WO2004031390A1 - Systeme de vecteur - Google Patents

Systeme de vecteur Download PDF

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Publication number
WO2004031390A1
WO2004031390A1 PCT/GB2003/004260 GB0304260W WO2004031390A1 WO 2004031390 A1 WO2004031390 A1 WO 2004031390A1 GB 0304260 W GB0304260 W GB 0304260W WO 2004031390 A1 WO2004031390 A1 WO 2004031390A1
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Prior art keywords
disease
use according
vector system
eoi
vector
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PCT/GB2003/004260
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English (en)
Inventor
Nicholas Mazarakis
Mimoun Azzouz
Katie Binley
Stuart Naylor
Susan Kingsman
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Oxford Biomedica (Uk) Limited
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Priority claimed from GB0223076A external-priority patent/GB0223076D0/en
Priority claimed from GB0318213A external-priority patent/GB0318213D0/en
Application filed by Oxford Biomedica (Uk) Limited filed Critical Oxford Biomedica (Uk) Limited
Priority to JP2005500061A priority Critical patent/JP2006502240A/ja
Priority to EP03753721A priority patent/EP1551974A1/fr
Priority to AU2003271883A priority patent/AU2003271883A1/en
Priority to US10/716,725 priority patent/US20040076613A1/en
Publication of WO2004031390A1 publication Critical patent/WO2004031390A1/fr
Priority to US10/838,906 priority patent/US20040266715A1/en
Priority to US11/583,427 priority patent/US20070213290A1/en
Priority to US11/810,007 priority patent/US20080131400A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6072—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses
    • C12N2810/6081—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses rhabdoviridae, e.g. VSV

Definitions

  • the present invention relates to a vector system.
  • the present invention relates to a vector system capable of delivering an entity of interest (“EOI”) - such as a nucleotide sequence of interest (“NOI”) - to a target site, such as for the treatment of diseases affecting the central nervous system (CNS).
  • ECI entity of interest
  • NOI nucleotide sequence of interest
  • the present invention relates to a viral vector system capable of delivering a nucleotide sequence of interest ("NOI”) to a target site.
  • NOI nucleotide sequence of interest
  • the present invention relates to a lentiviral vector system capable of delivering a nucleotide sequence of interest ("NOI”) to a target site.
  • NOI nucleotide sequence of interest
  • the present invention relates to a retroviral vector useful in gene therapy.
  • Gene therapy includes any one or more of: the addition, the replacement, the deletion, the supplementation, the manipulation etc. of one or more nucleotide sequences in, for example, one or more targeted sites - such as targeted cells. If the targeted sites are targeted cells, then the cells may be part of a tissue or an organ. General teachings on gene therapy may be found in Molecular Biology (Ed Robert Meyers, Pub VCH, such as pages 556-558).
  • gene therapy also provides a means by which any one or more of: a nucleotide sequence, such as a gene, can be applied to replace or supplement a defective gene; a pathogenic gene or gene product can be eliminated; a new gene can be added in order, for example, to create a more favourable phenotype; cells can be manipulated at the molecular level to treat cancer (Schmidt-Wolf and Schmidt-Wolf, 1994, Annals of Hematology 69;273-279) or other conditions - such as immune, cardiovascular, neurological, inflammatory or infectious disorders; antigens can be manipulated and/or introduced to elicit an immune response - such as genetic vaccination.
  • a nucleotide sequence such as a gene
  • retroviruses have been proposed for use in gene therapy.
  • retroviruses are RNA viruses with a life cycle different to that of lytic viruses.
  • a retrovirus infects a cell, its genome is converted to a DNA form.
  • a retrovirus is an infectious entity that replicates through a DNA intermediate. More details on retro viral infection etc. are presented later on.
  • the gene env encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction leads ultimately to fusion of the viral membrane with the cell membrane.
  • SU surface glycoprotein
  • TM transmembrane
  • the SU and TM proteins are not always required for the assembly of enveloped virion particles as such, they do play an essential role in the entry process.
  • the SU domain binds to a receptor molecule - often a specific receptor molecule - on the target cell. It is believed that this binding event activates the membrane fusion-inducing potential of the TM protein after which the viral and cell membranes fuse.
  • a cleavage event - resulting in the removal of a short portion of the cytoplasmic tail of TM - is thought to play a key role in uncovering the full fusion activity of the protein (Brody et al 1994 J. Virol. 68: 4620-4627, Rein et al 1994 J. Virol. 68: 1773-1781).
  • This cytoplasmic "tail" distal to the membrane-spanning segment of TM remains on the internal side of the viral membrane and it varies considerably in length in different retroviruses.
  • the specificity of the SU/receptor interaction can define the host range and tissue tropism of a retrovirus. In some cases, this specificity may restrict the transduction potential of a recombinant retroviral vector. For this reason, many gene therapy experiments have used MLV.
  • a particular MLV that has an envelope protein called 4070A is known as an amphotropic virus, and this can also infect human cells because its envelope protein "docks" with a phosphate transport protein that is conserved between man and mouse. This transporter is ubiquitous and so these viruses are capable of infecting many cell types. In some cases however, it may be beneficial, especially from a safety point of view, to specifically target restricted cells.
  • mice ecotropic retrovirus which unlike its amphotropic relative normally only infects mouse cells, to specifically infect particular human cells.
  • Replacement of a fragment of an envelope protein with an erythropoietin segment produced a recombinant retrovirus which then bound specifically to human cells that expressed the erythropoietin receptor on their surface, such as red blood cell precursors (Maulik and Patel 1997 "Molecular Biotechnology: Therapeutic Applications and Strategies” 1997. Wiley-Liss Inc. pp 45.).
  • Pseudotyping can confer one or more advantages. For example, with the lentiviral vectors, the ewv gene product of the HIV based vectors would restrict these vectors to infecting only cells that express a protein called CD4. But if the env gene in these vectors has been substituted with env sequences from other RNA viruses, then they may have a broader infectious spectrum (Verma and Somia 1997 Nature 389:239-242).
  • brain is a difficult and complex organ to target (Raymon H.K. et al (1997) Exp. Neur. 144: 82-91).
  • the usual route is by injection of vectors to the striatum (Bilang-Bleuel et al (1997) Proc. Acad. Natl. Sci. USA 94:8818-8823; Choi-Lundberg et al (1998) Exp. Neurol.154:261-275) or to near the substantia nigra (Choi-Lundberg et al (1997) Science 275:838-841; Mandel et al (1997) ) Proc. Acad. Natl. Sci.
  • the substantia nigra lies deep in the brain and direct injection to this area can cause lesion of axons, resulting in damage.
  • the striatum in particular the caudate putamen, is a relatively easy target because it is larger and more dorsal than the substantia nigra. It has been used extensively for transplantation in Parkinson's disease, and there is currently thought to be less than 1% risk involved in the operation. Similar problems exist in relation to other parts of the CNS.
  • pseudotyping might alleviate some of the above-mentioned problems.
  • transduction and expression characteristics of pseudotyped vectors have not yet been fully determined and there remains the need to provide further and improved vectors.
  • WO02/36170 teaches the use of a wild-type rabies G protein to achieve retrograde transport, and particularly transduction of a TH positive neuron.
  • EOI entity of interest
  • CNS ChallengeNirus Standard
  • the present invention relates to a vector system that is capable of causing retrograde transport of an entity of interest ("EOI").
  • EAI entity of interest
  • vector system includes any vector that is capable of infecting or transducing or transforming or modifying a recipient cell with an EOI.
  • the EOI may be a chemical compound, a biological compound or combinations thereof.
  • the EOI may be a protein (such as a growth factor), a nucleotide sequence, an organic and/or an inorganic pharmaceutical (such as an analgesic, an anti- inflammatory, a hormone, a lipid), or combinations thereof.
  • the vector system of the present invention is capable of delivering the EOI to a site, wherein at that site the EOI may then be distributed and/or penetrate distant sites, e.g. through diffusion or retrograde transport.
  • the vector system will also comprise an EOI.
  • a vector system to transduce a target site, wherein the vector system travels to the target site by diffusion, and wherein the vector system is or comprises at least part of a rabies G envelope protein or a mutant, variant, homologue or fragment thereof, or a CVS envelope protein or a mutant, variant, homologue or fragment thereof, and further wherein the target site is at least part of the central nervous system.
  • a vector system comprising an EOI to biodistribute the EOI, wherein the vector system is or comprises at least part of a rabies G envelope protein or a mutant, variant, homologue or fragment thereof, or a CVS envelope protein or a mutant, variant, homologue or fragment thereof.
  • a vector system to transduce a target site, wherein the vector system travels to the target site by retrograde transport, and wherein the vector system is or comprises at least part of a CVS envelope protein or a mutant, variant, homologue or fragment thereof, and further wherein the target site is at least part of the central nervous system.
  • a vector system to transduce an in utero target site or a target site in a neonate, wherein the vector system is or comprises at least part of a rabies G envelope protein or a mutant, variant, homologue or fragment thereof, or a CVS envelope protein or a mutant, variant, homologue or fragment thereof.
  • the vector system can be a non-viral system or a viral system, or combinations thereof.
  • the vector system itself can be delivered by viral or non- viral techniques.
  • Viral vector or viral delivery systems include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, he ⁇ es viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
  • Non-viral delivery or non-viral vector systems include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • the at least part of the rabies G protein (or a mutant, variant, homologue or fragment thereof), and/or the at least part of the CVS protein (or a mutant, variant, homologue or fragment thereof) may be used to encapsulate or enshroud an EOI.
  • the at least part of the rabies G protein (or a mutant, variant, homologue or fragment thereof), or the at least part of the CVS protein (or a mutant, variant, homologue or fragment thereof) may form a matrix around the EOI.
  • the matrix may contain other components - such as a liposome type entity.
  • the vector system is a viral vector system.
  • the vector system is a retroviral vector system and, preferably, a lentiviral vector system.
  • a particular type of vector system - such as viral vector system, preferably a retroviral vector system, more preferably a lentiviral vector system - according to the present invention is capable of transducing one or more sites which are distant from the site of administration due to retrograde transport of the vector system.
  • Administration to a single target site may cause transduction of a plurality of target sites.
  • the vector system may travel to the or each target by retrograde transport, diffusion or biodistribution, optionally in combination with anterograde transport.
  • the present invention relates to:
  • the present invention relates to a new use of a vector system.
  • the vector system can be a non-viral system or a viral system.
  • the vector system is a viral vector system.
  • the vector system is a retroviral vector system and, preferably, a lentiviral vector system.
  • retrovirus includes: murine leukaemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo- MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) and all other retroviridiae including lenti viruses.
  • MMV murine leukaemia virus
  • HCV human immunodeficiency virus
  • EIAV equine infectious anaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • the retroviral vector system is derivable from a lentivirus.
  • Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells (Lewis et al (1992) EMBO J. 3053-3058).
  • the lentivirus group can be split into "primate” and "non-primate".
  • primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEN), equine infectious anaemia virus (ELAN) and the more recently described feline immunodeficiency virus (FIN) and bovine immunodeficiency virus (BIN).
  • VMV visna/maedi virus
  • CAEN caprine arthritis-encephalitis virus
  • ELAN equine infectious anaemia virus
  • FIN feline immunodeficiency virus
  • BIN bovine immunodeficiency virus
  • genomic structure of some lentiviruses may be found in the art.
  • HIN and ELAN may be found from the ⁇ CBI Genbank database (i.e. Genome Accession ⁇ os. AF033819 and AF033820 respectively).
  • Details of HIV variants may also be found at http://hiv-web.lanl. gov.
  • Details of EIAV variants may be found through httpJ/www.ncbi.nlm.nih.gov.
  • a retrovirus initially attaches to a specific cell surface receptor.
  • the retroviral R ⁇ A genome is then copied to D ⁇ A by the virally encoded reverse transcriptase which is carried inside the parent virus.
  • This D ⁇ A is transported to the host cell nucleus where it subsequently integrates into the host genome.
  • the provirus is typically referred to as the provirus.
  • the provirus is stable in the host chromosome during cell division and is transcribed like other cellular genes.
  • the provirus encodes the proteins and other factors required to make more virus, which can leave the cell by a process sometimes called "budding".
  • Each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs).
  • LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes.
  • Encapsidation of the retroviral R ⁇ As occurs by virtue of apsi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5 'end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the site of transcription initiation is at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR.
  • U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
  • Some retroviruses have any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tat, rev, tax and rex.
  • gag encodes the internal structural protein of the virus.
  • Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid).
  • the pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome.
  • the env gene encodes the surface (SU) glycoprotein and the fransmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction leads ultimately to infection by fusion of the viral membrane with the cell membrane.
  • Retroviruses may also contain "additional" genes which code for proteins other than gag, pol and env.
  • additional genes include in HIN, one or more of vi vpr, vpx, vpu, tat, rev and nef.
  • E AN has (amongst others) the additional gene S2.
  • Proteins encoded by additional genes serve various functions, some of which may be duplicative of a function provided by a cellular protein.
  • EIAV EIAV
  • t ⁇ t acts as a transcriptional activator of the viral LTR. It binds to a stable, stem-loop R A secondary structure referred to as TAR. Rev regulates and co-ordinates the expression of viral genes through rev-response elements (RRE).
  • RRE rev-response elements
  • the mechanisms of action of these two proteins are thought to be broadly similar to the analogous mechanisms in the primate viruses.
  • the function of S2 is unknown.
  • an ELAN protein, Ttm has been identified that is encoded by the first exon of t ⁇ t spliced to the e «v coding sequence at the start of the transmembrane protein.
  • the vector system can be a non- viral system or a viral system.
  • the vector system is a viral vector system.
  • the vector system is a retroviral vector system and, preferably, a lentiviral vector system.
  • the vector system can be used to transfer an EOI to one or more sites of interest.
  • the transfer can occur in vitro, ex vivo, in vivo, or combinations thereof.
  • the delivery system is a retroviral delivery system which is a lentiviral vector system.
  • Retroviral vector systems have been proposed as a delivery system for inter alia the transfer of a ⁇ OI to one or more sites of interest. The transfer can occur in vitro, ex vivo, in vivo, or combinations thereof. Retroviral vector systems have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and R ⁇ A packaging (reviewed by Miller, 1992 Curr Top Microbiol Immunol 158:1-24).
  • vector system may also include a vector particle capable of transducing a recipient cell with an ⁇ OI.
  • a vector particle includes the following components: a vector genome, which may contain one or more NOIs, a nucleocapsid encapsidating the nucleic acid, and a membrane surrounding the nucleocapsid.
  • nucleocapsid refers to at least the group specific viral core proteins (gag) and the viral polymerase (pol) of a retrovirus genome. These proteins encapsidate the packagable sequences and are themselves further surrounded by a membrane containing an envelope glycoprotein.
  • RNA genome from a retroviral vector particle is reverse transcribed into DNA and integrated into the DNA of the recipient cell.
  • vector genome refers both to the RNA construct present in the retroviral vector particle and the integrated DNA construct.
  • the term also embraces a separate or isolated DNA construct capable of encoding such an RNA genome.
  • a retroviral or lentiviral genome should comprise at least one component part derivable from a retrovirus or a lentivirus.
  • the term "derivable” is used in its normal sense as meaning a nucleotide sequence or a part thereof which need not necessarily be obtained from a virus such as a lentivirus but instead could be derived therefrom.
  • the sequence may be prepared synthetically or by use of recombinant DNA techniques.
  • the genome comprises a psi region (or an analogous component which is capable of causing encapsidation).
  • the viral vector genome is preferably "replication defective" by which we mean that the genome does not comprise sufficient genetic information alone to enable independent replication to produce infectious viral particles within the recipient cell.
  • the genome lacks a functional env, gag or pol gene. If a highly preferred embodiment the genome lacks e «v, gag and pol genes.
  • the viral vector genome may comprise some or all of the long terminal repeats (LTRs).
  • LTRs long terminal repeats
  • the genome comprises at least part of the LTRs or an analogous sequence which is capable of mediating proviral integration, and transcription.
  • the sequence may also comprise or act as an enhancer-promoter sequence.
  • the packaging cell line produces the proteins required for packaging retroviral RNA but it cannot bring about encapsidation due to the lack of a psi region.
  • helper proteins can package the /wt-positive recombinant vector RNA to produce the recombinant virus stock. This can be used to transduce the NOI into recipient cells.
  • the recombinant virus whose genome lacks all genes required to make viral proteins can infect only once and cannot propagate. Hence, the NOI is introduced into the host cell genome without the generation of potentially harmful retrovirus.
  • the present invention also provides a packaging cell line comprising a viral vector genome which is capable of producing a vector system useful in the first aspect of the invention.
  • the packaging cell line may be transduced with a viral vector system comprising the genome or transfected with a plasmid carrying a DNA construct capable of encoding the RNA genome.
  • the present invention also provides a kit for producing a retroviral vector system useful in the first aspect of the invention which comprises a packaging cell and a retroviral vector genome.
  • the second approach is to introduce the three different DNA sequences that are required to produce a retroviral vector particle i.e.
  • transient triple fransfection (Landau & Littman 1992; Pear et al 1993).
  • the triple fransfection procedure has been optimised (Soneoka et al 1995; Finer et al 1994).
  • WO 94/29438 describes the production of producer cells in vitro using this multiple DNA transient fransfection method.
  • WO 97/27310 describes a set of DNA sequences for creating retroviral producer cells either in vivo or in vitro for re-implantation.
  • the components of the viral system which are required to complement the vector genome may be present on one or more "producer plasmids" for transfecting into cells.
  • the present invention also provides a kit for producing a retroviral vector system useful in the first aspect of the invention, comprising
  • the viral vector genome is incapable of encoding the proteins gag, pol and env.
  • the kit comprises one or more producer plasmids encoding env, gag and pol, for example, one producer plasmid encoding env and one encoding gag-pol.
  • the gag-pol sequence is codon optimised for use in the particular producer cell (see below).
  • the present invention also provides a producer cell expressing the vector genome and the producer plasmid(s) capable of producing a retroviral vector system useful in the present invention.
  • the retroviral vector system used in the first aspect of the present invention is a self-inactivating (SfN) vector system.
  • self-inactivating retroviral vector systems have been constructed by deleting the transcriptional enhancers or the enhancers and promoter in the U3 region of the 3' LTR. After a round of vector reverse transcription and integration, these changes are copied into both the 5' and the 3' LTRs producing a franscriptionally inactive provirus.
  • any promoter(s) internal to the LTRs in such vectors will still be franscriptionally active.
  • This strategy has been employed to eliminate effects of the enhancers and promoters in the viral LTRs on transcription from internally placed genes. Such effects include increased transcription or suppression of transcription.
  • This strategy can also be used to eliminate downstream transcription from the 3' LTR into genomic DNA. This is of particular concern in human gene therapy where it may be important to prevent the adventitious activation of an endogenous oncogene.
  • a recombinase assisted mechanism is used which facilitates the production of high titre regulated lentiviral vectors from the producer cells of the present invention.
  • recombinase assisted system includes but is not limited to a system using the Cre recombinase / loxP recognition sites of bacteriophage PI or the site- specific FLP recombinase of S. cerevisiae which catalyses recombination events between 34 bp FLP recognition targets (FRTs).
  • the site-specific FLP recombinase of S. cerevisiae which catalyses recombination events between 34 bp FLP recognition targets (FRTs) has been configured into DNA constructs in order to generate high level producer cell lines using recombinase-assisted recombination events (Karreman et al (1996) NAR 24:1616-1624).
  • a similar system has been developed using the Cre recombinase / loxP recognition sites of bacteriophage PI (see PCT/GB00/03837; Vanin et al (1997) J. Virol 71:7820-7826). This was configured into a lentiviral genome such that high titre lentiviral producer cell lines were generated.
  • producer/packaging cell lines By using producer/packaging cell lines, it is possible to propagate and isolate quantities of retroviral vector particles (e.g. to prepare suitable titres of the retroviral vector particles) for subsequent transduction of, for example, a site of interest (such as adult brain tissue).
  • Producer cell lines are usually better for large scale production of vector particles.
  • Transient fransfection has numerous advantages over the packaging cell method.
  • transient fransfection avoids the longer time required to generate stable vector- producing cell lines and is used if the vector genome or retroviral packaging components are toxic to cells.
  • the vector genome encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apoptosis, it may be difficult to generate stable vector-producing cell lines, but transient fransfection can be used to produce the vector before the cells die.
  • cell lines have been developed using transient infection that produce vector titre levels that are comparable to the levels obtained from stable vector-producing cell lines (Pear et al 1993, PNAS 90:8392-8396).
  • Producer cells/packaging cells can be of any suitable cell type.
  • Producer cells are generally mammalian cells but can be, for example, insect cells.
  • the term "producer cell” or “vector producing cell” refers to a cell which contains all the elements necessary for production of retroviral vector particles.
  • the producer cell is obtainable from a stable producer cell line.
  • the producer cell is obtainable from a derived stable producer cell line.
  • the producer cell is obtainable from a derived producer cell line.
  • derived producer cell line is a transduced producer cell line which has been screened and selected for high expression of a marker gene. Such cell lines support high level expression from the retroviral genome.
  • derived producer cell line is used interchangeably with the term “derived stable producer cell line” and the term “stable producer cell line.
  • the derived producer cell line includes but is not limited to a retroviral and/or a lentiviral producer cell.
  • the derived producer cell line is an HIV or EIAV producer cell line, more preferably an EIAV producer cell line.
  • envelope protein sequences, and nucleocapsid sequences are all stably integrated in the producer and/or packaging cell.
  • one or more of these sequences could also exist in episomal form and gene expression could occur from the episome.
  • packaging cell refers to a cell which contains those elements necessary for production of infectious recombinant virus which are lacking in the RNA genome.
  • packaging cells typically contain one or more producer plasmids which are capable of expressing viral structural proteins (such as gag-pol and env, which may be codon optimised) but they do not contain a packaging signal.
  • packetaging signal which is referred to interchangeably as “packaging sequence” or “psi” is used in reference to the non-coding, cw-acting sequence required for encapsidation of retroviral RNA strands during viral particle formation.
  • packetaging sequence or “psi” is used in reference to the non-coding, cw-acting sequence required for encapsidation of retroviral RNA strands during viral particle formation.
  • this sequence has been mapped to loci extending from upstream of the major splice donor site (SD) to at least the gag start codon.
  • SD major splice donor site
  • Packaging cell lines may be readily prepared (see also WO 92/05266), and utilised to create producer cell lines for the production of retroviral vector particles.
  • retroviruses a summary of the available packaging lines is presented in "Retroviruses" (as above).
  • simple packaging cell lines comprising a provirus in which the packaging signal has been deleted, have been found to lead to the rapid production of undesirable replication competent viruses through recombination.
  • second generation cell lines have been produced wherein the 3 'LTR of the provirus is deleted. In such cells, two recombinations would be necessary to produce a wild type virus.
  • a further improvement involves the introduction of the gag-pol genes and the env gene on separate constructs so-called third generation packaging cell lines. These constructs are introduced sequentially to prevent recombination during fransfection.
  • the packaging cell lines are second generation packaging cell lines.
  • the packaging cell lines are third generation packaging cell lines.
  • third generation cell lines a further reduction in recombination may be achieved by changing the codons.
  • This technique based on the redundancy of the genetic code, aims to reduce homology between the separate constructs, for example between the regions of overlap in the gag-pol and env open reading frames.
  • the packaging cell lines are useful for providing the gene products necessary to encapsidate and provide a membrane protein for a high titre vector particle production.
  • the packaging cell may be a cell cultured in vitro such as a tissue culture cell line. Suitable cell lines include but are not limited to mammalian cells such as murine fibroblast derived cell lines or human cell lines.
  • the packaging cell line is a human cell line, such as for example: HEK293, 293-T, TE671, HT1080.
  • the packaging cell may be a cell derived from the individual to be treated such as a monocyte, macrophage, blood cell or fibroblast.
  • the cell may be isolated from an individual and the packaging and vector components administered ex vivo followed by re-administration of the autologous packaging cells. It is highly desirable to use high-titre virus preparations in both experimental and practical applications. Techniques for increasing viral titre include using a psi plus packaging signal as discussed above and concentration of viral stocks.
  • high titre means an effective amount of a retroviral vector or particle which is capable of transducing a target site such as a cell.
  • the term "effective amount” means an amount of a regulated retroviral or lentiviral vector or vector particle which is sufficient to induce expression of the NOIs at a target site.
  • a high-titre viral preparation for a producer/packaging cell is usually of the order of 10 5 to 10 7 t.u. per ml.
  • the titer is expressed in transducing units per ml (t.u./ml) as titred on a standard D17 cell line).
  • the viral preparation is concentrated by ulfracentrifugation.
  • the resulting preparation should have at least 10 8 t.u./ml, preferably from 10 8 to 10 9 t.u./ml, more preferably at least 10 9 t.u./ml.
  • the expression products encoded by the NOIs may be proteins which are secreted from the cell. Alternatively the NOI expression products are not secreted and are active within the cell. For some applications, it is preferred for the NOI expression product to demonstrate a bystander effect or a distant bystander effect; that is the production of the expression product in one cell leading to the modulation of additional, related cells, either neighbouring or distant (e.g. metastatic), which possess a common phenotype.
  • cPPT central polypurine tract
  • the genome of the vector system used in the present invention comprises a cPPT sequence.
  • the viral genome may comprise a post-translational regulatory element and/or a translational enhancer.
  • the NOIs may be operatively linked to one or more promoter/enhancer elements. Transcription of one or more NOI may be under the control of viral LTRs or alternatively promoter-enhancer elements can be engineered in with the transgene.
  • the promoter is a strong promoter such as CMV.
  • the promoter may be a regulated promoter.
  • the promoter may be tissue-specific. In a preferred embodiment the promoter is glial cell-specific. In another preferred embodiment the promoter is neuron-specific.
  • a primate lentivirus minimal system can be constructed which requires none of the HIV/SIV additional genes vi vpr, vpx, vpu, tat, rev and nef for either vector production or for transduction of dividing and non-dividing cells. It has also been demonstrated that an EIAV minimal vector system can be constructed which does not require S2 for either vector production or for transduction of dividing and non-dividing cells.
  • the deletion of additional genes is highly advantageous. Firstly, it permits vectors to be produced without the genes associated with disease in lentiviral (e.g. HIV) infections. In particular, tat is associated with disease. Secondly, the deletion of additional genes permits the vector to package more heterologous DNA.
  • genes whose function is unknown, such as S2 may be omitted, thus reducing the risk of causing undesired effects.
  • Examples of minimal lentiviral vectors are disclosed in WO-A- 99/32646 and in WO-A-98/17815.
  • the delivery system used in the invention is devoid of at least tat and S2 (if it is an EIAV vector system), and possibly also vif, vpr, vpx, vpu and nef. More preferably, the systems of the present invention are also devoid of rev.
  • Rev was previously thought to be essential in some retroviral genomes for efficient virus production. For example, in the case of HIV, it was thought that rev and RRE sequence should be included. However, it has been found that the requirement for rev and RRE can be reduced or eliminated by codon optimisation (see below) or by replacement with other functional equivalent systems such as the MPMV system.
  • codon optimised gag-pol is REV independent, RRE can be removed from the gag-pol expression cassette, thus removing any potential for recombination with any RRE contained on the vector genome.
  • the viral genome of the first aspect of the invention lacks the Rev response element (RRE).
  • RRE Rev response element
  • system used in the present invention is based on a so-called “minimal" system in which some or all of the additional genes have be removed.
  • Codon optimisation has previously been described in WO99/41397. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.
  • viruses including HIV and other lentiviruses
  • Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.
  • Codon optimisation has a number of other advantages.
  • the nucleotide sequences encoding the packaging components of the viral particles required for assembly of viral particles in the producer cells/packaging cells have RNA instability sequences (INS) eliminated from them.
  • INS RNA instability sequences
  • the amino acid sequence coding sequence for the packaging components is retained so that the viral components encoded by the sequences remain the same, or at least sufficiently similar that the function of the packaging components is not compromised.
  • Codon optimisation also overcomes the Rev/RRE requirement for export, rendering optimised sequences Rev independent. Codon optimisation also reduces homologous recombination between different constructs within the vector system (for example between the regions of overlap in the gag-pol and env open reading frames). The overall effect of codon optimisation is therefore a notable increase in viral titre and improved safety.
  • codons relating to INS are codon optimised.
  • sequences are codon optimised in their entirety, with the exception of the sequence encompassing the frameshift site.
  • the gag-pol gene comprises two overlapping reading frames encoding the gag-pol proteins.
  • the expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused at least in part by ribosome-stalling RNA secondary structures.
  • Such secondary structures exist downstream of the frameshift site in the gag- pol gene.
  • the region of overlap extends from nucleotide 1222 downstream of the beginning of gag (wherein nucleotide 1 is the A of the gag ATG) to the end o ⁇ gag (nt 1503). Consequently, a 281 bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon optimised. Retaining this fragment will enable more efficient expression of the gag-pol proteins.
  • nt 1262 where nucleotide 1 is the A of the gag ATG.
  • the end of the overlap is at 1461 bp.
  • the wild type sequence has been retained from nt 1156 to 1465. Derivations from optimal codon usage may be made, for example, in order to accommodate convenient restriction sites, and conservative amino acid changes may be introduced into the gag-pol proteins.
  • codon optimisation was based on lightly expressed mammalian genes.
  • the third and sometimes the second and third base may be changed.
  • gag-pol sequences can be achieved by a skilled worker.
  • retroviral variants described which can be used as a starting point for generating a codon optimised gag-pol sequence.
  • Lentiviral genomes can be quite variable. For example there are many quasi-species of HIV- 1 which are still functional. This is also the case for EIAV. These variants may be used to enhance particular parts of the transduction process. Examples of HIV-1 variants may be found at http ://hiv-web . lanl. go v. Details of EIAV clones may be found at the NCBI database: http ://www.ncbi.nlm.nih. gov.
  • the strategy for codon optimised gag-pol sequences can be used in relation to any retrovirus. This would apply to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1 and HIV-2. In addition this method could be used to increase expression of genes from HTLV-1, HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV and other retroviruses.
  • HERV human endogenous retroviruses
  • Codon optimisation can render gag-pol expression Rev independent.
  • the genome also needs to be modified. This is achieved by optimising vector genome components.
  • these modifications also lead to the production of a safer system absent of all additional proteins both in the producer and in the transduced cell.
  • the packaging components for a retroviral vector include expression products of gag, pol and env genes.
  • efficient packaging depends on a short sequence of 4 stem loops followed by a partial sequence from gag and env (the "packaging signal").
  • packaging signal the partial sequence from gag and env
  • inclusion of a deleted gag sequence in the retroviral vector genome will optimise vector titre.
  • efficient packaging has been reported to require from 255 to 360 nucleotides of gag in vectors that still retain env sequences, or about 40 nucleotides of gag in a particular combination of splice donor mutation, gag and env deletions.
  • the retroviral vector genome includes a gag sequence which comprises one or more deletions, more preferably the gag sequence comprises about 360 nucleotides derivable from the N-terminus.
  • retroviral vector systems it is desirable to engineer particles with different target cell specificities to the native virus, to enable the delivery of genetic material to an expanded or altered range of cell types.
  • One manner in which to achieve this is by engineering the virus envelope protein to alter its specificity.
  • Another approach is to introduce a heterologous envelope protein into the vector particle to replace or add to the native envelope protein of the virus.
  • pseudotyping means inco ⁇ orating in at least a part of, or substituting a part of, or replacing all of, an env gene of a viral genome with a heterologous env gene, for example an env gene from another virus.
  • Pseudotyping is not a new phenomenon and examples may be found in WO 99/61639, WO-A-98/05759, WO-A-98/05754, WO-A- 97/17457, WO-A-96/09400, WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841- 847.
  • Pseudotyping can improve retroviral vector stability and transduction efficiency.
  • a pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV) has been described (Miletic et al (1999) J. Virol. 73:6114-6116) and shown to be stable during ultracentrifugation and capable of infecting several cell lines from different species.
  • the vector system may be pseudotyped with at least a part of a mutant rabies G envelope protein, or a CVS envelope protein or a mutant, variant, homologue or fragment thereof.
  • the retroviral delivery system used in the first aspect of the invention comprises a first nucleotide sequence coding for at least a part of an envelope protein; and one or more other nucleotide sequences derivable from a retrovirus that ensure transduction by the retroviral delivery system; wherein the first nucleotide sequence is heterologous with respect to at least one of the other nucleotide sequences; and wherein the first nucleotide sequence codes for at least a part of a rabies G envelope protein or a mutant, variant, homologue or fragment thereof, or at least a part of a CVS protein or a mutant, variant, homologue or fragment thereof.
  • a retroviral delivery system comprising a heterologous env region, wherein the heterologous env region comprises at least a part of a rabies G protein or a mutant, variant, homologue or fragment thereof or at least a part of a CVS protein or a mutant, variant, homologue or fragment thereof.
  • the heterologous env region may be encoded by a gene which is present on a producer plasmid.
  • the producer plasmid may be present as part of a kit for the production of retroviral vector particles suitable for use in the first aspect of the invention.
  • the vector system may be pseudotyped with at least a part of a mutant rabies G protein or a variant, homologue or fragment thereof.
  • rabies G protein provides vectors which, in vivo, preferentially transduce targeted cells which rabies virus preferentially infects. This includes in particular neuronal target cells in vivo.
  • rabies G from a pathogenic strain of rabies such as ERA may be particularly effective.
  • rabies TJ protein confers a wider target cell range in vitro including nearly all mammalian and avian cell types tested (Seganti et al, 1990 Arch Virol. 34,155-163; Fields et al, 1996 Fields Virology, Third Edition, vol.2, Lippincott-Raven Publishers, Philadelphia, New York).
  • the fropism of the pseudotyped vector particles may be modified by the use of a mutant rabies G which is modified in the extracellular domain.
  • Rabies G protein has the advantage of being mutatable to restrict target cell range.
  • the uptake of rabies virus by target cells in vivo is thought to be mediated by the acetylcholine receptor (AchR) but there may be other receptors to which in binds in vivo (Hanham et al, 1993 J. Virol. ,67, 530-542; Tuffereau et ⁇ /.,1998 J. Virol., 72, 1085-1091). It is thought that multiple receptors are used in the nervous system for viral entry, including NCAM (Thoulouze et al (1998) J.
  • Viruses in which amino acid 330 has been mutated are further attenuated (i.e. ERAdm), were reported as being unable to infect either motor neurons or sensory neurons after infra-muscular injection (Coulon et ⁇ /.,1998 J. Virol., 72, 273-278).
  • rabies G proteins from laboratory passaged strains of rabies may be used. These can be screened for alterations in tropism. Such strains include the following:
  • the ERA strain is a pathogenic strain of rabies and the rabies G protein from this strain can be used for transduction of neuronal cells.
  • the sequence of rabies G from the ERA strains is in the GenBank database (accession number J02293). This protein has a signal peptide of 19 amino acids and the mature protein begins at the lysine residue 20 amino acids from the translation initiation methionine.
  • the HEP-Flury strain contains the mutation from arginine to glutamine at amino acid position 333 in the mature protein which correlates with reduced pathogenicity and which can be used to restrict the tropism of the viral envelope.
  • WO 99/61639 discloses the nucleic and amino acid sequences for a rabies virus strain ERA (Genbank locus RAVGPLS, accession M38452).
  • the vector system may be pseudotyped with at least part of a CVS (Challenge Standard Virus) protein, and in particular the CVS glycoprotein G, or a mutant, variant, homologue or fragment thereof.
  • CVS Copper Standard Virus
  • CVS glycoproteins from laboratory passaged strains of CVS may be used. These can be screened for alterations in tropism.
  • ATCC deposit No. 40280 designated pKB3-JE-13, may conveniently be used in the present invention.
  • the vector system is or comprises at least part of a wild-type rabies G protein or a mutant, variant, homologue or fragment thereof and or a wild-type CVS protein or a mutant, variant, homologue or fragment thereof.
  • wild type is used to mean a polypeptide having a primary amino acid sequence which is identical with the native protein (i.e., the viral protein).
  • mutant is used to mean a polypeptide having a primary amino acid sequence which differs from the wild type sequence by one or more amino acid additions, substitutions or deletions.
  • a mutant may arise naturally, or may be created artificially (for example by site-directed mutagenesis).Preferably the mutant has at least 90% sequence identity with the wild type sequence.
  • the mutant has 20 mutations or less over the whole wild-type sequence. More preferably the mutant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.
  • variant is used to mean a naturally occurring polypeptide which differs from a wild-type sequence.
  • a variant may be found within the same viral strain (i.e. if there is more than one isoform of the protein) or may be found within a different strains.
  • the variant has at least 90% sequence identity with the wild type sequence.
  • the variant has 20 mutations or less over the whole wild-type sequence. More preferably the variant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.
  • homologue means an entity having a certain homology with the wild type amino acid sequence and the wild type nucleotide sequence.
  • homology can be equated with “identity”.
  • an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence.
  • the homologues will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • an homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence.
  • the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247- 50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, ⁇ -alanine*, L- ⁇ -amino butyric acid*, L- ⁇ -amino butyric acid*, L- ⁇ -amino isobutyric acid*, L- ⁇ -amino caproic acid # , 7- amino heptanoic acid*, L-methionine sulfone , L-norleucine*, L-norvaline*, p-nitro-L- phenylalanine*, L-hydroxyproline # , L-thioproline*, methyl derivatives of
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ - alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ - alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the ⁇ -carbon substituent group is on the residue's nitrogen atom rather than the ⁇ -carbon.
  • fragment indicates that the polypeptide comprises a fraction of the wild-type amino acid sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections.
  • the polypeptide may also comprise other elements of sequence, for example, it may be a fusion protein with another protein.
  • the polypeptide comprises at least 50%, more preferably at least 65%, most preferably at least 80% of the wild-type sequence.
  • the mutant, variant, homologue or fragment should be capable of transducing at least part of the brain, a motor neuron or cerebrospinal fluid (CSF0 when used to pseudotype an appropriate vector.
  • CSF0 cerebrospinal fluid
  • the mutant, variant, homologue or fragment should alternatively or in addition, be capable of conferring the capacity for retrograde transport on the vector system.
  • the vector delivery system used in the present invention may comprise nucleotide sequences that can hybridise to the nucleotide sequence presented herein (including complementary sequences of those presented herein).
  • the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and 0.1 SSC) to the nucleotide sequence presented herein (including complementary sequences of those presented herein).
  • the EOI is one or more NOIs (nucleotide sequences of interest) - wherein said NOIs may be delivered to a target cell in vivo or in vitro.
  • the vector system of the present invention is a viral vector system, then it is possible to manipulate the viral genome so that viral genes are replaced or supplemented with one or more NOIs which may be heterologous NOIs.
  • heterologous refers to a nucleic acid or protein sequence linked to a nucleic acid or protein sequence to which it is not naturally linked.
  • the term NOI includes any suitable nucleotide sequence, which need not necessarily be a complete naturally occurring DNA or RNA sequence.
  • the NOI can be, for example, a synthetic RNA/DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof.
  • the sequence need not be a coding region. If it is a coding region, it need not be an entire coding region.
  • the RNA/DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation.
  • the sequence is, comprises, or is transcribed from cDNA.
  • the retroviral vector genome may generally comprise LTRs at the 5' and 3' ends, suitable insertion sites for inserting one or more NOI(s), and/or a packaging signal to enable the genome to be packaged into a vector particle in a producer cell.
  • suitable primer binding sites and integration sites may even be suitable primer binding sites and integration sites to allow reverse franscription of the vector RNA to DNA, and integration of the proviral DNA into the target cell genome.
  • the retroviral vector particle has a reverse franscription system (compatible reverse transcription and primer binding sites) and an integration system (compatible integrase and integration sites).
  • the NOI may encode a protein of interest ("POI").
  • POI protein of interest
  • the vector delivery system could be used to examine the effect of expression of a foreign gene on the target cell (such as a TH positive neuron).
  • the retroviral delivery system could be used to screen a cDNA library for a particular effect on the brain, motor neuron or CSF.
  • suitable NOIs include those that are of therapeutic and/or diagnostic application such as, but not limited to: sequences encoding cytokines, chemokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppresser protein and growth factors, membrane proteins, vasoactive proteins and peptides, anti- viral proteins and ribozymes, and derivatives thereof (such as with an associated reporter group).
  • the NOIs may also encode pro-drug activating enzymes.
  • the expression products encoded by the NOIs may be proteins which are secreted from the cell. Alternatively the NOI expression products are not secreted and are active within the cell. In either event, it is preferred for the NOI expression product to demonstrate a bystander effect or a distant bystander effect; that is the production of the expression product in one cell leading to the killing of additional, related cells, either neighbouring or distant (e.g. metastatic), which possess a common phenotype.
  • the NOI or its expression product may act to modulate the biological activity of a compound or a pathway.
  • modulate includes for example enhancing or inhibiting biological activity. Such modulation may be direct (e.g. including cleavage of, or competitive binding of another substance to a protein) or indirect (e.g. by blocking the initial production of a protein).
  • the NOI may be capable of blocking or inhibiting the expression of a gene in the target cell.
  • the NOI may be an antisense sequence.
  • the inhibition of gene expression using antisense technology is well known.
  • the NOI or a sequence derived therefrom may be capable of "knocking out” the expression of a particular gene in the target cell.
  • the NOI may be capable of integrating in the genome of a neuron so as to disrupt expression of the particular gene.
  • the NOI may disrupt expression by, for example, introducing a premature stop codon, by rendering the downstream coding sequence out of frame, or by affecting the capacity of the encoded protein to fold (thereby affecting its function).
  • the NOI may be capable of enhancing or inducing ectopic expression of a gene in the target cell.
  • the NOI or a sequence derived therefrom may be capable of "knocking in” the expression of a particular gene.
  • the NOI encodes a ribozyme.
  • Ribozymes are RNA molecules that can function to catalyse specific chemical reactions within cells without the obligatory participation of proteins.
  • group I ribozymes take the form of infrons which can mediate their own excision from self-splicing precursor RNA.
  • Other ribozymes are derived from self-cleaving RNA structures which are essential for the replication of viral RNA molecules.
  • ribozymes can fold into secondary and tertiary structures that provide specific binding sites for substrates as well as cofactors, such as metal ions. Examples of such structures include hammerhead, hai ⁇ in or stem-loop, pseudoknot and hepatitis delta antigenomic ribozymes have been described.
  • Each individual ribozyme has a motif which recognises and binds to a recognition site in a target RNA.
  • This motif takes the form of one or more "binding arms” but generally two binding arms.
  • the binding arms in hammerhead ribozymes are the flanking sequences Helix I and Helix III which flank Helix II. These can be of variable length, usually between 6 to 10 nucleotides each, but can be shorter or longer. The length of the flanking sequences can affect the rate of cleavage.
  • reducing the total number of nucleotides in the flanking sequences from 20 to 12 can increase the turnover rate of the ribozyme cleaving a HIV sequence, by 10-fold (Goodchild, JVK, 1991 Arch Biochem Biophys 284: 386-391).
  • a catalytic motif in the ribozyme Helix II in hammerhead ribozymes cleaves the target RNA at a site which is referred to as the cleavage site. Whether or not a ribozyme will cleave any given RNA is determined by the presence or absence of a recognition site for the ribozyme containing an appropriate cleavage site.
  • Each type of ribozyme recognizes its own cleavage site.
  • the hammerhead ribozyme cleavage site has the nucleotide base triplet GUX directly upstream where G is guanine, U is uracil and X is any nucleotide base.
  • Hai ⁇ in ribozymes have a cleavage site of BCUGNYR, where B is any nucleotide base other than adenine, N is any nucleotide, Y is cytosine or thymine and R is guanine or adenine. Cleavage by hai ⁇ in ribozymes takes places between the G and the N in the cleavage site.
  • ribozyme may be induced in all cells, but will only exert an effect in those in which the target gene transcript is present.
  • the substance may suppress the biologically available amount of a polypeptide of the invention. This may be by inhibiting expression of the component, for example at the level of transcription, transcript stability, translation or post-translational stability.
  • An example of such a substance would be antisense RNA or double-stranded interfering RNA sequences which suppresses the amount of mRNA biosynthesis.
  • the NOI comprises an siRNA.
  • Post-franscriptional gene silencing (PTGS) mediated by double-stranded RNA (dsRNA) is a conserved cellular defence mechanism for controlling the expression of foreign genes. It is thought that the random integration of elements such as fransposons or viruses causes the expression of dsRNA which activates sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA.
  • RNAi RNA interference
  • the mechanism of RNAi involves the processing of long dsRNAs into duplexes of 21-25 nucleotide (nt) RNAs.
  • siRNAs small interfering or silencing RNAs
  • siRNAs small interfering or silencing RNAs
  • dsRNA >30bp has been found to activate the interferon response leading to shut-down of protein synthesis and non-specific mRNA degradation.
  • this response can be bypassed by using 21nt siRNA duplexes allowing gene function to be analysed in cultured mammalian cells.
  • an RNA polymerase III promoter e.g., U6, whose activity is regulated by the presence of tefracycline may be used to regulate expression of the siRNA.
  • the NOI comprises a micro-RNA.
  • Micro-RNAs are a very large group of small RNAs produced naturally in organisms, at least some of which regulate the expression of target genes. Founding members of the micro-RNA family are let-7 and lin-4.
  • the let-7 gene encodes a small, highly conserved RNA species that regulates the expression of endogenous protein-coding genes during worm development.
  • the active RNA species is transcribed initially as an ⁇ 70nt precursor, which is post- transcriptionally processed into a mature ⁇ 21nt form.
  • Both let-7 and lin-4 are transcribed as hai ⁇ in RNA precursors which are processed to their mature forms by Dicer enzyme.
  • the NOI comprises double-stranded interfering RNA in the form of a hai ⁇ in.
  • the short hai ⁇ in may be expressed from a single promoter, e.g., U6.
  • an effective RNAi may be mediated by inco ⁇ orating two promoters, e.g., U6 promoters, one expressing a region of sense and the other the reverse complement of the same sequence of the target.
  • effective or double-stranded interfering RNA may be mediated by using two opposing promoters to transcribe the sense and antisense regions of the target from the forward and complementary strands of the expression cassette.
  • the NOI may encode a short RNA which may act to redirect splicing ('exon-skipping') or polyadenylation or to inhibit translation.
  • the NOI may also be an antibody.
  • antibody includes a whole immunoglobuhn molecule or a part thereof or a bioisostere or a mimetic thereof or a derivative thereof or a combination thereof. Examples of a part thereof include: Fab, F(ab)' 2> and Fv. Examples of a bioisostere include single chain Fv (ScFv) fragments, chimeric antibodies, bifunctional antibodies.
  • Transduced target cells which express a particular gene, or which lack the expression of a particular gene have applications in drug discovery and target validation.
  • the expression system could be used to determine which genes have a desirable effect on target cells, such as those genes or proteins which are able to prevent or reverse the triggering of apoptosis in the cells. Equally, if the inhibition or blocking of expression of a particular gene is found to have an undesirable effect on the target cells, this may open up possible therapeutic strategies which ensure that expression of the gene is not lost.
  • the present invention may therefore be used in conjunction with disease models, such as experimental allergic encephalomyelitis, which is the animal model of Multiple Sclerosis, and experimental autoimmune neuritis which is the animal model of acute and chronic inflammatory demyelinating polyneuropathy.
  • disease models such as experimental allergic encephalomyelitis, which is the animal model of Multiple Sclerosis, and experimental autoimmune neuritis which is the animal model of acute and chronic inflammatory demyelinating polyneuropathy.
  • Other disease models are known to those skilled in the art.
  • An NOI delivered by the vector delivery system may be capable of immortalising the target cell.
  • a number of immortalisation techniques are known in the art (see for example Katakura Y et al (1998) Methods Cell Biol. 57:69-91).
  • the vector delivery system can be a non-viral delivery system or a viral delivery system.
  • the vector delivery system is a viral delivery vector system.
  • the vector delivery system is a retroviral vector delivery system.
  • immortalised is used herein to cells capable of growing in culture for greater than 10 passages, which may be maintained in continuous culture for greater than about 2 months. Immortalised motor and sensory neurons and brain cells are useful in experimental procedures, screening programmes and in therapeutic applications. For example, immortalised dopaminergic neurones may be used for transplantation, for example to treat Parkinson's disease.
  • An NOI delivered by the vector delivery system may be a selection gene, or a marker gene.
  • selectable markers have been used successfully in retroviral vectors. These are reviewed in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 444) and include, but are not limited to, the bacterial neomycin and hygromycin phosphofransferase genes which confer resistance to G418 and hygromycin respectively; a mutant mouse dihydrofolate reductase gene which confers resistance to methotrexate; the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine and aminopterin; the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; and the bacterial genes which confer resistance to puromycin or phleomycin. All of
  • An NOI delivered by the vector delivery system may be a therapeutic gene - in the sense that the gene itself may be capable of eliciting a therapeutic effect or it may code for a product that is capable of eliciting a therapeutic effect.
  • mimetic relates to any chemical which may be a peptide, polypeptide, antibody or other organic chemical which has the same binding specificity as the antibody.
  • the invention is useful for obtaining good distribution of an expressed protein, for example by administering the vector at one site, the protein may be released such that it affects other parts of the brain and nervous system.
  • the vector system used in the present invention is particularly useful in treating and/or preventing a disease which is associated with the death or impaired function of cells of the nervous tissue, such as neurons, CSF and/or brain cells including glial cells.
  • the vector system is useful in treating and/or preventing neurodegenerative disease's.
  • the vector system used in the present invention may be used to treat and/or prevent a disease which is associated with the death or impaired function of motor or sensory neurons.
  • Diseases which may be treated include, but are not limited to: pain; movement disorders such as Parkinson's disease, motor neuron diseases including amyotrophic lateral schlerosis (ALS or Lou Gehrig's Disease) and Huntington's disease; Alzheimer's Disease; Spinal Muscle Atrophy and Lysosomal Storage Diseases.
  • movement disorders such as Parkinson's disease, motor neuron diseases including amyotrophic lateral schlerosis (ALS or Lou Gehrig's Disease) and Huntington's disease
  • Alzheimer's Disease Spinal Muscle Atrophy and Lysosomal Storage Diseases.
  • Amyotrophic lateral schlerosis is a degenerative disorder of motorneurons with a yearly incidence of 1-2 per 100,000. It is characterised by degeneration of motorneurons in the spinal cord, brain stem and motor cortex which leads to wasting and weakness of limb, bulbar and respiratory muscles. Approximately 5-10% of ALS is familial. Genes whose mutations or haplotypes are thought to play a role in disease predisposition include SOD1, ALS2 and VEGF (Lambrechts et al. Nature Genetics 2003; published on line 6 July 2003 (10.1038/ngl211); Oosthuyse et al. Nature Genetics 2001; June; Vol 28 pages 131-138).
  • the vector system used in the present invention is useful in treating and/or preventing ALS.
  • the NOI may be capable of knockdown of SOD 1.
  • Other NOI(s) may encode molecules which prevent apoptosis and therefore prevent cells from dying. Suitable molecules include XIAP and NAIP.
  • NOI(s) may encode neurotrophic molecules which stimulate regeneration such as IGF-1, GDNF, VEGF and cardiotrophin (CT1).
  • Lysosomal Storage Diseases or Glycolipid Storage Disorders are genetic diseases that result when the rate of glycolipid synthesis is not balanced with the rate of degradation within the cells. As a result, undegraded glycolipids build up in the lysosomes. Such disorders include Fabry Disease, Niemann-Pick diseases, Gangliosidosis, Metachromatic Leukodystrophy and many types of Mucopolysaccharidosis.
  • SMA Spinal Muscular Atrophy
  • SMA is a disease of the anterior horn cells and is an autosomal recessive disease. Anterior horn cells are located in the spinal cord. SMA affects the voluntary muscles for activities such as crawling, walking, head and neck control and swallowing. Categories of SMA include: Type I SMA also called Werdnig- Hoffmann Disease, Type II, Type III, often referred to as Kugelberg-Welander or Juvenile Spinal Muscular Atrophy, Type JN (Adult Onset) and Adult Onset X-Linked SMA. This form also known as Kennedy's Syndrome or Bulbo-Spinal Muscular Atrophy. SMA is a common motor neuron disease in humans and its most severe form causes death by the age of 2 years.
  • the vector system used in the present invention is useful in treating and/or preventing SMA.
  • the ⁇ OI may be capable of encoding a gene for replacement of defective SM ⁇ 1 gene.
  • Other ⁇ OI(s) may encode molecules which prevent apoptosis and therefore prevent cells from dying. Suitable molecules include XIAP and NAIP.
  • NOI(s) may encode neurotrophic molecules which stimulate regeneration such as IGF-1, GDNF, neurotrophin-3 (NT-3), VEGF and cardiotrophin (CT1).
  • the vector system used in the present invention is useful in treating and/or preventing Parkinson's disease.
  • the NOI is capable of encoding a neuroprotective molecule.
  • the NOI(s) may encode molecules which prevent TH-positive neurons from dying or which stimulate regeneration and functional recovery in the damaged nigrostriatal system.
  • the NOI is capable of encoding an enzyme or enzymes responsible for L- DOPA or dopamine synthesis such as tyrosine hydroxylase, GTP-cyclohydrolase I, aromatic acid dopa decarboxylase, and vesicular monoamine transporter 2..
  • the vector system of the present invention may also be used in the treatment and/or prevention of an inflammatory neurological disorder including an autoimmune neurological disease.
  • MS Multiple Sclerosis
  • MS is a chronic inflammatory disease of the C ⁇ S and is presumed to have an autoimmune etiology.
  • MS is believed to be caused by blood-derived T cells specific for C ⁇ S antigens. These T cells induce the production in the C ⁇ S of antigen- nonspecific mononuclear cells able to destroy oligodendrocytes directly and/or by releasing substances toxic to myelin.
  • autoimmune neurological diseases include the Guillain-Barre syndrome, myasthenia gravis, acute disseminated encephalomyelitis, the stiff-man syndrome, autoimmune neuritis, motor dysfunction, chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy, paraproteinaemic neuropathy, autoimmune diseases of the neuromuscular junction and other disorders of the motor unit, inflammatory myopathy, autoimmune myositis, a parameoplastic neurological disorder, neurological complications of connective tissue diseases and vasculitis.
  • the nucleotide of interest delivered by the vector system used in the present invention encodes an anti-inflammatory molecule, such as an anti-inflammatory cytokine, or a molecule capable of upregulating the anti-inflammatory molecule.
  • an anti-inflammatory molecule such as an anti-inflammatory cytokine
  • a molecule capable of upregulating the anti-inflammatory molecule such as MS
  • Cytokines which may be useful in the treatment of MS and possible other disorders include IL-l ⁇ , IL-2, IL-4, IL-6, IL-ln, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ , p55TNFR-Ig, p75dTNFR, TGF- ⁇ , PDGF- ⁇ and NGF. More generally, it will be appreciated that anti-inflammatory cytokines may useful be delivered in accordance with the present invention in the treatment and/or prevention of neurological inflammatory diseases.
  • Another approach involves the delivery of a nucleotide of interest which inhibits, or encodes a molecule which inhibits, a pro-inflammatory molecule, such as an inflammatory cytokine.
  • a nucleotide of interest which inhibits, or encodes a molecule which inhibits, a pro-inflammatory molecule, such as an inflammatory cytokine.
  • inhibitors such as those described above, e.g. ribozymes, siRNA, antibodies and antisense sequences, is envisaged.
  • a further approach involves the delivery of myelin proteins and or growth factors for rebuilding and or regenerating the damaged neuron myelin sheath.
  • disorders include (but are not limited to) glaucoma or other disorders that are secondary to an elevation in intraocular pressure, neuronal dystrophies such as multiple sclerosis.
  • Suitable genes for expression include growth or survival factors such as erythropoietin or VEGF for the treatment of stroke, expression of neuroprotective factor such as PEDF, GDNF or neurotrophins for the treatment of optic neuropathies ( eg Leber's congenital disease).
  • the vector system is a lentiviral vector system because advantageously with the use of a lentiviral vector system having a rabies G pseudotype, one achieves high efficiency retrograde transport and long term expression. While both adenovirus and HSV and even AAV (to a lesser extent) do get retrogradely transported, the lentiviral vector system having a rabies G pseudotype achieves high efficiency retrograde transport through the selective transduction of neurons.
  • lentiviral vectors pseudotyped with rabies G specifically target motor neurons with high efficiency.
  • the use of lentiviral vectors avoids the toxicity issues common to the use of adenovirus and HSV, for example.
  • the present invention also provides the use of a vector delivery system in the manufacture of a pharmaceutical composition.
  • the pharmaceutical composition may be used to deliver an EOI, such as an NOI, to a target cell in need of same.
  • the vector delivery system can be a non-viral delivery system or a viral delivery system.
  • the vector delivery system is a viral delivery vector system.
  • the vector delivery system is a retroviral vector delivery system which is, preferably, a lentiviral vector delivery system.
  • the pharmaceutical composition may be used for treating an individual by gene therapy, wherein the composition comprises or is capable of producing a therapeutically effective amount of a vector system according to the present invention.
  • the method and pharmaceutical composition of the invention may be used to treat a human or animal subject.
  • the subject is a mammalian subject. More preferably the subject is a human.
  • a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
  • the composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • a pharmaceutically acceptable carrier diluent, excipient or adjuvant.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).
  • the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavemosally, intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the vector system used in the present invention may conveniently be administered by direct injection into the patient.
  • the system may be injected into the brain.
  • the system may be injected directly into any target area of the brain (for example, the striatum or substantia nigra).
  • the system can be injected into a given area, and the target area transduced by retrograde transport of the vector system.
  • Intramuscular injection is particularly preferred as the least invasive method of treatment.
  • the table above outlines preferred sites for administering therapy by injection and includes intraspinal, intrathecal, amygdala, DRG, skin, sites of lesions of he ⁇ etic neuralgia, cortex, striatum, nigra, hypothalamus, pituitary, glioma bed, retina, vitreus, muscle, spinal cord and intraventricular injection.
  • the present invention provides the use of a vector system comprising at least part of a CVS envelope protein or a mutant, fragment, variant of homologue thereof to transduce a target site, wherein the vector system travels to the site by retrograde transport.
  • a virus particle may travel in the same direction as a nerve impulse, i.e. from the cell body, along the axon to the axon terminals. This is known as anterograde transport.
  • Retrograde transport or transfer of a vector means that it is taken up by the axon terminals and travels towards the cell body.
  • This type of transport is often called “Fast transport” and is responsible for the movement of membranous organelles at 50-200 mm per day toward the synapse (anterograde) or back to the cell body (retrograde) (Hirokawa (1997) Curr Opin Neurobiol 7(5):605-614).
  • the precise mechanism of retrograde transport is unknown, however. It is thought to involve transport of the whole viral particle, possibly in association with an internalised receptor. The fact that the present vector systems can be specifically transported in this manner (as demonstrated herein) suggests that the env protein may be involved.
  • HSV, adenovirus and hybrid HSV/adeno-associated virus vectors have all been shown to be transported in a retrograde manner in the brain (Horellou and Mallet (1997) Mol Neurobiol 15(2) 241-256; Ridoux et al (1994) Brain Res 648:171-175; Constantini et al
  • Retrograde transport can be detected by a number of mechanisms known in the art.
  • a vector system expressing a heterologous gene is injected into the striatum, and expression of the gene is detected in the substantia nigra. It is clear that retrograde transport along the neurons which extend from the substantia nigra to the basal ganglia is responsible for this phenomenon. It is also known to monitor labelled proteins or viruses and directly monitor their retrograde movement using real time confocal microscopy (Hirokawa (1997) as above).
  • the present invention also provides the use of a vector system of the present invention to transduce a target site, which comprises the step of administration of the vector system to an administration site which is distant from the target site to achieve good penetration and biodistribution throughout the CNS.
  • administration to the one area of the brain may give rise to distribution of the EOI is different parts of the brain and/different cell types.
  • the target site may be any site of interest. It may or may not be anatomically connected to the administration site.
  • the target site may be capable of receiving vector from the administration site by axonal transport, for example anterograde or (more preferably) retrograde transport.
  • axonal transport for example anterograde or (more preferably) retrograde transport.
  • a number of potential target sites may exist which can be identified using tracers by methods known in the art (Ridoux et al (1994) as above).
  • CVS/EIAV vectors causes transgene expression in the globus pallidus, cortex, various thalamic nuclei, amygdala, hypothalamus, supraoptic nucleus, deep mesencepthalic nuclei, substantia nigra, caudal regions of the brainstem such as the nuclei of the brachium inferior colliculus, paraleminiscal nuclei, genie nuclei, parabrachial nuclei, ventral cochlear nuclei and facial nuclei.
  • a target site is considered to be "distant from the adminisfration" if it is (or is mainly) located in a different region from the administration site.
  • the two sites may be distinguished by their spatial location, mo ⁇ hology and/or function.
  • the basal ganglia consist of several pairs of nuclei, the two members of each pair being located in opposite cerebral hemispheres.
  • the largest nucleus is the co ⁇ us striatum which consists of the caudate nucleus and the lentiform nucleus.
  • Each lentiform nucleus is, in turn, subdivided into a lateral part called the putamen and a medial part called the globus pallidus.
  • the substantia nigra and red nuclei of the midbrain and the subthalamic nuclei of the diencephalon are functionally linked to the basal ganglia. Axons from the substantia nigra terminate in the caudate nucleus or the putamen.
  • the subthalamic nuclei connect with the globus pallidus.
  • the administration site is the striatum of the brain, in particular the caudate putamen.
  • Injection into the putamen can label target sites located in various distant regions of the brain, for example, the globus pallidus, amygdala, subthalamic nucleus or the substantia nigra. Transduction of cells in the pallidus commonly causes retrograde labelling of cells in the thalamus.
  • the (or one of the) target site(s) is the substantia nigra.
  • the vector system may transduce a target cell.
  • the target cell may be a cell found in nervous tissue, such as a sensory or motor neuron, astrocyte, oligodendrocyte, microglia or ependymal cell
  • the vector system is preferably administered by direct injection.
  • Methods for injection into the brain are well known in the art (Bilang-Bleuel et al (1997) Proc. Acad. Natl. Sci. USA 94:8818-8823; Choi-Lundberg et al (1998) Exp. Neurol.154:261-275; Choi-Lundberg et al (1997) Science 275:838-841; and Mandel et al (1997) ) Proc. Acad. Natl. Sci. USA 94:14083-14088). Stereotaxic injections may be given.
  • the viral preparation is concentrated by ultracentrifugation.
  • the resulting preparation should have at least 10 8 t.u./ml, preferably from 10 8 to 10 10 t.u./ml, more preferably at least 10 9 t.u./ml.
  • the titer is expressed in transducing units per ml (t.u./ml) as titred on a standard D17 cell line). It has been found that improved dispersion of transgene expression can be obtained by increasing the number of injection sites and decreasing the rate of injection (Horellou and Mallet (1997) as above). Usually between 1 and 10 injection sites are used, more commonly between 2 and 6. For a dose comprising 1-5 x 109 t.u./ml, the rate of injection is commonly between 0.1 and 10 ⁇ l/min, usually about 1 ⁇ l/min.
  • an NOI may be found in various areas of the brain, such as the ependymal and leptomeningeal cells, hippocampus, co ⁇ us collasum and septum, and the spinal cord.
  • the present invention also provides an immortalised cell of the CNS such as a sensory or motor neuron or brain cell and its use in transplantation methods. Grafting protocols using embryonic dopaminergic neurons, equivalent cells from other species, and neural progenitor cells are known (reviewed in Dunnett and Bjorklund (1999) Nature Vol 399 Supplement pages A32-39). Similar techniques could be used for grafting the cells of the present invention.
  • Figures 1 and 2 show the polynucleotide and amino acid sequences of ERA wild- type
  • Figure 3 shows the polynucleotide sequence of ERAdm
  • Figure 4 shows the polynucleotide sequence of CVS rabies virus glycoprotein
  • FIG. 5 shows the results of Example 1 and illustrates the transduction efficiency of
  • Figure 6 shows the of Example 1 and illustrates the expression of the marker gene LacZ in the spinal cord after injection of EIAV-LacZ into the CSF;
  • Figure 10 shows the results of Example 4 using CVS.
  • Figure 11 shows, following sub-retinal gene delivery of the pONY ⁇ .O CMVGFP virus,
  • GFP fluorescence is seen in the optic chiasm (A), in the axons of the optic tract (B) and in the cell bodies of the optic tract (C).
  • Figure 12a shows a diagram of a replacement vector comprising the SMN gene.
  • Figure 12b shows a diagram of pONY8.7NCSMN.
  • Figure 13 shows confocal analysis of SMN immunolabelling following in vitro transduction with Smart2SMN vector pseudotyped with rabies G envelope.
  • a and B Restoration of SMN protein in SMA fibroblast transduced with lentiviral vector-mediated expression of SMN.
  • C Untransduced cells.
  • D b-gal immunostaining in SMA fibroblast transduced with Smart2LacZ. Note the strong staining in the cytoplasm and nucleus in A and B.
  • Figure 14 shows a Western Blot confirming expression of SMN in transduced D17 fibroblasts.
  • D17 cells are transduced with Smart2SMN, SMN-HA and LacZ vectors.
  • Figure 15 shows SMN gene therapy in mild model of SMA.
  • Figure 16 shows immune response study in Type III mice after intramuscular injection of
  • Figure 17 shows SMN gene transfer in mouse model of type I SMA.
  • DRG cells (A) and spinal motor neurons (B) were transduced by retrograde transport following intramuscular injection of SMN expressing vectors (C) control.
  • EIAV vectors were pseudotyped with wild-type and 2 variants of the ERA strain of rabies-G envelopes.
  • the sequence of rabies virus strain ERA is shown in Figures 1 and 2 (SEQ ID NO: 1 and 2).
  • a single mutant of the wild-type ERA strain (ERAwt) was generated by replacing arginine at amino acid 333 with glutamine. This mutant, which is naturally occurring and apathogenic in adult mice, was termed ERAsm.
  • An additional substitution at amino acid 330 from K to N resulted in a double mutant of ERAwt named ERAdm. Both these envelopes were used to pseudotype the EIAV vectors expressing a marker gene LacZ.
  • the resulting fragment was cloned into pSA91 using appropriate restriction enzymes. Successful clones were sequenced and used to produce EIAV vectors using the fransient fransfection method.
  • CVS cDNA for CVS (Challenge Virus Standard) rabies virus glycoprotein was obtained from ATCC (ATCC number 40280 designation ⁇ KB3-JE-13). The fragment containing the complete coding sequence of the glycoprotein was excised using EcoRI, cloned into pSA91 and sequenced (Bk 1092 pg 75). The sequence is shown in Figure 4 (SEQ ID NO:4).
  • Example 1 Injection of EIAV pseudotyped with rabies-G or VSV-G envelopes into the cerebrospinal fluid (CSF) and treatment of MS using an intrathecal route for gene therapy
  • the expression of the marker gene LacZ can be demonstrated in different areas of the brain and spinal cord (Figure 5).
  • the rabies-G pseudotyped vectors are able to infect the ependymal and leptomeningeal cells (Figure 5 A-C). Strong bilateral transduction was also observed in the hippocampus (mainly in CA3), co ⁇ us collasum and septum ( Figure 5 D-I). The virus can also spread to the spinal cord ( Figure 6 A-F).
  • Lentiviral-mediated delivery of cytokines-encoding genes to the CSF in accordance with the present invention shows the following major advantages: i) the availability of high cytokine levels widely in the CNS; ii) long-term and persistent expression of exogenous genes after inco ⁇ oration into the DNA of the host cell; and iii) absence of the immune response to the viral particle.
  • Example 4 Injection of EIAV vectors pseudotyped with CVS envelope into the striatum Approximately 2 x 10 6 TU of each vector was slowly infused into the striatum of adult male Wistar rats (300g) using the stereotaxic coordinates AP 0 mm, ML 3.5 ram, DN 4.75mm and left for 2 or 4 weeks. The rats were then sacrificed and transcardially perfused with 4% paraformaldehyde. Following an overnight incubation in 4% paraformaldehyde, the brains were cryoprotected in 30% sucrose for at least 3 days, after which they were frozen and cut into 40 ⁇ m coronal sections. X-gal staining and immunohistochemistry were performed.
  • Retrograde transport was observed in the cortex, various thalamic nuclei, amygdala, hypothalamus, supraoptic nucleus, deep mesencephalic nuclei and substantia nigra. In addition retrograde transport to the caudal regions of the brainstem was observed. In this region, various nuclei such as the nuclei of the brachium inferior colliculus, paraleminiscal nuclei, genie nuclei, parabrachial nuclei, ventral cochlear nuclei and facial nuclei were positive for X-gal staining.
  • Example 5 Retrograde transport to the brain following subretinal delivery of a lentivirus vector pseudotyped with the Rabies envelope.
  • the transient three plasmid fransfection method is used to generate an EIAN virus vector based on the pO ⁇ Y ⁇ .O CMN GFP genome pseudotyped with the Rabies envelope (pSA91 ERAwt).
  • the virus (batch number OBM039) is titered biologically and estimated to be lxlOelO TU/ml.
  • a total of 4ul (2x2ul) is sub-retinally injected into C57/M-6J mice and tissues harvested at different time points for analysis of gene expression.
  • the optic nerve fibres from each eye cross over in a very specific way at the optic chiasm - fibres originating in the nasal part of the retina cross over to the opposite hemisphere, while those originating in the temporal retina do not, but continue to the same side of the brain. Therefore, sub-retinal delivery to a single eye can lead to retrograde transport to both cerebral hemispheres. Alternatively, if the sub-retinal injection is restricted to a particular region of the eye, either nasal or temporal, then a single cerebral hemisphere may be targeted.
  • Example 6 In vitro validation of SMA fibroblast Construction of Smart2SM ⁇ and pONY 8.7NCSMN vectors as shown in Figure 12 is described by Mazarakis et al., 2001. SMN gene was a gift from Dr. Arthur Burghes (Ohio State University, Ohio, USA). The Smart2SMN vector was pseudotyped with rabies-G envelope protein derived from ERA strain. SMA fibroblast represent an in vitro model of SMA. And were extracted from SMA patients type I.
  • the Smart2SMN vector pseudotyped with rabies-G envelope was used to transduce SMA fibroblast at an MOI of 50 and 100 essentially as described in Mazarakis et al. Human Molecular Genetics, 2001.
  • a Smart2LacZ ERAwt transduction and untransduced cells were used as negative controls. Immunocytochemistry was used to confirm expression of the SMN protein from pSMT2SMN ERAwt. Confocal microscopy demonstrated strong positive SMN staining in the cytoplasm. This experiment also demonstrates the use of EIAV to restore gems in the nucleus of SMA fibroblast ( Figure 13). The best results were obtained with an MOI 100. No such staining was seen in the negative controls.
  • Figure 14 shows a Western Blot using SMN antibody (Transduction Laboratories) recognising SMN and antibodies against HA tag which demonstrates expression of SMN in those cells transduced with the SMN vector.
  • SMN antibody Transduction Laboratories
  • SMN-1 gene replacement strategy using gene therapy can be used for rescuing motor neurons from cell death in an animal model of SMA and in SMA patients.
  • Type III mice display muscle weakness, motor neuron degeneration and a reduction in SMN protein level (an average of 4.5 nuclear gems were counted per motor neuron in the type III SMA mice versus 9.8 nuclear gems in the age-matched control).
  • the animal model of type I SMA represents a model of the severe form of SMA. These mice display motor neuron death, muscle weakness and die by postnatal day 14. The aim of this work was to extend mice survival using muscle delivery of LentiVector® expressing SMN gene.
  • Intracranial (brainstem) 5 ⁇ l Muscles of the thoracic trunk: 10 ⁇ l
  • Diaphragm muscle 10 ⁇ l
  • Intracranial (brainstem) 5 ⁇ l
  • Diaphragm muscle 10 ⁇ l
  • Tongue 10 ⁇ l
  • Intracranial (brainstem) 5 ⁇ l
  • Diaphragm muscle 10 ⁇ l Face muscles : 20 ⁇ l Tongue : 10 ⁇ l
  • Intracranial (brainstem) 5 ⁇ l
  • All the lentiviral vectors for these experiments are rabies-G pseudotyped so as to achieve retrograde transport of the virus and fransduction of motor neurons.
  • EIAV gene transfer in mouse model of type I SMA leads to widespread expression of the transgene, extending the survival of these mice.
  • SMN immunostaining demonstrates robust expression of the transgene, not only in spinal motor neurons but also in DRG neurons suggesting that intramuscular injection of Smart2SMN or pONY8.7NCSMN in type I mice leads to transduction of motor neurons and DRG cells by refrograde transport (Figure 17). No such staining was seen in mice injected with Smart2LacZ ( Figure 17).
  • Lentiviral vector-mediated expression of SMN gene in SMA type I mice extend the survival of these mice by 35% compared to control LacZ treated mice and 50% compared to untreated mice.
  • Smart2hVEGF treatment delay the onset of the disease and extend the survival of SOD1 transgenic mice compared to LacZ control mice. The onset of the disease was delayed by an average of 30 days. hVEGF-injected mice survive a minimum of 40 days longer that LacZ group. However, smart2XIAP do not show any efficacy in SOD1 transgenic mice. VEGF treatment also enhances the motor function in SOD1 mice compared to LacZ group. This result was based on rotarod and footprint tests.

Abstract

L'invention concerne l'utilisation d'un système de vecteur permettant d'effectuer une transduction vers un site cible. Ledit système de vecteur se déplace vers ledit site cible par diffusion. Ledit système de vecteur est ou comprend au moins une partie de protéine G d'enveloppe de la rage ou d'un mutant, d'un variant, d'un homologue ou d'un fragment de cette protéine. Le site cible est au moins une partie du système nerveux central.
PCT/GB2003/004260 1996-10-17 2003-10-03 Systeme de vecteur WO2004031390A1 (fr)

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JP2005500061A JP2006502240A (ja) 2002-10-04 2003-10-03 ベクター系
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AU2003271883A AU2003271883A1 (en) 2002-10-04 2003-10-03 Vector system
US10/716,725 US20040076613A1 (en) 2000-11-03 2003-11-19 Vector system
US10/838,906 US20040266715A1 (en) 1999-03-31 2004-05-03 Neurite regeneration
US11/583,427 US20070213290A1 (en) 1996-10-17 2006-10-19 Neurite regeneration
US11/810,007 US20080131400A1 (en) 2000-11-03 2007-06-04 Vector system

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GB0228314.1 2002-12-04
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WO2008054544A2 (fr) * 2006-05-22 2008-05-08 Immune Disease Institute, Inc. Procédé d'administration à travers la barrière hématoencéphalique
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JP2008500312A (ja) * 2004-05-27 2008-01-10 フラームス・インテルウニフェルシタイル・インステイチュート・フォール・ビオテヒノロヒー・ヴェーゼットウェー（ヴェーイーベー・ヴェーゼットウェー） 筋萎縮性側索硬化症の治療
JP2012111774A (ja) * 2004-05-27 2012-06-14 Vlaams Interuniversitair Inst Voor Biotechnologie Vzw (Vib Vzw) 筋萎縮性側索硬化症の治療
WO2008054544A2 (fr) * 2006-05-22 2008-05-08 Immune Disease Institute, Inc. Procédé d'administration à travers la barrière hématoencéphalique
WO2008054544A3 (fr) * 2006-05-22 2009-12-03 Immune Disease Institute, Inc. Procédé d'administration à travers la barrière hématoencéphalique
US8748567B2 (en) 2006-05-22 2014-06-10 Children's Medical Center Corporation Method for delivery across the blood brain barrier
US9757470B2 (en) 2006-05-22 2017-09-12 Children's Medical Center Corporation Peptides for assisting delivery across the blood brain barrier
EP2239330A1 (fr) * 2009-04-07 2010-10-13 Institut Pasteur Génération, régénération et protection de neurones
WO2010116258A1 (fr) * 2009-04-07 2010-10-14 Institut Pasteur Génération, régénération et protection des neurones
CN102459602A (zh) * 2009-04-07 2012-05-16 巴斯德研究所 神经元的生成、再生和保护
US8822665B2 (en) 2009-04-07 2014-09-02 Institut Pasteur Neuron generation, regeneration and protection
CN114450032A (zh) * 2019-08-08 2022-05-06 生物基因麻省公司 病毒载体生产的效价测定
EP4010033A4 (fr) * 2019-08-08 2023-05-24 Biogen MA Inc. Test d'activité biologique pour la production de vecteurs viraux

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