[go: up one dir, main page]

WO2007031727A2 - Systeme - Google Patents

Systeme Download PDF

Info

Publication number
WO2007031727A2
WO2007031727A2 PCT/GB2006/003367 GB2006003367W WO2007031727A2 WO 2007031727 A2 WO2007031727 A2 WO 2007031727A2 GB 2006003367 W GB2006003367 W GB 2006003367W WO 2007031727 A2 WO2007031727 A2 WO 2007031727A2
Authority
WO
WIPO (PCT)
Prior art keywords
ncs
vector
eiav
rarβ2
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2006/003367
Other languages
English (en)
Other versions
WO2007031727A3 (fr
Inventor
Liang-Fong Wong
Nicholas Mazarakis
Susan Kingsman
Ping Yip
Jonathan Corcoran
Stephen Mcmahon
Malcom Maden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kings College London
Oxford Biomedica UK Ltd
Original Assignee
Kings College London
Oxford Biomedica UK Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kings College London, Oxford Biomedica UK Ltd filed Critical Kings College London
Priority to EP06779383A priority Critical patent/EP1937820A2/fr
Publication of WO2007031727A2 publication Critical patent/WO2007031727A2/fr
Anticipated expiration legal-status Critical
Publication of WO2007031727A3 publication Critical patent/WO2007031727A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • 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
    • C12N2740/15045—Special targeting system for viral vectors
    • 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 system, in particular a system capable of delivering a protein or a nucleotide sequence to a target cell.
  • the system may be capable of delivering or causing the expression of neuronal calcium sensor- 1 (NCS-I ), for the promotion of neurite outgrowth.
  • NCS-I neuronal calcium sensor- 1
  • neurons of the adult CNS have limited intrinsic capacity to regenerate, e.g. following injury.
  • the present inventors have shown that exogenous delivery of the transcription factor retinoic acid receptor ⁇ 2 (RAR ⁇ 2) via a lentiviral vector into adult rat dorsal root ganglion (DRG) neurons promotes axonal regeneration of injured sensory axons and restores functional recovery in the rhizotomy model of spinal cord injury (Wong et al., Paper submitted). These results suggest that RAR ⁇ 2 can activate regeneration programs in injured neurons to promote axonal outgrowth.
  • RAR ⁇ 2 transcription factor retinoic acid receptor ⁇ 2
  • NCS-I Neuronal calcium sensor- 1 1
  • EP-A-I 250 931 and corresponding US2003/0159158
  • NCS-I is involved in the formation and function of the presynaptic nerve terminal part of the neuromuscular junction during synaptogenesis and in adult mammals.
  • Xiao-Liang Chen et al (2001) J Physiology, 532(3): 649-659 suggests that overexpression of rat NCS-I in rodent NG108- 15 cells enhances synapse formation and transmission.
  • NCS-I is upregulated by RAR ⁇ 2 and that overexpression of NCS-I, like RAR ⁇ 2, promotes neurite outgrowth in adult neurons.
  • NCS-I causes axonal outgrowth, making the expression of NCS-I in vivo of considerable therapeutic interest.
  • the present invention provides a system capable of delivering neuronal calcium sensor-1 (NCS-I), or a nucleotide sequence capable of encoding NCS-I, to a target cell, for promoting neurite outgrowth.
  • NCS-I neuronal calcium sensor-1
  • a nucleotide sequence capable of encoding NCS-I to a target cell, for promoting neurite outgrowth.
  • Delivery systems include a non-viral and viral vector systems.
  • Viral vector systems include retroviral and lentiviral systems.
  • the system may, for example be derivable from EIAV.
  • the vector system of the present invention may be capable of promoting neurite outgrowth and may be used, for example, for treating spinal cord injury.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a system according to the first aspect of the invention.
  • the composition may also comprise RAR ⁇ 2, or a nucleotide sequence capable of encoding RAR ⁇ 2.
  • the system of the first aspect of the invention, the composition of the second aspect of the invention, or an agent capable of promoting the expression or activity of NCS-I may be used to promote neurite outgrowth, for example, in the treatment of spinal cord injury to activate regeneration processes in injured neurons.
  • the present invention relates to a method of promoting neurite outgrowth by administering to a subject a system of the first aspect of the invention, a composition of the second aspect of the invention, or an agent capable of promoting the expression or activity of NCS-I.
  • the present invention relates to the use of a system according the first aspect of the invention to deliver NCS-I, or a nucleotide sequence capable of encoding NCS-I, to a cell in vitro.
  • the invention also provides a cell containing or expressing NCS- 1, produced by such a method.
  • Such a cell may be used for implanting or transplanting into a subject, for example to promote neuron regeneration. DESCRIPTION OF THE FIGURES
  • DRG neurons 500 neurons/well are plated on reduced laminin substrate (0.1 ⁇ g/ml) and transduced with lentiviral vectors expressing (a) control ⁇ -galactosidase (EIAV-LacZ) or (b) RAR ⁇ 2 (EIAV-RAR ⁇ 2) or at a multiplicity of infection of 10.
  • Vector expression of ⁇ - galactosidase ( ⁇ -gal) or RAR ⁇ 2 are indicated in green while neurite outgrowth is detected by ⁇ lll-tubulin in red. The average length of the longest neurite (c) and the number of neurons possessing neurites (d) in transduced cultures are measured.
  • RAR ⁇ 2 cultures are also incubated in an adenylate cyclase inhibitor 2', 5'-dideoxyadenosine DDA (100 ⁇ M; e) or a PKA inhibitor Rp-cAMP (50 ⁇ M; f).
  • cAMP levels are assessed in transduced cultures using a competitive immunoassay. Results are the mean ( ⁇ SEM) of at least 8 experiments, each carried out in triplicate,
  • DRGs transduced with EIAV-LacZ or EIAV-RAR ⁇ 2 vectors are co-cultured with transduced or na ⁇ ve spinal cord,
  • Neurite extension as detected by GAP43 staining red
  • EIAV-LacZ DRG neurons green
  • EIAV-RAR ⁇ 2 transduced spinal cord EIAV-LacZ neurons that are grown on the dorsal root (DR) projected fibers up to the DREZ but did not enter the spinal cord (SC) (indicated by white arrow).
  • the central and peripheral regions of the DREZ are delineated by GFAP staining in blue (marked by a white dotted line), (c) EIAV-RAR ⁇ 2 transduced DRG neurons (green) extended several neurites on EIAV-LacZ transduced cord and (d) neurons that are present in the DR projected extensions up to and beyond the central portion of the DREZ (indicated by white arrows). Neurons that are not transduced with EIAV-RAR ⁇ 2 do not project neurites on the spinal cord or beyond the DREZ (indicated by white arrowheads).
  • ⁇ -galactosidase expression is observed in the cuneate fasciculus and the dorsal horn of the spinal cord 3 weeks following infusion of EIAV-LacZ vector into the spinal cord,
  • Retrograde transport of the vector via the dorsal root resulted in ⁇ -galactosidase expression in the ipsilateral DRG.
  • RAR ⁇ 2 mRNA is detected in the ipsilateral DRG and not in the contralateral uninjected side (c & d respectively).
  • RAR ⁇ 2 protein is observed in the ipsilateral DRG and not in the contralateral DRG (e & f respectively) and in embryonic day 14 rat DRG (g).
  • ⁇ -galactosidase expression in the DRG is colocalized immunohistochemically with NF200 (marker for large-diameter myelinated neurons), CGRP (marker for small-diameter peptidergic neurons) or IB 4 (marker for small- diameter non-peptidergic DRG neurons), all indicated in green. Overlapping expression is illustrated in yellow. Scale bars: 200 ⁇ m (a-d); 50 ⁇ m (e-h).
  • FIG. 4 Axonal regeneration of injured sensory neurons across the DREZ
  • dorsal roots approach the cord from the left and the peripheral region of the DREZ is delineated by the presence of laminin (blue). Injured sensory afferents are labeled transganglionically with BDA tracer 10 days before termination of experiment, (a) At five weeks post-lesion, BDA-labeled axons (red) are present in the dorsal root up to, but not beyond, the DREZ in EIAV-LacZ animals, (b) By contrast, several BDA axons (indicated by white arrows) are observed in the DREZ after EIAV- RAR ⁇ 2 expression, (c) Quantification of axonal regeneration across the DREZ is assessed by counting the number of BDA fibers at respective distances from the DREZ (distance in graph represents peripheral part of the DREZ to the central region from left to right, with the DREZ denoted at 0
  • Results are the mean number of BDA fibers per section ( ⁇ SEM). *P ⁇ 0.05, two-way RM ANOVA, Tukey post-hoc), (d-f) NF200- (d), CGRP- (e) and IB 4 - (f) expressing afferents (green, indicated by white arrows) are detected central to the DREZ in EIAV-RAR ⁇ 2, however these afferents are undetectable in the DREZ in EIAV-LacZ animals (g-i respectively). Scale bar, 100 ⁇ m.
  • Peripheral afferent stimulation activates postsynaptic neurons in spinal cord Following noxious heat stimulation several Fos (a) and pERK (b) immunoreactive nuclei are detected in the laminae I-II of the dorsal horn (indicated by white arrows) in the spinal cord of EIAV-RAR ⁇ 2 animals. By contrast fewer Fos (c) and pERK (d) nuclei are detected in the EIAV-LacZ animals compared to unlesioned control animals (e, f).
  • the number of Fos- (g) and pERK (h) -positive nuclei is significantly higher in EIAV-RAR ⁇ 2 cords compared to EIAV-LacZ cords and EIAV-RAR ⁇ 2 cords with ablated dorsal roots. Results are expressed as mean number of immunoreactive nuclei per section ( ⁇ SEM). *P ⁇ 0.05, Students' 2-tailed unpaired ⁇ -test). Scale bars, 50 ⁇ m.
  • RAR ⁇ 2 promotes functional recovery after axonal regeneration
  • closed and open squares represent injured and uninjured forelimbs of EIAV- LacZ animals respectively
  • closed and open diamonds represent injured and uninjured forelimbs of EIAV-RAR ⁇ 2 animals respectively.
  • Animals are assessed in behavioral tasks before and for 4 weeks after rhizotomy.
  • RAR ⁇ 2 cDNA is previously isolated from a mouse brain cDNA library by PCR and cloned downstream of the human CMV promoter.
  • the transfer vector also contains a self-inactivating (SESf) LTR, the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a central polypurine tract (cPPT).
  • SESf self-inactivating
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • cPPT central polypurine tract
  • Control EIAV-LacZ contains the ⁇ -galactosidase gene in place of the RAR ⁇ 2 gene in an identical vector. Scale bars: 200 ⁇ m (a), 100 ⁇ m (b-e).
  • FIG. 8 Increased cAMP immunoreactivity in EIAV-RAR ⁇ 2 transduced DRG neurons.
  • cAMP immunoreactivity, indicated in green, is increased in ipsilateral DRGs of (a) EIAV- RAR ⁇ 2 treated animals compared to (b) control EIAV-LacZ animals and to (c, d) contralateral untransduced DRGs of the respective groups.
  • FIG. 10 Microarray and Q-PCR analyses demonstrate differential regulation of NCS-I in RAR ⁇ 2-transduced spinal cord.
  • EIAV .NCS-I promotes neurite outgrowth in adult neurons compared to control EIAV.LacZ in vitro.
  • Adult neurons from DRG (A, D), spinal cord (B, E) and cortex (C, F) are either transduced with control EIAV.LacZ (A-C) or EIAV.NCS-1 (D-F).
  • Vector expression of either ⁇ -galactosidase (1:300, Promega; Z3781) or NCS-I (1: 200, Abeam; ab 18060) is indicated in red while neurite outgrowth is detected by GAP 43 (1:1000, Chemicon; AB5220), a regeneration marker in green.
  • G The average length of the longest neurite is measured in neuronal cultures. Black bar; EIAV-LacZ transduced neurons, Blue bar; EIAV-NCS-I transduced neurons. Scale bar represents 100 ⁇ m.
  • the first aspect of the present invention relates to a system capable of delivering a nucleotide sequence to a target cell.
  • the vector system may be a non-viral system or a viral system.
  • Non-viral delivery systems include but are not limited to DNA transfection methods.
  • transfection includes a process using a non-viral vector to deliver a gene to a target mammalian cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or poly lysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • the vector system may be a viral vector system.
  • Viral vector or viral delivery systems include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors (including lentiviral vectors) and baculoviral vectors.
  • AAV adeno-associated viral
  • the vector system is a retroviral vector system.
  • retrovirus includes: murine leukemia virus (MLV), human immunodeficiency virus (HTV), 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 lentiviruses.
  • MMV murine leukemia virus
  • HTV human immunodeficiency virus
  • EIAV equine infectious anaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • FuSV Fujinami
  • 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 (ADDS), and the simian immunodeficiency virus (SrV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anaemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • the retroviral vector system is derivable from EIAV.
  • genomic structure of some lentiviruses may be found in the art.
  • details on HIV and EIAV may be found from the NCBI Genbank database (i.e. Genome Accession Nos. AF033819 and AF033820 respectively).
  • HTV variants may also be found at http ://hiv-web . lanl.gov.
  • Details of EIAV variants may be found through http://www.ncbi.nlm.nih.gov.
  • the sequences encoding retroviral Gag, Pol and Env proteins are introduced into the cell and stably integrated into the cell genome; a stable cell line is produced which is referred to as the packaging cell line.
  • 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.
  • the helper proteins can package the ⁇ /-positive recombinant vector RNA to produce the recombinant virus stock. This can be used to transduce a gene (i.e an NCS-I encoding gene) into recipient cells.
  • the present invention also provides a packaging cell line comprising a viral vector genome which is capable of producing a vector system 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 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. the env coding sequences, the gag-pol coding sequence and the defective retroviral genome containing the NCS-I -encoding gene into the cell at the same time by transient transfection and the procedure is referred to as transient triple transfection (Landau & Liftman 1992; Pear et al 1993).
  • the triple transfection 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 transfection method.
  • 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 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 of the present invention.
  • the retroviral vector system of the present invention is a self-inactivating (SIN) vector system (see, e.g., US Patents 6,924,123 and 7,056,699).
  • SI self-inactivating
  • 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 Pl 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 Pl (see PCT/GBOO/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 a DRG).
  • Producer cell lines are usually better for large-scale production of vector particles.
  • Transient transfection has numerous advantages over the packaging cell method.
  • transient transfection 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 transfection can be used to produce the vector before the cells die.
  • cell lines have been developed using transient infection which produces 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.
  • 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, czs-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, czs-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. As already mentioned, a summary of the available packaging lines is presented in "Retroviruses" (Coffin et al., supra).
  • simple packaging cell lines comprising a provirus in which the packaging signal has been deleted
  • second generation cell lines have been produced wherein the 3'LTR of the provirus is deleted.
  • 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 transfection.
  • 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.
  • 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 NCS-I at the target site.
  • cPPT central polypurine tract
  • This cis-acting element is located, for example, in the EIAV polymerase coding region element.
  • 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.
  • a primate lentivirus minimal system can be constructed which requires none of the HTV7SIV additional genes vif, 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.
  • additional genes is highly advantageous. Firstly, it permits vectors to be produced without the genes associated with disease in lentiviral (e.g. HTV) 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.
  • rev and RRE sequence should be included.
  • codon optimisation see below
  • replacement with other functional equivalent systems such as the MPMV system.
  • expression of the codon optimised gag- ol 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.
  • a viral vector system in a preferred embodiment lacks the Rev response element (RRE).
  • RRE Rev response element
  • the 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.
  • the 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 of 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.
  • gag- ol 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-I 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 HTV-I variants may be found at http ://hiv-web. lanl. gov. 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, HTV-I and HTV-2. In addition this method could be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV and human endogenous retroviruses (HERV), MLV and other retroviruses. Codon optimisation can render gag-pol expression Rev independent. In order to enable the use of anti-rev or RRE factors in the retroviral vector, however, it would be necessary to render the viral vector generation system totally Rev/RRE independent. Thus, the genome also needs to be modified. This is achieved by optimising vector genome components. Advantageously, 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.
  • codon optimisation can, of course, be applied to all or some parts of the system of the present invention.
  • 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 incorporating 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.
  • LCMV lymphocytic choriomeningitis virus
  • a viral vector system in accordance with the first aspect of the invention may be pseudotyped with any heterologous env protein.
  • the vector system may comprise a protein (or a mutant, variant, homologue or fragment thereof) from a virus which is capable of travelling by retrograde transport.
  • the system may comprise a protein from a rabies virus, herpes virus, adenovirus or from Ebola virus.
  • the vector system is a viral vector system, it may be pseudotyped with the envelope protein from such a virus.
  • the vector system is pseudotyped with at least a part of a rabies G protein or a mutant, variant, homologue or fragment thereof.
  • vector systems comprising Rabies G protein or a part thereof are capable of transducing (tyrosine hydroxylase) TH positive neurons - a subset which are difficult to transduce with conventional vectors (see WO02/36170).
  • Ca 2+ sensing proteins including the neuronal calcium sensor (NCS) proteins
  • NCS neuronal calcium sensor
  • Change their conformation on Ca 2+ binding which allows them to modulate the interaction with their targets, and thus act as effector molecules to transduce Ca 2+ signals into appropriate downstream events.
  • Neuronal calcium sensor- 1 (NCS-I), the mammalian ortholog of Drosphila frequenin, is a recently cloned and characterised member of the neuronal calcium sensor protein family (Burgoyne and Weiss, 2001 Biochem J 353:1-12).
  • This 22 kDa protein is highly conserved across species, with 100% amino acid sequence homology between rat, mouse, human, chicken and Xenopus species and only 25% divergence with C. elegans, or 28% with yeast (De Castro et al 1995 Biochem Biophys Res Commun 216: 133-140), suggesting a fundamental and highly conserved function (Burgoyne and Weiss, 2001 as above).
  • NCS-I is neuron specific, localizing in the cell bodies, dendrites and axons throughout the brain, spinal cord, dorsal root ganglia and peripheral nerves (Martone et ah, 1999 Cell Tissue Res 295:395-407; Olafsson et ah, 1997 Proc Natl Acad Sci U S A 92:8001-8005.; Averill et al., 2004 Neuroscience 123:419-27).
  • NCS-I has been shown to be present spatiotemporally in the embryonic rat spinal cord and olfactory system during development and to co-localise with GAP 43, a growth and regeneration marker (Kawasaki et ah, 2003. J Comp Neurol 460:465-475. ;Treloar et al., 2005. J Comp Neurol 482:201-216).
  • NCS-I has also been shown to co-localise spatiotemporally with synaptophysin, a synapse marker, in embryonic spinal cord, olfactory system and retina (Kawasaki et ah, 2003 as above;Pongs et a 1993.
  • NCS-I Xenopus frequenin overexpressed in spinal neurons resulted in a general enhancement of synaptic efficacy of the neuromuscular junction (Olafsson et ah, 1995 Proc Natl Acad Sci U S A 92:8001-8005).
  • Overexpression of NCS-I created acute short-term plastic changes in the synapses of hippocampal cells, unmasking somnolent synapses (Sippy et ah, 2003 Nat Neurosci 6:1031-1038).
  • Overexpression of NCS-I has been suggested to enhance associative learning and memory in Caenorhabditis elegans (Gomez et al (2001) Neuron 30; 241-248; EP 1250931).
  • NCS-I has been implicated in enhancing synaptic transmission and there has been interest in its possible mechanism of action (Hilfiker (2003) Biochem Soc Trans 31:828-
  • NCS-I Mammalian NCS-I is thought to interact with a phophatidylinositol 4-hydroxykinase (type III PI4K ⁇ ) and stimulate its activity (Hilfiker as above). PI4K ⁇ is thus thought to be an in vivo downstream target of NCS-I, and NCS-I may act by modulating the levels of phosphoinositides .
  • NCS-I possesses four EF-hand motifs (EF1-EF4), each consisting of a 12-amino acid loop within which Ca2+ is coordinated with ⁇ -helices on either side.
  • neuroneuronal calcium sensor-1 or “NCS-I” in the context of the present invention includes mutants and fragments of the wild-type protein as long as they retain the capacity of wild-type protein to stimulate neurite outgrowth.
  • retinoic acid ⁇ 2 or “RAR ⁇ 2” in the context of the present invention includes mutants and fragments of the wild-type protein as long as they retain the capacity of wild- type protein to stimulate neurite outgrowth.
  • wild type is used to mean a polypeptide having a primary amino acid sequence which is identical with the native protein (i.e. NCS-I or RAR ⁇ 2 from the subject species).
  • 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).
  • the mutant has at least 90%, 95% or 98% sequence identity (or homology) 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.
  • 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).
  • 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.
  • sequences may 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 derivative
  • 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, which will be well understood by those skilled in the art.
  • 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.
  • NCS-I has 190 amino acids.
  • a system of the present invention encoding a fragment of NCS-I preferably encodes at least 150, 160, 170, 180 or 185 amino acids of the wild-type sequence.
  • NCS-I is an N-terminally myristoylated protein that contains four EF-hand motifs, three of which are capable of binding Ca 2+ in the submicromolar range.
  • the fragment may contain at least three of the EF hands, the fragment may also contain the N-terminal myristoylation consensus sequence.
  • Human RAR ⁇ 2 has two isoforms: isoform 1, having 448 amino acids; and isoform 2, which lacks an exon in its 5' region so that translation begins at a downstream, in-frame start codon.
  • the encoded protein is shorter at the N terminus than isoform 1, having 336 amino acids.
  • a system of the present invention encoding a fragment of RAR ⁇ 2 preferably encodes at least 250, 175, 300, 325 or 330 amino acids of the wild-type sequence.
  • the mutant or fragment of NCS-I or RAR ⁇ 2 should be capable promoting neurite outgrowth when delivered to or expressed in a neuron.
  • the system of the present invention may comprise a nucleotide sequence encoding NCS-I and/or RAR ⁇ 2 (which term includes a mutant or fragment of the wild-type sequence).
  • the nucleotide sequence may be 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 is, comprises, or is transcribed from cDNA.
  • the system is capable of causing the target cell to encode both NCS-I and RAR ⁇ 2, it may comprise a separate construct (e.g. plasmid or vector) for each sequence or it may comprise a single construct capable of expressing both sequences.
  • the two or more genes may be operably linked by one or more internal ribosome entry sequences (IRES(s)).
  • IRES internal ribosome entry sequences
  • IRESs are an efficient way to co-express multiple genes from one vector
  • other methods are also useful, and may be used alone or in conjunction with IRESs. These include the use of multiple internal promoters in the vector (Overell et al., MoI Cell Biol. 8: 1803-8 (1988)), or the use of alternate splicing patterns leading to multiple RNA species derived from the single viral genome that expresses the different genes. This strategy has previously been used by itself for two genes (Cepko et al. Cell 37: 1053 (1984)).
  • sequence(s) may be or correspond to the wild-type nucleotide sequence(s) (i.e. the endogenous sequence encoding NCS-I and/or RAR ⁇ 2 in the subject species or the corresponding mRNA or cDNA).
  • wild-type nucleotide sequence(s) i.e. the endogenous sequence encoding NCS-I and/or RAR ⁇ 2 in the subject species or the corresponding mRNA or cDNA.
  • overexpression of NCS-I promotes neurite outgrowth.
  • overexpression is achieved by transducing a target cell with a NCS-I -encoding gene.
  • Overexpression of NCS-I may alternatively be achieved by delivering a factor (or a nucleotide sequence encoding a factor) which enhances the transcription or translation of NCS- 1.
  • the present invention also provides the use of such a system to promote neurite outgrowth.
  • the system may deliver a factor which for example is or encodes a transcription factor.
  • Another option is to activate NCS-I rather than cause its over expression. "Activating" factors may be necessary for NCS-I to become active, or may accentuate its activity.
  • the present invention also provides the use of a system capable of delivering an NCS-I activating factor to a target cell to promote neurite outgrowth.
  • the "activating" factor may, for example, be an anti-NCS-1 antibody, a ligand binding molecule, a calcium mimetic or a derivative of a calmodulin activator.
  • the spinal cord includes nerve cells, or neurons, and long nerve fibres called axons. Axons in the spinal cord carry signals downward from the brain (along descending pathways) and upward toward the brain (along ascending pathways). Dendrites are branched extensions of neurons that may receive signals from other nerve cells.
  • Neurite outgrowth is the process by which the neuron grows out axons and dendrites, in order to form functional networks with surrounding cells and other neurons.
  • NCS-I promotes both neurite outgrowth and process production by neurons.
  • the target cell for delivery of an NCS-I expressing gene may be a neuron, in particular an adult neuron.
  • An adult neuron is a substantially terminally differentiated neuron.
  • the system may, for example deliver the NCS-I encoding gene to the dorsal root ganglion, spinal cord or to a cortical neuron.
  • the system may activate regeneration processes in neurons, for example, injured neurons. Injured (or damaged) neurons include diseased neurons.
  • stimulation of neuronal growth may improve neural function after disease or injury to the nervous system: i) to promote the regeneration of axons that have been damaged so that they reestablish functional connections and thereby restore some of the lost function; and ii) to promote the sprouting (growth) of intact neurons that have survived or been spared by the injury or disease and the establishment of functional connections by these sprouts to restore some of the lost function (promoting synaptic plasticity).
  • the system of the present invention may be used to treat and/or prevent a condition which results in, or is likely to cause, neuron damage. It may be used to treat and/or prevent a condition which is likely to be prevented or ameliorated by neuron generation or regeneration.
  • the system may be used to treat one or more of the following conditions: spinal cord injury, avulsion injury, brachial plexus injury, traumatic brain injury, stroke, and a neurodegenerative disease.
  • a spinal cord injury usually begins with a sudden, traumatic blow to the spine that fractures or dislocates vertebrae.
  • the damage begins at the moment of injury when displaced bone fragments, disc material, or ligaments bruise or tear into spinal cord tissue. Most injuries to the spinal cord don't completely sever it. Instead, an injury is more likely to cause fractures and compression of the vertebrae, which then crush and destroy the axons, extensions of nerve cells that carry signals up and down the spinal cord between the brain and the rest of the body.
  • An injury to the spinal cord can damage a few, many, or almost all of these axons. Major damage can result in complete paralysis.
  • Avulsion injury is a nerve injury in which traction produces a ripping of the nerve roots.
  • Major causes include automotive (in particular motorbike) accidents, lacerations, gunshot wounds and nerve cancer.
  • the brachial plexus is a network of spinal nerves that originates in the back of the neck, extends through the axilla (armpit) and gives rise to nerves to the upper limb. Injuries to the brachial plexus affect the nerves supplying the shoulder, upper arm, forearm and hand, causing numbness, tingling, pain, weakness, limited movement or even paralysis of the upper limb. Although injuries can occur at any time, many brachial plexus injuries happen during birth. The baby's shoulders may become impacted during the birth process, causing the brachial plexus nerves to stretch or tear. Brain injury can occur in many ways. Traumatic brain injuries typically result from accidents in which the head strikes an object.
  • Traumatic brain injury can significantly affect many cognitive, physical, and psychological skills. Physical problems can include ambulation, balance, coordination, fine motor skills, strength, and endurance.
  • a stroke also referred to as a cerebrovascular accident or CVA
  • CVA cerebrovascular accident
  • Neurodegenerative diseases are hereditary and sporadic conditions characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral nervous system structures.
  • Neurodegenerative diseases include: Lewy Body Disease, Motor Neuron Disease, Multiple System Atrophy, Parkinsons Disease, Postpoliomyelitis Syndrome, Prion Diseases, Shy- Drager Syndrome, Cockayne Syndrome, Huntington Disease, Lafora Disease, Neurofibromatoses, Tourette Syndrome, Tuberous Sclerosis Amyotrophic Lateral Sclerosis, Creutzfeldt- Jakob Syndrome, Kuru and Scrapie Alzheimer Disease.
  • the present invention also provides the use of a system in the manufacture of a pharmaceutical composition.
  • the pharmaceutical composition may be used to deliver NCS-I and/or a nucleotide sequence expressing NCS-I to a target cell in a subject.
  • the system may be a delivery system, such as a non-viral delivery system or a viral 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 subject.
  • 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 vector system used in the present invention may be administered by direct injection into the subject.
  • it may be injected directly into the CNS.
  • the vector system may be administered to a site which is distant to the CNS and may then travel to the CNS by retrograde transport.
  • NCS-I protein For administration of NCS-I protein, there are many available systems known in the art.
  • US 2005/0137134 describes a method for infusion to the putamen (within the brain) using an implantable pump, and also makes reference to many other known drug delivery apparatus, catheters and combinations thereof which have been developed for dispensing medical substances to specific sites in the body.
  • Laske et al (1997 - J. Neurosurg. 87:586-594) also describes a method for interstitial infusion to the brain.
  • the present invention also provides a method for delivering NCS-I, or a nucleotide sequence capable of encoding NCS-I, to a cell in vitro using a system of the invention.
  • the cell may, for example, be a supporting cell such as an olfactory ensheathing cell or a Schwann cell.
  • the cell may be implanted into a subject, for example to promote neuron regeneration.
  • the cell may be implanted into the CNS of the subject.
  • the cell may be introduced into the spinal cord of the subject.
  • the cell to which NCS-I or its encoding sequence is delivered may be derivable from the subject (making implantation an ex vivo procedure), or it may be from an alternative source (such as from a suitable donor or a cell line etc).
  • Example 1 Exogenous delivery of RAR ⁇ 2 into adult rat DRG neurons promotes axonal regeneration
  • RAR ⁇ 2 has been shown to activate neurite outgrowth in embryonic DRG neurons and adult mouse spinal cord explants (Concoran et al (2000) J. Cell Sci 113:2567-2574; (2002) J. Cell Sci. 115:3779-3786).
  • a)RAR ⁇ 2 enhances neurite outgrowth in DRG neurons via activation of cAMP
  • EIAV minimal equine infectious anemia virus
  • a minimal equine infectious anemia virus (EIAV)-based lentiviral vector (Mazarakis et al (2001) Hum MoI Genet 10:2109-21; Wong et al (2004) MoI Ther 9:101-11) expressing RAR ⁇ 2 (EIAV- RAR ⁇ 2) is constructed.
  • EIAV- RAR ⁇ 2 a minimal equine infectious anemia virus
  • ⁇ -galactosidase ⁇ -gal
  • EIAV-RAR ⁇ 2 neurons show decreased neurite lengths (Figs. Ie & h).
  • DDA 5'- dideoxyadenosine
  • EIAV-RAR ⁇ 2 neurons show decreased neurite lengths (Figs. Ie & h).
  • This blocking of RAR ⁇ 2-enhanced regeneration is dose-dependent and at higher DDA concentrations (50 and 100 ⁇ M) neurite outgrowth is completely abrogated.
  • PKA cAMP-dependent protein kinase
  • b)RAR ⁇ 2 induces axonal regeneration on the nonpermissive spinal cord Transduction of DRG neurons in vitro thus promotes neurite outgrowth on a minimal substrate.
  • the CNS environment contains inhibitory factors such as myelin and its derivatives that can restrict regeneration.
  • the following example investigates neurite outgrowth in a co-culture system. After adult rat spinal cord and DRG neurons are transduced with either EIAV-LacZ or EIAV-RAR ⁇ 2 in vivo, the transduced DRG neurons are grown on spinal cord sections which provided an inhibitory substrate for neurite outgrowth.
  • EIAV-LacZ transduced DRG neurons grown either on na ⁇ ve spinal cord or on EIAV-RAR ⁇ 2 transduced spinal cord do not extend many neurites on the spinal cord (Fig. 2a). In the occasional neuron that settles on the peripheral section of the dorsal root, neurite outgrowth is observed but these projections extended up to the DREZ and do not extend into the spinal cord (Fig. 2b). By contrast, EIAV-RAR ⁇ 2 neurons grown either on na ⁇ ve or on EIAV-LacZ cord often extend projections on the spinal cord (Fig. 2c). Furthermore neurons that are grown on the dorsal root project neurites up to and beyond the DREZ into the spinal cord (Fig. 2d).
  • RAR ⁇ 2 through the induction of cAMP, stimulated neurite outgrowth of DRG neurons, it does not address regeneration in vivo.
  • the following example is to determine if RAR ⁇ 2 expression is sufficient to promote regeneration of injured sensory axons from the dorsal root into spinal cord to establish functional connectivity.
  • Adult DRG neurons express low levels of RAR ⁇ 2 mRNA and protein and in order to deliver RAR ⁇ 2 to the DRG neurons, lentiviral vectors are used. High gene transfer efficiency to DRG neurons in vivo is obtained at three weeks after unilateral injection of 3 - 6 x 10 6 transducing units of rabies-G pseudotyped EIAV-LacZ into the spinal cord.
  • the transduction efficiency of DRG neurons are as follows: 46 ⁇ 2% co-expressed NF200, 33 ⁇ 5% co-expressed CGRP and 22 ⁇ 7% expressed IB 4 .
  • EIAV vectors pseudotyped with the rabies-G glycoprotein can mediate efficient gene transfer to all classes of DRG neurons after injection into the spinal cord. Injection of the EIAV vector into the spinal cord does not induce a significant inflammatory response, as is evident by normal Nissl staining and the lack of increased markers for macrophages (0X42), microglia (EDl) and T cells (CD8) (Fig. 7). Furthermore, no increased vascular density in the injected spinal cord is observed (Fig. 7e).
  • e " Regenerated axons enhanced post-synaptic activity of second-order neurons in the spinal cord
  • the regenerating sensory fibers need to find and activate the appropriate second-order neuronal targets in the spinal cord.
  • Noxious heat stimuli is applied to the forelimbs of both EIAV-LacZ and EIAV-RAR ⁇ 2 treated animals after injury to test for such functional activation.
  • Noxious heat stimuli to forelimbs normally induces c-fos activation and phosphorylation of the extracellular signal-regulated kinase ERK in post-synaptic neurons in superficial laminae of the dorsal horn Hunt et al (1987) Nature 328: 632-4).
  • EIAV-RAR ⁇ 2 treated animals with ablated dorsal roots are not significantly different from EIAV-LacZ treated animals (Figs. 5g, h).
  • Adhesive tape-removal tasks assessed sensory (awareness of the tape) and motor (ability to remove tape) function.
  • the injured forelimb of EIAV-LacZ rats shows significantly increased latencies in sense and removal tasks (50.7 + 18.1s and 54.0 + 16.4s); in contrast EIAV-RAR ⁇ 2 treatment produces recovery of function for sense and removal tasks (6.3 ⁇ 4.2s and 16.5 ⁇ 4.4s; P ⁇ 0.05 2-way RM ANOVA followed by Tukey's post-hoc test, Figs. 6a, b).
  • the lesioned forelimb of EIAV-LacZ treated rats is significantly impaired in its ability to reach and grasp food pellets in a staircase apparatus (Montoya et al (1991) J. Neurosci Methods 36:219-28).
  • EIAV-RAR ⁇ 2 In comparison the lesioned forelimb of EIAV-RAR ⁇ 2 demonstrates significant improvement (food displacement scores were: 0.8 + 0.5 (EIAV-LacZ injured) and 2.8 + 0.9 (EIAV-RAR ⁇ 2 injured), and 3.3 ⁇ 0.7 (EIAV-LacZ control) and 4.5 ⁇ 1.1 (EIAV-RAR ⁇ 2 control); P ⁇ 0.05 2-way RM ANOVA followed by Tukey's post-hoc test, Fig. 6c). To test for locomotion the number of footslips made by the forelimbs are recorded when rats crossed a horizontal ladder or a narrow beam.
  • EIAV vector genomes are constructed from pSMART2 lentiviral vectors as previously described (Bienemann et al (2003) MoI Ther 7:588-96).
  • the RAR ⁇ 2 or LacZ gene is inserted under the control of a minimal hCMV promoter in an EIAV transfer vector containing a 5' cPPT element and a 3' WPRE enhancer (vector map illustrated in Supplementary Fig. If).
  • Viral vector stocks pseudotyped with the rabies-G envelope glycoprotein are prepared by triple plasmid transient transfection of HEK293T cells as previously described (Mazarakis et al (2001), as above; Azzouz et al (2002) J Neurosci 22:10302-12; Bienemann et al (2003) as above; Mitrophanous et al (1999) Gene Ther 6: 1808-18) .
  • the titer of EIAV-LacZ is determined by transduction of dog osteosarcoma Dl 7 cells (4 x 10 8 TU ml 4 ) while the titer of EIAV-RAR ⁇ 2 (8 x 10 s TU ml "1 ) is calculated by determining the normalized viral RNA genome copy number using real-time quantitative polymerase chain reaction analysis and comparing it to EIAV-LacZ as previously described (Martin-Rendon et al (2002) MoI Ther 5: 566-570).
  • DRG cultures are prepared as previously described (Gavazzi et al (1999) J Neurosci 11: 3405-14).
  • Adult male Wistar rats (200-25Og) are sacrificed according to institutional and UK Home Office Regulations and the DRGs are dissected and transferred to Ham's F12 medium (Gibco, UK).
  • DRGs are desheathed, their roots trimmed and then digested in 0.125% collagenase (Sigma, UK) at 37 0 C for 2 hr, after which they are mechanically dissected by trituration with a PlOOO Gilson pipette in ImI modified Bottenstein and Sato's culture medium (BS) in Ham's F12.
  • the resulting cell suspension is centrifuged at 600 rpm for 8 min through a cushion of 15% bovine serum albumin (BSA, Sigma).
  • BSA bovine serum albumin
  • the dissociated neurons are resuspended in lOO ⁇ l of calcium- and magnesium-free HBSS (Gibco) containing 50 ⁇ g/ml DNase (Type I, Sigma) and 250 ⁇ g/ml soybean trypsin inhibitor (Type II, Sigma) and diluted in modified BS culture medium to a final concentration of approximately 1600 cells/ml.
  • Cells (500 per well) are plated in each well per eight-well plate (Labtek), which are precoated with poly-L-lysine (2 mg/ml; Sigma) and O.l ⁇ g/ml solution of EHS laminin (Sigma) for at least 2 hr at 37 0 C prior to plating.
  • Neurite outgrowth from dissociated DRG neurons is affected by low cell density and low laminin concentrations (data not shown).
  • Neuronal cultures are incubated at 37 0 C in a humidified atmosphere containing 5% CO 2 .
  • Viral transduction is carried out by adding the appropriate viral vectors at a multiplicity of infection (MOI) of 10 at DIVO.
  • MOI multiplicity of infection
  • cultures are incubated from DIVl in 2', 5'-dideoxyadenosine (DDA, Sigma) or Rp-adenosine-3 ',5 '-cyclic monophosphorothioate (sodium salt, Rp- cAMP, Sigma) at the concentrations indicated.
  • neurons are either harvested for measurement of cAMP using a competitive immunoassay, according to manufacturer's instructions (Amersham, UK), or fixed for 30 min in 4% paraformaldehyde for assessment of neurite outgrowth.
  • Cells are permeabilized with methanol at -2O 0 C for 3 min and washed with PBS.
  • Cells are incubated at room temperature for at least 2 hr with a combination of mouse ⁇ m tubulin (1:1000) with either rabbit ⁇ -galactosidase (1:300, Europa Biolabs) or rabbit RAR ⁇ (1:50, Santa Cruz). After further rinsing in PBS, the cultures are incubated at room temperature for 1 hr with a mixture of Alexa488 (1:1000, Molecular Probes) and Alexa546 (1:1000, Molecular Probes). Following further PBS washes, cells are mounted with FluorSaveTM reagent (Calbiochem, UK) and observed under a Zeiss microscope. The length of the longest neurite for the first 150-220 neurons encountered when scanning the slide in a systematic manner is determined using an image analysis program (SigmaScan Pro 4.01), and expressed as mean length + SEM (Gavazzi et al (1999) as above).
  • transduced DRG neurons are prepared for culture as described above.
  • Cryosections of the spinal cord are prepared according to the method of Golding et ⁇ /.(1999-Glia 26:309-323). Briefly, the transduced or naive cervical cord is removed and placed between two sterile glass plates on dry ice. The weight of the glass plate is sufficient to gently compress the cord so that cut longitudinal sections contained the dorsal root and the dorsal root surface of the spinal cord in the same plane.
  • mice ⁇ -galactosidase (1: 600, Promega)
  • mouse RAR ⁇ (1: 300, Chemicon)
  • rabbit GAP43 (1: 1500, Chemicon)
  • goat GFAP (1 : 300, Santa Cruz).
  • the cultures are incubated at room temperature for 1 hr with a mixture of Cy3 (1: 600), AMCA (1: 300) and FITC (1:300).
  • Animals are irreversibly anaesthetized and perfused transcardially with heparinised 0.9% NaCl solution and 4% paraformaldehyde in 0.1 M phosphate buffer.
  • the cervical cords with attached DRGs are dissected, frozen and sectioned (20 ⁇ m) on a cryostat.
  • in situ hybridization is carried out using non-radioactive ribroprobes specific to RAR ⁇ 2 as previously described (Zelent et al (1989) Nature 339: 714-7; Rattray and Michael "In situ hybridization-a practical approach” 1998) and development is performed after 2 h hybridization.
  • Sections are analyzed by X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) staining and immunohistochemistry.
  • RAR ⁇ is also detected in 20 ⁇ m sections of embryonic day 14 DRGs.
  • Primary antibodies are used as follows: mouse NF200 (1: 400, N52, Sigma), rabbit CGRP (1:8000, Sigma), Griffonia simplicifolia Isolectin B4 (10 ⁇ g/ml, Sigma), rabbit ⁇ - galactosidase (1:300, Europa Labs), rabbit RAR ⁇ (1:50, Santa Cruz), rabbit laminin (1 :200, Sigma), rabbit Fos (1:10000, Oncogene Sciences) and rabbit pERK (1:200, Sigma).
  • inflammatory response markers For inflammatory response markers (Supplementary Fig. 1), antibodies used are NeuroTrace fluorescent Nissl (1:100, Molecular Probes), 0X42 (1:100, Chemicon), EDl (1:1000, Chemicon), CD8 (1:100, Serotec) and Glutl (1:1000, Serotec).
  • cAMP staining (Supplementary Fig. 2) is performed with rabbit anti-cAMP (1:1000, Chemicon).
  • Extra- avidin conjugated to fluorescein isothiocyanate (1:200, Sigma) is used to detect BDA- labeled axonal tracts.
  • Quantitative analysis of axonal regeneration is carried out by counting BDA-labeled fibers at measured intervals within a 1-mm square grid graticule in the peripheral and central regions of DREZ (delineated by laminin staining) by a blinded experimenter. At least 36 random sections are counted for each animal. For transduction efficiency, immunopositive cells are counted in 6 sections per animal and expressed as a percentage of ⁇ -galactosidase positive cells. For Fos and pERK quantification, immunopositive cells are counted in 36 random sections per animal.
  • the paw reaching test assesses the rats' ability in reaching and grasping food pellets in a staircase apparatus, and measures side bias, maximum forelimb extension and grasping skill (Montoya et al (1991) J Neurosci Methods 36:219-228). Rats are placed in the staircase apparatus for 10 min and food displacement is scored.
  • rats are trained to cross a horizontal beam (2.5 cm X 100 cm) or ladder (18 cm X 100 cm with each rung 5 cm apart) and the number of forelimb footslips (off the beam or below the plane of the ladder) is recorded, hi footprint analysis (adapted from Kunkel-Bagden et al (1993) as above), the forepaws are covered with ink to measure walking patterns during continuous locomotion across a wooden runway, and stride lengths and widths are calculated. Behavioral responses between treated and control groups are compared using 2-way RM ANOVA followed by Tukey post hoc test.
  • Example 2 - NCS-I expression is differentially regulated by overexpression of RAR ⁇ 2 in adult spinal cord.
  • the results of Experiment 1 suggest that RAR ⁇ 2 can activate regeneration programs in injured neurons to promote axonal outgrowth.
  • microarray analyses are performed to identify genes that are differentially regulated by overexpression of RAR ⁇ 2 in adult spinal cord.
  • NCS-I is upregulated by 2.49+0.7 fold in RAR ⁇ 2-transduced cord compared to LacZ-transduced cord ( Figure 10).
  • lentiviral vectors encoding NCS-I are constructed.
  • Rat NCS-I is PCR amplified from a rat brain cDNA library and cloned into EIAV transfer vectors.
  • Lentiviral vectors encoding NCS-I are prepared via a standard 3-plasmid transient transfection method and titres are determined at 5.8 x 10 8 integration units/ml.
  • EIAV-NCS-I is used to transduce various types of adult neurons (dorsal root ganglion, spinal cord and cortical).
  • NCS-I overexpression enhances neurite outgrowth compared to control LacZ transduced neurons transduced neurons ( Figures 1 IA-F). Significantly longer neurites are observed in NCS-I transduced neurons compared to control neurons ( Figure HG; PO.001, Student's t-test, from 3 independent experiments).
  • overexpression of NCS-I in adult dorsal root ganglion (DRG) neurons increases the percentage of process bearing neurons to 53.2 ⁇ 5.0% from 23.5 ⁇ 4.6 % in control cultures.
  • DRG dorsal root ganglion
  • NCS-I Overexpression of NCS-I on its own, via a lentiviral vector, promotes neurite outgrowth in adult DRG, spinal cord and cortical cultures. Neurons are known to have limited intrinsic capacity to regenerate in the adult central nervous system. Overexpression of NCS-I, on its own or as part of the signalling cascade initiated by RAR ⁇ 2, boosts this intrinsic growth capacity and stimulates neurite outgrowth.
  • Concentrated viral preparations of EIAV .NCS-I and EIAV .LacZ vectors are produced using a transient transfection system.
  • Viral vector stocks of EIAV .NCS-I and EIAV.LacZ, are produced by transient transfection of human embryonic kidney 293 T cells plated on 10 cm dishes (3.5 x 10 6 cells/dish). 3 DNA components; 2 ⁇ g vector plasmid, 2 ⁇ g of gag/pol plasmid (pONY3.1) and 1-2 ⁇ g of plasmid encoding envelope glycoprotein are added to a mix containing FuGENE 6TM and OptiMEM. Sodium butyrate (10 mM final concentration) is added after 16h transfection. Superaatants are harvested 24-42 hours after transfection and filtered.
  • the supernatants are ultracentrifugated at 6000 x g at 4 0 C for at least 18 hours, followed by ultracentrifugation at 50 000 x g at 4 0 C for 90 min. After the virus has been in formulation buffer for 2-3 hours at 4 0 C, it is aliquoted and stored at -8O 0 C.
  • the biological titres of the viral preparations are determined by transducing canine osteosarcoma cells. After 2-3 days incubation, the cells are incubated in 5-bromo-3- indolyl- ⁇ -D-galactosidase (X-gal) solution and blue colonies are counted.
  • RNA titres of the viral preparations are also calculated by determining the number of viral RNA genomes per ml of viral stock solution using quantitative PCR analysis. Vectors are also tested qualitatively (mycoplasma, endotoxin and sterility testing) prior to use in in vivo experiments.
  • NCS-I transduced neurons in culture can induce an enhanced neurite outgrowth on inhibitory substrates.
  • the present inventors have shown that NCS-I overexpressed in adult DRG and cortical neurons can significantly increase the length of neurites and stimulate neuritogenesis in culture.
  • NCS-I can reproduce similar results if grown on an inhibitory substrates such as on spinal cord cryosections (Golding et al., 1999) and on myelin substrate (Zheng et al., 2005) since these same DRG and cortical neurons in vivo will not extend axons into these inhibitory environments.
  • DRG are desheathed, attached roots trimmed away and placed into 0.125% collagenase for
  • the DRG neurons are mechanically dissociated and separated from debris by centrifugation in 15% bovine serum albumin solution.
  • the DRG neurons are in Bottenstein and Sato's medium until ready to plate onto either spinal cord cryosections or onto myelin substrate at a density of 300 neurons per well.
  • the adult cortical neurons are similarly prepared as follows. Briefly, the cortices are cut into 0.5 mm longitudinally sections using a Mcllwain tissue chopper, white matter trimmed away then dissociated in 2 mg/ml papain. The cortical neurons are mechanically dissociated and separated from debris by centrifugation in four 1-ml steps of Optiprep in B27/HibernateA medium. Fractions containing neurons are collected, washed and resuspended in B27/NeurobasalA medium, ready for plating at a density of approximately 2000 neurons per well.
  • Cover slips are either covered with spinal cords cryosections (10 ⁇ m) or with 25 ⁇ l of 4 ⁇ g/ml myelin solution and left overnight to dry before plating with neurons.
  • CNS myelin substrate CNS myelin extract are prepared in advance and stored at -8O 0 C until required.
  • EIAV .NCS-I pseudotyped with rabies virus (ERA) envelope is added to the media at MOI 100. It has been demonstrated that EIAV vectors pseudotyped with ERA mediate strong transduction of all types of DRG neurons and CNS neurons (Mazarakis et al., 2001; Yip et al., 2004, as above). The neurons are studied for up to a further 3 days in vitro before they are prepared for histology by fixation in 4% paraformaldehyde and permeabilised with cold methanol.
  • Outcome measures involve cultures double immunostained for GAP43 (growth and regeneration marker) and for PGP9.5 (pan neuronal marker) or for markers of specific subpopulations (NF200, P2X3, CGRP). For each time point and myelin concentration at least 100 neurons are assessed for: the length of the longest neurite, percentage of cells with neurites. Survival is also checked under different conditions.
  • NCS-I can restore sensory and motor function in a spinal cord injury model.
  • EIAV vector pseudotyped with a VSV-G envelope produce strong anterograde transgene expression (Mazarakis et al., 2001; Yip et al., 2004, as above).
  • SCI is induced by performing a bilateral dorsal column crush at the level of C4 using fine forceps.
  • regeneration of the descending motor system are assessed behaviourally, electrophysically and anatomically using established protocols. These include grid and beam walking, rearing, forelimb pellet retrieval (staircase test), forelimb grip strength, and footprint analysis, which are determined once per week (Kunkel-Bagden et al., 1993 as above).
  • NCS-I promotes sprouting of intact axons in adult animals.
  • VSV-G EIAV.NCS-1 or EIAV.LacZ is injected into the sensorimotor cortex, and the animals are subjected to weekly behavioural tasks, BDA tracing, electrophysiology and then sacrificed for histology as described in ci and ii, above.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un système capable d'amener sur une cellule cible un détecteur 1 de calcium neuronal (NCS-1), ou une séquence nucléotide codant NCS-1, de façon à favoriser l'excroissance des neurites. Ce système convient à la fabrication d'une composition pharmaceutique destinée au traitement d'un état tel que les lésions de la moelle épinière.
PCT/GB2006/003367 2005-09-13 2006-09-11 Systeme Ceased WO2007031727A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP06779383A EP1937820A2 (fr) 2005-09-13 2006-09-11 Systeme pour delivrer de neuronal calcium sensor-1 (ncs-1)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0518674.7 2005-09-13
GB0518674A GB0518674D0 (en) 2005-09-13 2005-09-13 System

Publications (2)

Publication Number Publication Date
WO2007031727A2 true WO2007031727A2 (fr) 2007-03-22
WO2007031727A3 WO2007031727A3 (fr) 2008-05-15

Family

ID=35221413

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2006/003367 Ceased WO2007031727A2 (fr) 2005-09-13 2006-09-11 Systeme

Country Status (3)

Country Link
EP (1) EP1937820A2 (fr)
GB (1) GB0518674D0 (fr)
WO (1) WO2007031727A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017162798A1 (fr) * 2016-03-23 2017-09-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Ciblage du capteur de calcium neuronal 1 pour le traitement du syndrome de wolfram
CN112852976A (zh) * 2021-03-17 2021-05-28 湖北省农业科学院畜牧兽医研究所 蛋鸡ncs1基因中与后期产蛋性状相关的分子标记及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE384133T1 (de) * 2000-03-30 2008-02-15 Oxford Biomedica Ltd Retinolsäure-rezeptor beta-2 und gentherapievektoren für die behandlung von neurologischen erkrankungen
EP1250931A1 (fr) * 2001-04-17 2002-10-23 F. Hoffmann-La Roche Ag Nouvelle utilisation du neuronal calcium sensor-1 (ncs-1) pour le traitement de troubles du système nerveux central et pour le développement d'agents thérapeutiques

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017162798A1 (fr) * 2016-03-23 2017-09-28 INSERM (Institut National de la Santé et de la Recherche Médicale) Ciblage du capteur de calcium neuronal 1 pour le traitement du syndrome de wolfram
US10639384B2 (en) 2016-03-23 2020-05-05 Inserm (Institut National De La Sante Et De La Recherche Medicale) Targeting the neuronal calcium sensor 1 for treating wolfram syndrome
CN112852976A (zh) * 2021-03-17 2021-05-28 湖北省农业科学院畜牧兽医研究所 蛋鸡ncs1基因中与后期产蛋性状相关的分子标记及其应用
CN112852976B (zh) * 2021-03-17 2023-10-31 湖北省农业科学院畜牧兽医研究所 蛋鸡ncs1基因中与后期产蛋性状相关的分子标记及其应用

Also Published As

Publication number Publication date
EP1937820A2 (fr) 2008-07-02
GB0518674D0 (en) 2005-10-19
WO2007031727A3 (fr) 2008-05-15

Similar Documents

Publication Publication Date Title
US20080131400A1 (en) Vector system
EP1337655B1 (fr) Vecteurs lentiviraux pour le traitement de maladies neurodégénératives
EP2292778B1 (fr) Utilisation de séquences lentivirales de structure triplex pour l'import nucléaire de séquences nucléotidiques
ES2634424T3 (es) Vector multicistrónico lentivírico
EP1080216B1 (fr) Systeme d'apport retroviral
WO2008071959A1 (fr) Vecteurs lentiviraux comprenant des micro-arn
JP2008303215A (ja) ベクターシステム
JP2008503230A (ja) 非組み込み型且つ非複製型組換えレンチウイルス、その調製および使用
US6800281B2 (en) Lentiviral-mediated growth factor gene therapy for neurodegenerative diseases
US6818209B1 (en) Retroviral delivery system
US10744162B2 (en) Genetically modified muscle cells which express neurotrophic factors
EP1813676B1 (fr) Vecteurs rétroviraux pseudotypés avec hémagglutination du virus de la grippe pour fournir des gènes
WO2007031727A2 (fr) Systeme
EP1392838B1 (fr) Pseudotypage de vecteurs hiv par des enveloppes de virus mokola
EP1425403B1 (fr) Vecteurs lentiviraux dans le traitement de la douleur
HUE028512T2 (en) Gene transfer to airway epithelial stem cell using RNA virus spike protein pseudotyped lentiviral vector
WO2004031390A1 (fr) Systeme de vecteur
JP2022525136A (ja) パーキンソン病を処置するための遺伝子治療組成物および方法
DE102020106710A1 (de) Wpre-mutantenkonstrukte, zusammensetzungen und zugehörige verfahren
Cattaneo Combinatorial gene therapy for epilepsy
GB2337520A (en) Retroviral delivery system
Levin Muscle Lim Protein in naïve and injured neurons
Golden Viral vector-mediated transduction of Schwann cells expressing a bifunctional neurotrophin facilitates myelination and axon growth after spinal cord injury

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2006779383

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2006779383

Country of ref document: EP