • 55% of the mice immunized with spinal cord homogenate or purified myelin showed good evidence of regeneration of corticospinal tract axons as revealed by the presence of double-labeled neurons in the sensory-motor cortex (Figs. If, lg, 2).
  • 50% " 16% of the motor neurons were double-labeled (i.e., regenerated neurons).
  • the serum from the immunized mice was tested in an in vitro neurite growth assay (described above) for their ability to block the inhibitory activity of myelin.
  • the serum of three ofthe animals that showed axon regeneration in vivo were tested and compared to the serum of an uninjected normal control mouse.
  • the sera ofthe injected mice were able to stimulate neurite growth on the inhibitory myelin substrate as compared to control serum.
  • the serum ofthe mice was also used for biochemical studies such as Western blotting, ELISA, and immunohistochemistry.
  • FIG. 6 Another animal model that can be used to assess the effectiveness ofthe immunogenic agents is an optic nerve lesion in rat. Recently, it has been shown that microlesions in the CNS reduce the extent ofthe glial scar and allow axons access to CNS white matter distal to the lesion (Davies etai, (1997) Nature 390:680-683). Microlesions of optic nerve were therefore used to axotomize RGC axons (Fig. 6). Regenerating RGC axons were visualized by anterograde labeling with cholera toxin and immunofluorescent detection ofthe cholera protein in longitudinal cryostat sections ofthe optic nerve. Some sections were double-labeled with glial fibrillary acidic protein (GFAP) to better resolve the lesion site.
  • GFAP glial fibrillary acidic protein
  • a spinal cord homogenate was made from 2 adult Sprague Dawley rat spinal cords.
  • the spinal cords were removed from adult rats, frozen in liquid nitrogen, and pulverized into powder with a mortar and pestal.
  • Twenty ml of PBS was added to the spinal cord powder, the solution was homogenized with a Dounce teflon-in-glass homogenizer, and the protein concentration was determined by protein assay (Biorad kit). The protein concentration was adjusted to 1.5 mg/ml.
  • the spleens were fixed and examined for enlargement. Also, the blood was collected from rats receiving injection of spinal cord homogenate with adjuvant or adjuvant alone. The blood was left overnight in the refrigerator, and the serum separated from the red cells by centrifugation.
  • Immunoreactivity ofthe serum to spinal cord homogenate, myelin protein, or MAG was tested by ELISA.
  • spinal cord homogenate, myelin, or MAG was dried overnight into 96 well plates.
  • Serum diluted 1 : 10 was added to the plates, and the plates were incubated for 2 hours at 37EC. The plates were washed and treated with anti-rat antibody conjugated with alkaline phosphatase.
  • a color reaction was obtained using 1 tablet p nitropheny phosphate (Sigma) made up fresh in substrate solution (6.4 ml 0.2M sodium carbonate, 18.5 ml 0.2M sodium bicarbonate, 0.2 ml 1 M MgC12, made up to 100 ml). After development of color, the reaction was stopped with 50 ⁇ l 3 M NaOH added to a well containing 100 ⁇ l of substrate solution. The optical density was read with an ELISA plate reader.
  • Rats were anesthetized with 0.6 ml/kg hypnorm, 2.5 mg kg diazepan, and 35 mg/kg ketamin.
  • the left optic nerve was exposed and the sheath was slit longitudinally to allow a 10.0 suture to be passed around the nerve separately from the sheath.
  • the optic nerve was crushed 1 mm from the globe by constriction for 60 seconds (Fig. 6).
  • Retrograde labeling of RGCs from the superior colliculus (not shown), and anterograde labeling (eg., Ctn) verified that RGC axons were axotomized by the microlesion.
  • Comparison of control optic nerves to those treated with the immunogenic composition revealed axon regeneration in the latter group (Figs. 7, 8, 9, 10). Numerous axons were observed to extend long distances of over 1 mm in the optic nerve. These axons could be easily identified as regenerating axons by their twisted path of growth, and by the identification of growth cones at the distal end of these fibers.

Abstract

La présente invention concerne des compositions comprenant des agents immunogènes possédant des propriétés d'inhibition de croissance de neurones, ces compositions entraînant la génération d'anticorps polyréactifs se liant à des inhibiteurs de croissance de l'axone, ce qui favorise la régénération de l'axone. Ces agents comprennent de la glycoprotéine associée à la myéline (MAG), ainsi que des protéines arrétine ou protéoglycane. On peut utiliser ces compositions pour restaurer des transmissions nerveuses, notamment une fonction motrice et sensorielle après des lésions de la moelle épinière ou des lésions du nerf optique. On peut également les utiliser pour traiter des affections impliquant des lésions de l'axone telles qu'une sclérose en plaques et un accident vasculaire cérébral. On peut enfin utiliser les compositions selon l'invention comme vaccins prophylactiques avant que ne survienne une maladie ou une lésion du système nerveux central.
PCT/CA1999/000304 1998-04-16 1999-04-16 Molecules inhibant la croissance neuronale ou leurs derives utilises pour immuniser des mammiferes et ainsi favoriser la regeneration de l'axone WO1999053945A1 (fr)

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WO1999060021A2 (fr) * 1998-05-19 1999-11-25 Yeda Research And Development Co. Ltd. Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation
WO2001066120A1 (fr) * 2000-03-10 2001-09-13 Department Of Human Physiology, Flinders University School Of Medicine Amelioration de l'assistance a la croissance nerveuse
WO2002062383A2 (fr) * 2001-02-08 2002-08-15 Smithkline Beecham P.L.C. Nouvelle methode de traitement
WO2004014953A3 (fr) * 2002-08-06 2004-05-06 Glaxo Group Ltd Anticorps
WO2004083363A2 (fr) * 2003-03-19 2004-09-30 Glaxo Group Limited Methode de traitement
US7560102B2 (en) 1998-05-19 2009-07-14 Yeda Research And Development Co., Ltd Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
EP2163561A1 (fr) 2000-01-12 2010-03-17 Yale University Blocage de la croissance axonale à médiation par récepteur NOGO
US7772200B2 (en) 2005-07-21 2010-08-10 Alnylam Pharmaceuticals, Inc. iRNA agents targeted to the Rho-A gene

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7560102B2 (en) 1998-05-19 2009-07-14 Yeda Research And Development Co., Ltd Method for reducing neuronal degeneration so as to ameliorate the effects of injury or disease
WO1999060021A3 (fr) * 1998-05-19 2000-06-15 Yeda Res & Dev Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation
WO1999060021A2 (fr) * 1998-05-19 1999-11-25 Yeda Research And Development Co. Ltd. Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation
EP2163561A1 (fr) 2000-01-12 2010-03-17 Yale University Blocage de la croissance axonale à médiation par récepteur NOGO
WO2001066120A1 (fr) * 2000-03-10 2001-09-13 Department Of Human Physiology, Flinders University School Of Medicine Amelioration de l'assistance a la croissance nerveuse
WO2002062383A3 (fr) * 2001-02-08 2003-04-10 Smithkline Beecham Plc Nouvelle methode de traitement
WO2002062383A2 (fr) * 2001-02-08 2002-08-15 Smithkline Beecham P.L.C. Nouvelle methode de traitement
EP1645285A3 (fr) * 2001-02-08 2010-04-14 SmithKline Beecham Limited Utilisation thérapeutique d'anticorps anti-mag pour traiter l'accident ischèmique
EP1645285A2 (fr) * 2001-02-08 2006-04-12 Smithkline Beecham Plc Utilisation thérapeutique d'anticorps anti-mag pour traiter l'accident ischèmique
WO2004014953A3 (fr) * 2002-08-06 2004-05-06 Glaxo Group Ltd Anticorps
US7612183B2 (en) 2002-08-06 2009-11-03 Glaxo Group Limited Humanised anti-mag antibody or functional fragment thereof
US8071731B2 (en) 2002-08-06 2011-12-06 Glaxo Group Limited Humanised anti-MAG antibody or functional fragment thereof
EP2110139A2 (fr) * 2003-03-19 2009-10-21 Glaxo Group Limited Méthode d'identification d'anticorps anti MAG (myelin associated glycoprotein)
WO2004083363A2 (fr) * 2003-03-19 2004-09-30 Glaxo Group Limited Methode de traitement
WO2004083363A3 (fr) * 2003-03-19 2004-12-02 Glaxo Group Ltd Methode de traitement
EP2110139A3 (fr) * 2003-03-19 2010-04-21 Glaxo Group Limited Méthode d'identification d'anticorps anti MAG (myelin associated glycoprotein)
US8017115B2 (en) 2003-03-19 2011-09-13 Glaxo Group Limited Therapeutical use of anti-myelin associated glycoprotein (MAG) antibodies
US7772200B2 (en) 2005-07-21 2010-08-10 Alnylam Pharmaceuticals, Inc. iRNA agents targeted to the Rho-A gene
EP2230305A1 (fr) 2005-07-21 2010-09-22 Alnylam Pharmaceuticals Inc. Modulation de l'arni du gène rho-a et utilisations de celle-ci

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