Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to pharmaceutical use of secreted, injected bacterial effector proteins.
• the present invention relates to manufacture and use of such proteins and combination and conjugation of the proteins with carriers.
• a number of neurological disorders arise from neuronal trauma that stimulates nerve damage due to internal processes such as apoptosis. It is known to treat such disorders using a superoxide dismutase in combination with a components that targets the enzyme to neurons. However, further active compounds for treatment of neuronal disease are desired.
• WO 98/56817 describes pharmaceutical compositions comprising a non- pathogenic organism expressing the YopJ protein, and YopJ protein combined with a carrier, for delivery of YopJ to gastrointestinal cells from the gut.
• the delivery mechanism disclosed in this document is via the normal bacterial Type III secretion system - that is, one step from bacterium to target cell.
• the present invention has as an object the provision of new pharmaceutical compositions for a variety of uses.
• a further object is to provide new pharmaceutical compositions for treatment of neuronal cells.
• a first aspect of the invention thus lies in a pharmaceutical composition, comprising a bacterial injected effector secreted by the type III or IV secretion pathway.
• a carrier can be provided to target the effector protein to a target cell, optionally targeting the effector to a cell selected from an epithelial cell, a neuronal cell, a secretory cell, an immunological cell, an endocrine cell, an inflammatory cell, an exocrine cell, a bone cell and a cell of the cardiovascular system.
• Another means of delivery of the effector is via a conjugate of the effector protein and the carrier, the two suitably linked by a linker.
• One particularly preferred linker is cleavable, in that it can be cleaved after entry into the target cell so as to release the effector from the carrier.
• This linker can be a di- sulphide bridge or a peptide sequence including a site for a protease found in the target cell.
• the linker is composed of two cooperating proteins, a first cooperating protein associated with the effector and the second associated with the cell targetting component. These respective parts can be administered separately and combine in vivo to link the effector to the cell targetting component.
• An example of such a two-part linker is botulinum toxin C2 1 in cooperation with C2 2 .
• a composition comprises a neuronal cell targeting component, linked by a cleavable linker to the effector protein.
• the neuronal cell targeting component comprises a first domain targeting the effector to a neuronal cell and a second domain that translocates the effector into the cytosol of the neuronal cell.
• compositions of the invention can be by combining a type III effector protein with a pharmaceutically acceptable carrier.
• the effector protein may be on its own or may be chemically linked with a (targetting) carrier.
• Another preparation method is to express a DNA that encodes a polypeptide having a first region that corresponds to the effector protein and a second region that codes for the carrier.
• a third region, between the first and second regions, which is cleaved by a proteolytic enzyme present in the target cell is optionally included.
• the second domain is selected from (a) domains of clostridial neurotoxins that translocate polypeptide sequences into cells, and (b) fragments, variants and derivatives of the domains of (a) that substantially retain the translocating activity of the domains of (a).
• the linker is cleaved in the neuronal cell so as to release the effector protein from the targeting component, thus enabling the effector to have effect in the cell without being hindered by attachment to the targeting component.
• the invention provides a method of delivering a bacterial type III effector protein to a neuronal cell comprising administering a composition of the invention.
• the first domain may suitably be selected from (a) neuronal cell binding domains of clostridial toxins; and (b) fragments, variants and derivatives of the domains in (a) that substantially retain the neuronal cell binding activity of the domains of (a).
• the second domain is suitably selected from (a) domains of clostridial neurotoxins that translocate polypeptide sequences into cells, and (b) fragments, variants and derivatives of the domains of (a) that substantially retain the translocating activity of the domains of (a).
• the second domain is further suitably selected from:-
• translocation domain that is not a H N domain of a clostridial toxin and is not a fragment or derivative of a H N domain of a clostridial toxin
• a construct comprises effector protein linked by a disulphide bridge to a neuronal cell targeting component comprising a first domain that binds to a neuronal cell and a second domain that translocates the effector protein into the neuronal cell.
• This construct is made recombinantly as a single polypeptide having a cysteine residue on the effector protein which forms a disulphide bridge with a cysteine residue on the second domain.
• the effector protein is covalently linked, initially, to the second domain. Following expression of this single polypeptide, effector protein is cleaved from the second domain leaving the effector protein linked only by the disulphide bridge to the rest of the construct.
• binding and translocation domains reside in further choices for the binding and translocation domains, and one such aspect provides a non-toxic polypeptide, for delivery of the effector protein to a neuronal cell, comprising:- a binding domain that binds to the neuronal cell, and a translocation domain that translocates the effector protein into the neuronal cell, wherein the translocation domain is not a H N domain of a clostridial neurotoxin and is not a fragment or derivative of a H N domain of a clostridial toxin.
• the binding domain is suitably comprised of or derived from clostridial heavy chain fragments or modified clostridial heavy chain fragments.
• modified clostridial heavy chain fragment means a polypeptide fragment that retains similar biological functions to the corresponding heavy chain of a botulinum or tetanus neurotoxin but differs in its amino acid sequence and other properties compared to the corresponding heavy chain.
• botulinum toxin C 2 is not a neurotoxin as it has no neuronal specificity, instead it is an enterotoxin and suitable for use in the invention to provide a non-aggregating translocation domain.
• a yet further aspect of the invention provides a polypeptide, for delivery of a effector protein to a neuronal cell, comprising:- a binding domain that binds to the neuronal cell, and a translocation domain that translocates the effector protein into the neuronal cell, wherein the polypeptide has reduced affinity to neutralising antibodies to tetanus toxin compared with the affinity to such antibodies of native tetanus toxin heavy chain.
• the system delivers a wide range of protein effectors capable of modulating host cell function in such a way as to allow the persistence or spread of the pathogen in the host.
• These effectors modulate a number of signalling pathways and one pathogen may export several effectors that regulate different pathways either concurrently or during different phases of its life cycle.
• Table 1 lists a number of type III effectors that have been identified to date.
• the type IV secretion system shows a remarkable degree of similarity to the type III system in that it forms a needle-like structure through which effector proteins are injected into the host cell cytoplasm.
• the proteins involved in the structure ofthe needle are different for the two systems and the effectors are also divergent.
• the effectors function to modulate cellular signalling to establish and maintain the intracellular niche and/or promote invasion and proliferation.
• the system is described as essential in a number of important bacterial pathogens including Legionella pneumophila, Bordetella pertussis, Actinobacillus actinomycetemcomitans, Bartonella henselae,
• YopE is a 23kDa effector which is translocated into the cytosol of cells during infection by Y.pseudotuberculosis and other strains. Studies in vitro have shown that it acts as a GAP for RhoA, Cdc42 and Rad , but not for Ras (Black and Bliska, (2000) Molecular Microbiology 37:515-527). A point mutation within the arginine finger motif causes a loss of GAP activity and this correlates directly with its biological activity in cells.
• YopE appears to have a greater specificity for Cdc42 (Andor et al (2001) Cellular Microbiology 3:301-310).
• the GAP activity of SptP shows greater specificity for Cdc42 and Rad compared to RhoA.
• SptP, ExoS and ExoT are bifunctional enzymes with additional enzymatic domains (SptP, tyrosine phosphatase; ExoS, ExoT, ADP- ribosyltransferase). In the case of ExoS this activity blocks the activation of Ras GTPase allowing a co-ordinated modulation of different signalling pathways
• GEFs guanine nucleotide exchange factors
• SopE and SopE2 from Salmonella typhimurium and related proteins act as guanine nucleotide exchange factors (GEFs) for a range of GTPases (Hardt et al (1998) Ce//93:815). GEFs function by enhancing the rate of replacement of bound GDP by GTP causing the activation of the GTPase. This effectively upregulates the activity of specific GTPases in the cell.
• Native SopE is a 240 amino acid protein which is injected into the host cell cytosol by S.typhimurium.
• the N-terminal 77 amino acids of the protein act as a secretion signal and are dispensable for the biological activity of the protein (Hardt et a/ (1998) Cell 93:815).
• SopE acts as a GEF for CDc42, Rad , Rac2, RhoA, and RhoG.
• Cellular GEFs show a high degree of specificity for particular GTPases and it is likely that SopE will show greater specificity in vivo. This specificity is likely to vary according to cell type and delivery route.
• the type IV effector, RalF, from Legionella pneumophila is a further exchange factor affecting the function of small GTPases.
• the target is the ADP ribosylation factor (ARF) family and this is the first example of a bacterial effector that targets this family (Nagai ef al (2002) Science 295;679-682).
• YopT causes a shift in the electrophoretic mobility of RhoA but not Cdc-42 or Rac (Zumbihl ef al (1999) dournal of Biological Chemistry 274:29289-29293). It is still not known whether this represents a direct modification of RhoA by YopT or whether other cellular factors are involved. The specificity of YopT for RhoA offers significant therapeutic possibilities.
• YopO/YpkA from Yersinia spp are protein kinase related to eukaryotic serine/threonine kinases (Galyov ef a/( 1993) Nature 361 :730-732).
• YopO/YpkA causes a similar cell rounding to that observed for other effectors such as YopE suggesting a role in modulating GTPase function.
• RhoA and Racl have been shown to bind to YopO and YpkA suggesting that these are the intracellular targets for the kinase (Barz C et al (2000) FEBS Letters 482: 139- 143).
• the type IV effector CagA from Helicobacter pylori also affects the cytoskeleton of infected cells and its activity is dependent on its phosphorylation by intracellular kinases.
• CagA functions via the SHP-2 tyrosine phosphatase to modulate downstream signalling.
• YopJ from Yersinia pestis is another translocated effector with a wide range of homologs including AvrA from Salmonella spp. and a variety of effectors from plant pathogens.
• YopJ has been shown to inactivate a broad range of mitogen-activated protein kinase kinases (MKKs) (Orth et a/ (1999) Science 285:1920-1923) causing apoptosis in macrophages.
• MKKs mitogen-activated protein kinase kinases
• YopJ is suggested to act as a ubiquitin-like protein protease causing increased turnover of signalling molecules via removal of a Sumo-1 tag from the MKK (Orth ef a/ (2000) Science 290:1594-1597).
• AvrA shows no activity despite its homology to YopJ suggesting that the specificity ofthe proteins may be different (Schesser K ef al (2000) Microbial Pathogenesis 28:59-70). In neuronal cells these different specificities may offer potential therapeutic applications for modulating MKKs involved in apoptosis or inflammatory responses.
• Salmonella in common with many other pathogens, establishes a specialised intracellular compartments. Salmonella has a dedicated type III secretion system that secretes proteins into the host cell cytosol from within this compartment and the effectors secreted by this system (including SpiC, SopE/E2, SseE,F,G,J, PipA.B, SifA,B)maintain the integrity of this compartment.
• Tetanus toxin is structurally very similar to botulinum neurotoxins but its primary site of action is the central nervous system where it blocks the release of inhibitory neurotransmitters from central synapses (Renshaw cells). As described for the botulinum toxins above, it is domains within the heavy chain of tetanus toxin that bind to receptors on neuronal cells.
• botulinum type A heavy chain binds to a different receptor to botulinum type F heavy chain and these two ligands are non-competitive with respect to their binding to neuronal cells (Wadsworth et al. (1990), Biochem J.
• clostridial neurotoxin serotypes so far characterised (tetanus, botulinum A, B, C ⁇ D, E and F), all appear to recognise distinct receptor populations on neuronal cells.
• the clostridial neurotoxin heavy chains provide high affinity binding ligands that recognise a whole family of receptors that are specific to neuronal cells.
• the present invention also provides constructs for the delivery of type III effector proteins specifically to neuronal cells.
• the mechanism by which the type III effector protein is delivered to the cell by these constructs is completely different to that used by the host bacteria. Instead of being injected directly into the cellular cytosol, specific constructs of the invention deliver the type III effector protein to cells via a number of sequentially acting biologically active domains and by a process resembling receptor-mediated endocytosis. Surprisingly, when delivered by this completely different mechanism, the type III effector proteins are biologically active within the cellular cytosol.
• the type III effector-containing construct may also contain 'linker proteins' by which these domains are interconnected.
• the type III effector moiety is linked to the translocation domain via a disulphide bridge.
• the clostridial H c fragments bind with high affinity to receptors on the cell surface and provide high specificity to neuronal cells.
• the clostridial neurotoxins are internalised via an acidic endosome which triggers the translocation of the type III effector moiety across the membrane and into the cytosol.
• Ligands suitable to target an agent to immunological cells include Epstein Barr virus fragment/surface feature or idiotypic antibody (binds to CR2 receptor on
• B-lymphocytes and lymph node follicular dendritic cells B-lymphocytes and lymph node follicular dendritic cells.
• SigD is incorporated into a construct of the invention and can be used to promote neuronal cell survival under stress. By targeting the appropriate intracellular signalling pathway, it is possible to simultaneously regulate a number of pathways to improve the prospects for neuronal survival.
• SigD also known as SopB
• Akt protein kinase
• membrane fusion events are classified either as secretory processes for the release of material from the plasma membrane, or as endocytic processes that move material from the plasma membrane to the lysosomal system. This simplified classification does not take into account retrograde and anterograde processes, which occur within these pathways, or multiple points of communication between the two pathways.
• the underlying mechanism in all membrane fusion events can be broken down into 4 component phases:
• the vesicle is transported to the acceptor membrane along cytoskeletal fibres (e.g. microtubules).
• cytoskeletal fibres e.g. microtubules
• the targeting of the membrane fusion event between secretory vesicles and the plasma membrane allow the control of secretion from cells.
• Effectors that alter regulation of specific Rab proteins can regulate secretion. Effector proteins can be applied to either increase or decrease secretion from a specific cell type. In a therapeutic context this is valuable for the treatment of a wide range of disorders including muscle spasms (blephorospasm, torticolis etc) hypersecretion disorders (COPD, bronchitis, asthma).
• constructs are provided for inhibition or promotion of secretion, containing a type III effector and a targetting moiety.
• Specific effectors for this purpose are selected from SpiC,
• the effector proteins of two intracellular pathogens existing in membrane bound vesicles are also not necessarily compatible.
• enhancement of Rab ⁇ a activity by Salmonella in macrophages is correlated with enhanced survival (Cell Microbiology 3;473-).
• increases in Rab ⁇ a expression/activity accelerates intracellular destruction of Listeria monocytogenes in macrophages (J. Biological Chemistry 274;11459).
• the Salmonella effector proteins that are likely to be involved in Rab ⁇ a recruitment e.g. SopE2, SpiC or other SPI-2 secreted effectors
• anti-microbial therapy could involve treating one intracellular pathogen with a second pathogen on the basis that the two intracellular compartments and requirements of the organisms would not be compatible.
• treatment of TB infected macrophages with Salmonella might be expected to result in provoked "vacuole” lysosome fusion within the macrophage leading to the eradication of the TB.
• the type and fate of the super-infecting pathogen would have to be carefully chosen so as not to exacerbate the infectivity or spread of the original organism.
• a refinement of the superinfection strategy would therefore focus on the targeted delivery of effector molecules to specific target cells as described by this invention.
• This could either utilise a highly attenuated pathogen (e.g. Salmonella that only secretes SopE2 or SptP) or targeted protein delivery (e.g. using a toxin delivery domain, antibody or similar cell targeting ligands).
• pathogen e.g. Salmonella that only secretes SopE2 or SptP
• targeted protein delivery e.g. using a toxin delivery domain, antibody or similar cell targeting ligands.
• Protective antigen from Bacillus anthracis would be capable of targeting effectors to macrophages for the treatment of a wide range of bacterial pathogens.
• carbohydrate moieties will enable specific targeting of pools of macrophages via the mannose receptor (e.g Vyas et al, International Journal of Pharmaceutics (2000) 210p1 -14).
• a cell surface marker specific for infected cells would offer an ideal target for delivery systems.
• the cell type infected by the pathogen would determine the choice of delivery ligand whilst the precise fate of the cell compartment would determine the choice of effector (e.g. cell apoptosis, lysis, endosome-lysosome fusion, endosome acidification etc).
• a key benefit of this type of therapy is that the effector proteins are not intrinsically toxic to the cell and therefore delivery of the protein to uninfected target cells is unlikely to have any deleterious effects. In this case, whilst desirable, the precise specificity of the targeting ligand is not essential for successful therapy.
• the construct may consist of the following:- the SpiC effector moiety fused to a domain capable of interacting with protective antigen; the protective antigen from Bacillus anthracis; where the construct is either co-administered or where the SpiC moiety is administered after the protective antigen.
• the constructs of the invention are preferably produced either wholly or partially by recombinant technology.
• the construct ofthe invention will be produced as a single multi-domain polypeptide comprising from the N-terminus:- the type III effector moiety; a linker peptide; the translocation domain; and the binding domain.
• the C-terminus ofthe type III effector protein is fused to the N-terminus of the translocation domain via the linker peptide.
• linker peptide is the sequence CGLVPAGSGP which contains the thrombin protease cleavage site and a cysteine residue for disulphide bridge formation.
• the latter single chain fusion protein may then be treated with thrombin to give a dichain protein in which the type III effector is linked to the translocation domain by a disulphide link.
• linker peptide in which the translocation domain does not contain a free cysteine residue near its C-terminus, such as is the case when the translocation domain is a fusogenic peptide, the linker peptide contains both cysteine residues required for the disulphide bridge.
• linker peptide is the amino acid sequence: CGLVPAGSGPSAGSSAC.
• the construct may consist of polypeptide containing (from the N-terminus) the following domains:- the SigD type III effector moiety; linker peptide (sequence CGLVPAGSGP) to enable attachment of the
• SigD effector to the translocation domain via a disulphide bridge; the translocation domain from diphtheria toxin (residues 194-386); and the binding domain (H c domain) from botulinum type A neurotoxin (residues 872-1296).
• the constructs of the invention may also be produced using chemical cross- linking methods.
• Various strategies are known by which type III effector proteins can be linked to a polypeptide consisting of the translocation domain and binding domain using a variety of established chemical cross-linking techniques. Using these techniques a variety of type III effector constructs can be produced.
• the type III effector protein is, for example, derivatised with the cross-linking reagent N-succinimidyl 3-[2-pyridyldithio] propionate.
• the derivatised type III effector is then linked to a peptide containing a translocation domain and binding domain via a free cysteine residue present on the translocation domain.
• Protein effectors can be altered to allow their delivery to intracellular compartments other than their usual site of action. For example, mitochondrial or nuclear targeting signals could be added to direct the effector to these compartments. By covalently linking the effector to the targeting domain the effector can be retained in the endosome/lysosome compartment, which would not normally be accessible by bacterial delivery. Effectors can be targeted to specific membrane locations via lipid modifications including myristoylation, palmitoylation, orthe addition of short proteins domains that might include SH2, SH3, WW domains, fragments of Rab proteins or synaptojanin-like domains. Those practised in the art would recognise that these targeting strategies offer an advantage for certain therapeutic strategies.
• Constructs of the invention may be introduced into either neuronal or non- neuronal tissue using methods known in the art. By subsequent specific binding to neuronal cell tissue, the targeted construct exerts its therapeutic effects. Ideally, the construct is injected near a site requiring therapeutic intervention.
• Translocation in relation to translocation domain, means the internalization events that occur after binding to the cell surface. These events lead to the transport of substances into the cytosol of target cells.
• Injected effector secreted by type III or type IV secretion system means bacterial proteins that are injected into host cells (mammalian, plant, insect, fish or other) via a modified pilus or "needle-like" injection system frequently referred to as type III or type IV secretion systems" and the term embraces fragments, modifications and variations thereof that retain the essential effector activity.
• the invention thus uses modification of intracellular signalling for promoting neuronal growth.
• Many of the effectors and inhibitors that control the development of the growth cone act through common intracellular signalling pathways that modulate the phosphorylation state of cytoskeletal components and that ultimately determine whether the axon grows or collapses.
• the appropriate manipulation of intracellular signalling is therefore a powerful approach for eliminating the need for multiple inhibitors of the many factors shown to induce apoptosis and growth cone collapse.
• the up-regulation of transcription factors that inhibit apoptosis is an example of manipulation of intracellular signalling to promote neural regeneration.
• T7 polymerase promoter site e.g. pET28, pET30 or derivatives (Novagen Inc, Madison, WI)
• a fusion with maltose binding protein e.g. pMALc2x (NEB)
• pMALc2x e.g. pMALc2x
• the recombinant proteins expressed from pET vectors contain amino-terminal histidine (6-His) and T7 peptide tags allowing proteins to be purified by affinity chromatography on either a Cu 2+ charged metal chelate column.
• Expression cultures were grown in Terrific Broth containing 30microg/ml kanamycin and 0.5% (w/v) glucose to an OD 600 of 2.0 and protein expression was induced with 500microM IPTG for 2 hours. Cells were lysed by either sonication or suitable detergent treatment (e.g.
• Clostridium botulinum C2 toxin component 1 Clostridium botulinum C2 toxin component 1 25
• Table 1 Examples of type III and type IV effectors and their activity.
• YopT Yersinia spp Inactivates Rho GTPases by Stimulate nerve regrowth direct following damage
• ExoS N-terminal domain
• GTPase activating protein for Stimulate nerve regrowth
• Pseudomonas aeuruginosa Rho GTPases
• ExoS C-terminal domain
• ADP-ribosyltranferase Block Ras/Rap signalling P. aeuruginosa modifies Ras and Rap pathways
• SptP N-terminal domain GAP activity for Rac 1/ Cdc Salmonella spp 42
• YpkO/YopO Yersinia spp Serine/threonine kinase modifies RhoA/Rac
• YopP/YopJ Yersinia spp Blocks activation of various Induction of apoptosis in AvrXv/AvrBsT Xanthomonas MAP kinase pathways tumour cells campestris Block release of inflammatory mediators from damaged cells
• SopB/SigA/SigD Salmonella Activate AKT kinase Block apoptosis in spp damaged/ageing
• SpiC S.entehca Block endosome fusion Prevent neurotransmitter release from pain fibres
• IpaB Induces apoptosis by direct Induction of apoptosis in SipB activation of caspase 1 glioma/neuroblastoma cells
• YopM Yersinia spp PopC Leucine rich repeat protein. Upregulation of genes Ralstonia solanacearum Possible transcription factors involved in cell cycle and cell growth (YopM) or other genes.

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  • 2002-05-21 WO PCT/GB2002/002384 patent/WO2002096467A2/en not_active Ceased
  • 2002-05-21 EP EP02726319A patent/EP1390400A2/en not_active Withdrawn
  • 2002-05-21 US US10/478,516 patent/US20040208889A1/en not_active Abandoned
  • 2002-05-21 CN CNB028105052A patent/CN100439394C/zh not_active Expired - Fee Related
  • 2002-05-21 JP JP2002592975A patent/JP2004533250A/ja active Pending
• 2009
  • 2009-09-02 JP JP2009202601A patent/JP2009286794A/ja active Pending

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