ES2352654T3 - FUSION PROTEINS. - Google Patents

Abstract

A polypeptide set forth in SEQ ID NO .: 43 or a polypeptide comprising at least one transport agent region covalently linked to an active agent region, wherein the transport agent region is a proline-rich region, in where the transport agent region is at the carboxy terminal end of said polypeptide and the active agent region is at the amino terminal end of said polypeptide, and said active agent region is selected from the group consisting of ADP-ribosyl C3 transferase and a fragment thereof that retains ADP-ribosyl transferase activity, where the proline-rich region is in SEQ ID NO .: 48.

Description

FIELD OF THE INVENTION

The present invention relates to a conjugate or fusion-like proteins (polypeptides) comprising, for example, C3 (see below) (i.e., C3-like protein, C3 chimeric proteins). Although, hereinafter, the fusion-type proteins of the present invention will be discussed particularly in relation to use to facilitate axon regeneration and neuroprotection, it is understood that fusion proteins can be exploited in other contexts.

The present invention in particular belongs to the field of mammalian nervous system repair (for example repair of a central nervous system (CNS) injury site or a peripheral nervous system (SNP) site of injury), axon regeneration and reinnervation axon, neurite growth and protection from neurodegeneration and ischemic damage.

The GTPases of the Rho family regulate axon growth and regeneration. Inactivation of Rho with C3 Clostridium botulinum exotransferase (simply as referred to hereinafter C3) can stimulate regeneration and reinnervation of injured axons; C3 is a purified toxin from Clostridium botulinum (see Saito et al., 1995, FEBS Lett 371: 105-109; Wilde et al 2000. J. Biol. Chem. 275: 16478). The C3 family compounds of Clostridium botulinum inactivate Rho by ADP-ribosylation and thus act as antagonists of the Rho effect or function (Rho antagonists).

The present invention in particular relates to a means of intracellular delivery of the C3 protein (eg C3 itself or other active analogues such as C3-like transferases - see below) or other Rho antagonists to repair damage to the nervous system. , to avoid ischemic cell death, and to treat various diseases where Rho inactivation is required. The delivery media can take the form of chimeric C3-like Rho antagonists (i.e. conjugates). These conjugated antagonists provide a significant improvement over C3 compounds (alone) because they are 3 to 4 orders of magnitude more potent with respect to stimulating axon growth on inhibitory substrates than on recombinant C3 alone. Examples of these Rho antagonists have been made as recombinant proteins created to facilitate penetration of the cell membrane (i.e. to improve cellular uptake of antagonists), improve dose response when applied to neurons to stimulate growth on substrates growth inhibitors, and to inactivate Rho. Examples of these Rho conjugated antagonists are described below in connection with the designations C3APL, C3APLT, C3APS, C3-TL, C3TS, C3Basic1, C3Basic2 and C3Basic3.

BACKGROUND OF THE INVENTION

Traumatic injury to the spinal cord results in permanent functional impairment. Most of the deficits associated with spinal cord injury result from the loss of axons that are damaged in the central nervous system (CNS). Similarly, other CNS diseases are associated with axonal loss and retraction, such as stroke, human immunodeficiency virus (HIV) dementia, prion diseases, Parkinson's disease, Alzheimer's disease, multiple sclerosis, and glaucoma. Common to all these diseases is the loss of axonal connections with their targets, and cell death. The ability to stimulate axon growth of the diseased or affected neuronal population would enhance recovery from loss of neurological functions, and protection from cell death may limit the degree of damage. For example, after a white matter stroke, axons are damaged and lost, even though the neural cell bodies are alive, and the gray matter stroke kills many neuronal and non-neuronal (glial) cells. Treatments that are effective in causing reinnervation of injured axons are equally effective in treating some types of stroke (Boston life sciences, Sept. 6, 2000 Press release). Neuroprotective agents are frequently tested as potential compounds that can limit damage after stroke. Compounds that show growth promotion and neuroprotection are especially good candidates for the treatment of stroke and neurodegenerative diseases. Similarly, although the following discussion will relate generally to the supply of Rho antagonists, etc. With traumatic damage to the nervous system, this invention can also be applied to damage of unknown origin, such as during stroke, multiple sclerosis, HIV dementia, Parkinson's disease, Alzheimer's disease, prion diseases, or other CNS diseases where damage axons in the CNS environment. Also, Rho is an important target for the treatment of cancer and metastasis (Clark et al (2000) Nature 406: 532-535), and hypertension (Uehata et al. (1997) Nature 389: 990) and it is reported that the RhoA has a cardioprotective role (Lee et al. FASEB J. 15: 1886-1884). Therefore, the new C3-like protein is expected to be useful for a variety of diseases that require inhibition of Rho activity.

The use of various Rho antagonists has been proposed as agents to stimulate the regeneration of axons (cut), that is, nerve injuries; please see, for example, Canadian Patent Applications us. 2,304,981 (McKerracher et al) and 2,300,878 (Strittmatter). These patent application documents propose the use of known Rho antagonists such as for example C3, chimeric C3 proteins, etc. (see below) as well as substances selected from known trans-4-amino (alkyl) -1-pyridylcarbamoylcyclohexane compounds (also see below) or Rho kinase inhibitors for use in axon regeneration. C3 inactivates Rho by ADP-ribosylation and is quite non-toxic to cells (Dillon and Feig (1995) Methods in Enzymology: Small GTPases and their regulators Part. B.256: 174-184).

Although the following discussion will be generally related or directed to CNS repair, the techniques described here can be extended for use in many other diseases including, but not limited to, cancer, metastasis, hypertension, heart disease, stroke, diabetic neuropathy, and neurodegenerative disorders such as stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). Treatment with Rho antagonists will be used to improve the axon growth rate of peripheral nerves and are therefore effective for repair of peripheral nerves after surgery, for example after replanting amputated limbs. Also, treatment with our fusion compounds (proteins) is expected to be effective for the treatment of various peripheral neuropathies due to its axon growth promoting effects.

As previously mentioned, traumatic spinal cord injury results in permanent functional impairment. Axon regeneration does not occur in the adult mammalian CNS due to substrate-linked growth inhibitory proteins that block axon growth. Many compounds, such as trophic factors, enhance neuronal differentiation and stimulate axon growth in tissue culture. However, many factors that enhance growth and differentiation are not capable of promoting regenerative axon growth on inhibitory substrates. To demonstrate that a compound known to stimulate axon growth in tissue culture more accurately reflects the potential for therapeutic use for axon regeneration in the CNS, it is important for cell culture studies to include the demonstration that a compound can allow axon growth on growth inhibitory substrates. An example of differentiation and trophic factors that stimulate growth in permissive substrates in tissue culture are neurotrophins such as nerve growth factor (NGF) and brain derived growth factor. NGF, however, does not promote growth on inhibitory substrates (Lehmann, et al. (1999) 19: 7537-7547) and it is not effective in promoting axon regeneration in vivo. Brain-derived neurotrophic factor (BDNF) is not effective in promoting regeneration in vivo (Mansour-Robaey, et al. J. Neurosci. (1994) 91: 1632-1636). BDNF does not promote neurite growth on growth inhibitory substrates (Lehmann et al supra).

Objective intracellular signaling mechanisms involving Rho and Rho kinase have been proposed to promote axon regeneration (see, for example, the aforementioned Canadian Patent application no. 2,304,981 (McKerracher et al)). To demonstrate that Rho inactivation promotes axon regeneration on growth inhibitory substrates, recombinant C3, a protein that inactivates Rho by ADP ribosylation of the effector domain, is used. While such a C3 protein can effectively promote regeneration, it has been noted that such a C3 protein does not easily penetrate cells, and therefore high doses can be applied for being effective.

The high dose of recombinant C3 that needs to promote functional recovery presents a practical restriction or limitation on the use of C3 in vivo to promote regeneration (Lehmann, et al. (1999) J. Neurosci. 19: 7537-7547; Morii, N and Narumiya, S. (1995) Methods in Enzymology, Vol 256 part B, p.196-206 In tissue culture studies, for example, it has been determined that the minimum amount of C3 that can be used to induce Growth on inhibitory substrates is 25 ug / ml (Lehmann, et al. (1999) J. Neurosci. 19: 7537-7547; Morii, N and Narumiya, S. (1995) Methods in Enzymology, Vol 256 part B, pg. If the cells are not shredded, this dose is still ineffective It has been estimated, for example, that at least 40 µg of C3 per 20 g mouse needs to be applied to the spinal cord of the injured mouse or the rat optic nerve (McKerracher, Canadian Patent Application No .: 2,325,842). Calculate the doses that will be required to treat an adult human in an e For the equivalent dose-by-weight scale used for rat and mouse experiments, it would be necessary to apply 120 mg / kg C3 (ie alone) to the injured human spinal cord. The large amount of recombinant C3 protein needs to create significant manufacturing problems, due to large-scale protein purification and costs. This also limits the variant dose that can be tested due to the large amount of protein required for minimal effective doses.

Another related limitation regarding the use of C3 to promote repair of the injured CNS is that it does not essentially penetrate the plasma membrane of living cells. In tissue culture studies when C3 is applied the biological test effects have been microinjected directly into the cell (Ridley and Hall (1992) Cell 70: 389-399), or applied by shredding the cells by breaking the membrane of plasma (Lehmann, et al. (1999) J. Neurosci. 19: 7537-7547, Jin and Strittmatter (1997) J. Neurosci. 17: 6256-6263). In the case of axon injury in vivo, the C3 protein is likely able to enter the cell because the injured axons easily take up substances from their environment. However, the C3-like proteins of the present invention also act similarly on surrounding damaged neurons and help them make new connections as well, thus facilitating recovery. After incomplete SCI, there is plasticity of motor systems attributed to cortical and subcortical levels, including the spinal cord circuit (Raineteau, O., and Schwab, M. E. (2001) Nat Rev Neurosci 2: 263-73). This plasticity can be attributed to reinnervation of collateral or dendritic axons and synaptic resistance or weakening. Additionally, it has been shown that the preservation of a few ventrolateral fibers can translate into significant differences in locomotor performance since these fibers are important in the initiation and control of locomotor pattern through central spinal pattern generators (Brustein, E. , and Rossignol, S. (1998) J Neurophysiol 80: 1245-67). It is well documented that reorganization of preserved collateral cortical spinal fibers occurs after spinal cord injury and this contributes to functional recovery (Weidner et al, 2001 Proc. Natl. Acad. Sci. 98: 3513-3518). The process of reorganization and reinnervation of preserved fibers will be improved by treatment with C3-like proteins capable of entering neurons without injury. This would spontaneously improve axon plasticity and dendritic remodeling known to aid functional recovery.

Other methods of C3 delivery in vitro have made a recombinant protein that can be taken up by a receptor mediated mechanism (Boquet, P. et al. (1995) Meth. Enzymol. 256: 297-306). The disadvantage of this method is that the cells that need the treatment can express the necessary receptor. Finally, the addition of a C2II-binding protein to the tissue culture medium, together with a C21N-C3 fusion toxin, allows uptake of C3 by receptor mediated endocytosis (Barthe et al. (1998) Infection and Immunity 66 : 1364). The disadvantage of this system is that much of the C3 in the cell will be restricted within a membrane compartment. More importantly, two different proteins can be added separately to transport what occurs (Wahl et al. 2000. J. Cell Biol. 149: 263), which makes this system difficult to apply for the treatment of disease in vivo .

WO 99/23113 describes the use of Rho antagonists, or Rho related proteins as therapeutic targets for agents designed to block growth inhibition by myelin or myelin proteins. One embodiment pertains to the use of Rho antagonists that promote axon regeneration in the central nervous system. Therapeutic or antagonistic agents can be small molecules, proteins or peptides, or any agent that binds Rho or its family members to inactivate this pathway. Regarding the use of C3 transferase, we describe C3 ADP-ribosyl transferase, Clostridium botulinum C3, and C3 fragments, and the use of plasmid pGEX2T-C3 encoding the G3-C3 fusion protein to prepare recombinant C3 with cleavage posterior fusion protein by thrombin.

B. I. Kazmierczak et al. "Rho GTPase activity modulates Pseudomonas aeruginosa internationalization by epithelial cells", Cellular Microbiology (2001), 3 (2), pp. 85-98 describes a C3 ADP-ribosyltransferase membrane permeable fusion construct that is described as "TAT-C3" and as a membrane permeable C3 fusion protein, using an 11-amino acid sequence of the HIV TAT protein TAT fused to the C3 N-Terminal to confer membrane permeability on the protein.

SUMMARY OF THE INVENTION The problems are solved according to the subject matter of claims 1-3 and 7. According to the present invention a polypeptide comprising an agent

Therapeutically active is provided where the active agent can be delivered through a cell wall membrane, the fusion protein (polypeptide) which comprises at least one transport subdomain or functional group (i.e. transport agent region) in addition to an active agent functional group (i.e., active agent region). More particularly, as discussed herein, according to the present invention a conjugate or fusion protein is provided wherein the therapeutically active agent is one capable of facilitating (to facilitate) the growth of axon (or dendrite, or neurite) (by regeneration example) ie a conjugate or fusion protein in the form of a conjugated Rho antagonist.

The present invention according to one aspect thereof provides a polypeptide [eg capable of suppressing inhibition of neuronal axon growth at a site of central nervous system (CNS) injury or a site of peripheral nervous system injury ( SNP)] comprising at least one transport agent region covalently attached to an active agent region, where the transport agent region is a proline-rich region, where the transport agent region is at the extreme the carboxy terminal of said polypeptide and the active agent region is at the amino terminal end of said polypeptide, and said active agent region is selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains an activity ADPribosyl transferase, where the proline-rich region is in SEQ ID NO .: 48. The transport agent region is capable of facilitating (ie facilitating) the uptake of the region n of active agent in a mammalian (i.e. human or animal) tissue or cell, and the active agent region is a region of active therapeutic agent capable (i.e. has the ability or is capable) of facilitating axon growth by example on growth inhibitory substrates (for example regeneration), in vivo (in a mammal (for example, human or animal)) or in vitro (in cell culture), including a derivative or homologue thereof (i.e. their equivalents pharmaceutically acceptable chemicals - derivative or pharmaceutically acceptable homolog). In accordance with the present invention the active agent region may be a C3 ADP-ribosyl transferase region. In accordance with the present invention C3 ADP-ribosyl transferase can be selected from the group consisting of ADP-ribosyl transferase (eg ADP-ribosyl transferase C3) derived from Clostridium botulinum and a recombinant ADPribosyl transferase (eg recombinant ADPribosyl transferase C3) that includes the entire C3 coding region, or only a part (fragment) of the C3 coding region that retains ADP-ribosyl transferase activity, or C3 analogs (derivatives) that retain ADP-ribosyl transferase activity, or enough of the C3 coding region for being able to effectively inactivate Rho. The active agent can also be selected from other known ADP-ribosyl transferases that act on Rho (Wilde et al. 2000 J. Biol. Chem. 275-16478-16483; Wilde et al 2001. J. Biol. Chem. 276: 9537 -9542). The present invention in a further aspect provides the use of a fusion protein (polypeptide) as described herein (eg including its pharmaceutically acceptable chemical equivalents) to suppress inhibition of neuronal axon growth. The present invention in a further aspect relates to a pharmaceutical composition (eg, to suppress inhibition of neuronal axon growth), the pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier, and an effective amount of a fusion protein (polypeptide) as described here (eg including its pharmaceutically acceptable chemical equivalents). The present invention is further provided for the use of a fusion protein (polypeptide) as described herein (eg including its pharmaceutically acceptable chemical equivalents) for the manufacture of a pharmaceutical composition (eg to suppress inhibition of neuronal axon growth ). Also disclosed is a method of preparing a drug delivery construct, a conjugate or fusion protein (polypeptide) as defined above comprising -culturing a host cell (bacterial or eukaryotic) under conditions that provide expression of the delivery construct of drug, the conjugate or fusion protein (polypeptide) within the cell; (the drug, conjugate or fusion protein (polypeptide) delivery construct can also be expressed to be

6 produced in animals, such as, for example, the production of recombinant proteins in the milk of farm animals), and - recover the drug, conjugate or fusion protein (polypeptide) supply construct by a purification step. Purification of the drug, conjugate or fusion protein (polypeptide) delivery construct can be done by affinity methods, ion exchange chromatography, size exclusion chromatography, hydrophobicity, or any other purification technique typically used for purification. of protein. Preferably, the purification step will be carried out under non-denaturing conditions. On the other hand, if a denaturing step is required, the protein can be renatured using techniques known in the art. Also described is the expression of a drug, conjugate, or fusion protein (polypeptide) delivery construct in a mammalian cell, which, when used with a signal sequence, will allow expression and secretion of the fusion protein in extracellular medium. . Another expression system (yeast cells, bacterial cells, insect cells, etc.) may be suitable for expressing (producing) the drug, conjugate or fusion protein (polypeptide) delivery construct of the present invention as discussed herein. . The present invention in particular provides a fusion protein (polypeptide) selected from the group consisting of C3APLT (SEQ ID NO .: 37) and SEQ ID NO .: 43 and their pharmaceutically acceptable chemical equivalents. In accordance with a further aspect, the present invention provides a pharmaceutical composition comprising a polypeptide selected from the group consisting of C3APLT (seq ID NO.:37), and SEQ ID NO .: 43, and a pharmaceutically acceptable carrier. In accordance with the present invention, the pharmaceutical composition may further comprise a biological bioadhesive, such as, for example, fibrin (fibrin glue). In a further aspect the present invention provides a pharmaceutical composition comprising a polypeptide comprising at least one (one or more) transport agent region and one active agent region, said active agent region is selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADPribosyl transferase C3 activity, and a pharmaceutically acceptable carrier. In accordance with the present invention, the transport agent region is at the carboxy-terminal end of said polypeptide and the active agent region is at the amino-terminal end of said polypeptide. In accordance with the present invention, the pharmaceutical composition may further comprise a biological bioadhesive, such as, for example, fibrin (fibrin glue). In a further aspect, the present invention provides a polypeptide comprising at least one (one or more) transport agent region and one active agent region, said active agent region is selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains a C3 ADP-ribosyl transferase activity (wherein the transport agent region is capable of facilitating uptake of the active agent region in (within the cell or cell membrane) a cell). In accordance with the present invention, the carboxy terminal transport functional group region is a proline rich region. In a further aspect, the present invention relates to a polypeptide consisting of an amino terminal active agent functional group and a carboxy terminal transport functional group region, wherein said amino terminal active agent functional group is

you can select from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity.

In accordance with the present invention, the carboxy terminal transport functional group region is a proline rich region.

In accordance with the present invention, the transport agent region can be fused to ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity according to recombinant DNA technology (eg, cloning of the DNA sequence of the transport agent region in box with the DNA sequence of the C3 analog ADP-ribosyl transferase or an ADP-ribosyl transferase C3 whether or not it comprises a spacer DNA sequence (multiple site cloning, linker) or any other DNA sequence that would not interfere with the activity of the C3-like protein once expressed).

In a further aspect, the present invention relates to the use of a polypeptide selected from the group consisting of C3APLT (SEQ ID NO.:37), and SEQ ID NO .: 43, for the manufacture of a pharmaceutical composition.

In other aspects, the present invention relates to the use of a polypeptide comprising at least one (one or more) transport agent region and one active agent region, for the manufacture of a pharmaceutical composition, or to facilitate ( facilitates) axon growth or to treat (in treatment of) nerve injury (for example, nerve injury arising from traumatic nerve injury or nerve injury caused by disease), or to prevent (decrease, inhibit ( partially or totally)) cell apoptosis (cell death, such as after CNS ischemia), or to suppress (decrease) inhibition of neuronal axon growth, or to treat stroke-related ischemic damage, or to suppress (decrease) Rho activity, or to regenerate (regenerate) the injured axon (help the injured axon to partially or fully regain its function), or to help (help) neurons to make new connections (developing axon, dendrite, neurite) with other (surrounding) cells (neuronal cells), in a mammal, (eg, human, animal), wherein said active agent region is selected from the group consisting of ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogs.

In accordance with the present invention, the transport agent region is at the carboxy terminus of the polypeptide (i.e., protein) and C3 ADP-ribosyl transferase or a fragment thereof that retains C3 ADP-ribosyl transferase activity. it is at the amino terminus of the polypeptide (ie, protein).

A method of suppressing inhibition of neuronal axon growth (eg, in a mammal, (eg, human, animal)) is disclosed which comprises administering (eg, supplying) a member selected from the group consisting of a construct of drug delivery, a drug conjugate, a fusion protein, and a polypeptide (eg, including their pharmaceutically acceptable chemical equivalents), said polypeptide comprises at least one (one or more) transport agent region and one active agent region selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity (directly) at (a) a site of injury to the central nervous system (CNS)

or a site of injury to the peripheral nervous system (PNS) (of a patient), in an amount effective to counteract such inhibition. Such an application may be useful for the treatment of a wide variety of peripheral neuropathies, such as diabetic neuropathy.

Recombinant Rho antagonists are described that comprise C3 enzymes with a proline-rich region added to the C3 coding sequence to facilitate their return to tissue or cells for repair and / or growth promotion in the CNS, even in the absence of damage to traumatic axon. Examples of proline-rich regions are given below.

A method of facilitating axon growth (eg, in a mammal, (eg, human, animal)) is disclosed which comprises supplying a polypeptide comprising at least one transport agent region and one selected active agent region. from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADPribosyl transferase C3 activity directly at a site of central nervous system (CNS) injury or a site of peripheral nervous system (SNP) injury, in an effective amount to facilitate such growth.

In a further aspect, a method of treating nerve injury (eg, in a mammal, (eg, human, animal)) is disclosed which comprises supplying a polypeptide comprising at least one transport agent region and one region of active agent selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity directly to (at) a central nervous system (CNS) site of injury or site of injury of the peripheral nervous system (SNP).

In accordance with a further aspect, a method for regenerating the injured axon (eg, in a mammal, (eg, human, animal)) is disclosed comprising supplying a polypeptide comprising at least one transport agent region and an active agent region selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity directly at a site of central nervous system (CNS) injury or an injury site of the peripheral nervous system (SNP) (eg, in a mammal), in an amount effective to regenerate said injured axon.

In accordance with a further aspect, a method is described to help neurons to make a new cellular connection (developing axon, dendrite, neurite with other (surrounding) cells (neuronal cells) which comprises supplying a polypeptide or conjugate comprising at least a transport agent region and an active agent region selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof that retains ADPribosyl transferase C3 activity directly at a site of central nervous system (CNS) injury or a site of injury to the peripheral nervous system (PNS).

In a further aspect, a method is described for preparing a polypeptide comprising at least one (one or more) transport agent region and one active agent region, wherein said transport agent region can be selected from the group that it consists of SEQ ID NO .: 48 and wherein said active agent region can be selected from the group consisting of ADP-ribosyl transferase C3 and a fragment thereof which retains ADP-ribosyl transferase C3 activity, said method comprises:

-cultivate a host cell under conditions that is provided for

expression of the polypeptide within the cell; and

-recover the polypeptide by a purification step.

Polypeptide purification can be done by affinity methods, ion exchange chromatography, size exclusion chromatography, hydrophobicity, or any other purification technique typically used for protein purification. Preferably, the purification step will be carried out under non-denaturing conditions. On the other hand, if a denaturing step is required, the protein can be renatured using techniques known in the art.

In yet another aspect, the present invention relates to the use of a polypeptide comprising at least one transport agent region and one active agent region, said active agent region being selected from the group consisting of ADPribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity for the manufacture of a medicament (or a pharmaceutical composition) to suppress inhibition of neuronal axon growth.

In accordance with the present invention, the polypeptide can be selected from the group consisting of C3APLT (SEQ ID NO.:37), and seq ID NO .: 43.

In a further aspect, the present invention relates to the use of a polypeptide comprising at least one transport agent region and one active agent region, said active agent region being selected from the group consisting of ADPribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity for the manufacture of a medicament (or pharmaceutical composition) to facilitate axon growth.

In accordance with the present invention, the polypeptide can be selected from the group consisting of CAAPLT (SEQ ID NO.:37), and SEQ ID NO .: 43.

In still a further aspect the present invention relates to the use of a polypeptide comprising at least one (one or more) transport agent region and one active agent region, said active agent region is selected from the group consisting of of ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity for the manufacture of a medicament (or pharmaceutical composition) to treat nerve injury (eg, in a mammal, (eg, human , animal)).

In accordance with the present invention, the polypeptide can be selected from the group consisting of C3APLT (SEQ ID NO.:37), and SEQ ID NO .: 43.

In accordance with the present invention, the transport agent region discussed here is a proline-rich region (eg proline-comprising region).

In accordance with the present invention, the proline-rich region discussed here can be selected from the group consisting of SEQ ID NO .: 48 (APLT) and its analogs.

Transport of a functional functional group across the cell membrane (intracellular delivery) can be facilitated (increased) when it is bound (eg, genetically fused, chemically crosslinked, etc.) to SEQ ID NO: 48. describes to provide a method for intracellular delivery of a cargo functional group, the method comprises exposing the cell to a delivery agent comprising a cargo functional group and a transport functional group, said transport functional group is selected from the group consisting of SEQ ID NO: 48 and its analogs and wherein said transport functional group allows the delivery agent to be released into the cell (ie, through cell membranes). An example of a functional cargo group that can be delivered through the cell membrane is ADP-ribosyl transferase C3 and its analogs. Other examples of a functional charge group are mentioned here. The method also comprises bringing the delivery agent comprising a cargo functional group and a transport functional group (SEQ ID NO: 48 and its analogs) around a target cell in a form (eg, concentration) sufficient to allow uptake of the supply agent through the cell. For example, in the case of in vitro delivery (for example, cell culture), the delivery agent (in a pharmaceutically acceptable carrier, diluent, excipient, etc.) can be added directly to the extracellular medium (for example, cell culture medium ) of adherent cells (i.e. cell lines or primary cells) or cells in suspension. Alternatively, cells can be harvested and concentrated before being contacted with the delivery agent. Intracellular delivery can be monitored by techniques known in the art, such as, for example, immunofluorescence, immunohistochemistry, or by the intrinsic properties of the cargo functional group (eg, its enzymatic activity).

In vivo delivery (in a mammal) can develop eg by exposing (i.e. contacting) tissue, a site of nerve injury, an open wound, etc. with the delivery agent (in a pharmaceutically acceptable carrier, diluent, excipient, fibrin gel etc.) in an amount sufficient to promote the biological effect of the cargo functional group (eg, recovery, healing of wounded tissue, etc.). Additionally, in vivo delivery can be developed by other methods known in the art such as, for example, injection via intramuscular (IM), subcutaneous (SC), intradermal (ID), intravenous (IV) routes. ) or intra-peritoneal (IP) or administration to the mucosal membranes including the oral and nasal cavity membranes using any suitable means. Alternatively, cells from a mammal can be isolated and treated (exposed) ex-vivo (eg, in gene therapy techniques) with the delivery agent before being reinfused into the same individual or a compatible individual.

The term "Rho antagonists" as used herein includes, but is not restricted to, (known) C3, including C3 chimeric proteins, and as Rho antagonists.

The term "C3 protein" refers to ADP-ribosyl transferase C3 isolated from Clostridium botulinum or a recombinant ADPribosyl transferase.

The term "C3-like protein", "C3ADP-ribosyl transferase-like protein", "ADP-ribosyl transferase C3 analog", "C3-like transferase" or "C3 chimeric protein" as used herein refers to any protein ( polypeptide) having similar biological activity (eg, equal, substantially similar), tao ADP-ribosyl transferase C3. Examples of such a C3-like protein include, for example, but are not restricted to C3APL, C3APLT, C3APS, C3-TL, C3-TS, C3Basic1, C3Basic2, and C3Basic3 and the protein defined in SEQ ID NO .: 20.

The term "nerve injury site" refers to a site of traumatic nerve injury or nerve injury caused by disease. The site of nerve injury may be a single nerve (eg, sciatic nerve) or a nerve tract comprised of many nerves (eg, damaged region of the spinal cord). The site of nerve injury can be in the central nervous system or peripheral nervous system or in any necessary repair region. The nerve injury site can form as a result of stroke-related damage. The nerve injury site may be in the brain as a result of surgery, brain tumor removal, or therapy after a cancerous injury. The nerve injury site can result from stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), diabetes, or any other type of neurodegenerative disease.

The term "cargo" refers to a molecule that is different from the transport functional group and that is not inherently capable of (1) entering a cell (eg, cell compartment) or (2) is not inherently capable of entering a cell (eg example, cell compartment) in a useful proportion. The term "cargo" as used herein refers to a molecule per se, ie, prior to conjugation, or to the functional group of cargo of a transport polypeptide cargo conjugate. Examples of "charge" include, but are not limited to, small molecules and macromolecules such as polypeptides, nucleic acids (polynucleotides), polysaccharides, and chemicals.

As used herein, the term "delivery agent" relates to an agent comprising a cargo functional group and a transport functional group. Examples of cargo functional group were discussed above and include for example ADP-ribosyl transferase C3 and a fragment thereof that retains ADP-ribosyl transferase C3 activity. Examples of transport functional groups include for example SEQ ID NO: 48 and its analogs.

"Polynucleotide" generally refers to any polyribonucleotide or polideoxyribonucleotide, which can be unmodified DNA or RNA, or modified RNA or DNA. "Polynucleotides" include, without limitation, single and double stranded DNA, DNA that is a mixture of single and double stranded regions, single and double stranded RNA, and RNA that is a mixture of single and double stranded regions, hybrid molecules that comprise DNA and RNA that can be single-stranded, more typically double-stranded or a mixture of single or double-stranded regions. Additionally, "polynucleotide" refers to triple chain regions comprising RNA or DNA or both RNA AND DNA. The term polynucleotide also includes DNA and RNA that contain one or more modified bases and DNA and RNA with modified structures for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications have been made for DNA and RNA; thus the "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. The "polynucleotide" includes but is not limited to linear, closed-end molecules. The "polynucleotide" also encompasses relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptides" refers to any peptide or protein that comprises two or more amino acids linked to each other by peptide linkages or modified peptide linkages (ie, peptide isosters). "Polypeptide" refers to short chains, commonly referred to as peptides, oligopeptides, or oligomers, and with long chains generally referred to as proteins. As described above, polypeptides can contain different amino acids than the 20 amino acids that encode the gene.

As used herein the term "analogs" relates to additional mutants, variants, chimeras, fusions, deletions, and any other type of modifications made relative to a given polypeptide. The term "analog" is synonymous with homologous, derivative and chemical equivalent.

or biological equivalent.

As used herein, the term "homologous" sequence relates to the nucleotide or amino acid sequence derived from the DNA sequence or polypeptide sequence of C3APL, C3APLT, C3APS, C3-TL, C3-TS, C3Basic1, C3Basic2 and C3Basic3 .

As used herein, the term "heterologous" sequence relates to the DNA sequence or amino acid sequence of a heterologous polypeptide and includes the sequence different from that of C3APL, C3APLT, C3APS, C3-TL, C3-TS, C3Basic1, C3Basic2 and C3Basic3.

As used herein the term "proline rich region" refers to a region of a protein with 5% or more (up to 100%) proline in this sequence. In some cases, a "proline rich region" can have between 5% and 15% prolines. Additionally, a "proline rich region" refers to a region, of a protein containing more prolines than is generally observed in naturally occurring proteins (eg, proteins encoded by the human genome). "Proline-rich region" of the present invention functions as a transport agent region.

As the term "to help the neuron to make new connections with other cells" or "to help neurons to make a new cellular connection" is used here it means that after treating cells (eg neurons) or tissue with a construction of drug delivery, a conjugate, a fusion protein, a polypeptide or a pharmaceutical composition of the present invention, neurons can grow (develop) eg new dendrite, new axon

either new neurite (ie cell bud), or existing dendrites, axon or neurite (ie cell bud) are induced to grow to a greater degree.

12 As used herein, the term "vector" refers to a DNA or RNA molecule that automatically replicates into whose previous DNA or RNA fragments are inserted and then propagated in a host cell for expression or amplification of the Previous DNA or RNA. The term "vector" comprises and is not limited to a plasmid (eg, linearized or not) that can be used to transfer DNA sequences from one organism to another. The term "pharmaceutically acceptable carrier" or "adjuvant" and "physiologically acceptable carrier" and the like are understood to refer to an acceptable carrier or adjuvant that can be administered to a patient, together with a compound of this invention, and which does not destroy its pharmacological activity. Additionally, as used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered medium. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases, and the like. As used herein, "pharmaceutical composition" means therapeutically effective amounts (doses) of the agent along with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. A "therapeutically effective amount" as used herein refers to that amount that provides a therapeutic effect for a given condition and regimen of administration. Such compositions are liquid or lyophilized or otherwise dry formulations and include diluents or various buffer content (eg Tris-HCl., Acetate, phosphate), pH and ion resistance, additives such as albumin or gelatin to prevent absorption detergents on the surfaces (eg Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (eg, glycerol, polyethylene glycerol), anti-oxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, thimerosal, benzyl alcohol, parabens), mass forming substances, or tonicity modifiers ( for example, lactose, mannitol), covalent adhesion of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or in particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or in liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte phantoms, or spheroplasts. Such compositions will influence physical state, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled or sustained release compositions include formulation and lipophilic deposits (eg, fatty acids, waxes, oils). Particulate compositions coated with polymers (eg poloxamers or poloxamins) are also encompassed by the invention. Other embodiments of the compositions of the invention incorporate particulate forms of protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, and oral routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly,

intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, intratumorally, or more preferably, directly to a site of central nervous system (CNS) injury or a site of peripheral nervous system (PNS) injury.

Additionally, the term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (dose) effective in treating a patient, who has, for example, a nerve injury. It is also understood herein that a "pharmaceutically effective amount" can be interpreted as an amount giving a desired therapeutic effect, taken in one dose or in any dosage or route, or taken alone or in combination with other therapeutic agents. In the case of the present invention, a "pharmaceutically effective amount" can be understood as an amount of ADP-ribosyl transferase C3 or ADP-ribosyl transferase C3 analogs (eg, fusion proteins) of the present invention which can eg suppress (for example, totally or partially) inhibition of neuronal axon growth, facilitate axon growth, prevent cell apoptosis, suppress Rho activity, help regenerate the injured axon, or that can help neurons make new ones connections with other cells.

As can be appreciated, a number of modifications can be made to the polypeptides of the present invention, such as for example the active agent region (eg, ADP-ribosyl transferase C3 or a fragment thereof that retains ADPribosyl transferase C3 activity. or the transport agent region without detrimentally affecting the biological activity of the polypeptides or fragments The polypeptides of the present invention comprise for example, those containing amino acid sequences modified by natural processes, such as post-translational processing, or by techniques Chemical modification techniques known in the art Modifications may occur in a polypeptide including the polypeptide structure, amino acid side chains and amino or carboxy terminus It will be appreciated that the same type of modification may be present in the same or several degrees at various sites in a given polypeptide. Also, a given polypeptide can c have many types of modifications. Polypeptides can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. Branched, cyclic and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. Modifications include for example, without limitation, acetylation, acylation, the addition of the acetomidomethyl group (mAb), AD Pribosylation, amidation, covalent binding to fiavin, covalent binding to a heme functional group, covalent binding of a nucleotide or nucleotide derivative, binding covalent of a lipid or lipid derivative, covalent binding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation , hydroxylation, iodination, methylation, myristolation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenolation, sulfation, transfer of RNA mediated by the addition of amino acids to proteins such as arginylation and ubiquitination (for reference see, Protein-structure and molecular proterties, 2nd Ed., TE Creighton, WH Freeman and Company, New-York, 1993).

Another type of polypeptide modification may comprise, for example, amino acid insertion (i.e., addition), deletion and substitution (i.e., replacement), conservative or non-conservative (eg, D-amino acids, deamino acids) in the polypeptide sequence wherein such changes substantially alter the overall biological activity of the polypeptide. The polypeptides of the present invention comprise, for example, biologically active mutants, variants, fragments, chimeras, and the like; Fragments that span amino acid sequences that have truncations of one or more amino acids, where the truncation can originate from the amino terminal (Terminal N), carboxy terminal (Terminal C), or from within the protein. Analogues of the invention involve insertion or substitution of one or more amino acids. Variants, mutants, fragments, chimeras, and the like may have the biological properties of the polypeptides of the present invention comprising eg (without being restricted by the current examples) to facilitate neuronal axon growth, to suppress inhibition of axon growth neuronal, to facilitate neurite growth, to inhibit apoptosis, to treat nerve injury, to regenerate the injured axon and / or to act as a Rho antagonist.

When this can be exemplified (Example 13: reverse Tat sequence), in some case, the order of the amino acids in a particular polypeptide is not critical. As for the transport agent region described here, the transport function of this region can be preserved even if the amino acids are not in their original order (as found in nature) (sequence).

Examples of substitutions may be those, which are conservative (that is, where one residue is replaced by another of the same general type). As understood, naturally occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. Additionally, three of the encoded amino acids are aromatic. It may be of use that the encoded polypeptides differ from the determined polypeptide of the present invention that they contain substituted codons for the amino acids, which are from the same group as that of the amino acid being replaced. Thus, in some cases, the basic amino acids Lys, Arg and His can be interchangeable; the acidic amino acids Asp and Glu can be interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and Asn can be interchangeable; the nonpolar aliphatic amino acids Gly, Ala, Val, Ile, and Leu are interchanged but due to the size of Gly and Ala are more closely related and Val, Ile and Leu are more closely related to each other, and the aromatic amino acids Phe, Trp and Tyr can be interchangeable.

It should be noted that if polypeptides are made synthetically, amino acid substitutions can also be made, which are not naturally encoded by DNA. For example, alternative residues include omega amino acids of the Formula NH2 (CH2) nCOOH where n is 2-6. There are neutral non-polar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine can be substituted for Trp, Tyr or Phe; Citrulline and methionine sulfoxide are nonpolar neutrals, cysteic acid is acidic, and ornithine is basic. Proline can be substituted with hydroxyproline and retain the conformational properties.

Mutants or variants are known in the art that can be generated by substitution mutagenesis and retain the biological activity of the polypeptides of the present invention. These variants have at least one amino acid residue in the removed protein molecule molecule and a different residue inserted in its place (one or more nucleotides in the DNA sequence is changed to a different one using known molecular biology techniques, giving an amino acid different after translation of messenger RNA corresponding to a polypeptide). For example, a site of interest for substitutional mutagenesis may include but is not restricted to sites identified as active sites, or immunological sites. Other sites of interest are identical to those, for example, where particular residues obtained from various species are obtained. These positions may be important for biological activity. Examples of substitutions identified as "conservative substitutions" are shown in Table 1. If such substitutions result in an undesired change, then other types of substitutions, called "example substitutions" in Table 1, or as further described herein in With reference to the amino acid classes, products are introduced and detected.

by amino acid substitution, insertion, or deletion. For example, modifying a

polypeptide can result in an increase in the biological activity of the polypeptide, it can modulate

its toxicity may result in changes in bioavailability or stability, or it may modulate its

5 immunological activity or immunological identity. Substantial modifications based on or

Immunological identity are carried out by selecting substitutions that differ significantly in

its effect on maintaining (a) the structure of the polypeptide structure in the area of the substitution, by

example, as a sheet or helical conformation. (b) the charge or hydrophobicity of the molecule in

the target site, or (c) the mass of the side chain. Naturally occurring residues are divided into 10 groups based on common side chain properties:

(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile)

(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr)

(3) Acidic: Aspartic Acid (Asp), Glutamic Acid (Glu) 15 (4) Basic: Asparagine (Asn), Glutamine (Gln), Histidine (His), Lysine (Lys),

Arginine (Arg)

(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); and

(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), PhenylAlanine (Phe) Non-conservative substitutions will require exchanging a member of one of these

20 classes to another. TABLE 1. Preferred amino acid substitution

Original waste

Example substitution Conservative substitution

Wing (A)

Val, Leu, Ile Val

Arg (R)

Lys, Gln, Asn Lys

Asn (N)

Gln, His, Lys, Arg Gln

Asp (D)

Glu Glu

Cys (C)

To be To be

Gln (Q)

Asn Asn

Glu (E)

Asp Asp

Gly (G)

Pro Pro

His (H)

Asn, Gln, Lys, Arg Arg

Ile (I)

Leu, Val, Met, Ala, Phe, norleucine Leu

Leu (L)

Norleucine, Ile, Val, Met, Ala, Phe Ile

Lys (K)

Arg, Gln, Asn Arg

Mef (M)

Leu, Phe, Ile Leu

Phe (F)

Leu, Val, Ile, Ala Leu

Pro (P)

Gly Gly

Be (S)

Thr Thr

Thr (T)

To be To be

Trp (W)

Tyr Tyr

Tyr (Y)

Trp, Phe, Thr, Ser Phe

Val (V)

Ile, Leu, Met, Phe, Ala, norleucine Leu

16 Amino acid sequence inserts (eg, additions) include amino and / or carboxyl terminal fusions ranging in length from one of the residues to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single amino acid residues or multiple. Other insertion variants include fusion of the N- or C-Terminal of the protein to a homologous or heterologous polypeptide that forms a chimera. Chimeric polypeptides (i.e., chimeras, analog polypeptide) comprise the sequence of the polypeptides of the present invention fused to the homologous or heterologous sequence. Said homologous or heterologous sequence encompasses those that, when formed in a chimera with the polypeptides of the present invention, retain one or more immunological or biological properties. Another type of homologous fusion generated chimera includes new polypeptides formed by repeating two or more polypeptides of the present invention. The repeat number can be, for example, between 2 and 50 units (ie repeats). In some cases, it may be useful to have a new polypeptide with a repeat number of more than 50. For example, it may be useful to fuse (using crosslinking techniques or recombinant DNA technology techniques) polypeptides such as C3APL, C3APLT, C3APS, C3-TL, C3-TS, C3Basic1, C3Basic2, and C3Basic3 themselves (eg, C3APLT fused to C3APLT) or to another polypeptide of the present invention (eg, C3APLT fused to C3APL). Additionally, a transport agent can be repeated more than once in a polypeptide comprising C3 ADP-ribosyl transferase or a fragment thereof that retains ADP-ribosyl transferase C3 activity. The transport agent region is in its carboxy terminal region. Repeating a transport agent region can affect (eg, increase) the uptake of ADPribosyl transferase C3 or a fragment thereof that retains ADP-ribosyl transferase C3 activity by a desired cell. Heterologous fusion includes new polypeptides made by infusing the polypeptides of the present invention with heterologous polypeptides. Such polypeptides can include but are not limited to bacterial polypeptides (eg, betalactamase, glutathione-S-transferase, or an enzyme encoded by the E.coli trp site), yeast protein, viral proteins, phage proteins, serum albumin bovine, chemotactic polypeptides, immunoglobulin constant region (or other immunoglobulin regions), albumin, or ferritin. Another type of polypeptide modification includes deletions from the amino acid sequence (eg, truncations). They generally range from about 1 to 30 residues, more preferably approximately 1 to 10 residues, and typically approximately 1 to 5 residues. Mutants, Variants, and Analogous Proteins Mutant polypeptides will possess one or more mutations, which are deletions (eg, truncations), insertions (eg, additions), or substitutions of amino acid residues. Mutants can be naturally-occurring (that is, purified or isolated from a natural source) or synthetic (for example, by developing site-directed mutagenesis in the encoding DNA or by other synthetic methods such as chemical synthesis). It is thus evident that the polypeptides of the invention can be naturally occurring or recombinant (which is to say prepared from recombinant DNA techniques). A protein at least 50% identical, as determined by methods known to those of skill in the art (eg, the methods described by Smith, TF and Waterman MS (1981) Ad. Appl.Math., 2: 482-489 , or Needleman, SB and Wunsch, CD (1970) J.Mol.Biol., 48: 443453), for those polypeptides of the present invention, for example C3APLT, is included in the

invention, as are proteins of at least 70% or 80% and more preferably at least 90% identical to the protein of the present invention. This is generally over a region of at least 5, preferably at least 20 contiguous amino acids.

"Variant" as the term used herein, is a polynucleotide or polypeptide that differs from the reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs from the nucleotide sequence of another, reference polynucleotide. Changes in the variant nucleotide sequence may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusion, and truncation in the polypeptide encoded by the reference sequence, as discussed here. A typical variant of a polypeptide differs from one amino acid sequence to another, reference polypeptide. Generally, the differences are limited to those of the reference polypeptide sequence and the variants are closely similar in general and, in many regions, identical. A variant and reference polypeptide may differ in amino acid by one or more substitutions, additions, deletions, or therefore any combination. An inserted or substituted amino acid residue may or may not be one encoded by the genetic code. A polynucleotide or polypeptide variant may be naturally occurring such as an allelic variant, or it may be a variant not known to occur naturally. Unnaturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis.

Amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into DNA, or by in vitro synthesis of the desired polypeptide. Such a variant includes, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. A combination of removal, insertion, and substitution can be made to arrive at a final construct, which provides that the final protein product possesses the desired biological activity, or characteristics. Amino acid changes can also alter posttranslational processes such as changing the number or position of glycosylation sites, altering membrane anchoring characteristics, altering intracellular location by inserting, deleting, or otherwise affecting the transmembrane sequence of the native protein, or modify its susceptibility to proteolytic division.

Unless otherwise indicated, the recombinant DNA techniques used in the present invention are standard procedures, known to those of skill in the art. Examples of such techniques are explained in the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glsobre and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and

F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, which includes all updates so far) and are incorporated herein by reference.

It is understood herein, that if a "range" or "group of substances" is mentioned with respect to a particular characteristic (eg, amino acid groups, temperature, pressure, time, and the like) of the present invention, the present invention relates to and is explicitly incorporated here and each specific member and combination of sub-ranges or sub-groups there. Thus, any specific range or group is understood as a short form of reference to each and any member of a range or group individually as well as each and all possible sub

18 ranges or sub-groups covered there; and similarly with respect to any sub-ranges or sub-groups there. Thus, for example, -with respect to a sequence comprising up to 50 base units is understood as specifically incorporating each and all individual units, as well as sub-range of units; -with respect to a reaction time, a time of 1 minute or more is understood as specifically incorporating each and all the individual times, as well as subrange, above 1 minute, such as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hours to 20 hours etc .; -with respect to polypeptides, an analogous polypeptide comprising a particular sequence and having an addition of at least one amino acid at its amino terminal or its carboxy terminal is understood as specifically incorporating each and every individual possibility, such such as one, two, three, ten, eighteen, forty, etc .; -with respect to polypeptides, an analogous polypeptide having at least 90% of its amino acid sequence identical to a particular amino acid sequence is understood as specifically incorporating each and every individual possibility (excluding 100%), such as for example , an analogous polypeptide having 90%, 90.5%, 91%, 93.7%, 97%, 99%, etc., of its amino acid sequence identical to the particular amino acid sequence. -Regarding polypeptides, an analogous polypeptide that has at least 70% of its amino acid sequence identical to the particular amino acid sequence is understood as specifically incorporating each and every individual possibility (excluding 100%), such as for example , an analogous polypeptide having 70%, 72.3%, 20 73%, 88.6%, 98% etc., of its identical amino acid sequence a particular amino acid sequence. -Regarding polypeptides, an analogous polypeptide having at least 50% of its amino acid sequence identical to a particular amino acid sequence is understood as specifically incorporating each and every individual possibility 25 (excluding 100%), such as for example, an analogous polypeptide having 50%, 54%, 66.7%, 70.2%, 84%, 93% etc., of its amino acid sequence identical to that particular amino acid sequence. -Regarding polypeptide, a polypeptide comprising at least one transport agent region is understood as specifically incorporating each one and all the individual possibility, such as for example a polypeptide having one, two, five, ten, etc., transport agent regions. -and similarly with respect to other parameters such as low pressures, concentrations, elements, etc ... It is also understood here that "g" or "gm" is a reference for the unit of weight of grams; 35 and that "C", or "° C" is a reference to the Celsius temperature unit. Table 2: Abbreviations

Abbreviation

Full name

C3

ADP-ribosyl transferase C3

NGF

Nerve growth factor

BDNF

Brain derived neurotrophic factor

C or ° C

Celsius degrees

ml

milliliter

µl or ul

Microliter

µM or uM

micromolar

mM

millimolar

M

cool

N

normal

CNS

Central Nervous System

SNP

Peripheral nervous system

HIV

Human immunodeficiency virus

HIV-1

Human immunodeficiency virus type-1

kDa

kilodalton

GST

Glutathione S-transferase

MTS

Membrane transport sequence

SDS-PAGE

Sodium Dodecyl Sulfate Gel Electrophoresis

PBS

Phosphated saline buffer

OR

Unit

BBB

Basso Behavior Recovery Scale, Beattie Breshnahan

IPTG

Isopropyl β-D-thiogalactopyranoside

Rpm

Rotation by minutes

DTT

dithiothreitol

PMSF

Phenylmethylsulfonyl fluoride

NaCl

Sodium chloride

MgCl2

Magnesium chloride

HBSS

Hank's Balanced Salt Solution

NaOH

Sodium hydroxide

CSPG

Chondroitin Sulfate Proteoglycan

PKN

Protein kinase N

RSV

Rous sarcoma virus

MMTV

Mouse mammary tumor virus

LTR

Long Terminal Repeat

HL

Hind leg

FL

Foreleg

neo

neomycin

hygro

hygromycin

IN 1

Monoclonal antibody called IN-1

ADP

Adenosine diphosphate

ATP

Adenosine triphosphate

32P

Phosphorus 32 isotope

DHFR

Dihydrofolate reductase

PCR

Polymerase chain reaction

C3-like proteins are described, which may have additional amino acids added to the carboxy terminus of C3 proteins. Examples of such proteins include: C3APL: (C3 antenapedia-long) created by hybridization sequence of the transcription factor antenapedia to the 3 'end of the sequence encoding the cDNA

C3. The 60-amino acid long antennapedia sequence that contains the antennapedia homeodomain is used;

C3APLT: (C3 antenapedia-truncated) created by hybridization sequences of the transcription factor antenapedia at the 3 'end of the sequence encoding the C3 nDNA. This clone with a structure change mutation gives a proline-rich transport peptide with good transport activity. This sequence is truncated ie it is shorter than C3APL.

C3APS: An 11 short amino acid sequence from antenapedia that has transmembrane transport properties fuses to the carboxy terminus of C3 to create C3APS;

C3-TL: C3 Tat-longs created by using Tat amino acids 27 to 72 at the carboxy terminus of the C3 protein; C3-TS: C3 Tat-shorts created by using the amino acids YGRKRRQRRR for

C3 protein; C3Basic1: a randomized basic load sequence added to the C3 terminal of C3; C3Basic2: a randomized basic load sequence added to C terminal of C3; C3Basic3: C3 Tat-shorts created by using the reverse sequence of Tat amino acids

RRQRRKKR for protein C3.

The Rho antagonist fusion proteins (C3-like proteins) of the present invention have been found to be effective in stimulating CNS repair after spinal cord injury. The increased cellular permeability of the new Rho antagonist (new chimeric C3) will now allow treatment of stroke victims and neurodegenerative disease due to the Rho signaling pathway is important in repair after stroke (Hitomi, et al. (2000) 67: 1929 -39. Trapp et al 2001. Mol.Cell. Neurosci. 17: 883-84). Treatment with Rho antagonists in the adhesive delivery system can be used to improve the speed of axon growth in the SNP. Also, it is evidenced in the literature now Rho signaling links with rings of Alzheimer's disease formation through its ability to inactivate PKN that then phosphorylates tau and neurofilaments (Morissette, et al. (2000) 278: H1769-74 ., Kawamata, et al. (1998) 18: 7402-10., Amano, et al. (1996) 271: 648-50., Watanabe, et al. (1996) 271: 645-8.). Therefore, Rho antagonists are expected to be useful in the treatment of Alzheimer's disease. The new chimeric C3 drugs must be able to spread easily and therefore can promote the repair of diseases that are neurodegenerative. Examples include, but are not limited to stroke, traumatic brain injury, Parkinson's disease, Alzheimer's disease, and ALS. Furthermore, it will now be well established that Rho signaling antagonists are effective in the treatment of other diseases. These include, but are not limited to eye diseases such as glaucoma (Honjo, et al. (2001) 42: 137-44., Rao, et al. (2001)

42: 1029-1037.), Cancer cell migration and metastasis (Sahai, et al. (1999) 9: 136-45., Takamura, et al. (2001) 33: 577-81., Imamura, et al . (2000) 91: 811-6.). The effect of the Rho signaling pathway on smooth muscle relaxation is well established. This has led to the identification of Rho signaling antagonists as effective in the treatment of hypertension (Chitaley, et al. (2001) 3: 139-144., Somlyo (1997) 389: 908-911, Uehata, et al. (1997) 389: 990-994), asthma (Nakahara, et al. (2000) 389: 103-6., Ishizaki, et al. (2000) 57: 976-83), and vascular disease (Miyata, et al . (2000) 20: 2351-8., Robertson, et al. (2000) 131: 5-9.) As well as erectile dysfunction of the penis (Chitaley, et al. (2001) 7: 119-22.). Rho is also important as a cardioprotective protein (Lee et al. 2001. FASEB J. 15: 1886-1894).

21 Rho GTPases include members of the Rho, Rac and Cdc42 family of proteins. Our invention relates to members of the Rho family of the Rho class. Rho proteins consist of different variants encoded by different genes. For example, PC-12 cells express RhoA, RhoB, and RoC (Lehmann et al 1999 supra); PC-12 cells: pheochromocytome cell line (Greene A and Tischler, AS PNAS 73: 2424 (1976). To inactivate Rho proteins within cells, Rho antagonists of the type C3 family are effective because they inactivate all forms of Rho (eg RhoA, Rho B etc.) In contrast, gene therapy techniques, such as introducing a dominant negative RhoA family member into a diseased cell, will only inactivate that specific RhoA family member. Recombinant C3 proteins, or C3 proteins that retain ribosylation activity are also effective in our delivery system and are recovered by this invention.In addition, Rho kinase is a well-known target for active Rho, and inactivating Rho kinase has the same effect as inactivating Rho, at least in terms of neurite or axon growth (Kimura and Schubert (1992) Journal of Cell Biology. 116: 777-783, Keino-Masu, et al. (1996) Cell.87 : 175-185, Matsui, et al (1996) EMBO J.15: 2208-2216, Matsui, et al. (1998) J. Cell Biol. 140: 647-657, Ishizaki (1997) FEBS Lett. 404: 118-124), the biological activity that relates to this invention. The C3 polypeptides of the present invention include biologically active fragments of C3; the fragments encompass amino acid sequences that have truncations of one or more amino acids, where the truncation can originate from the amino terminal, carboxy terminal, or from within the protein. Fragments containing C3 Glu (173) are included in this invention (Saito et al. 1995. FEBS Lett. 371-105). Analogs involve an insertion or substitution of one or more amino acids. Fragments and analogs will have the biological property of C3 which is capable of inactivating GTPase Rho in Asn (41) in Rho. Chimeric polypeptides comprising C3 amino acid sequences fused to heterologous amino acid sequences are also encompassed by the invention. Such heterologous sequences encompass those which, when formed in a C3 chimera, retain one or more immunological properties of C3. A host cell transformed or transfected with nucleic acids encoding the C3 protein or the C3 chimeric protein is also encompassed by the invention. Any host cell that produces a polypeptide that has at least one of the biological properties of C3 can be used. Specific examples include bacteria, yeast, plant, insect or mammalian cells. Additionally, the C3 protein can be produced in transgenic animals. Transfected or transformed host cells and transgenic animals are obtained using materials and methods that are routinely available to one skilled in the art. Host cells may contain nucleic acid sequences that have the full-length gene for protein C3 including a leader sequence and a C-terminal membrane anchor sequence (see below), or alternatively they may contain nucleic acid sequences that they lack one or both of the leader sequences and the Terminal C membrane anchor sequence. Additionally, nucleic acid fragments, variants, and analogs encoding a polypeptide capable of retaining C3 biological activity may also be resident in host expression systems. . C3 is produced as a 26 kDa protein. The full length C3 protein inactivates Rho via Rho rib DP-asparagine 41 of Rho A (Han et al. (2001) J. Mol. Biol. 305: 95). Altered, elongated or truncated C3 proteins or C3-derived peptides that retain the ability to ribosylate Rho are included in this invention and can be used to make fusion proteins.

The crystal structure of C3 has been determined by giving insight with elements of the C3 protein that can be changed without affecting ribosylating activity (Han et al. (2001) J. Mol. Biol. 305: 95).

The Rho antagonist which is a recombinant protein can be made according to methods present in the art. The proteins of the present invention can be prepared from bacterial cell extracts, or through the use of recombinant techniques. In general, C3 proteins according to the invention can be produced by transformation (transfection, transduction, or infection) of a host cell with all or part of a C3-encoding DNA fragment in a suitable expression vehicle. Suitable expression vehicles include: plasmids, virus particles, and phages. For insect cells, baculovirus expression vectors are suitable. The entire expression vehicle, or a part thereof, can be integrated into the genome of the host cell. In some circumstances, it is desirable to employ an inducible expression vector.

Those of skill in the field of molecular biology will understand that any one of a wide variety of expression systems can be used to provide the recombinant protein. The precise host cell used is not critical to the invention. C3 and C3-like proteins can be produced in a prokaryotic host (eg, E. coli or B. subtilis) or in a eukaryotic host (eg, Saccharomyces or Pichia; mammalian cells, eg, COS cells, NIH 3T3, CHO, BHK, 293, or HeLa; or insect cells).

Proteins and polypeptides can also be produced in plant cells. Viral expression vectors (eg, cauliflower mosaic virus and tobacco mosaic virus) and plasmid expression vectors (eg, Ti plasmid) are also suitable for plant cells. Such cells are available from a wide range of sources (eg, the American Type Culture Collection, Rockland, Md.). Transformation and transfection methods and choice of expression vehicle will depend on the selected host system.

Host cells that harbor the expression vehicle can be grown in standard nutrient medium adapted as needed for activation of a selected gene, repression of a selected gene, selection of transformants, or amplification of a selected gene. An expression system is a mouse 3T3 fibroblast host cell transfected with a pMAMneo expression vector (Clontech, Palo Alto, Calif.). The pMAMneo provides an RSV-LTR enhancer linked to a dexamethasone inducible MMTV-LTR promoter, an SV40 origin of replication that allows replication in mammalian systems, a selectable neomycin gene, and SV40 cleavage and polyadenylation sites. DNA encoding a C3 or C3-like protein could be inserted into the pMAMneo vector in an orientation designed to allow expression. Recombinant C3 or C3-like protein can be isolated as described below. Other preferable host cells that can be used in conjunction with the pMAMneo expression vehicle include COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively).

C3 polypeptides can be produced as fusion proteins. For example, expression vectors can be used to create lacZ fusion proteins. PGEX vectors can be used to express the above polypeptides as glutathione Stransferase (GST) fusion proteins. In general, such fusion proteins are soluble and can be easily purified from smooth cells by adsorption on glutathione-agarose beads followed by elution in the presence of free glutathione. PGEX vectors are designed to include thrombin or factor Xa protease cleavage sites such that the cloned target gene product can be released from the GST functional group. Another strategy for making fusion proteins is to use the His tag system.

23 In an insect cell expression system, the Autographa californica AcNPV) nuclear polyhedrosis virus, which grows in Spodoptera cells, is used as a vector to express the above genes. A C3 coding sequence can be individually cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter, eg, the polyhedrin promoter. Successful insertion of a gene encoding a C3 or C3-like protein (polypeptide) will result in the inactivation of the polyhedrin gene and the production of the non-occluded recombinant virus (i.e., virus lacking the proteinaceous coating by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which it is expressed in the inserted gene (see, Lehmann et al for an example to make the recombinant protein MAG). In mammalian host cells, a number of viral based expression systems can be used. In cases where an adenovirus is used as an expression vector, the C3 nucleic acid sequence can be linked to an adenovirus transcription / translation complex, eg, the last promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing a C3 gene product in infected hosts. Specific initiation signals may also be required for efficient translation of inserted nucleic acid sequences. These signals include the ATG start codon. In cases where an entire native C3 gene or cDNA, including its own start codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be required. In other cases, exogenous translational control signals, including, perhaps, the ATG start codon, can be provided. Additionally, the start codon can be in phase with the reading structure of the desired codon sequence to ensure translation of the entire insert. These exogenous translational control signals and start codons can be of a variety of origins, natural and synthetic. Expression efficiency can be improved by the inclusion of appropriate translation enhancing elements, transcription terminators. Additionally, a host cell can be selected to modulate the expression of the inserted sequences, or modify and process the gene product in a specific desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important to the function of the protein. Different host cells have specific characteristics and mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed protein above. For this purpose, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, choroid plexus cell lines. Alternatively, a C3 protein can be produced by a stably transfected mammalian cell line. A suitable number of vectors suitable for stable transfection of mammalian cells are available to the public; Construction methods for such cell lines are also publicly available. In one example, the cDNA encoding the C3 protein can be cloned into an expression vector that includes the dihydrofolate reductase gene (DHFR). The integration of the plasmid and, therefore, the C3 or protein similar to the gene that

encoding C3 on the host cell chromosome is selected by including 0.01-300 µM of methotrexate in the cell culture medium (as described in Ausubel et al., supra). This dominant selection can be carried out on most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are known in the art; Such methods generally involve extended culture in medium containing gradually increased levels of methotrexate. DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV (A). Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line (eg, CHO DHFR cells, ATCC Accession No. CRL 9096) are among the preferred host cells for selection of DHFR from a stably transfected cell line or DHFR-mediated gene amplification.

A number of other selection systems can be used, including but not limited to herpes simplex virus thymidite kinase, hopoxanthin-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes can be employed in tk, hgprt, or aprt cells, respectively. Additionally, gpt can be used, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro, which confers resistance to hygromycin.

Alternatively, any fusion protein can be easily purified by using an antibody specific for the expressed fusion protein. For example, a system described in Janknecht et al. (1981) Proc. Natl. Acad. Sci. USA 88, 8972, allows for the ready purification of non-denaturing fusion proteins in human cell lines. In this system, the gene of interest is subcloned into a vaccine recombination plasmid such that the open gene reading frame is translationally fused to an amino terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccine virus can be loaded onto Ni2 + nitriloacetic acid-agarose columns, and histidine tag proteins are selectively eluted with imidazole-containing buffers.

Alternatively, C3, the C3-like protein or its portion (fragment), can be fused into an Fc immunoglobulin domain. Such a fusion protein can be easily purified using a protein A column.

To test Rho antagonists for activity, a tissue culture bioassay system is used. This bioassay is used to define the activity of Rho antagonists that will be effective in promoting axon regeneration in spinal cord injury, stroke, or neurodegenerative disease.

Neurons do not grow neurites on inhibitory myelin substrates. When neurons are placed on inhibitory substrates in tissue culture, they remain surrounded. When an effective Rho antagonist is added, neurons are able to grow neurites on myelin substrates. The time it takes for neurons to grow neurites after the addition of a Rho antagonist is the same as if neurons have been plated in the growth of the permissive substrate such as laminin or polylysine, typically 1 to 2 days in cell culture . The results can be visually classified. If needed, a quantitative assessment of neurite growth can be developed. This involves measuring neurite length in a) control cultures where neurons are plated on myelin substrates and left untreated b) in positive control cultures, such as polylysine placed neurons c) or treated cultures with different concentrations of the test antagonist.

25 To test C3 in tissue culture, the best concentration has been found to be 25-50 ug / ml. (Lehmann et al, 1999. J. Neurosci. 19: 7537-7547; Jin & Strittmatter, 1997. J. Neurosci. 17: 6256-6263). Thus, high concentrations of this Rho antagonist are needed when compared to growth factors used to stimulate neurite growth. Growth factors, such as nerve growth factor (NGF) are used in concentrations of 1-100 ng / ml in tissue culture. However, growth factors are not capable of inhibiting growth through myelin. Our tissue culture experiments are conducted in the presence of growth factor BDNF for retinal ganglion cells, or NGF for PC cells.

12. When growth factors have been tested in vivo, typically highest possible concentrations are used, in the ug / ml range. They, too, are frequently added to the CNS with the use of prolonged supply pumps (eg Ramer et al, supra). For in vivo experiments the highest possible concentrations are used when working with C3 storage with a frozen 1 mg / ml solution.

The Rho C3 antagonist is stable at 37 ° C for at least 24 hours. The stability of C3 in tissue culture is tested with the following experiment. C3 is diluted in tissue culture medium, left in the incubator at 37 ° C for 24 hours, then added to the bioassay system described above, using retinal ganglion cells as the type of test cell. These cells are capable of spreading neurites on inhibitory substrates when treated with C3 storage for 24 hours at 37 ° C. Therefore, the minimum stability is 24 hours. This is maintained by projecting stability based on amino acid composition (see sequence data, below).

A compound with a Rho antagonist can be confirmed in one of the following ways:

a) The cells are cultivated in a growth inhibition substrate as was done

above, and the candidate antagonist Rho is exposed;

b) The cells from step a) are homogenized and an assay is carried out.

regime. This assay is based on the ability of Rho bound to GST-Rhotektina to bind

GTP. The GST-Rhotektin or recombinant GST rhotektin (GSTRBD) binding domain is

added to the cell homogenate made from cultured cells as in a). It has been found that

inhibitory substrates activate Rho, and that is Rho activated it is placed under

using GST-RBD. Rho antagonists will block Rho activation, and therefore

an effective Rho antagonist will block detection of Rho when the cell is cultured as

described by a) above;

c) An alternative method for this regimen test could be used in the

GTPase activating protein, Rho-GAP as bait in the assay to activate

Rho regimen, as described (Diekmann and Hall, 1995. In Methods in Enzymology Vol.

256 part B 207-215).

Another method of confirming that the compound is a Rho antagonist is as follows: When added to Rho inactivating living cell antagonists by ADP-ribosylation of the effector domain it can be identified by detecting a molecular weight change in Rho (Lehmann et al, 1999 supra). The change in molecular weight can be detected after treatment of cells with the Rho antagonist by homogenizing the cells, separating the proteins in the cell homogenate by SDS polyacrylamide gel electrophoresis. Proteins are transferred to nitrocellulose paper, then Rho is detected with antibodies specific to Rho using a Western blot technique.

Another method to confirm that the compound is a Rho kinase antagonist is as follows:

GST tag or any suitable tag on Hela cells or other suitable cell type

by transfection;

b) The kinase is purified from the cell homogenates by

immunoprecipitation using antibodies directed against the specific tag (for

example, myc tag or GST tag);

c) The immunoprecipitates recovered from b) are incubated with [32P] ATP and histone

type 2 as a substrate in the presence or absence of the Rho kinase inhibitor. In the

absence of Rho kinase inhibitor activity, phosphorylated histone Rho kinase.

In the presence of the phosphorylation activity of the Rho kinase inhibitor the

Rho kinase (i.e. histone phosphorylation), and as such the compound is identified as

a Rho kinase antagonist.

In turn now for the lateral transport of the conjugates, known methods are available for adding transport sequences that allow proteins to penetrate cells; examples include membrane translocation sequence (Rojas (1998) 16: 370-375), Tat mediated protein delivery (Vives (1997) 272: 16010-16017), polyargine sequences (Wender et al. 2000, PNAS 24: 13003-13008) and antenapedia (Derossi (1996) 271: 18188-18193). Examples of known transport agents, functional groups, subdomains and the like are also shown for example in Canadian patent document no. 2,301,157 (conjugates containing the homeo domain of antenapedia) as well as in US Patent 5,652,122, 5,670,617, 5,674,980, 5,747,641, and 5,804,604 (conjugates containing amino acids of the HIV Tat protein (hereinafter the Tat HIV protein refers simply to Tat); the full contents of which these patent documents are incorporated herein by reference.

A 16 amino acid region in the third alpha-helix of the homeodomain antennapedia has been shown to allow proteins (made as fusion proteins) to cross cell membranes (PCT International Publication Number WO 99/11809 and Canadian Application No .: 2,301,157 (Crisanti et al,) incorporated herein by reference). Fusion proteins have been generated that comprise C3 and that have an antennapedia homeodome sequence located at the carboxy terminus of the fusion protein. The biological activity (eg, promoting axon growth) of these fusion proteins is demonstrated in primary mammalian cells such as neurons. Similarly, the HIV Tat protein is shown to be capable of crossing cell membranes (Frankel A.D. et al., Cell, 55: 1189). This has been shown here using a sequence spanning 27 to 72 amino acids of HIV Tat, that Tat mediated delivery of the biologically active C3 protein is possible in neuronal cells and more specifically, in primary neuronal cells.

In addition to HIV Tat and the antenna-mediated transport of the C3 protein and the analogs, novel transport sequences are described (ie, polypeptide polypeptide functional group, transport agent region, etc.).

Various receptor-mediated transport strategies have been used to treat and improve ADP ribosylase function: these methods include originating the C2 and C3 sequences (Wilde, et al. (2001) 276: 9537-9542.) And the use of receptor mediated transport with the diphtheria toxin receptor (Aullo, et al. (1993) 12: 921-31; Boquet, P. et al. (1995) Meth. Enzymol. 256: 297-306).). These methods have not been shown to dramatically increase the power of C3. Furthermore, these proteins require receptor mediated transport. This means that the cells can express the receptor, and can express sufficient amounts of the receptor to significantly improve transport. Furthermore, when C3 enters the cell by endocytosis, it will lock into a membrane compartment, and therefore most of it will not be available to inactivate Rho. In the case of diphtheria toxin, not all cells express the appropriate receptor, limiting its potential use. The clinical significance for any of these has not been proven or shown. A C2 / C3 fusion protein has also been made to treat and improve the effectiveness of C3. In this case, the addition of a C2II binding protein to the tissue culture medium is necessary, along with the C2-C3 fusion toxin that enables C3 uptake by receptor-mediated endocytosis (Barthe et al. (1998) Infection and Immunity 66: 1364). The disadvantage of this system is that much of the C3 in the cell will be restricted within a membrane compartment. More importantly, two different proteins can be added separately for the transport that occurs (Wahl et al. 2000. J. Cell Biol. 149: 263), which makes the system difficult to apply in vivo for the treatment of the disease. Furthermore, none of the methods to inactivate Rho with C3 or C3 analogs (C3-like protein) has been shown to be sufficient to induce growth inhibition in tissue culture, or to promote recovery after in vivo damage to the tissue. CNS.

One strategy that can be used in accordance with the present invention is to exploit the homeopathic antenna domain that is capable of transporting proteins across the plasma membrane by a receptor independent mechanism (Derossi (1996) 271: 18188-18193); an alternate strategy is to exploit Tat-mediated supply (Vives (1997) 272: 16010-16017, Fawell (1994) 91: 664-668, Frankel (1988) 55: 1189-1193).

Antenapedia strategies have been used for protein translocation in neurons (Derossi (1996) 271: 18188-18193). Antenapedia, for example, has been used to transport biotin-labeled peptides to demonstrate the efficacy of the technique; see US Patent No. 6,080,724 (the full contents of this patent are incorporated herein by reference). Antenapedia improves the growth and branching of neurons in vitro (Bloch-Gallego (1993) 120: 485-492). Homeoproteins are transcription factors that regulate the development of body organization, and the antennae is a Drosophila homeoprotein. Tat on the other hand is a regulatory protein of the human immunodeficiency virus (HIV). This is a highly basic protein that is found in the nucleus and can carry reporter genes in the cell. Furthermore, Tat-linked proteins can penetrate cells after intraperitoneal injection, and Tat can still cross the brain's blood barrier to enter cells within the brain (Schwarze, et al. (1999) 285: 1569-72) .

Other transport sequences are known that have been tested in other contexts, (that is, to show that they work through the use of reporter sequences). A transport peptide, a 12 mer, AAVLLPVLLAAP, is rich in proline. This is done as a GSTMTS fusion protein and is derived from the h region of the Kaposi FGF signal sequence (Royas et al. 1998-Nature Biotech. 16: 370-375. Another example is the sperm alpha-fertiline peptide, HPIQIAAFLARIPPISSIGTCILK (This is reviewed in Pécheur, J. Sainte-Marie, A. Bienvenüe, D. Hoekstra. 1999. J. Membrane Biol. 167: 1-17.) It should be noted however that the propensity for rupture of the alpha helix of proline residues (Pro) is not a general rule, as the putative sperm alpha fertilizer fusion peptide exhibits a high helical alpha content in the presence of liposomes, however the Pro-Pro sequence is required for the properties efficient fusion proteins. The C3APLT fusion protein we tested meets the requirement of having two prolines to make an effective transport peptide. Therefore, proline-rich sequences and random sequences that are prone to helix cleavage they act as transports Effective doctors would also be effective if merged into C3.

28 In the context of axon growth on inhibitory substrates, axon regeneration after injury, or axon regeneration in the brain or spinal cord, the method of these transport sequences are not used. In particular, it should be noted that the ability of antenapedia to enhance growth is tested with neurons placed on laminin-coated slides. Laminin supports axon growth and replaces growth inhibition (David, et al. (1995) 42: 594-602) so this is not a suitable substrate to test the potential for regeneration. Over the past 20 years there has been a tremendous wealth of literature on substances that promote axon growth under such favorable tissue culture conditions, but none of these has led to clinical advances in the treatment of spinal cord injury. The antennae effect is shown to act similarly to growth factors. Growth factors do not overcome growth inhibition by CNS growth substrate inhibitors (Lehmann, et al. (1999) 19: 7537-7547, Cai, et al. (1999) 22: 89-101). Growth factors applied in vivo do not support regeneration, only shoots (Schnell, et al. (1994) 367: 170-173). The transport sequence can be added to the N-terminal (amino terminal) sequence of the C3 protein. Alternatively, the transport sequence can be added at the C-terminus (carboxy-terminus) of the C3 protein; Because terminal C is already very basic, this should further improve transport properties. This is probably one of the reasons why C3APLT shows activity in addition to its basic load and proline-rich sequences. The new chimeric C3 can be used to treat spinal cord injury to promote functional repair. We have shown that C3APLT and C3APS can bring growth inhibition on complex inhibitory substrates including myelin and chondroitin sulfate proteoglycan mixture. Additionally, we demonstrate that C3APLT can promote functional recovery after application to the injured spinal cord in adult mice. The new chimeric protein can be used to promote axon regeneration and reduce scarring after CNS injury. Scarring is a barrier to nerve regeneration. The advantage of the new chimeric C3 is the ability to treat injured axons after a significant delay between injury and treatment. Also, the new recombinant protein may be useful in the treatment of chronic injury. Chimeric C3 can also be used to treat neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease where penetration of the Rho antagonist into the affected neuronal population is required for effective treatment. Chimeric C3 (fusion proteins) will also be of benefit for the treatment of stroke and traumatic brain injury. Furthermore, much evidence suggests efficacy in treating cancer cell migration. Rho antagonists are also useful in the treatment of disease involving smooth muscle, such as vascular disease, hypertension, asthma, and penile dysfunction. For the treatment of spinal cord injury, the Rho conjugate antagonists of the present invention may be useful in conjunction with cell transplantation. Many different cell transplants have been extensively tested for their potential to promote regeneration and repair, including, but not limited to, Schwann cells, fibroblasts modified to express growth factors, fetal spinal cord transplants, macrophages, embryonic cells, or stem cells. adults, and olfactory bulb envelope neuroglia. C3 fusion proteins can be used in conjunction with neurotrophils, apoptosis inhibitors, or other agents that prevent cell death. They can also be used in conjunction with cell adhesion molecules such as L1, laminin, and artificial growth matrices that promote the growth of

axon. The chimeric C3 constructs of the present invention can also be used in conjunction with the use of antibodies that block inhibitory protein substrates to promote axon growth. Examples of such antibody methods are the use of IN-1 or related antibodies (Schnell and Schwab (1990) 343: 269-272) or through the use of therapeutic vaccine methods (Huang (1999) 24: 639-647) .

BRIEF DESCRIPTION OF THE FIGURES

In the drawings illustrating example embodiments of the present invention and reference examples:

Figure 1 illustrates the normal C3 dose response with and without grinding;

Figure 2 illustrates ADP ribosylation by C3APLT and C3APS, but not C3

after passively adding the compounds to PC-12 cells;

Figure 3A illustrates that C3APLT penetrates cells;

Figure 3B illustrates a lower level of cell penetration by C3 when

compared to Figure 3A;

Figure 4 illustrates the effectiveness of C3APLT and C3APS at low doses;

Figure 5 illustrates the effectiveness of C3APLT and C3APS at low doses;

Figure 6 illustrates the effectiveness of C3APLT in stimulating axon regeneration.

of primary neurons;

Figure 7 illustrates the effectiveness of C3APLT in promoting recovery.

functional after spinal cord injury;

Figure 8 illustrates the effectiveness of Tat transport sequences in improving the

growth as C3-Tat chimeras (C3-TL and C3-TS);

Figures 9A and 9B illustrate axon regeneration after spinal cord injury.

spinal and C3APLT treatment;

Figure 10 illustrates the effectiveness of C3APLT in preventing cell death after

spinal cord injury, thus showing that it is neuroprotective,

Figure 11 illustrates a comparison of C3APLT and C3Basic3 to promote

neurite growth, and;

Figure 12 illustrates that C3APLT promotes neurite growth of

Retinal neurons plated with inhibitory myelin or CSPG substrates. The neurons of

retina plated on myelin substrates (dark bars) or CSPG (dotted bars) and

they are dealt with C3-05. Figure 12A illustrates the percentage of cells with neurites greater than

1 cell body diameter (neurite growth); Figure 12 B illustrates the length of the

longest neurite per cell (neurite length).

Referring to Figure 1, PC-12 cells are plated on inhibitory myelin substrates (0). Unmodified C3 added to the tissue culture medium at a concentration of 0.00025-50 ug / ml does not significantly improve neurite growth over the untreated control (gray bars). C3 is only effective in stimulating neurite growth for plated cells in myelin substrates after charge scraping (black bars). This Figure demonstrates the limit or non-penetration into cells when passively added to the tissue culture medium. Please see Example 4 below for the techniques.

Referring to Figure 2, this Figure provides a demonstration that C3APLT and C3APS, ADP ribosylate Rho. The Western blot shows RhoA in untreated cells (line 1), and cells treated with C3APLT (line 2) or C3APS (line 3). When Rho is ADP ribosylated by C3 this

3. Please see Example 4 below for the techniques.

Referring to Figure 3, this Figure shows intracellular activity after C3APLT treatment. Detection that the new C3 fusion penetrates cells. Immunocytochemistry with the anti-C3 antibody from PC-12 cells are plated on myelin plates and treated with C3 (A) or C3APLT (B). The cells in A (Figure 3A) are not immunoreactive because C3 does not penetrate the cells. The cells in B (Figure 3B) are immunoreactive and they are capable of spreading the neurites in myelin substrates. Please see Example 4 below for techniques.

Returning to Figure 4, this Figure shows that C3-antenapedia fusion proteins promote growth on inhibitory substrates. The percentage of neurons that grow neurites is counted for each treatment. The dose response experiment shows that C3APLT and C3APS promote more neurite growth per cell than PC12 control cells plated in myelin (0). PC-12 cells are plated in myelin and charge scraping with unmodified C3 (C3 50) is left untreated (0) or treated with various concentrations of C3APLT. Compared to C3 used at 25 ug / ml, C3APS is effective in stimulating more cells to grow neurites at 0.0025 ug / ml, a dose 10,000 X less. Please see Example 4 below for techniques.

Figure 5 shows a dose response experiment showing that C3APLT and C3APS cause large neurites to grow when cells are plated on inhibitory substrates. The length of the neurites is measured for each treatment. PC-12 cells are plated in myelin and charge scraped with unmodified C3 (C3 50), left untreated (0), or treated with various concentrations of C3APLT. Compared to C3 used at 25 ug / ml, C3APS is effective in stimulating more cells to increase neurite growth at 0.0025 ug / ml, a dose 10,000 X less. Please see Example 4 below for techniques.

As seen in Figure 6 it shows the primary neurons growing on inhibitory substrates after C3APLT treatment. Rat retinal ganglion cells are plated on myelin substrates and treated with different concentrations of C3APLT. The concentrations of

0.025 and above the significantly larger neurites promoted. This dose is 1000X lower than that of C3 necessary to promote myelin growth.

Referring to Figure 7, this Figure shows behavioral recovery after treatment of adult mice with C3APLT in a dose response experiment. Mice receive a dorsal hemisection of the spinal cord and are left untreated (transection), treated with fibrin alone (fibrin), or treated with fibrin plus C3APLT at the indicated concentrations given in ug / mouse. Each dot represents an animal. The BBB classification (see Example 6 for details) is evaluated 24 hours after treatment. Animals treated with C3APLT exhibit a significant improvement in behavioral recovery compared to untreated animals. The effective dose of 0.5 µg is 100X less than the unmodified C3 used (see previous experiments shown in Canadian Patent Application 2,325,842). Please see Example 6.

Referring to Figure 8, this Figure shows axon growth promotion by C3-Tat chimeric proteins. The dose-response experiment shows that C3-TS and C3-TL promote more neurite growth by cells than control PC-12 cells plated in myelin. PC-12 cells are plated in myelin and charge scraping with unmodified C3 (charge scraping) left untreated (myelin) or treated with various concentrations of C3-TS (gray bars) or C3-TL ( black bars). Compared to C3 using at 25 ug / ml, C3-TL is effective in stimulating more cells to grow neurites at 0.0025 ug / ml, a 10,000 X dose of less than C3.

Referring to Figure 9A and 9B, these Figures show axon regeneration in the injured spinal cord, ie anatomical regeneration after C3APLT treatment. The section of the spinal cord After antegrade labeled with horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP). A) Reinnervation of axons cut in the dorsal white matter. The arrows show regenerate distal axons in the lesion. B) the same section 3 mm from the site of injury. Arrows show regenerate axons.

Referring to Figure 10, this Figure shows that C3-APLT protects neurons from cell death after spinal cord injury. Apoptotic (dry) cells are counted after TCTNEL marking (see Example 16). 2mm rostral for injury (Rostral) at the site of injury (injury) and 2mm caudal for the site of injury (caudal). The bars show average Tunel positive cell counts of 4 animals treated with fibrin only after spinal cord injury as the control (white bars), or with C3APLT in fibrin at 1 µg (black bars). C3APLT treatment showed significantly reduced numbers of Tunel-labeled cells (dry cells). The uninjured spinal cord samples are also processed and these spinal cords do not show Tunel marking, as expected.

Referring to Figure 11, this Figure shows that C3APLT and C3Basic3 promote rapid neurite growth compared to untreated cells when cells are plated in plastic as part of a rapid bioassay (see Example 4).

Referring to Figure 12A and 12B, to further support the ability of C3-like chimeric proteins to promote neurite growth on inhibitory substrates, we examined the responses of plated primary cultures on inhibitory substrates for C3APLT treatment. Purified retinal ganglion cells (RGC) in myelin, or CSPG substrates are plated and treated with various concentrations of C3APLT. During great care the RGC dissection is taken in order to treat the limit amount of mechanical manipulation of the cells, however the isolation protocol requires that some of the shredding takes place in order to dissociate and separate the cells. When RGCs are plated on inhibitory substrates, they maintain a similar close appearance on plated myelin PC-12 cells. Treatment of RGCs with C3APLT promotes neurite growth and increases neurite length in myelin and CSPG substrates. In contrast to the wide range of concentrations, a narrower range of C3APLT treatment shows that effective in other PC-12 experiments, 0.025 ug / ml to 50 ug / ml promotes the growth of neurites and increases the length of neurites in myelin. For RGCs plated on CSPG substrates, effective concentration ranges of 0.0025ug / ml to 50ug / ml are observed.

DETAILED DESCRIPTION

Method to elaborate the C3APL, C3APLT, and C3APS

C3APL is the name given to the protein made by linking the cDNA that encodes C3 (Dillon and Feig (1995) 256: 174-184) with the cDNA that encodes the homeodomain antenna (Bloch-Gallego (1993) 120: 485- 492). The stop codon at the 3 'end of the DNA is replaced with an EcoR I site by polymerase chain reaction (PCR) using the primers (oligonucleotides) 5'GAA TTC TTT AGG ATT GAT AGC TGT GCC 3' (SEQ ID NO: 1) and 5'GGT GGC GAC CAT CCT CCA AAA 3 '(SEQ ID NO: 2). The PCR product is subcloned into a pSTBlue-1 vector (Novagen, city), then cloned into a pGEX-4T vector using the BamH I and Not I restriction site. This vector is called pGEX-4T / C3. The antenatal sequence used to add to the 3 'end of C3 in pGEX-4T / C3 is created by PCR of the vector pET-3a (Bloch-Gallego (1993) 120: 485-492, Derossi (1994) 269: 10444-10450) , is subcloned into a blunt pSTBlue-1 vector, then cloned into pGEX4T / C3, using the EcoR I and Sal I restriction sites, creating pGEX-4T / C3APL. Another clone (C3APLT) with a structure change mutation is selected, and the protein is made and tested. When cultures tested positive despite the mutation, the clone is re-sequenced by another company to confirm the mutation, and this clone is called C3APLT. To confirm the C3APLT sequence, the coding sequence for both strands is sequenced. The sequence of this clone is given in Examples 16 and 17 (C3APLT nucleotide sequence; SEQ ID NO: 42, C3APLT amino acid sequence; SEQ ID NO: 43).

A shorter version of Antenapedia (pGEX-4T / C3APS) is also made. This chimeric sequence is made by ligating oligonucleotides that encode the short peptide antenapedia (Maizel (1999)

126: 3183-3190) in the vector pGEX-4T / C3 that cuts with EcoR I and Salt I. Recombinant cDNAs C3APLT and C3APS are separately transformed into bacteria, and then recombinant proteins are produced, a bacterial homogenate is obtained by sonication, and the homogenate purified by centrifugation. Glutathione-agarose beads (Sigma) are added to the purified lysate and placed on a rotary plate for 2-3 hours, then washed extensively. To remove the glutathione S transferase sequence from the recombinant protein, 20 U (unit) of Thrombin is added, the beads are left on a rotator overnight at 4 ° C. After thrombin cleavage, the beads are loaded onto a 20 ml empty column, and the proteins eluted with PBS (phosphate buffered saline). Aliquots containing the recombinant protein are pooled and 100 µl of p-aminobenzanidine beads (Sigma) are added and allowed to mix for 45 minutes at 4 ° C to remove thrombin, then the recombinant protein is isolated from the beads by centrifugation . Sample purity is determined by sodium dodecyl sulfate pliacrylamide electrophoresis (SDS-PAGE), and bioactivity bioassay with PC-12 cells is performed (See Lehmann et al supra).

Other possible methods for making bioactive chimeric proteins include ion exchange chromatography. Therefore, the GST tag is not required and can be removed. The cDNA can then be cloned into a high expression bacterial vector, such as pET, as given in Example 16.

The Rho antagonist is a recombinant protein and can be made according to methods present in the art. The proteins of the present invention can be prepared from bacterial cell extracts, or through the use of recombinant techniques by transformation, transfection, or infection of a host cell with all or part of a C3-encoding DNA fragment with a derived transport sequence of antenapedia in a suitable expression vehicle. Those skilled in the art of molecular biology will understand that any one of a wide variety of expression systems can be used to provide the recombinant protein. The precise host cell used is not critical to the invention.

Any fusion protein can be easily purified using more traditional affinity purification or column chromatography techniques. Affinity techniques include, but are not restricted to GST (gluationia-S-transferase), or the use of an antibody specific for the expressed fusion protein, or the use of a histidine tag. Alternatively, the recombinant protein can be fused to an Fc immunoglobulin domain. Such a fusion protein can be easily purified using a protein A column. It is envisioned that the small molecule mimics of the antagonists described above are also encompassed by the invention.

Bioactivity test of C3APLT, C3APS, C3-TL and C3-TS

33 To test the efficacy of C3APLT, C3APS, C3-TL and C3-TS, a number of experiments were carried out with PC-12 cells, a neuronal cell line, growth on growth inhibitory substrates (see Lehmann et al supra). PC-12 cells are placed on myelin substrates as described (Lehmann et al, supra). C3, C3APLT, C3APS, C3-TL or C3-TS are added in different concentrations without crushing (please refer to Figures 4, 5 and 8 for the concentrations used). C3 passively added to the culture medium in this way is not capable of promoting neurite growth in growing inhibitory substrates because cells can be minced for C3 to enter cells and to be active (Figure 1). C3APLT and C3APS are capable of Rho ribosilar ADP to cause a change in the molecular weight of RhoA (Figure 2). C3APLT and C3APS are capable of promoting neurite growth and entering neurons after being passively added to the culture medium (Figure 3, Figures 4 and 5). The dose response experiment where concentrations of 0.25ng / ml, 2.5 ng / ml, 25 ng / ml, 250 ng / ml and 2.5 µg / ml and 25 µg / ml are tested and show that C3APLT and C3APS they help more neurons differentiate neurites at doses 10,000 times less than C3 (Figure 4). Dose response experiments where concentrations of 0.25ng / ml, 2.5 ng / ml, 25 ng / ml, 250 ng / ml and 2.5 µg / ml and 25 µg / ml are tested and show that C3APLT is capable of promoting large growth of neurite when added to a minimum concentration of 0.0025 ug / ml (Figure 5). These concentrations of 2.5 ng / ml and 25 ng / ml for C3APLT and C3APS, represent 10,000 and 1,000 times less than the required dose with C3, respectively. Furthermore, at the highest concentration tested, 50 ug / ml, these two new Rho antagonists do not exhibit toxic effects on PC-12 cells, and are capable of stimulating neurite growth on growth inhibitory substrates. C3-TL and C3-TS are also tested in concentrations of 0.25ng / ml, 2.5 ng / ml, 25 ng / ml, 250 ng / ml and 2.5 µg / ml and 25 µg / ml and are found to be able to promote neurite, grow on myelin substrates at significantly lower doses of C3 (Figure 8). C3Basic3 is tested at 50 ug / ml in a rapid growth assay (Figure 11). To verify the ability of C3APLT and C3APS to promote the growth of primary neurons, primary retinal cultures are prepared, and neuronal plates are plated on myelin substrates as described with respect to Example 5. In the absence of C3APLT treatment or C3APS, the cells remain round and are unable to grow neurites. When treated with C3APLT or C3APS, retinal neurons are able to extend large neurites into inhibitory myelin substrates (Figure 6). The ability of C3APLT and C3APS to promote growth in a different type of growth from the relevant inhibitory substrate is then tested with the type of growth of inhibitory proteins found in glial scars. The chamber slides are coated with a mixture of chondroitin sulfate proteoglycans (Chemicon), and then plated with retinal neurons (results presented in Figure 12). Neurons are not capable of extending neurites on proteoglycan substrates, but when treated with C3APLT or C3APS, they extend large neurites. These studies demonstrate that C3APLT and C3APS can be used to promote neurite growth in myelin and in proteoglycans, the major classes in inhibitory substrates that prevent repair after CNS injury. C3APLT testing ability to promote regeneration and functional recovery after spinal cord injury. To test whether C3APLT can promote repair after spinal cord injury, fully adult mice are used (as described with respect to Example 6). A dorsal hemisection is performed at T8 (thoracic spinal level 8), and mice are treated with different

amounts (Figure 7) of C3APLT in a fibrin glue as described (McKerracher, US patent pending patent (supply patent)). In previously known experiments with C3, it was found that 40-50 µg is needed to promote anatomical regeneration in the optic nerve (Lehmann et all supra). We tested different doses (see Figure 7) of C3APLT ranging from 1 µg to 50 µg and animals were evaluated for behavioral recovery according to the BBB scale (Basso (1995) 12: 1-21).

The day after the surgery and the application of C3APLT, the behavior test begins. Animals are placed in an open field environment consisting of a rubber blanket approximately 4 ’X 3’ in size. Animals stop moving randomly, animal movement is video recorded. For each test, two observers classified the animals by the ability to move the ankle, knee and hip joints in the early recovery phase. Previously C3 treatment of mice is seen to lead to observable functional recovery 24 hours after treatment. In C3APLT-treated mice, functional recovery can be observed as early as 24 hours after spinal cord injury (Figure 7). Untreated mice exhibit a recovery score for function according to the BBB scale averaging 0, where C3-treated mice are able to walk and have a BBB rating averaging 8 (Figure 7). At concentrations greater than 50 ug, approximately 50% of the C3APLT-treated mice died within 24 hours. However, of the surviving mice, they exhibit good long-term functional recovery. These results demonstrate that C3APLT effectively promotes early functional recovery after spinal cord injury, and that it is effective at much lower doses of C3. However, at high concentrations, C3APLT appears to exhibit toxicity, and therefore care is required for clinical use.

Qualitative observations from the videotapes show that only animals that received C3APLT reach the last stage of recovery after 30 days of treatment. Untreated control animals typically do not pass between the early recovery phase. These results indicate that the application of C3APLT improves long-term functional recovery after spinal cord injury compared to no treatment, injury alone, or fibrin adhesive alone.

To test whether early recovery is due to neuroprotection, sections of the spinal cord for apoptosis are examined by Tunel marking following the manufacturer's instructions (Roche Diagnostic). C3APLT is capable of reducing the number of dry cells observed at the site of injury. Therefore, C3APLT must be an effective neuroprotective agent for the treatment of ischemia, such as after stroke.

EXAMPLE 1. METHOD FOR DEVELOPING C3APLT AND C3APS PROTEINS

C3APL (amino acid sequence: SEQ ID NO .: 4) and C3APLT (amino acid sequence; SEQ ID NO: 37) are the names given for the cDNA encoded proteins made by binding the C3 transferase functional domain and region homeobox of the transcription factor called antenapedia (Bloch-Gallego (1993) 120: 485-492) in the following form. A cDNA encoding C3 (Dillon and Feig (1995) 256: 174-184) cloned into plasmid vector pGEX-2T is used for the C3 portion of the chimeric protein. The stop codon at the 3 'end of DNA is replaced with an EcoR I site by polymerase chain reaction using primers 5'GAA TTC TTT AGG ATT GAT AGC TGT GCC 3' (SEQ ID NO: 1) and 5 ' GGT GGC GAC CAT CCT CCA AAA 3 '(SEQ ID NO: 2). The PCR product is subcloned into a pSTBlue-1 vector (Novagen, city), then cloned into a pGEX-4T vector using the BamH I and Not I restriction site. This vector is called pGEX-4T / C3. The pGEX-4T vector has a 5 'glutathione S transferase (GST) sequence for use in affinity purification. The antenapedia sequence used to add to the 3 'end of C3 in pGEX-4T / C3 is created by PCR of the vector pET-3a (Bloch-Gallego (1993) 120: 485-492, Derossi (1994) 269: 10444-10450 ). The primers used are 5'GAA TCC CGC AAA CGC GCA AGG CAG 3 ’(SEQ m NO: 7) and 5’TCA GTT CTC CTT CTT CCA CTT CAT GCG 3’ (SEQ ID NO: 8). The PCR product obtained from the reaction is subcloned into a blunt pSTBlue-1 vector, then cloned into pGEX-4T / C3, using the EcoR I and Sal I restriction sites, creating pGEX-4T / C3APL and C3APLT. C3APLT is selected for the presence of a structure change mutation giving a functional group of transport region in prolines.

A shorter version of antenapedia (pGEX-4T / C3AP-short) (C3APS amino acid sequence; SEQ ID NO .: 6) is also made. This chimeric sequence is made by ligating the oligonucleotides that encode the short peptide antenapedia (Maizel (1999) 126: 3183-3190) in the vector pGEX-4T / C3 that cuts with EcoR I and Sal I. For short sequences pGEX-4T / C3AP of the oligos made are 5'AAT TCC GCC AGA TCA AGA TTT GGT TCC AGA ATC GTC GCA TGA AGT GGA AGA AGG 3 '(SEQ ID NO: 9) and 5'GGC GGT CTA GTT CTA AAC CAA GCT CTT AGC AGC GTAEP 1377 319 B1 24 5 10 15 20 25 3035 404550 55 GTT CACCTT CTT CCA GCT 3 '(SEQ ID NO: 10). The two strains hybridize by mixing equal amounts of the oligonucleotides, heating at 72 ° C for 5 minutes, and then left at room temperature for 15 minutes. Oligonucleotides in the pGEX4T / C3 vector are ligated and the clones are collected and analyzed.

To prepare the recombinant C3APLT proteins (SEQ ID NO .: 37) and C3APS (SEQ ID NO .: 6), the plasmids containing the corresponding cDNAs (pGEX-4T / C3APLT and pGEX4T / C3AP-short) are transformed into bacteria, the competent blue strain XL-1 E. coli. Bacteria are grown in L broth (10g / L Bacto-Tryptone, 5g / L Yeast Extract, 10g / L NaCl) with ampicillin at 50 ug / ml (BMC-Roche), in a shaker incubator for 1 hr at 37 ° C and 300 rpm. Isopropyl β-Dthiogalactopyranoside (IPTG), (Gibco) is added in a final concentration of 0.5 mM to induce the production of recombinant protein and the culture is grown for an additional 6 hours at 37 ° C and 250 rpm. Globules of bacteria are obtained by centrifugation in 250 ml centrifuge bottles at 7000 rpm for 6 minutes at 4 ° C. Each bead is resuspended in 10 ml of Buffer A (50mM Tris, pH 7.5, 50mM NaCl, 5mM MgCl2, 1mM DTT) plus 1mM PMSF. All resuspended blood cells are pooled and transferred to a 100 ml plastic beaker on ice. The remaining buffer A with PMSF is added to the pooled sample. The bacteria sample is sonicated 6 x 20 seconds using a Branson Sonifier 450 probe sonicator. The bacteria and probe are pooled on ice for 1 minute between sonications. The sonicate is centrifuged in a Sorvall SS-34 rotor at 16,000 rpm for 12 minutes at 4 ° C to clarify the supernatant. The supernatant is transferred into fresh SS-34 tubes and centrifuged at 12,000 rpm for 12 minutes at 4 ° C. Up to 20 ml of glutathione-agarose beads (Sigma) are added to the purified lysate and placed on a rotating plate for 2-3 hours. The beads are washed 4 times with buffer B, (Buffer A, NaCl is 150mM, not PSMF) then 2 times with buffer C (Buffer B + 2.5mM CaCl2). The final wash is poured into beads created in a thick suspension. To remove the glutathione S transferase sequence from the recombinant protein, 20U Thrombin (Bovine, plasminogen free, Calbiochem) is added, the beads are left to rotator overnight at 4 ° C. After thrombin cleavage the globules are loaded onto an empty 20 ml column. Approximately 20 1 ml aliquots are collected by elution with PBS. Samples from each 0.5 ul aliquot are nitrocellulose stained and Amido Black stained to determine protein peak. The aliquots containing the fusion proteins are pooled and 100 µl beads of p-aminobenzamidine agarose (Sigma) are added and allowed to mix for 45 minutes at 4 ° C. This last step removes thrombin from the recombinant protein sample. The recombinant protein is centrifuged to remove the blood cells and then concentrated using a centriprep-10 concentrator (Amicon). Salt is removed from the concentrated recombinant protein with a PD-10 column (Pharmacia, containing Sephadex G-25M) and ten 0.5 ml aliquots are collected. A blot blot is made on these samples to determine the protein peak, and the appropriate aliquots are pooled, filtered and sterilized, and stored at -80 ° C. A protein assay (DC assay, Biorad) is used to determine the concentration of recombinant protein. The purity of the sample is determined by SDS-PAGE, and bioactivity assay with PC-12 cells.

EXAMPLE 2: TEST OF EFFICACY OF C3APLT AND C3APS IN TISSUE CULTIVATION

To test the ability of C3APLT and C3APS to overcome growth inhibition, PC-12 cells are plated on myelin, a growth inhibitory substrate. Myelin is purified from bovine brain (Norton and Poduslo (1973) 21: 749-757). In some other experiments chondroitin sulfate proteoglycan substrates (CSPG) are made from a purchased protein composition (Chemicon). Before coating slides or plates from a 96-well plate, they are coated with poly-L-lysine (0.025µg / ml) (Sigma, St. Louis, MO), washed with water, and allowed to dry. Myelin is stored as a 1mg / ml solution at -80 ° C, then thawed at 37 ° C, and centrifuged. Myelin is plated at 8 ug / wells in an 8-well chamber of Lab-Tek slides (Nuc, Naperville, IL). The myelin solution is allowed to dry overnight in a sterile tissue culture cabinet. The next morning the substrate is gently washed with phosphate buffered saline, and then the cells in the medium are added to the substrate. PC-12 cells (Lehmann et al., 1999) are grown in DMEM with 10% horse serum (HS) and 5% fetal bovine serum (FBS). Two days before use of PC-12 cells it is differentiated by 50 ng / ml of nerve growth factor (NGF). After the cells are primed, 5 ml of trypsin is added to the culture dish to dissociate the cells, the cells are pelleted and resuspended in 2 ml of DMEM with 1% HS and 50 ng / ml of nerve growth factor. Approximately 5,000 to 7,000 cells are then placed in 8-well chamber-coated myelin on Lab-Tek slides (Nuc, Naperville, IL). The cells are placed on the test substrates at 37 ° C for 3-4 hours to allow the cells to settle. The original medium is carefully removed by aspiration, taking care not to interrupt the cells and replace with DMEM with 1% HS, 50ng / ml NGF and various amounts of C3, C3APLT, or C3APS, depending on the desired dose. After two days, the cells are fixed (4% paraformaldehyde and 0.5% glutaraldehyde). For control experiments with unmodified C3, PC-12 cells primed with NGF are triptinized to disunite them from the culture dish, cells are washed once with load stripping buffer (114mM KCL, 15mM NaCl, 5.5mM MgCl2 , and 10 mM Tris-HCL) and then the cells are peeled with a rubber spatula in

0.5 ml of peeling buffer in the presence of 25 or 50 µg / ml of C3. Cells are pelleted and resuspended in 2ml DMEM, 1% HS, and 50 ng / ml nerve growth factor before plating. At least four experiments are analyzed for each treatment. For each well, twenty images with a 20X objective are collected using a Zeiss Axiovert microscope. For each image, the numbers of the cells with and without neurites are counted and the lengths of the neurites are determined. Although myelin is dense phase, cells plated on myelin substrates are immunostained with anti-βIII tubulin antibody prior to analysis. Quantitative analysis of neurite growth is with the help of Northern Eclipse software (Empix Imaging, Mississauga, Ontario, Canada). Data analysis and statistics are done with Microsoft Excel.

For a rapid bioassay, compounds are tested in tissue culture as described above, except that cells are plated in the plastic tissue culture than in the inhibitory substrates. For these experiments the plates are fixed and the neurites are counted five hours after plating the cells. The test compounds (C3APLT and C3Basic3) are able to promote faster growth in plastic tissue culture than in plated cells without treatment (Figure 11).

To examine ADP ribosylation by C3, C3APLT, and C3APS, compounds are added to PC-12 cell cultures, as described above. Cells are harvested by centrifugation, prepared cell homogenates, and proteins separated by SDS polyacrylamide gel electrophoresis. Proteins are then transferred to nitrocellulose and Western blot probe with anti-RhoA antibody (Santa Cruz).

EXAMPLE 3: TESTING THE CAPACITY OF C3APLT AND C3APS TO VOID THE INHIBITION OF MULTIPLE GROWTH INHIBITING PROTEINS.

Myelin substrates are made as described in Example 2 and plated on tissue culture chamber slides. Puppies of rat P1 to P3 are decapitated, the heads are washed in ethanol and the eyes are removed and placed in a petri dish with Hanks' buffered saline solution (HBSS, from Gibco). A hole is cut in the cornea, crystalline is removed, and the retina is ejected. Typically, four retinas are used per preparation. The retinas are removed into a 15 ml tube and the volume is brought to 7 ml. An additional 7 ml of the cleavage enzymes and papain are added. The dissociation enzyme solution is made as follows: 30 mg of DL is added to a 15 ml tube (Sigma DL cysteine hydrochloride), and 70 ml of HBSS, 280 ul of 10mg.ml of bovine serum albumin are added and the solution is mixed and the pH is adjusted to 7 with 0.3N NaOH. The dissociated solution is filter sterilized and kept frozen in 7 ml aliquots, and 12.5 units of papain per ml (Worthington) is added before use. After adding the dissociation solution to the retina, the tube is incubated for 30 minutes on a rocking tray at 37 ° C. The retinas are then gently crushed, spun, and washed with HBSS. Ele HBSS is replaced in the growth medium (DMEM (Gibco), 10% fetal bovine serum, and 50 ng / ml brain derived neurotrophic factor (BDNF) vitamins, penicillin-streptomycin, in the presence or absence of C3APLT or C3APS Cells are plated onto the myelin or CSPG test substrates on chamber slides as described in Example 2, above.Quantitative analysis is completed as described by Example 2 above.Neurons are visualized by fluorescent microscopy with anti-βIII tubulin antibody, which detects the growth of retinal ganglion cells (RGC) The results are presented in Figure 6.

EXAMPLE 4. TREATMENT OF INJURED SPINAL CORD WITH C3APLT AND MEASUREMENT OF RECOVERY OF MOTOR FUNCTION IN TREATED MICE.

Adult Balb-c mice are anesthetized with 0.6 ml / kg Hypnorm, 2.5 mg / kg diazepam, and 35 mg / kg ketamine. This gives approximately 30 minutes of anesthetic, which is sufficient for the entire operation. A segment of the thoracic spinal column is exposed by removing the vertebra and the spinal process with “micro-geon” (Fine Science Tools). A spinal cord injury is then made dorsally, extending into the central canal with fine scissors, and the injury is re-cut with a fine knife. This injury makes all control animals paraplegic. The paravertebral muscles are closed with absorbable sutures, and the skin is closed with 2.0 silk sutures. After surgery, the bladder is manually overridden every 8-10 hours until all animals are back in control, typically 2-3 days. Food is placed in the cage for easy access, and a sponge with water is used for easy accessibility of the water after surgery. Also, animals that receive subcutaneous injection of Buprenorphine (0.05 to 0.1 mg / kg) every 8-12 hours for the first 3 days. Any animals that lose 15-20% of body weight are killed.

Rho antagonists (C3 or C3-like proteins) are delivered locally to the site of injury by a fibrin-based tissue adhesive delivery system (McKerracher, Canadian Patent Application No. 2,325,842). Recombinant C3APLT is mixed with fibrinogen and thrombin in the presence of CaCl2, fibrinogen is cleaved by thrombin, and the resulting fibrin monomers polymerize in a three-dimensional matrix. We added C3APLT as part of a fibrin adhesive, which polymerizes within approximately 10 seconds to be placed on the injured spinal cord. We tested C3APLT that is applied at the site of the spinal cord injury after the injury is done. For control we injected fibrin adhesives alone, or transected into the marrow without further treatment. For the behavioral test, the BBB classification method is used to examine locomotion in an open field environment (Basso (1995) 12: 1-21). The results are presented in Figure 7. The environment is a rubber blanket approximately 4 ’X 3’ in size, and the animals are placed on the blanket and videotaped for approximately 4 minutes. Care is not taken to stimulate the peroneal region or touch animals excessively during the recording session. Video tapes are digitized and viewed by two observers who assign BBB ratings. The BBB classification, modified for mice, is as follows:

Classification Description

1. There is no observable hind leg movement (HL). 2 Slight movement of one or more joints.

3 Extensive movement of one joint and / or slight movement of the other

joint.

4 Extensive movement of two joints.

5 Slight movement of all three joints of the HL.

6 Light movement of two joints and extensive movement of the

third.

7 Extensive movement of two joints and light movement of the

third.

8 Extensive movement of all three joints of the HL walk without

weight support.

9 Extensive movement of all three joints, walking with

weight support.

10 Frequent to consistent dorsal progression with weight support.

11 Frequent plantar progression with weight support.

12 Plantar progression consistent with weight support, without coordination.

13 Consistent plantar progression with consistent weight support,

occasional FL-HL coordination. 14 Consistent plantar progression with consistent weight support, frequent FL-HL coordination. 15 Consistent plantar progression with consistent weight support, consistent FL-HL coordination; the predominant leg position during locomotion is

39 tub internally or externally, or consistent FL-HL coordination with occasional dorsal progression. 16 Consistent plantar progression with consistent weight support, consistent FL-HL coordination; the predominant position of the leg is parallel to the body; frequent to consistent toe drag, or finger curl, trunk instability. 17 Consistent plantar progression with consistent weight support, consistent FL-HL coordination; the predominant position of the leg is parallel to the body, without toe drag, some trunk instability. 18 Consistent plantar progression with consistent weight support, consistent FL-HL coordination; the predominant position of the leg is parallel to the body, without finger drag and consistent stability in locomotion. EXAMPLE. 5 TREATMENT OF INJURED MOUSE SPINAL CORD WITH C3APLT AND EVALUATION OF ANATOMIC RECOVERY. Mice that receive a spinal cord injury and are treated as controls or with C3APLT, as described for Example 4, are evaluated for morphological changes in the scar and for axon regeneration. To study axon regeneration, corticospinal axons are identified by antegrade marking. For antegrade mark studies, animals are anesthetized as before, and the skull is removed over the motor cortex. The fine glass micropipette (approximately 100 um in diameter) is injected into the cerebral cortex with 2-4 ul of horseradish peroxidase conjugated to wheat germ agglutinin (2%), a marker taken by nerve cells and it is transported anterograde in the axon extending into the spinal cord. After injection of the antegrade tracer, the skull is replaced, and the skin is closed with 5-0 of suture thread. Animals are sacrificed with chloral hydrate (4.9 mg / 10 g) after 48 hours, and perfused with 4% paraformaldehyde in phosphate buffer as a fixative. The spinal cord is removed, cryoprotected with sucrose, and cryostat sections are placed on microscope slides for histological examination. EXAMPLE 1 for reference. DNA DETAILS AND C3TL PROTEIN SEQUENCE. Tat coding sequence is obtained by polymerase chain reaction of plasmid SVCMV-TAT (form obtained from Dr. Eric Cohen, Universite de Montreal) containing the complete HIV-1 Tat coding sequence. To isolate the Tat protein transport sequence, PCR is used. The first primer (5'GAATCCAAGCACCAGGAAGTCAGCC 3 '(SEQ ID NO .: 11)) and the second primer (5' ACC AGCCACCACCTTCTGATA 3 '(SEQ ID NO .: 12)) correspond to amino acids 27 to 72 of the HIV protein Tat. After verification and purification, the PCR product is subcloned into a blunt pSTBlue-1 vector. This transport segment of the Tat protein is then cloned into pGEX-4T / C3 at the 3 'end of C3, using the EcoR I and Sac I restriction sites. The new C3-Tat fusion protein is called C3-TL. Recombinant protein is made as described in Example 1. C3-TL DNA sequence (SEQ ID NO .: 13)

image 1

The TL transport peptide sequence itself is as follows: (SEQ ID NO .: 46)

image 1

The C3-TL protein sequence (SEQ ID NO .: 14)

image2

Molecular Weight 32721.40 Daltons 291 Amino Acids 43 Strongly Basic Amino Acids (+) (K, R) 21 Strongly Acidic Amino Acids (-) (D, E)

10 82 Hydrophobic Amino Acids (A, I, L, F, W, V) 104 Polar Amino Acids (N, C, Q, S, T, Y)

9,688 Isoelectric Point

22,655 Charge at PH 7.0 The total number of transferred bases is 876

15% A = 37.44 [328]% G = 17.58 [154]% T = 28.31 [248]% C = 16.67 [146]

41 Reference EXAMPLE 2. C3TS DNA PROTEIN AND SEQUENCE DETAILS. It is also a shorter Tat construct (C3-TS). To make the C3 fusion protein shorter the following oligonucleotides are 5'AAT TCT ATG GTC GTA AAA AAC GTC GTC 5 AAC GTC GTC GTG 3 '(SEQ ID NO .: 15) and 5' GAT ACC AGC ATT TTT TGC AGC ACT TGC AGC AGC ACA GCT 3 '(SEQ ID NO .: 16). The two oligonucleotide strains hybridize by combining equal amounts of the oligonucleotides, heating at 72 ° C for 5 minutes, and then allowing the oligonucleotide solution to cool to room temperature for 15 minutes. The oligonucleotides are ligated into the vector pGEX4T / C3 at the 3 'end of C3. Construction is sequenced. All plasmids are transformed into blue XL-1 competent cells. Recombinant protein is made as described in Example 1. C3-TS nucleotide sequence (SEQ ID NO .: 17)

image 1

The TS transport peptide sequence itself is as follows: (SEQ ID NO .: 47) 15 YGAKKRRQRRRVDSSGPHRD

The C3-TS protein sequence (SEQ ID NO .: 18)

image 1

Molecular Weight 26866.62 Daltons 238 Amino Acids 36 Strongly Basic Amino Acids (+) (K, R) 21 Strongly Acidic Amino Acids (-) (D, E) 71 Hydrophobic Amino Acids (A, I, L, F, W, V) 78 Polar Amino Acids ( N, C, Q, S, T, Y) 9,802 Isoelectric Point

15.212 Charge at PH 7.0. The total number of transferred bases is 717% A = 38.91 [279]

5% G = 17.43 [125] % T = 28.45 [204] % C = 15.20 [109]

EXAMPLE 6. The following examples illustrate how a modifying sequence can be modified without

10 affect the effectiveness of the transferred protein. The example shows modifications to C3Basic3 that could affect activity. The sequences may include the complete GST sequence, as shown here including the start site, which could not be enzymatically removed. Also, the transport sequence shown in this example has changes in the amino acid composition surrounding the active sequence due to the difference in cloning strategy, and the

15 His tag. However, the active region is: R R K Q R R K R R. It is in sequence contained in C3Basic3, and the transport sequence active in the sequence below. Also note that the C-terminal region of the protein after this active region differs from C3Basic3. This is because the cloning strategy changes, the restriction sites differ, and therefore 3 'terminal non-essential amino acids for the transport sequence are transplanted and included in the protein.

20 Nucleic acid sequence: (SEQ ID NO .: 19) 1413 base pairs Single strain Linear sequence

image3

image 1

Amino acid sequence (SEQ ID NO .: 20) 479 linear amino acids, single strain

image2

Molecular Weight 53813.02 Daltons 470 Amino Acids 68 Strongly Basic Amino Acids (+) (K, R) 55 Strongly Acidic Amino Acids (-) (D, E)

10 149 Hydrophobic Amino Acids (A, l, L, F, W, V) 121 Polar Amino Acids (N, C, Q, S, T, Y)

9,137 Isoelectric Point

14.106 Charge at PH 7.0 The total number of transferred bases is 1413

15% A = 34.61 [489]% G = 19.75 [279]% T = 29.51 [417]% C = 15.99 [226]% ambiguous = 0.14 [2]

44% A + T = 64.12 [906]% C + G = 35.74 [505] Davis, Botstein, Roth Temp. Fusion C. 79.20 EXAMPLE 7. ADDITIONAL C3 CHEMICAL PROTEINS THAT COULD BE EFFECTIVE IN STIMULATING CNS REPAIR. The following sequences can be added to the amino terminus or carboxy terminus of C3 or a truncated C3 that retains its enzymatic activity. 1) Polyarginine sequences as described (Wender, et al. (2000) 97: 13003-8.). This can be 6 to 9 or more arginines. 2) Mixed poly-lysine sequences 3) Poly-histidine sequences 4) Mixed arginine and lysine sequences. 5) Basic amino acid resistances that do not contain basic amino acid resistances where the added sequence retains the transport characteristics. 6) Sequences of 5-15 amino acids that contain at least 50% of basic amino acids. 7) Sequences greater than 15 -30 amino acids that contain at least 30% of basic amino acids. 8) Sequences greater than 50 amino acids that contain at least 18% of basic amino acids. 9) Any of the above where amino acids are chemically modified, such as by the addition of cyclohexyl side chains, other side chains, different alkyl spacers. 10) Sequences that have proline residues with a propensity to break the helix to act as effective transporters. EXAMPLE 8. ADDITIONAL C3 CHEMICAL PROTEINS WHICH COULD BE EFFECTIVE IN STIMULATING CNS REPAIR. C3Basicl: C3 fused to a randomly designed basic tail C3Basic2: C3 fused to a randomly designed basic tail C3 Basic3: C3 fused to the reverse Tat sequence We designed the following DNA encoding a chimeric C3 with membrane transport properties. The protein is designated C3Basicl. This sequence is designed with C3 fused to a random basic sequence. The construction is made to encode the peptide given below. K R R R R P K K R R R A K R R (SEQ ID NO.:21) Construction is done by synthesizing the two oligonucleotides given below, hybridizing them, and aligning them in the pGEX-4T / C3 vector with an added histidine tag.

image 1

C3Basic1 DNA sequence (SEQ ID NO .: 24) C3Basic1 protein sequence (SEQ ID NO .: 25)

image 1

image 1

Molecular Weight 29897.03 Daltons

5 263 Amino acids 44 Strongly Basic Amino Acids (+) (K, R) 23 Strongly Acidic Amino Acids (-) (D, E) 75 Hydrophobic Amino Acids (A, I, L, F, W, V) 79 Polar Amino Acids (N, C, Q, S, T, Y)

10 10,024 Isoelectric Point

22.209 Charge at PH 7.0 Davis, Botstein, Roth Temp. Fusion C. 78.56 EXAMPLE 9. ADDITIONAL C3 CHEMICAL PROTEIN WHICH COULD BE EFFECTIVE TO STIMULATE CNS REPAIR.

15 We have designed the following DNA encoding a chimeric C3 with membrane transport properties. The protein is designated C3Basic2. This sequence is designed with C3 fused to a random basic sequence. The construction is made to encode the peptide given below.

K R R R R K K R R Q R R R (SEQ ID NO.:26) The construct is made by synthesizing the two oligonucleotides given below, hybridizing them, and ligating them into the vector pGEX4T / C3 with an added histidine tag.

image 1

C3Basic2 DNA sequence (SEQ ID NO .: 29)

image2

C3Basic2 protein sequence (SEQ ID NO .: 304)

image 1

Molecular Weight 29572.61 Daltons

260 amino acids

10

42 Strongly Basic Amino Acids (+) (K, R)

23 Strongly Acidic Amino Acids (-) (D, E)

74 Hydrophobic Amino Acids (A, I, L, F, W, V)

80 Polar Amino Acids (N, C, Q, S, T, Y)

9,956 Isoelectric Point

fifteen

20,210 Charge at PH 7.0

Davis, Botstein, Roth Temp. Fusion C. 78.45

EXAMPLE

10. PROTEIN CHEMICAL C3 ADDITIONAL THAN COULD TO BE

EFFECTIVE TO STIMULATE CNS REPAIR.

47 We have designed the following DNA encoding a chimeric C3 with membrane transport properties. The protein is designated C3Basic3. This sequence is designed with C3 fused to a reverse Tat sequence. Construction is done to encode the given peptide RRKQRRKRR (SEQ ID NO.:31) Construction is done by synthesizing the two given oligonucleotides, hybridizing them, and ligation them into the vector pGEX4T / C3 with an added histidine tag, then subcloned in pGEX4T / C3. 5 'AGA AGG AAA CAA AGA AGA AAA AGA AGA 3' (SEQ ID NO .: 32) 5 'TCT TCC TTT GTT TCT TCT TTT TCT TCT 3' (SEQ ID NO .: 33) C3Basic3 DNA sequence (SEQ ID NO .: 34)

image4

C3Basic3 protein sequence (SEQ ID NO .: 35)

image 1

Molecular Weight 29441.47 Daltons 15 260 Amino Acids

39 Strongly Basic Amino Acids (+) (K, R)

23 Strongly Acidic Amino Acids (-) (D, E)

76 Hydrophobic Amino Acids (A, I, L, F, W, V)

80 Polar Amino Acids (N, C, Q, S, T, Y) 9,833 Isoelectric Point 17,211 Charge at PH

20 7.0 Davis, Botstein, Roth Temp. Fusion C. 78.29 EXAMPLE 11. SEQUENCES FOR C3APLT

48 One of the clones that has been selected from the C3APL subcloning into pGEX that encodes a protein that is not the expected size but has good biological activity. This clone has a structure change mutation that leads to truncation, and this clone is called C3APLT. The clone is sequenced again and the chromatograms are analyzed to confirm the sequence. To confirm the C3APLT sequences, the coding sequence of both pGEX-4T / C3APLT strains are sequenced by full-length double-stranded sequencing of the clone (BioS & T, Montreal, Quebec). The DNA sequence for C3APLT is as follows: (SEQ ID NO .: 36)

image 1

The sequence of the APLT transport peptide itself is as follows (SEQ ID NO .: 48): VMNPANAQGRHTPGTRL The protein sequence for C3APLT is as follows: (SEQ ID NO .: 37)

image 1

15 Molecular Weight 27574.42 Daltons 248 Amino Acids 33 Strongly Basic Amino Acids (+) (K, R) 21 Strongly Acidic Amino Acids (-) (D, E) 76 Hydrophobic Amino Acids (A, I, L, F, W, V)

20 80 Polar Amino Acids (N, C, Q, S, T, Y)

9,636 Isoelectric Point

12.379 Loading at PH 7.0 EXAMPLE 12. SUBCLONATION AND SEQUENCES FOR C3APLT IN PET (p = ET3aC3APLT)

25 C3 has been reported for stably expressing in E. coli using pGEX-series and pET-series vectors (eg, Dillon and Feig, 1995 Meth. Enzymol. 256: 174-184. Small GTPases and Their Regulators. Part B. Rho Family. WE Balch, CJ Der, and A. Hall, eds.; Lehmann et al., 1999 supra; Han et al., 2001. J. Mol. Biol. 395: 95-107). Fusion proteins are well expressed in the pGEX vector, for synthesis and testing. However, for large-scale production it is more efficient to synthesize recombinant proteins without an affinity tag that increases the size of the protein produced. Also, it is more economical to synthesize proteins on a large scale by affinity chromatography using automated FPLC systems. The polymerase chain reaction is used to transfer the recombinant C3APLT construct into pET T7 polymerase based on the E. coli expression system system (reviewed by Studier et al., 1990. Meth. Enzymol. 185: 60

89. Gene Expression Technology. D.V. Goeddel, ed.). A similar PCR scope is appropriate for other series of fusion protein constructs with C3-based transport sequences. The pET3a vector DNA is obtained from Dr. Jerry Pelletier, McGill University. PCR primers are obtained from Invitrogen. The primer on top (5 ') is 5'-GGA TCT GGT TCC GCG TCA TAT GTC TAG AGT CGA CCT G-3 (37 b) (SEQ ID NO.:38). The Nde I site that is introduced into the primer is highlighted to replace the BamHI site in pGEX4T-C3APLT. The lower primer is 5'-CGC GGA TCC ATT AGT TCT CCT TCT TCC ACT TC-3 '(32 b) (SEQ ID NO.:39). This primer introduces two changes to the DNA strand encoding pGEX4T-C3APLT, replacing the EcoRI site of pGEX4T-.C3APLT with a BamH I site (underlined) and replacing a TGA stop codon with the strong TAAT stop sequence (the sequence ATTA in italics in the complementarity primer). Compared to pGEX4TC3APLT, the predicted Terminal N sequence of pET3a-C3APLT is Met-Ser different from Gly-Ser-Ser, a loss of a Serine, and a replacement for Met for Gly. There are no changes in amino acid sequences at C3APLT Terminal C.

The C3APLT target gene is amplified using Pfu polymerase (Invitrogen / Canadian Life Technologies) with manufacturer-recommended buffer, DNA and deoxyribonucleotide concentrations. PCR is carried out as follows: 95 ° C for 5 minutes, 10 cycles of 94 ° C for 2 minutes followed by 56 ° C for 2 minutes then extension to 70 ° C for 2 minutes, then 30 cycles of 94 ° C for 2 minutes followed by 70 ° C. Complete reactions are stored at 4 ° C. The QIAEXII kit (Qiagen) is used to purify the silica from agarose gel containing the DNA band. The purified PCR product DNA and vector is digested with BamH I and Nde I (obtained from New England BioLabs) following the manufacturer's instructions. Digestion products are separated from external DNA by agarose gel electrophoresis and purified with the QIAE3GI kit. The insert and DNA vector are incubated overnight at 16 ° C with T4 DNA DNA ligase according to the directions provided by the manufacturer (New England BioLabs). Competent E. coli (DH5a, obtained from Invitrogen / Canadian Life Technologies) are transformed with the ligation mixture.

DNA from purified colonies is prepared using the Qiagen plasmid midi kit, and the complete insert and binding sequences are verified by full-length double-stranded sequencing of the clone (BioS & T, Montreal, Quebec) with the 5 'AAA TTA ATA CGA CTC primer primer ACT ATA GGG 3 '(24 bases) (SEQ ID NO .: 40) and the T7 5' rear termination sequencing primer GCT ACT TAT TGC TCA GCG G 3 '(19 bases) (SEQ ID NO .: 41). The sequence of the C3APLT cDNA in pET is given in SEQ ID NO .: 42. The amino acid sequence is given in SEQ. ID NO .: 43 (pET3a-C3APLT).

EXAMPLE 13: SEQUENCE MODIFICATIONS.

Any of the sequences given in Examples 6, 7, 8 and 9, 11 and 12 and Reference EXAMPLES 1 and 2 can be modified to retain C3 enzyme activity and effective transport sequences. For example, the DNA encoded amino acids at the 3 'end of the sequence that represent the translation of the restriction sites used in cloning can be removed without affecting activity. Some of the amino acids in Terminal amino can also be removed without affecting activity. The minimum amount of sequence necessary for the biological activity of the C3 portion of the fusion protein is not known but can be easily determined by known techniques. For example, by increasing more than the 5 'end of the cDNA encoding C3, it can be removed, and the resulting proteins made and tested for biological activity. Similarly, the increased amounts of the 3 'end can be removed and the fragments for biological activity. Then, the central region for fragment testing can be tested by retaining C3 activity. Therefore, the C3 portion of the protein can be truncated to include just the amino acids necessary for activity. Alternatively, mutations can be made in the C3 coding regions, and the resulting proteins are tested for activity. The transport sequences can be modified to add or remove one or more amino acids or to completely change the transport peptide, but retain the transport characteristics in terms of effective dose compared to C3 in our tissue culture bioassay (Example 2) . New transport sequences for biological activity can be tested to improve the efficiency of C3 activity by plating neurons and testing them on inhibitory substrates, as described in Example 2.

As previously discussed, it has been determined in tissue culture studies that the minimum amount of C3 can be used to induce growth on inhibitory substrates is 25 ug / ml (Lehmann, et al. (1999) J. Neurosci. 19 : 7537-7547; Morii, N and Narumiya, S. (1995) Methods in Enzymology, Vol 256 part B, pg. 196-206 If the cells are not crushed, this dose is still ineffective (Figure 1). In the context of the present invention, it has been determined, for example, that at least 40 µg of mouse C3 / 20g needs to apply an injured mouse spinal cord or rat optic nerve (McKerracher, Canadian Patent Application No .: 2,325,842). To calculate the doses that might be required to treat a human adult in an equivalent dose-by-weight dose used for rat and mouse experiments, it would be necessary to apply 120 mg / kg of C3 (ie alone) to the injured human spinal cord. amount of recombinant C3 protein needed creates significant problems p For manufacturing, due to protein purification and large-scale costs. It also limits the varying dose that can be tested due to the large amount required for minimum effective doses.

The fusion proteins of the present invention are much more effective than C3 (i.e. alone) in promoting neurite growth in the myelin substrate. For example, the concentrations of C3APLT and C3APS, 10,000 and 1,000 times less than the concentration required for C3, can be used with comparable (similar) effects without exhibiting toxic effects (eg, in PC12 cells). C3-TL and C3-TS are also capable of promoting neurite growth in myelin substrates at significantly lower doses than C3. The in vivo results also indicate that less doses of the fusion proteins may be required to promote regeneration and functional recovery after spinal cord injury in mice. Thus, the fusion proteins of the present invention represent a significant improvement and advantage over C3 in manufacturing costs and doses required for treatment.

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<210> SEQ ID NO .: 1

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used to remove the stop codon of ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA.

<400> SEQ ID NO .: 1 gaattcttta ggattgatag ctgtgcc 27

<210> SEQ ID NO .: 2

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used to remove the stop codon of ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA.

<400> SEQ ID NO .: 2

ggtggcgacc atcctccaaa a 21 5 <210> SEQ ID NO .: 3

<211> LENGTH: 888

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: The C3APL nucleotide sequence: nucleotide sequence (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... ( 888) includes the Antenapedia sequences

<400> SEQ ID NO .: 3

image 1

image 1

<210> SEQ ID NO .: 4

<211> LENGTH: 295

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3APL amino acid sequence: amino acid sequence (1) ... (231) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (295) includes the Antenapedia sequences.

<400> SEQ ID NO .: 4

image 1

image 1

<210> SEQ ID NO .: 5

<211> LENGTH: 774

<212> TYPE: DNA 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: The C3APS nucleotide sequence: nucleotide sequence (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... (774 ) includes the Antenapedia sequences.

10 <400> SEQ ID NO .: 5 <210> SEQ ID NO .: 6

image 1

image 1

<211> LENGTH: 257

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3APS amino acid sequence: sequence (1) ... (231) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (257) includes the Antenapedia sequences.

10 <400> SEQ ID NO .: 6

image 1

image 1

image 1

<210> SEQ ID NO .: 7

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the Antenapedia amplification sequence

<400> SEQ ID NO .: 7 gaatcccgca, aacgcgcaag gcag 24

<210> SEQ ID NO .: 8

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the Antenapedia amplification sequence

<400> SEQ ID NO .: 8 tcagttctcc ttcttccact tcatgcg 27

<210> SEQ ID NO .: 9

<211> LENGTH: 54

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the cloning of the Antenapedia sequences

<400> SEQ ID NO .: 9 aattccgcca gatcaagatt tggttccaga atcgtcgcat gaagtggaag aagg 54

<210> SEQ ID NO .: 10

<211> LENGTH: 54

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the cloning of the Antenapedia sequences

<400> SEQ ID NO .: 10 ggcggtctag ttctaaacca agctcttagc agcgtagttc accttcttcc agct

54

<210> SEQ ID NO .: 11

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

59

<223> OTHER INFORMATION: Oligonucleotide used

sequence corresponding to amino acid 27-72 of HIV-1 Tat

<400> SEQ ID NO .: 11

gaatccaagc atccaggaag tcagcc 26 5 <210> SEQ ID NO .: 12

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

10 <223> OTHER INFORMATION: Oligonucleotide used sequence corresponding to amino acid 27-72 of HIV-1 Tat

<400> SEQ ID NO .: 12

accagccacc accttctgat a 21

<210> SEQ ID NO .: 13 15 <211> LENGTH: 876

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

in amplifying a

in amplifying a

<223> OTHER INFORMATION: C3-TL nucleotide sequence: sequence of

Nucleotide (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... (876) includes the sequences of HIV-1 Tat

<400> SEQ ID NO .: 13

image 1

image 1

<210> SEQ ID NO .: 14

<211> LENGTH: 291

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3-TL amino acid sequence: sequence (1) ... (231) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (291 ) includes the HIV-1 Tat sequences.

<400> SEQ ID NO .: 14

image2

image 1

<210> SEQ ID NO .: 15

<211> LENGTH: 39

<212> TYPE: DNA 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide used in the cloning of HIV-1 Tat sequences

<400> SEQ ID NO .: 15 10 aattctatgg tcgtaaaaaa cgtcgtcaac gtcgtcgtg 39

<210> SEQ ID NO .: 16

<211> LENGTH: 39

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence 15 <220> FEATURE:

<223>. OTHER INFORMATION: Oligonucleotide used in the cloning of HIV-1 Tat sequences

<400> SEQ ID NO .: 16

gataccagca ttttttgcag cagttgcagc agcacagct 39 20 <210> SEQ ID NO .: 17

<211> LENGTH: 756

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: The C3-TS nucleotide sequence: nucleotide sequence (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... (756) includes HIV-1 Tat sequences

<400> SEQ ID NO .: 17

image2

image 1

<210> SEQ ID NO .: 18

<211> LENGTH: 251

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of C3-TS: sequence (1) ... (231) includes the sequences of ADP-ribosyl transferse C3 (Clostridium botulinum), amino acid sequence (232) ... (251 ) includes the HIV-1 Tat sequences.

10 <400> SEQ ID NO .: 18 <210> SEQ ID NO .: 19

image 1

image 1

<211> LENGTH: 1413

<212> TYPE: DNA 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Nucleotide sequence (1) ... (672) includes glutathione S-transferase sequences, nucleotide sequence (673) ... (1365) includes ADP-ribosyl transferase C3 sequences (Clostridium botulinum ), nucleotide sequence (1366) ... (1413) includes a

10 sequence encoding a random amino acid sequence.

<400> SEQ ID NO .: 19

image 1

image 1

image 1

<210> SEQ ID NO .: 20

<211> LENGTH: 470

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence (1) ... (224) includes glutathione S-transferase sequences, amino acid sequence (225) ... (455) includes ADP-ribosyl transferase C3 sequences (Clostridium botulinum ), amino acid sequence (456) ... (470) includes a sequence

10 random amino acid.

<400> SEQ ID NO .: 20

image 1

image 1

image 1

<210> SEQ ID NO .: 21

<211> LENGTH: 16

<212> TYPE: PRT <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3Basicl Random Basic Amino Acid Sequence

<400> SEQ ID NO .: 21

image2

<210> SEQ ID NO .: 22

<211> LENGTH: 48

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence 10 <220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide used in the cloning of a random amino acid sequence in C3Basicl

<400> SEQ ID NO .: 22

aagagaaggc gaagaagacc taagaagaga cgaagggcga agaggaga 48 15 <210> SEQ ID NO .: 23

<211> LENGTH: 48

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

20 <223> OTHER INFORMATION: Oligonucleotide used in the cloning of a random amino acid sequence in C3Basicl

<400> SEQ ID NO .: 23 ttctcttccg cttcttctgg attcttctct gcttcccgct tctcctct 48

<210> SEQ ID NO .: 24 25 <211> LENGTH: 792

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: The C3Basicl nucleotide sequence: the sequence

30 (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... (792) includes a sequence that encodes a random amino acid sequence and a Histidine tag .

<400> SEQ ID NO .: 24

image 1

image 1

<210> SEQ ID NO .: 25

<211> LENGTH: 263

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3Basicl amino acid sequence: amino acid sequence (1) ... (231) includes the sequence of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (2630 includes a random amino acid sequence and a label

Histidine.

<400> SEQ ID NO .: 25

image 1

image 1

<210> SEQ ID NO .: 26

<211> LENGTH: 13

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: The random amino acid sequence of C3Basic2

<400> SEQ ID NO .: 26

image 1

<210> SEQ ID NO .: 27

<211> LENGTH: 39

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the cloning of a random amino acid sequence in C3Basic2

<400> SEQ ID NO .: 27 aagcgtcgac gtagaaagaa acgtagacag cgtagacgt 39

<210> SEQ ID NO .: 28

<211> LENGTH: 39

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the cloning of a random amino acid sequence in C3Basic2

<400> SEQ ID NO .: 28 ttcgcagctg catctttctt tgcatctgtc gcatctgca 39

<210> SEQ ID NO .: 29

<211> LENGTH: 783

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: The nucleotide sequence of C3Basic2: nucleotide sequence (1) ... (693) includes the sequences of ADP-ribosyl-transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... (783) includes a sequence encoding a random amino acid sequence and a Histidine tag.

<400> SEQ ID NO .: 29

image 1

image 1

<210> SEQ ID NO .: 30

<211> LENGTH: 260

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of C3Basic2, sequence (1) ... (231) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (260) includes a random amino acid sequence and a Histidine tag.

10 <400> SEQ ID NO .: 30

image 1

image 1

<210> SEQ ID NO .: 31

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Reverse Tat HIV-1 Amino Acid Sequence of C3Basic3

<400> SEQ ID NO .: 31

image 1

<210> SEQ ID NO .: 32

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide used in the cloning of a reverse HIV Tat sequence in C3Basic3

<400> SEQ ID NO .: 32 5 agaaggaaac aaagaagaaa aagaaga 27

<210> SEQ ID NO .: 33

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequences 10 <220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide used in the cloning of a reverse HIV Tat sequence in C3Basic3

<400> SEQ ID NO .: 33

tcttcctttg tttcttcttt ttcttct 27 15 <210> SEQ ID NO .: 34

<211> LENGTH: 771

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

20 <223> OTHER INFORMATION: The nucleotide sequence of C3Basic3: nucleotide sequence (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... ( 771) includes a sequence encoding reverse HIV-1 Tat amino acid sequence and a Histidine tag

<400> SEQ ID NO .: 34

image3

image 1

<210> SEQ ID NO .: 35

<211> LENGTH: 256

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of C3Basic3: amino acid sequence (1) ... (231) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (256) includes a reverse Tat HIV-1 amino acid sequence and a

10 Histidine label

<400> SEQ ID NO .: 35

image 1

image 1

<210> SEQ ID NO .: 36

<211> LENGTH: 887

<212> TYPE: DNA <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: The C3APLT nucleotide sequence: nucleotide sequence (1) ... (693) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (694) ... (747 ) includes a sequence encoding a proline-rich region

<400> SEQ ID NO .: 36

image 1

image 1

<210> SEQ ID NO .: 37

<211> LENGTH: 248

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of C3APLT: amino acid sequence (1) ... (231) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (232) ... (248) includes a proline-rich region

10 <400> SEQ ID NO .: 37

image 1

image 1

<210> SEQ ID NO .: 38

<211> LENGTH: 37

<212> TYPE: DNA 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide used in the amplification and cloning of C3APLT in the pET vector

<400> SEQ ID NO .: 38 10 ggatctggtt ccgcgtcata tgtctagagt cgacctg 37

<210> SEQ ID NO .: 39

<211> LENGTH: 32

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence 15 <220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in the amplification and cloning of C3APLT in the pET vector

<400> SEQ ID NO .: 39 cgcggatcca ttagttctcc ttcttccact tc 32

<210> SEQ ID NO .: 40

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in C3APLT sequencing

<400> SEQ ID NO .: 40 aaattaatac gactcactat aggg 24

<210> SEQ ID NO .: 41

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: Oligonucleotide used in C3APLT sequencing

<400> SEQ ID NO .: 41 gctagttatt gctcagcgg 19

<210> SEQ ID NO .: 42

<211> LENGTH: 888

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223>

OTHER INFORMATION: The C3APLT nucleotide sequence in a pET vector: nucleotide sequence (1) ... (690) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), nucleotide sequence (691) ... ( 744) includes a sequence encoding a proline rich region

<400> SEQ ID NO .: 42

image 1

image 1

<210> SEQ ID NO .: 43

<211> LENGTH: 247

<212> TYPE: PRT 5 <213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of C3APLT in a pET vector: amino acid sequence (1) ... (230) includes the sequences of ADP-ribosyl transferase C3 (Clostridium botulinum), amino acid sequence (2310 ... (247) includes a proline-rich region

10 <400> SEQ ID NO .: 43

image 1

image 1

<210> SEQ ID NO .: 44

<211> LENGTH: 64

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3APL Antenapedia Amino Acid Sequence

<400> SEQ ID NO .: 44

image 1

10 <210> SEQ ID NO .: 45

<211> LENGTH: 19

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE: 15 <223> OTHER INFORMATION: C3APS Antenapedia Amino Acid Sequence

<400> SEQ ID NO .: 45

image 1

<210> SEQ ID NO .: 46

<211> LENGTH: 60

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: C3-TL HIV-1 Tat amino acid sequence

<400> SEQ ID NO .: 46

image 1

<210> SEQ ID NO .: 47

<211> LENGTH: 20

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of HIV-1 Tat from C3-TS

<400> SEQ ID NO .: 47

image 1

10 <210> SEQ ID NO .: 48

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

15 <223> OTHER INFORMATION: Amino acid sequence of the proline-rich region of C3APLT

<400> SEQ ID NO .: 48

image 1

SEQUENCE LISTING

<110> BioAxone Therapeutics inc.

<120> FUSION PROTEINS

<130> 06746-004-WO-03

<150> CA 2,367,636

<151> 2002-01-15

<150> CA 2,362,004

<151> 2001-11-13

<150> CA 2,342,970

<151> 2001-04-12

<160> 48

<170> PatentIn version 3.1

<210> 1

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used to remove the stop codon of ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA.

<400> 1 gaattcttta ggattgatag ctgtgcc 27

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used to remove the stop codon of ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA.

<400> 2 ggtggcgacc atcctccaaa a 21

<210> 3

<211> 888

<212> DNA

<213> Artificial Sequence

<220>

<223>

The C3APL: sequence includes ADP-ribosyl transferase C3 (Clostrid ium botulinum) and the Antenapedia sequence.

<220>

<221> CDS

<222> (1) .. (888)

<223>

<400> 3

<210> 4

<211> 295

<212> PRT

<213> Artificial Sequence

<220>

<223>

The C3APL: sequence includes ADP-ribosyl transferase C3 (Clostrid ium botulinum) and the Antenapedia sequence.

image 1

image 1

<400> 4

image2

image 1

<210> 5

<211> 774

<212> DNA 5 <213> Artificial Sequence

<220>

<223> The C3APS sequence: Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and the Antenapedia sequence.

<220> 10 <221> CDS

<222> (1) .. (774)

<223>

<400> 5

image 1

<210> 6

<211> 257

<212> PRT

<213> Artificial Sequence

<220>

<223>

The C3APS sequence: Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and the Antenapedia sequence.

<400> 6

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the amplification of the Antenapedia sequence

<400> 7 gaatcccgca aacgcgcaag gcag 24

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the amplification of the Antenapedia sequence

<400> 8 tcagttctcc ttcttccact tcatgcg 27

<210> 9

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of the Antenapedia sequences

<400> 9 aattccgcca gatcaagatt tggttccaga atcgtcgcat gaagtggaag aagg 54

<210> 10

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of the Antenapedia sequences

<400> 10 ggcggtctag ttctaaacca agctcttagc agcgtagttc accttcttcc agct 54

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used in the amplification of a sequence that corresponds to amino acid 27-72 of HIV-1 Tat

image 1

image 1

<400> 11 gaatccaagc atccaggaag tcagcc 26

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence

<220> <223> Oligonucleotide used in the amplification of a sequence corresponding to the

HIV-1 Tat amino acid 27-72

<400> 12

accagccacc accttctgat a 21 5 <210> 13

<211> 876

<212> DNA

<213> Artificial Sequence

<220> 10 <223> The C3-TL sequence: Includes ADP-ribosyl transferase C3 (Clostridium botulinum)

and the HIV-1 Tat sequence.

<220>

<221> CDS

<222> (1) .. (876) 15 <223>

<400> 13 <210> 14

image 1

image 1

<211> 291

<212> PRT

<213> Artificial Sequence

<220>

<223> The C3-TL sequence: Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and the HIV-1 Tat sequence.

<400> 14

image4

image 1

<210> 15

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of HIV-1 Tat sequences

<400> 15 aattctatgg tcgtaaaaaa cgtcgtcaac gtcgtcgtg 39

<210> 16

<211> 39 5 <212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of HIV-1 Tat sequences

<400> 16 10 gataccagca ttttttgcag cagttgcagc agcacagct 39

<210> 17

<211> 756

<212> DNA

<213> Artificial Sequence 15 <220>

<223> The C3-TS sequence: Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and the HIV-1 Tat sequence.

<220>

<221> CDS 20 <222> (1) .. (756)

<223>

<400> 17

image 1

<210> 18

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> The C3-TS sequence: Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and the HIV-1 Tat sequence.

<400> 18

image2

<210> 19

<211> 1413

<212> DNA

<213> Artificial Sequence 5 <220>

<223> Includes GST sequences, the ADP-ribosyl transferase C3 sequence (C. botulinum) and a random amino acid sequence.

<220>

<221> CDS 10 <222> (1) .. (1413)

<223>

<400> 19

image 1

image 1

image 1

<210> 20

<211> 470

<212> PRT

<213> Artificial Sequence

<220>

<223> Includes GST sequences, ADP-ribosyl transferase C3 sequence (C. botulinum) and a random amino acid sequence.

<400> 20

image4

image 1

image 1

<210> 21

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> C3Basicl Random Basic Amino Acid Sequence

<400> 21

image 1

10 <210> 22

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used in the cloning of a random amino acid sequence in C3Basicl

<400> 22 aagagaaggc gaagaagacc taagaagaga cgaagggcga agaggaga. 48

<210> 23

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used in the cloning of a random amino acid sequence in C3Basicl

<400> 23 ttctcttccg cttcttctgg attcttctct gcttcccgct tctcctct 48

<210> 24

<211> 792

<212> DNA

<213> Artificial Sequence

<220>

<223>

The C3Basicl: sequence includes the ADP-ribosyl transferase C3 sequence (Clostridium botulinum) and a sequence encoding a random amino acid sequence and a Histidine tag.

<220>

<221> CDS

<222> (1) .. (792)

<223>

<400> 24

<210> 25

<211> 263

<212> PRT

<213> Artificial Sequence

<220>

<223>

The C3Basicl: sequence includes the ADP-ribosyl transferase C3 sequence (Clostridium botulinum) and a sequence encoding a random amino acid sequence and a Histidine tag.

image 1

<400> 25

image 1

image 1

<210> 26

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> The C3Basic2 Random Amino Acid Sequence

<400> 26

image 1

10 <210> 27

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used in the cloning of a random amino acid sequence in C3Basic2

<400> 27 aagcgtcgac gtagaaagaa acgtagacag cgtagacgt 39

<210> 28

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223>

Oligonucleotide used in the cloning of a random amino acid sequence in C3Basic2

<400> 28 ttcgcagctg catctttctt tgcatctgtc gcatctgca 39

<210> 29

<211> 783

<212> DNA

<213> Artificial Sequence

<220>

<223>

The C3Basic2: sequence includes the ADP-ribosyl-transferase C3 (Clostridium botulinum) sequences and a sequence encoding a random amino acid sequence and a Histidine tag.

<220>

<221> CDS

<222> (1) .. (783)

<223>

<400> 29

image 1

image 1

<210> 30

<211> 260

<212> PRT 5 <213> Artificial Sequence

<220>

<223> The C3Basic2: sequence includes the ADP-ribosyl-transferase C3 (Clostridium botulinum) sequences and a sequence encoding a random amino acid sequence and a Histidine tag.

10 <400> 30

image 1

image 1

<210> 31

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

116

<223> Reverse Tat HIV-1 Amino Acid Sequence of C3Basic3

<400> 31

image 1

<210> 32 5 <211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of a reverse HIV Tat sequence into 10 C3Basic3,

<400> 32 agaaggaaac aaagaagaaa aagaaga 27 210> 33

<211> 27 15 <212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in cloning a reverse HIV Tat sequence into C3Basic3

20 <400> 33 tcttcctttg tttcttcttt ttcttct 27

<210> 34

<211> 771

<212> DNA 25 <213> Artificial Sequence

<220>

<223> The C3Basic3 sequence: includes the sequences of ADP-ribosyl-transferase C3 (C. botulinum) and a sequence encoding a reverse Tat HIV-1 amino acid sequence and a Histidine tag

30 <220>

<221> CDS

<222> (1) .. (771)

<223>

<400> 34

image 1

<210> 35

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> The sequence of C3Basic3: includes the sequences of ADP-ribosyl-transferase C3 (C. botulinum) and the sequence encoding reverse His-1 Tat amino acid sequence and a Histidine tag

<400> 35

image 1

<210> 36

<211> 887

<212> DNA

<213> Artificial Sequence 5 <220>

<223> The C3APLT: sequence includes the ADP-ribosyl transferase C3 (Clostridium botulinum) sequences and a sequence encoding a proline rich region.

<220>

<221> CDS 10 <222> (1) .. (747)

<223>

<400> 36

image 1

image 1

<210> 37

<211> 248

<212> PRT

<213> Artificial Sequence

<220>

<223> The C3APLT: sequence includes the ADP-ribosyl transferase C3 (Clostridium botulinum) sequences and a sequence encoding a proline rich region.

<400> 37

image4

image 1

<210> 38

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of C3APLT in the pET vector

<400> 38 ggatctggtt ccgcgtcata tgtctagagt cgacctg 37

<210> 39

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in the cloning of C3APLT in the pET vector

<400> 39 cgcggatcca ttagttctcc ttcttccact tc 32

<210> 40

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide used in C3APLT sequencing

<400> 40 aaattaatac gactcactat aggg 24

<210> 41

<211> 19

<212> DNA

<213> Artificial Sequence

<220> <223> Oligonucleotide used in C3APLT sequencing

<400> 41 gctagttatt gctcagcgg 19

<210> 42 5 <211> 888

<212> DNA

<213> Artificial Sequence

<220>

<223>

The C3APLT sequence in a pET vector: includes the sequences of ADP-ribosyl 10 transferase C3 (Clostridium botulinum) and a sequence encoding a proline-rich region.

<220>

<221> CDS

<222> (1) .. (744)

<223>

15 <400> 42

image 1

image 1

<210> 43

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<223> The C3APLT sequence in a pET: vector includes the ADP-ribosyl transferase C3 (Clostridium botulinum) sequences and a sequence encoding a proline rich region.

<400> 43

image4

image 1

<210> 44

<211> 64

<212> PRT

<213> Artificial Sequence

<220>

<223> C3APL Antenapedia Amino Acid Sequence

<400> 44

image 1

10 <210> 45

<211> 19

<212> PRT

<213> Artificial Sequence

<220> 15 <223> C3APS Antenapedia Amino Acid Sequence

<400> 45

image 1

<210> 46

<211> 60

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino Acid Sequence of HIV-1 Tat from C3-TL

<400> 46

image 1

10 <210> 47

<211> 20

<212> PRT

<213> Artificial Sequence

<220> 15 <223> Amino acid sequence of HIV-1 Tat from C3-TS

<400> 47

image 1

<210> 48

<211> 17 20 <212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of the proline-rich region of C3APLT

<400> 48

image3

Claims (9)

one.

A polypeptide set forth in SEQ ID NO .: 43 or a polypeptide comprising at least one transport agent region covalently linked to an active agent region, wherein the transport agent region is a proline-rich region, in where the transport agent region is at the carboxy terminal end of said polypeptide and the active agent region is at the amino terminal end of said polypeptide, and said active agent region is selected from the group consisting of ADP-ribosyl C3 transferase and a fragment thereof that retains ADP-ribosyl transferase activity, where the proline-rich region is in SEQ ID NO .: 48.

2.

The use of a polypeptide as defined in claim 1 for the manufacture of a medicament for the treatment of glaucoma.

3.

The use of a polypeptide as defined in claim 1 for the manufacture of a pharmaceutical composition.

Four.

The use of a polypeptide according to claim 1 for the manufacture of a pharmaceutical composition to suppress inhibition of neuronal axon growth.

5.

The use of a polypeptide according to claim 1 for the manufacture of a pharmaceutical composition to facilitate axon growth.

6.

The use of a polypeptide according to claim 1 for the manufacture of a pharmaceutical composition for treating nerve injury.

7.

A pharmaceutical composition comprising: a) a polypeptide according to claim 1 and b) a pharmaceutically acceptable carrier.

8.

A pharmaceutical composition according to claim 7 further comprising a biological bioadhesive, insofar as it relates to SEQ ID NO .: 48 the pharmaceutical composition comprises the polypeptide consisting of SEQ ID. NO .: 37 (C3APLT).

9.

A pharmaceutical composition according to claim 7 further comprising fibrin, insofar as it relates to SEQ ID NO .: 48 the pharmaceutical composition comprises the polypeptide consisting of SEQ ID. NO .: 37 (C3APLT).