JP2006502240A - Vector system - Google Patents

Abstract

Use of a vector system to transduce a target site, the vector system being moved to the target site by diffusion, and the vector system comprising a rabies virus G coat protein or a mutant, variant thereof A homologue or fragment or a CVS coat protein or a mutant, variant, homologue or fragment thereof, or at least part thereof, and the target site is at least part of the central nervous system Use to. In a broad aspect, the present invention relates to a vector system that can provide retrograde transport of a substance of interest (“EOI”).

Description

The present invention relates to vector systems. In particular, the present invention delivers a substance of interest (“EOI”) — eg, a nucleotide sequence of interest (“NOI”) — to a target site, such as for the treatment of a disease affecting the central nervous system (CNS). To a vector system that can be

  In a preferred embodiment, the present invention relates to a viral vector system that can deliver a nucleotide sequence of interest (“NOI”) to a target site.

  In a further preferred aspect, the present invention relates to a lentiviral vector system capable of delivering a nucleotide sequence of interest (“NOI”) to a target site.

  More particularly, the present invention relates to retroviral vectors useful for gene therapy.

BACKGROUND OF THE INVENTION Gene therapy includes any one or more of, for example, the addition, substitution, removal, supplementation, manipulation, etc. of one or more nucleotide sequences at one or more target sites—such as target cells. If the target site is a target cell, such cell can be part of a tissue or organ. An overview on gene therapy is taught in Molecular Biology (Robert Meyers, Pub VCH, p556-558 et al.).

  As another example, gene therapy also provides a means by which any one or more of the following can be performed: replacement or complementation of defective genes by use of nucleotide sequences such as genes; pathogenic genes or gene products Removal; addition of new genes, for example creation of a more favorable phenotype; treatment of cancer by manipulating cells at the molecular level (Schmidt-Wolf, Schmidt-Wolf) 1994, Anals of Hematology 69; p273-279) or other conditions—eg treatment of immune, cardiovascular, nervous system, inflammatory or infectious diseases—antigen manipulation and / or Or induction of an immune response by introduction-eg gene Chin inoculation.

  In recent years, it has been proposed to use retroviruses for gene therapy. In essence, retroviruses are RNA viruses that have a different life cycle than lytic viruses. When a retrovirus infects a cell, its genome is converted to a DNA type. In other words, retroviruses are infectious agents that replicate via DNA intermediates. More details on retrovirus infection will be given later.

  Regarding the gene structure of the viral vector, the gene env encodes the virion surface (SU) glycoprotein and transmembrane (TM) protein, which is a complex that specifically interacts with the receptor protein of the cell. Form. This interaction ultimately causes the viral membrane to fuse with the cell membrane.

  The env protein can bind to the receptor without being cleaved, but the cleaving event itself is necessary to activate the potential fusion ability of the protein, and this activation is also dependent on the viral host cell. Necessary for intrusion. In general, both the SU protein and the TM protein are glycosylated at a plurality of sites, but TM is not glycosylated in some viruses represented by MLV.

  The SU protein and the TM protein are not necessarily required for the construction of virion particles having an outer skin, but play an important role in the invasion process. In doing so, the SU domain binds to the receptor molecule of the target cell—often a specific receptor molecule. It is believed that this binding event activates the potential membrane fusion inducing ability of the TM protein, and then the virus membrane and cell membrane are fused. In some viruses, particularly MLV, the split event—which results in the removal of a short part of the cytoplasmic tail of TM—is considered to play a key role in fully elucidating the fusion action of this protein. (Brody, et al., 1994, Journal of Byrology (J. Virol.) 68: p4620-4627, Rein, et al., 1994, Journal of Virology. (J. Virol.) 68: p 1773-1781). This cytoplasmic “tail” distal to the transmembrane segment of TM remains inside the viral membrane, and its length varies considerably with various retroviruses.

  Thus, the specificity of this SU / receptor interaction can determine the host range and tissue affinity of retroviruses. In some cases, this specificity may limit the potential transduction ability of the recombinant retroviral vector. Therefore, MLV has often been used in gene therapy experiments so far. A specific MLV with a coat protein of 4070A is called an amphotropic virus, because this coat protein "combines" with a phosphate transport protein that is conserved between humans and mice. Can also be infected. This transporter is ubiquitous and thus these viruses can infect many cell types. However, specifically targeting a limited number of cells may be considered beneficial, particularly from a safety standpoint. To this end, several research groups have engineered mice to specifically infect specific human cells against allotrophic retroviruses that normally infect only mouse cells, unlike those of both closely related species. is doing. That is, a recombinant retrovirus was produced by replacing the coat protein with an erythropoietin segment, and this was specifically bound to human cells expressing erythropoietin receptor on the surface, such as erythroid progenitor cells ( Maurik, Patel, 1997, “Molecular Biotechnology: Therapeutic Applications and Strategies, 1997—Wyley, Wyle. Inc.), p45 ".

  Replacement of the env gene with a heterologous env gene is an example of a technique or method called pseudotyping. Doing pseudotyping can provide one or more advantages. For example, in the case of a lentivirus, infection of this vector is limited only to cells expressing a protein called CD4 due to the env gene product of this HIV-derived vector. However, when the env gene of this vector is replaced with an env sequence from another RNA virus, this vector is considered to have a broader spectrum of infection (Verma, Sonia, 1997). , Nature 389: p239-242).

  More generally, delivering therapeutic molecules to the CNS has become an important challenge in the treatment of neurodegenerative diseases. Constraints to overcome include (i) the presence of the brain blood barrier, (ii) side effects associated with systemic administration, and (iii) instability of this molecule.

  For example, one problem with gene therapy in the treatment of Parkinson's disease is that the brain is a complex organ that is difficult to target (Raymon, HK et al. K. et al.) (1997), Experimental Neurology 144: p82-91). Common administration methods include injection of vectors into the striatum (Bilang-Bleuel et al. (1997) Proceedings of the National Academy of Sciences (Pro. Natl. Acad. Sci.) USA, 94: p8818-8823; Choi-Lundberg, et al. (1998), Experimental Neurology (Exp.Neur.) 154: p261-275). Or injection near the substantia nigra (Choi-Lundberg, et al. (1997) Science 275: p838-841; Mandel, et al. (1997) Ding Of the National Academy of Sciences (Pro. Natl. Acad. Sci. USA, 94: p14083-14088). For these parts of the brain, for example, their location and / or size is an obstacle and it is technically difficult to inject directly. Because the substantia nigra is deep in the brain, direct injection into this area can damage axons and cause injury. The striatum, especially the caudate nuclear putamen, is larger and more dorsal than the substantia nigra, making it a relatively easy target. It has been widely used for transplantation in Parkinson's disease, and it is currently believed that the risk factor for its enforcement is less than 1%. Similar problems exist for other parts of the CNS.

  Therefore, it is desirable to find a mechanism for transducing brain parts and other parts of the CNS that are difficult to reach by direct injection. It would also be desirable to find an administration method for head gene therapy that minimizes the number and complexity of intracerebral injections. In addition, it is also desirable to achieve sufficient penetration and distribution throughout the nervous system after administration.

  Although pseudotyping has been thought to be able to solve the above problems to some extent, the characteristics of transduction and expression of pseudotyped vectors have not yet been fully elucidated and there are still other improved vectors. Is needed.

  For example, in Mazarakis et al. (2001) Human Molecular Genetics 10 (19): p2109-2121, the lentiviral vector pseudotyped with VSV G is the injection site. It has been reported that surrounding muscle cells were transduced but did not produce expression in any of the spinal cord cells.

  World Patent No. 02/36170 discloses the use of wild type rabies virus G protein to achieve retrograde transport, particularly transduction for TH-positive nerves. We are able to achieve good biodistribution of the target substance (EOI) by a mechanism other than retrograde transport using rabies virus G protein and CVS (Challenge Virus Standard) coat protein I found. Thus, it should be understood that this allows targeting sites through administration sites other than those available by retrograde transport mechanisms. Without wishing to be bound by any theory, we believe that this high level of distribution can be achieved by a diffusion mechanism. In contrast, VSV G pseudotyping was found not to cause such biodistribution, confirming that the results presented herein were surprising. It should be understood that good biodistribution is important because access to various parts of the central nervous system can be made through limited administration sites. This is particularly beneficial when it is necessary to penetrate the EOI to sites that are not easily accessible. It was also found that a pseudotyped EIAV vector can provide particularly good effects.

  We have also found that CVS (countervirus standard) coat protein can be used to achieve retrograde transport and transduction of CNS cells. We are convinced that this is the first time that we have demonstrated the benefits of lentiviral pseudotyping using CVS proteins.

  In addition, we have found that pseudotyping using rabies virus G coat protein and CVS coat protein can provide special benefits in intrauterine administration or administration to newborns. In these cases, it has been found that good transduction can be achieved with muscle cells, which is surprising given the poor transduction in mature cells. It was also found that transport to motor and sensory nerves, such as retrograde transport, was improved. These results are particularly advantageous when treatment needs to be given early in life, for example in the case of spinal muscular atrophy.

DESCRIPTION OF THE INVENTION As a broad aspect, the present invention relates to a vector system capable of providing retrograde transport of a substance of interest (“EOI”).

  As used herein, the term “vector system” includes any vector capable of infecting recipient cells with EOI or effecting transduction or transformation or modification thereto. .

  The EOI can be a chemical compound, a biological compound, or a combination thereof. For example, the EOI can be a protein (such as a growth factor), a nucleotide sequence, an inorganic and / or organic drug (such as an analgesic, anti-inflammatory, hormonal, or lipid agent), or a combination thereof.

  The vector system of the present invention can deliver an EOI to a site, after which the EOI can be dispersed, eg, by diffusion or retrograde transport, and / or penetrated to a remote site.

  In general, the vector system can also include an EOI.

  In one aspect of the invention, the vector system is transferred to the target site by diffusion, and the vector system is a rabies virus G coat protein or mutant, variant, homologue or fragment thereof or CVS coat protein or mutant thereof, To transduce a target site, characterized in that it is or at least part of a variant, homologue or fragment, characterized in that the target site is at least part of the central nervous system The use of the vector system is provided.

  In another aspect of the invention, the vector system is at least part of a rabies virus G coat protein or mutant, variant, homologue or fragment thereof or CVS coat protein or mutant, variant, homologue or fragment thereof. Also provided is the use of a vector system comprising EOI to disperse EOI in the body, characterized in that it comprises.

  In yet another aspect of the invention, the vector system is transferred to the target site by retrograde transport, and the vector system is at least part of a CVS coat protein or a mutant, variant, homologue or fragment thereof, There is provided the use of a vector system for transducing a target site comprising any of these and further characterized in that the target site is at least part of the central nervous system.

  In yet another aspect of the present invention, the vector system is at least part of a rabies virus G coat protein or mutant, variant, homologue or fragment thereof or CVS coat protein or mutant, variant, homologue or fragment thereof. There is provided the use of a vector system for transducing an in utero target site or a neonatal target site, characterized in that it comprises or includes it.

  The vector system can be non-viral or viral or a combination thereof. Furthermore, the vector system itself can be delivered by viral or non-viral methods.

  Viral vectors or viral delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes simplex virus vectors, retrovirus vectors and baculovirus vectors. is not. Non-viral delivery systems or non-viral vector systems include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles and combinations thereof.

  In the non-viral vector system of the present invention, at least part of the rabies virus G protein (or mutant, variant, homologue or fragment thereof) and / or CVS protein (or mutant, variant, homologue or fragment thereof) ) Can be used to encapsulate or encapsulate EOI. Thus, in some embodiments, at least a portion of a rabies virus G protein (or mutant, variant, homologue or fragment thereof) or CVS protein (or mutant, variant, homologue or fragment thereof). At least a portion can form a matrix enclosing the EOI. In this case, the matrix can contain other components such as liposome-type substances.

  In some preferred embodiments, the vector system is a viral vector system.

  In some further preferred embodiments, the vector system is a retroviral vector system, preferably a lentiviral vector system.

  Also, certain types of vector systems--for example, viral vector systems, preferably retroviral vector systems, more preferably lentiviral vector systems--are remote from the site of administration by retrograde transport of this vector system. It has also been found that one or more sites can be transduced.

  When administered to a single target site, multiple target sites can be transduced. The vector system can be moved to its target or to each target by retrograde transport, diffusion or biodistribution combined with antegrade transport as needed.

In another broad aspect, the present invention provides:
(I) a method of treating and / or preventing a disease using the vector system as described above;
(Ii) use of this vector system in the manufacture of a pharmaceutical composition for treating and / or preventing a disease;
(Iii) a method for analyzing the effect of a target protein in cells using this vector system;
(Iv) a method for analyzing the function of a gene or protein using this vector system;
(V) cells transduced with this vector system;
(Vi) immortalized cells produced by transduction using this vector system;
(Vii) use of such immortalized cells in the manufacture of therapeutic agents; and (viii) a method of transplantation using the immortalized cells.

Detailed Description of the Invention The present invention relates to a novel use of vector systems.

  The vector system can be non-viral or viral.

  In some preferred embodiments, the vector system is a viral vector system.

In some further preferred embodiments, the vector system is a retroviral vector system, preferably a lentiviral vector system.
The concept of using a viral vector for retroviral gene therapy is known (Verma, Somia, (1997) Nature 389: p239-242).

  There are many types of retroviruses. In this application, the term “retrovirus” refers to murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor 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), Ebelson murine leukemia virus (A-MLV), avian bone marrow Included are staphylococcal virus-29 (MC29), and chicken erythroblastosis virus (AEV), and all other retroviruses including lentiviruses.

  For a detailed list of retroviruses, see Coffin, et al. ("Retroviruses" 1997, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, J. M. Coffin, SM Hughes, H. E. Barmouth (ed. Pp. 758-763).

  In a preferred embodiment, the retroviral vector system can be derived from a lentivirus. Lentiviruses also belong to the retrovirus family, but can infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO Journal (EMBO J.), p3053. -3058).

  The lentivirus group can be classified into “primates” and “non-primates”. Examples of primate lentiviruses include human immunodeficiency virus (HIV), human acquired immunodeficiency syndrome (AIDS) pathogens, and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype “late virus” visna / maedivirus (VMV), as well as similar goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV) and recently reported Feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

  Details of the genomic structure of some lentiviruses can be determined in the art. For example, details regarding HIV and EIAV can be obtained from the NCBI Genbank database (ie, genome accession numbers AF033819 and AF033820, respectively). For details of HIV mutant strains, see http: // hiv-web. lanl. gov. You can also ask for it. For more information on EIAV mutants, see http: // www. nebi. nlm. nih. gov. Can be obtained from

  In the course of infection, retroviruses first bind to specific cell surface receptors. Next, upon entry into the recipient host cell, the retroviral RNA genome is copied into the form of DNA by the virus-encoded reverse transcriptase carried in the original virus. This DNA is transferred to the nucleus of the host cell, where it is then integrated into the host genome. At this stage, this is usually called a provirus. This provirus is stable in the host chromosome during cell division and is transcribed like other cellular genes. This provirus encodes the proteins and other factors necessary to propagate the virus, and the propagated virus can leave the cell through a process sometimes called "budding".

  Each retroviral genome contains genes called gag, pol and env that code for virion proteins and enzymes. These genes are sandwiched at both ends by a region called a terminal repeat (LTR). These LTRs are involved in proviral integration and transcription. They also serve as enhancer-promoter sequences. In other words, these LTRs can control the expression of viral genes. The encapsidation of retroviral RNA is performed by a psi sequence at the 5 'end of the viral genome.

  These LTRs themselves have the same sequence and can be divided into three elements called U3, R and U5. U3 is derived from a unique sequence at the 3 'end of this RNA. R is derived from a repetitive sequence at both ends of the RNA, and U5 is derived from a unique sequence at the 5 'end of the RNA. The size of these three elements can vary considerably between the various retroviruses.

  In this viral genome, the transcription start site is at the boundary between U3 and R in one LTR, and the poly (A) addition (termination) site is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of this provirus, which contain a promoter and multiple enhancer sequences that are reactive to cellular and possibly viral transcriptional activation proteins. . Some retroviruses have a gene encoding a protein involved in the regulation of gene expression, ie any one or more of tat, rev, tax and rex.

  With respect to the structural genes gag, pol and env itself, gag encodes the internal structural protein of this virus. The gag protein is proteolytically processed to become mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), which includes DNA polymerase, the associated RNase H and integrase (IN) that mediate genomic replication. The env gene encodes the surface (SU) glycoprotein and transmembrane (TM) protein of the virion, which form a complex that specifically interacts with the cell receptor protein. By this interaction, the virus membrane and the cell membrane are finally fused to establish infection.

  Retroviruses also contain “another” genes that encode proteins other than gag, pol and env. Examples of other genes include one or more of vif, vpr, vpx, vpu, tat, rev and nef in HIV. EIAV has (among other things) S2 as another gene.

  Proteins encoded by other genes perform various functions, some of which can be compared to the functions of cellular proteins. For example, in EIAV, tat functions as a transcriptional activator of the viral LTR. This binds to a stable stem-loop RNA secondary structure called TAR. rev regulates and regulates the expression of viral genes via the rev responsive element (RRE). The mechanism of action of these two proteins is thought to be largely similar to similar mechanisms in primate viruses. The function of S2 is not known. Furthermore, the EIAV protein Ttm encoded by the first exon of tat linked to the first env coding sequence of the transmembrane protein has been identified.

Vector system The vector system can be non-viral or viral.

  In some preferred embodiments, the vector system is a viral vector system.

  In some further preferred embodiments, the vector system is a retroviral vector system, preferably a lentiviral vector system.

  The vector system can be used to transfer EOI to one or more sites of interest. This transfer can occur in vitro, ex vivo, in vivo, or a combination thereof.

  In a highly preferred embodiment, the delivery system is a retroviral delivery system that is a lentiviral vector system.

  Retroviral vector systems have been proposed, among other things, as delivery systems for transferring NOI to one or more sites of interest. This transfer can occur in vitro, ex vivo, in vivo, or a combination thereof. Retroviral vector systems have also been used in research on various aspects of the retroviral life cycle, such as receptor utilization, reverse transcription and RNA packaging (Miller, 1992, Current Topics. In Microbiology and Immunology (reviewed in Curr Top Microbiol Immunol 158: 1-24)).

  The term “vector system” as used herein can also include vector particles capable of transducing NOI into recipient cells.

  A vector particle includes the following components: a vector genome that can contain one or more NOIs, a nucleocapsid that encloses a nucleic acid, and a membrane that surrounds the nucleocapsid.

  The term “nucleocapsid” means at least the group-specific viral core protein (gag) and the viral polymerase (pol) of the retroviral genome. These proteins form capsids for stuffable sequences, and are themselves surrounded by a membrane containing a coat glycoprotein.

  Once inside the cell, the RNA genome of the retroviral vector particle is reverse transcribed into DNA and incorporated into the DNA of the recipient cell.

  The term “vector genome” refers to both the RNA structure within the retroviral vector particle and the integrated DNA structure described above. The term also encompasses a separate or isolated DNA construct that can encode such an RNA genome. The genome of the retrovirus or lentivirus must contain at least one component derivable from the retrovirus or lentivirus. The term “inducible” is not necessarily required to be obtained from a virus, such as a lentivirus, but is used in its ordinary sense to mean a nucleotide sequence or portion thereof that can be derived from these. Yes. For example, this sequence can be made synthetically or by using recombinant DNA techniques. The genome preferably has a psi region (or similar component that can lead to encapsidation).

  Preferably, the viral vector genome is a “replication defect” meaning that the genome alone does not contain sufficient genetic information and thus cannot produce infectious viral particles in recipient cells by independent replication. Type ". In a preferred embodiment, this genome lacks a functional env, gag or pol gene. In a highly preferred embodiment, this genome lacks the env, gag and pol genes.

  The viral vector genome may include some or all of a plurality of terminal repeat sequences (LTRs). Preferably, the genome includes at least a portion of these LTRs, or similar sequences that can mediate proviral integration and transcription. The sequence can also include or perform an enhancer-promoter sequence.

  When separately expressing the components necessary to produce retroviral vector particles using different DNA sequences co-introduced into the same cell, retroviral particles with defective retroviral genomes containing therapeutic genes It is known that it is obtained (e.g. Miller, 1992 review). This cell is called the producer cell (see below).

  Two methods are generally used to produce producer cells. One of them is a method of introducing a sequence encoding retrovirus Gag, Pol and Env protein into a cell and stably integrating it into the genome of this cell, which is a stable cell called a packaging cell line. A stock is obtained. This packaging cell line produces the proteins necessary to pack retroviral RNA, but lacks the psi region and cannot provide encapsidation. However, when a vector genome (having a psi region) is introduced into this packaging cell line, this helper protein is packed with psi-positive recombinant vector RNA, resulting in a recombinant virus stock. This can be used to transduce NOI into recipient cells. This recombinant virus, which lacks all the genes necessary for the genome to produce viral proteins, cannot propagate because it can only be infected once. Therefore, retroviruses that can be harmful even if the NOI is introduced into the genome of the host cell are not produced. For an overview of available packaging stocks, see “Retroviruses” (Cold Spring Harbor Laboratory Press, 1997, JM Coffin). , SM Hughes, edited by HE Barmus, p449).

  The present invention also provides a packaging cell line comprising a viral vector genome that can provide a vector system useful in the first aspect of the invention. For example, the packaging cell line can be transduced with a viral vector system containing the genome or transfected with a plasmid having a DNA construct capable of encoding the RNA genome. The present invention also provides a kit for making a retroviral vector system useful for the first aspect of the invention comprising a packaging cell and a retroviral vector genome.

  The second method involves the defective retroviral genome comprising three DNA sequences necessary to produce a retroviral vector particle: an env coding sequence, a gag-pol coding sequence, and one or more NOIs. At the same time by transient transfection, and this method is called transient triple transfection (Landau, Littman, 1992; Peer et al. (Pear et al), 1993). This triple co-transfection method has been improved to be optimal (Soneoka et al, 1995; Finer et al, 1994). World Patent No. 94/29438 discloses a method for producing producer cells in vitro using this multiple DNA transient transfection method. World Patent No. 97/27310 discloses a series of DNA sequences for generating retroviral producer cells in vivo or in vitro for reimplantation.

  The viral system components necessary to complement the vector genome can be present on one or more “producer plasmids” for transfection into cells.

The present invention also provides:
(I) a viral vector genome that cannot encode one or more proteins required to produce vector particles;
(Ii) a first of the invention comprising one or more producer plasmids capable of encoding the above proteins not encoded by (i), and optionally (iii) a cell suitable for conversion to a producer cell A kit for producing a retroviral vector system useful in the embodiments of is provided.

  In a preferred embodiment, the viral vector genome lacks the ability to encode the proteins gag, pol and env. Preferably, the kit comprises one or more producer plasmids encoding env, gag and pol, eg, a producer plasmid encoding env and one encoding gag-pol. This gag-pol sequence is preferably a codon optimized for use in a particular producer cell (see below).

  The present invention also provides a producer cell expressing the vector genome and the producer plasmid that can provide a retroviral vector system useful in the present invention.

  The retroviral vector system used in the first aspect of the invention is preferably a self-inactivating (SIN) vector system.

  Self-inactivating retroviral vector systems have been constructed, for example, by removing the transcription enhancer or this enhancer and promoter within the U3 region of the 3 'LTR. After a single reverse transcription and integration of the vector, these changes are copied into both the 5'LTR and the 3'LTR, resulting in a provirus with no transcriptional activity. However, any promoter within these LTRs in such a vector will still have transcriptional activity. This method has been used to eliminate the influence of enhancers and promoters within the viral LTR on transcription from genes located within. Such effects include enhanced transcription or inhibited transcription. This method can also be used to eliminate downstream transcription from the 3 'LTR into the genomic DNA. This is of particular interest in human gene therapy where it is important to prevent accidental activation of endogenous oncogenes.

  Preferably, a recombinase assisted mechanism that promotes the production of highly titered lentivirus from the producer cells of the present invention is used.

  As used herein, the term “recombinase assist system” includes S. catalysis of a recombination event between the Cre recombinase / loxP recognition site of bacteriophage P1, or the 34 bp FLP recognition target (FRT). Examples include, but are not limited to, systems that use the Servisier site-specific FLP recombinase.

  In order to generate high level producer cell lines that utilize recombinase-assisted recombination events, S. catalyzes recombination events between 34 bp FLP recognition targets (FRT). Servisier site-specific FLP recombinases have been placed in DNA constructs (Karreman et al (1996) NAR 24: p1616-1624). A similar system has been developed using the Cre recombinase / loxP recognition site of bacteriophage P1 (PCT / GB00 / 03837; Vanin et al (1997) Journal of Virology (J. Virol). 71: p7820-7826). By placing this in the lentivirus genome, a high titer lentivirus producer cell line was generated.

  By using a producer / packaging cell line, a large amount of lentiviral vector particles are propagated and isolated (eg, making this lentiviral vector particle of the appropriate titer) and then this is, for example, ( It can transduce a target site (such as mature brain tissue). Producer cell lines are usually suitable for mass production of vector particles.

  Transient transfection methods have a number of advantages over packaging cell methods. In this regard, transient transfection methods can avoid the increased time required to generate stable vector-producing cell lines, and the vector genome or retroviral packaging components This can be used if it is harmful to cells. If the vector genome encodes a harmful gene, that is, a gene that interferes with host cell replication, such as inhibiting the cell cycle, or a gene that induces apoptosis, it is difficult to produce a stable vector-producing cell line. Although it is conceivable, transient transfection methods allow the vector to be produced before the cells die. Cell lines have also been developed that utilize transient infections that produce vector titer levels comparable to those obtained with stable vector-producing cell lines (Pear et al, 1993, PNAS 90: p8392). 8396).

  The producer / packaging cell can be of any suitable cell type. Producer cells are usually mammalian cells, but may be, for example, insect cells.

  As used herein, the term “producer cell” or “vector producing cell” refers to a cell that contains all the elements necessary for the production of retroviral vector particles.

  This producer cell is preferably obtained from a stable producer cell line.

  In addition, the producer cell is preferably obtained from an induced stable producer cell line.

  Furthermore, the producer cell is preferably obtained from an induced producer cell.

  As used herein, the term “induced producer cell line” means a transduced producer cell that has been screened and selected for high expression of the marker gene. Such cell lines are capable of high level expression from the retroviral genome. The term “derived producer cell line” is used interchangeably with the terms “derived stable producer cell line” and “stable producer cell line”.

  Preferably, the derived producer cell line includes, but is not limited to, a retrovirus producer cell and / or a lentivirus producer cell.

  This derived producer cell line is preferably an HIV or EIAV producer cell line, more preferably an EIAV producer cell line.

  It is preferred that the coat protein sequence and the nucleocapsid sequence are all stably integrated into the producer and / or packaging cell. However, it is also possible for one or more of these sequences to exist as an episome, and gene expression can be performed from this episome.

  As used herein, the term “packaging cell” refers to a cell that lacks the RNA genome and contains elements necessary for the production of infectious recombinant virus. Usually such packaging cells contain one or more producer plasmids capable of expressing viral structural proteins (such as gag-pol and env which may be codon-optimized). Does not contain signal.

  The term “packaging signal”, also referred to as “packaging sequence” or “psi”, is used in reference to non-coding cis-acting sequences necessary for encapsidation of retroviral RNA strands during virion formation. In HIV-1, this sequence maps to a locus from upstream of the major splice donor site (SD) to at least the gag start codon.

  Packaging cell lines can be readily prepared (see also World Patent No. 92/05266) and can be used to create producer cell lines for the production of retroviral vector particles. As already mentioned, an overview of available packaging strains is described in “Retroviruses” (above).

  Further, as described above, it was found that in a simple packaging cell line containing a provirus from which a packaging signal was removed, a virus having an undesirable replication ability was rapidly produced by recombination. In order to improve safety, a second generation cell line from which the 3'LTR of this provirus was removed was prepared. In such cells, two recombination may be required to produce wild type virus. In order to further improve safety, it is necessary to introduce the gag-pol gene and the env gene using another construct called a third generation packaging cell line. By sequentially introducing such constructs, recombination during the transfection process is prevented.

  Preferably, the packaging cell line is a second generation packaging cell line.

  Preferably, the packaging cell line is a third generation packaging cell line.

  In this split-construct third generation packaging cell line, recombination can be further reduced by changing codons. This method, based on the redundancy of the genetic code, aims to reduce the homology between different constructs, for example between overlapping regions within the gag-pol and env reading frames.

  This packaging cell line is useful for obtaining the gene product required for encapsidation and produces a membrane protein for high titer vector particle formation. The packaging cell can 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 mouse fibroblast derived cell lines or human cell lines. Preferably, the packaging cell line is a human cell line such as, for example, HEK293, 293-T, TE671, HT1080.

  Alternatively, the packaging cell line can be a cell from the individual to be treated, such as a mononuclear cell, macrophage, blood cell or fibroblast. The autologous packaging cells can be re-administered after the cells are isolated from the individual and the packaging and vector components are administered thereto ex vivo.

  It is highly desirable to use high titer virus formulations for both experimental and practical applications. Methods for increasing virus titer include using psi and the above packaging signal and concentrated virus stock.

  As used herein, the term “high titer” refers to an effective amount of a retroviral vector or particle that can be transduced to a target site, such as a cell.

  As used herein, the term “effective amount” refers to the amount of regulated retroviral or lentiviral vector or vector particle sufficient to induce the expression of NOI at the target site. means.

High titer formulations for producer / packaging cells are typically 10 ^{5 to} 10 ⁷ t. u. Degree. (This titer is expressed in transducing units per ml (tuu / ml) quantified using the standard D17 cell line). To transduce tissues such as the brain, it is necessary to use a very small volume and thus the viral preparation is concentrated by ultracentrifugation. The resulting formulation is at least 10 ⁸ t. u. / Ml, preferably 10 ^{8 to} 10 ⁹ t. u. / Ml, more preferably at least 10 ⁹ t. u. Must have a titer of / ml.

  The expression product encoded by the NOI can be a protein secreted from the cell. Alternatively, the NOI expression product is not secreted but shows activity in the cell. For some applications, it is preferred that the NOI expression product exhibits a spatial bystander effect or a distant bystander effect, which produces the expression product in a cell. By this is meant that other nearby cells with a common phenotype or distant (eg metastasized) are subject to regulation.

  The presence of a sequence called Central Polypurine Tract (cPPT) can improve the efficiency of gene delivery to non-dividing cells (World Patent No. 00/31200). This cis-acting element is, for example, within the EIAV polymerase coding region element. The vector-based genome used in the present invention preferably contains a cPPT sequence.

  Additionally or alternatively, the viral genome can include post-translational regulatory elements and / or translation enhancers.

  The NOI can be operably coupled to one or more promoter / enhancer elements. One or more NOI transcripts can be placed under the control of the viral LTR, or promoter-enhancer elements can be designed with this transgene. This promoter is preferably a strong promoter such as CMV. This promoter can be a regulated promoter. This promoter can be tissue specific. In a preferred embodiment, the promoter is glial cell specific. In another preferred embodiment, the promoter is nerve specific.

Minimal system A minimal system of primate lentivirus that does not require the addition of the HIV / SIV genes vif, vpr, vpx, vpu, tat, rev and nef for vector production or transduction of dividing and non-dividing cells. It has been shown that it can be built. It has also been shown that a minimal EIAV vector system can be constructed that does not require S2 for vector production or transduction for dividing and non-dividing cells. The ability to exclude the addition of genes is extremely advantageous. First, this can produce vectors that do not have genes associated with diseases of lentivirus (eg, HIV) infection. In particular, tat is associated with disease. Second, the ability to exclude the addition of genes allows the vector to be packed with more heterologous DNA. Third, the ability to exclude genes with unknown functions such as S2 can reduce the risk of producing undesirable results. Examples of minimal lentiviral vectors are disclosed in WO99 / 32646A and 98 / 17815A.

  Accordingly, it is preferred that the delivery system used in the present invention does not have at least tat and S2, and if possible vif, vpr, vpx, vpu and nef (if this is an EIAV vector system). More preferably, the system of the present invention is also free of rev. Previously, rev was considered essential for efficient virus production in some retroviral genomes. For example, in the case of HIV, it was thought necessary to include rev and RRE sequences. However, the need for rev and RRE has been found to be reduced or eliminated by codon optimization (see below) or replacement by other functionally equivalent systems such as the MPMV system. Since cog-optimized expression of gag-pol is REV-independent, RRE can be removed from this gag-pol expression cassette and thus any recombination potential by any RRE on the vector genome. Can be eliminated.

  In a preferred embodiment, the viral genome of the first aspect of the invention is one that lacks a Rev response element.

  In a preferred embodiment, this system used in the present invention is based on a so-called “minimal” system in which the addition of the gene is partially or completely eliminated.

Codon optimization Codon optimization has already been disclosed in WO 99/41397. Different cells use different specific codons. This codon bias is consistent with the relative abundance bias of a particular tRNA in that cell type. Expression can be enhanced by adjusting this to match the relative abundance of the corresponding tRNA by changing codons within the sequence. For the same reason, it is possible to reduce expression by deliberately selecting codons known to be rare in the corresponding tRNA in a particular cell type. Therefore, it is possible to increase the degree of translation control.

  Many viruses, including HIV and other lentiviruses, utilize a number of rare codons, but by changing these to correspond to common mammalian codons, the packaging component in mammalian producer cells Expression can be increased. Codon usage tables for mammalian cells and various other organisms are known in the art.

  There are many other benefits of codon optimization. By altering the sequence, the RNA instability sequence (INS) can be eliminated from the nucleotide sequence encoding the packaging component of the viral particle necessary for the construction of the viral particle in the producer / packaging cell. At the same time, the amino acid sequence coding sequence of the packaging component maintains sufficient similarity such that the viral component encoded by the sequence is not altered or at least the function of the packaging component is not impaired. So that it is held. Also, codon optimization overturns the need for Rev / RRE for export, and the optimized sequence becomes Rev independent. In addition, codon optimization reduces homologous recombination between the various constructs in this vector system (eg, between overlapping regions in the gaa-pol and env reading frames). Thus, the overall effect of codon optimization is to significantly increase virus titer and improve safety.

  In one embodiment, codon optimization is performed only for codons related to INS. However, in a more preferred and practical embodiment, codon optimization is performed over the entire sequence except for sequences containing frameshift sites.

  The gag-pol gene contains two overlapping reading frames encoding the gag-pol protein. The expression of both proteins depends on the frameshift of the translation process. This frameshift occurs as a result of ribosome “slippage” during the translation process. This shift is believed to be caused, at least in part, by RNA secondary structures that ribosome-stale. Such secondary structure exists downstream of the frameshift site in the gag-pol gene. In the case of HIV, the overlapping region extends from the 1222th nucleotide downstream of the gag start point (in this case, the first nucleotide is A of gagATG) to the end point of gag (1503rd nt). Therefore, it is preferable not to perform codon optimization for the 281 bp fragment covering this frame shift site and the overlapping region of the two reading frames. By leaving this fragment as it is, the efficiency of gag-pol protein expression can be improved.

  In the case of EIAV, the starting point of duplication is the 1262th nucleotide (in this case, the first nucleotide is A of gagATG). The end point of duplication is at the 1461th bp. In order to ensure the conservation of the frameshift site and the gag-pol overlap region, the wild type sequence from the 1156th to 1465th nt was maintained as it was.

  For example, in order to incorporate convenient restriction enzyme recognition sites, induction from optimal codon usage can be performed and conservative amino acid changes can be introduced into the gag-pol protein.

  In a highly preferred embodiment, readily expressed mammalian genes were used for codon optimization. The third base, in some cases the second and third bases can be changed.

  It should be understood that due to the degeneracy of the genetic code, a skilled engineer can obtain numerous gag-pol sequences. A number of retroviral variants have also been reported that can be used as a starting point for generating codon optimized gag-pol sequences. The lentiviral genome may be highly mutated. For example, HIV-1 has many quasispecies that maintain functionality. This is also true for EIAV. These mutants can be used to enhance specific parts of the transduction process. For examples of HIV-1 mutants, see http: // hiv-web. lanl. gov. It is published in. For more information on EIAV clones, see NCBI database: http: // www. nebi. nfm. nih. gov. It is published in.

  The method of optimizing the codons of the gag-pol sequence can be used for any retrovirus. This is applicable to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1 and HIV-2. Furthermore, this method could be used to enhance the expression of HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retrovirus (HERV), MLV and other retrovirus genes.

  Codon optimization can make gag-pol expression Rev independent. However, to allow the use of anti-rev or RRE factors in retroviral vectors, it will be necessary to make the viral vector production system completely Rev / RRE independent. Therefore, the genome also needs to be modified. This is achieved by optimizing the vector genomic components. Advantageously, such modifications also provide a highly safe system in which there is no excess protein in producers and transduced cells.

  The packaging component of the aforementioned retroviral vector includes the expression products of gag, pol and env genes. Further, the efficiency of packaging is determined by a short sequence of four stem loops followed by a partial sequence from gag and env (“packaging signal”). Therefore, the deletion of the gag sequence in this retroviral vector genome (in addition to the complete gag sequence of the packaging construct) will optimize the vector titer. Become. To date, for efficient packaging, vectors that still retain the env sequence have 255 to 360 nucleotides of gag, or splice donor mutations, gag and env deletions. Certain combinations have been reported to require about 40 nucleotides of gag. Surprisingly, it was found that deleting all nucleotides except about 360 N-terminal in gag increased the vector titer. Accordingly, the retroviral vector genome preferably includes a gag sequence having one or more deletions, and more preferably, the gag sequence includes about 360 nucleotides derived from its N-terminus.

Pseudotyping When designing retroviral vector systems, particles with different target cell specificities for wild-type viruses can be devised to enable delivery of genetic material to a wide range or different cell types It is desirable to do. One way to achieve this is to design the viral coat protein to change its specificity. Another method is to introduce a heterologous coat protein into the vector particle to replace or add to the viral wild-type coat protein.

  The term pseudotyping means incorporating a heterologous env gene, eg, an env gene from another virus, into at least part of the viral genome, or replacing part or all of this genome. Pseudotyping is not a new matter. Examples include World Patent No. 99/61639, World Patent No. 98 / 05759A, World Patent No. 98 / 05754A, World Patent No. 97 / 17457A, World Patent. No. 96/09400 A, World Patent No. 91/00047 A and Mebation et al. 1997 Cell 90: p841-847.

  Pseudotyping can improve the stability and transduction efficiency of retroviral vectors. A pseudotype of murine leukemia virus packed in lymphocytic choriomeningitis virus has been reported (Miletic et al. (1999) Journal of Virology 73: p 6114-6116). It has been shown to be stable during centrifugation and capable of infecting several cell lines from different species.

  The vector system of the present invention can be pseudotyped using at least a part of the mutant rabies virus G coat protein or CVS coat protein or mutants, variants, homologues or fragments thereof.

  A retroviral delivery system for use in the first aspect of the present invention comprises a first nucleotide sequence encoding at least a portion of a coat protein and a retrovirus that ensures transduction by the retroviral delivery system. One or more other nucleotide sequences obtained from In this case, the first nucleotide sequence is heterologous to at least one of the other nucleotide sequences, and the first nucleotide sequence is a rabies virus G coat protein or a mutant thereof. , At least part of a variant, homologue or fragment, or at least part of a CVS protein or mutant, variant, homologue or fragment thereof.

  Accordingly, the heterologous env region comprises at least a portion of a rabies virus G protein or a mutant, variant, homologue or fragment thereof, or CVS protein or a mutant, variant, homologue or fragment thereof. There is provided the use of a retroviral delivery system that is intended to comprise at least a portion.

  The heterologous env region can be encoded by a gene present on the producer plasmid. This producer plasmid can be part of a kit for making retroviral vector particles suitable for use in the first aspect of the invention.

Rabies virus G protein The vector system of the present invention can be pseudotyped using at least a part of a mutant rabies virus G protein or a variant, homologue or fragment thereof.

  For the rabies virus G protein and mutants thereof, see World Patent No. 99/61639 and Rose et al., 1982 Journal of Virology (J. Virol.) 43: p361-364, Hanham. (Hanham et al.), 1993 Journal of Virology (J. Virol.) 67: p530-542, Tuffero et al. (1998) Journal of Virology 72: p1085-1910, Cusera. (Kucera et al.), 1985 Journal of Virology 55: p158-162, Dietzschold et al., 1983 PNAS 80: p70-74, Say. (Seif et al.), 1985 Journal of Virology 53: p926-934, Clon et al. (1998) Journal of Virology 72: p273-278, Tuffero et al. (Tuffereau et al.). ) 1998 Journal of Birology 72: p1085-1910, Burger et al., 1991 Journal of General Virology (J. Gen. Virol.) 72: p273-278, Gordan et al. ( Gaudin et al), 1995 Journal of Virology 69: p5528-5534, Benmansour et al, 1991 Journal of Viro. Rosie 65: p4198-4203, Luo et al., 1998 Microbiology. Immunol. 42: p187-193, Collection 1997 Archives of Virology (Coll 1997 Arch) Virol 142: p2089-2097, Luo et al., 1997 Virus Res 51: p35-41, Luo et al., 1998 Microbiology and Immunology. 42: p187-193, Collection 1995 Archives of Virology 140: p827-851, Tsuchiya et al. (Tuch) ya et al), 1992 Viras Research 25: p1-13, Morimoto et al, 1992 Virology 189: p203-216, Gaudin et al, 1992 Virology 187: p627 -632, Witt et al., 1991 Virology 185: p681-688, Dietzschold et al. ), 1978 Journal of General Virology 40: p131-139, Dietzhold et al., 1978 Developments in Biological Standardization (Dev Biol Stand) 40: p45-55. Dietzschold et al., 1977 Journal of Virology 23: p286-293, and Otvos et al, 1994 Biochim Biophys Acta 1224: p68-76. The rabies virus G protein is also described in EP 0445625 A.

  By using the rabies virus G protein, a vector can be obtained that selectively transduces in vivo the target cells that are selectively infected by the rabies virus. Such cells include in particular neural target cells in vivo. For vectors targeting nerves, rabies virus G from rabies virus pathogenic strains such as ERA is considered particularly effective. On the other hand, the target cells in vitro of rabies virus G protein will be a broader range, including almost all mammalian and avian cell types tested (Seganti et al., 1990 Archives Arch Virol. 34: p155-163; Fields et al., Fields Virology, 3rd edition, Volume 2, Lippincott-Raven Publisher (Lippincott-Raven) Publishers), Philadelphia, New York).

  The directivity of pseudotyped vector particles can be improved by using a mutant rabies virus G in which the extracellular domain is modified. The rabies virus G protein has the advantage that the range of target cells can be limited by mutation. Uptake of rabies virus by target cells in vivo is thought to be mediated by the acetylcholine receptor (AchR), although there are other receptors that bind in vivo (Hanham et al. 1993, Journal of Birology (J. Virol.) 67: p530-542; Tuffero et al. (1998 Journal of Birology 72: p1085-1910). Virus invasion in the central nervous system includes NCAM (Thoulouze et al. (1998) Journal of Biology 72 (9): p7181-90) and p75 neurotrophin receptor (C. Tüfero et al. ( (Tuffereau C et al.) (1998) EMBO Journal (EMBO J) 17 (24): p7250-9) is considered to be used.

  As a result of examining the influence of the mutation at the antigenic site III of the rabies virus G protein on the virus directivity, it is reported that this region is not considered to be involved in the binding of the virus to the acetylcholine receptor. (Kusera et al., 1985 Journal of Virology 55: p158-162; Dietzshold et al., 1983 Proceedings of the National Academy of Sciences. (Pro. Natl. Acad. Sci.) 80: p70-74; Seif et al., 1985 Journal of Virology 53: p926-934; Clon et al. 1998, Jar Le Of Bairoroji 72:; (. Tuffereau et al) p273-278 Teyuffuero addition, 1998 Journal of Bairoroji 72: p1085-10910). For example, by mutating the 333 amino acid arginine of this mature protein to glutamine (ie, ERAsm), viral entry in vivo is limited to olfactory and peripheral neurons, while at the same time spreading to the central nervous system. It has been reported that it can be reduced. It has also been reported that these viruses were able to enter motor and sensory nerves as efficiently as wild type viruses, but transneuronal transfer did not occur (Coulon et al. 1989, Journal of Birology 63: p3550-3554). The virus mutated at amino acid position 330 was further attenuated (ie, ERAADM) and was reported to fail to infect motor or sensory nerves after intramuscular injection (Coulon et al. .) 1998, Journal of Birology 72: p273-278).

  As another option, or in addition, a rabies virus G protein from a rabies virus strain passaged in the laboratory can be used. Such proteins can be screened for directional changes. Such strains include those in the table below:

For example, the ERA strain is a pathogenic strain of rabies virus, and the rabies virus G protein from this strain can be used for transduction of neurons. The rabies virus G sequence from this ERA strain is in the Genbank database (accession number J02293). This protein has a signal peptide of 19 amino acids, and the mature protein starts with a lysine residue at the 20th amino acid from the translation initiation methionine. In the HEP-Flury strain, the 333th amino acid of the mature protein is mutated from arginine to glutamine, and this mutation is associated with attenuated pathogenicity, limiting the direction of the coat protein of the virus. Can be used.

  World Patent No. 99/61639 discloses the nucleic acid and amino acid sequences of the rabies virus strain ERA (Genbank Locus RAVGPLS, Accession M38452).

CVS
The vector system of the present invention is pseudotyped using at least a part of a CVS (Challenge Virus Standard) protein, in particular CVS glycoprotein G, or a mutant, variant, homologue or fragment thereof. Can do.

  CVS is disclosed in US Pat. No. 5,348,741.

  It should also be understood that CVS glycoproteins from CVS strains passaged in the laboratory can be used. Such glycoproteins can be screened for directional changes.

  The ATTC deposit number 40280, also referred to as pKB3-JE-13, can be conveniently used in the present invention.

Mutants, variants, homologues and fragments The vector system comprises wild type rabies virus G protein or mutants, variants, homologues or fragments thereof and / or wild type CVS protein or mutants, variants, homologues thereof. Or at least part of or including a fragment.

  The term “wild type” is used to mean a polypeptide having the same primary amino acid sequence as a natural protein (ie, a viral protein).

  The term “mutant” is used to mean a polypeptide having a primary amino acid sequence that differs from the wild type sequence by the addition, substitution or deletion of one or more amino acids. Mutants can occur in nature or can be made artificially (eg, by site-directed mutagenesis). This mutant preferably has at least 90% identity with the wild type sequence. Preferably, the number of mutation sites in this mutant is 20 or less relative to the entire wild type sequence. More preferably, the mutation site of this mutant is 10 or less, and most preferably 5 or less, relative to the entire wild type sequence.

  The term “variant” is used to mean a naturally occurring polypeptide that differs from a wild-type sequence. Variants can be found within the same virus strain (ie, if one or more isoforms are present in the protein) or can be found within different strains. This variant preferably has at least 90% sequence identity with the wild type sequence. Preferably, this variant has no more than 20 mutation sites relative to the entire wild type sequence. More preferably, the variant mutation sites are 10 or less, and most preferably 5 or less, relative to the entire wild type sequence.

  As used herein, the term “homologue” means a substance having a certain homology with wild-type amino acid sequences and wild-type nucleotide sequences. Here, “homology” can be regarded as equivalent to “identity”.

  In the present context, a homologous sequence shall comprise an amino acid sequence which can be at least 75, 85 or 90% identical to the subject sequence, preferably 95 or 98%. Usually, this homologue contains the same active site as the target amino acid sequence. Although homology can be considered in terms of similarity (ie, amino acid residues have similar chemical properties / functions), in the context of the present invention, homology is considered in terms of sequence identity. It is preferable to express.

  In this context, a homologous sequence shall comprise a nucleotide sequence which can be at least 75, 85 or 90% identical to the subject sequence, preferably 95 or 98%. Usually, this homologue will contain the same sequence as the subject sequence, encoding the active site, etc. Although homology can be considered in terms of similarity (ie, amino acid residues have similar chemical properties / functions), in the context of the present invention, homology is considered in terms of sequence identity. It is preferable to express.

  Homology can be compared with the naked eye, and more usually using sequence comparison programs that are always available. According to such a commercially available computer program,% homology between two or more sequences can be calculated.

  % Homology can be calculated for adjacent sequences. That is, one sequence is aligned in parallel with another sequence, and each amino acid in one sequence is directly compared, residue by residue, simultaneously with the corresponding amino acid in the other sequence. This is called an “ungapped” alignment. Usually, such gap-free alignment is performed only for a relatively short number of residues.

  This is a very simple and consistent method, but, for example, if there is an insertion or deletion in one place in an otherwise identical sequence pair, the subsequent amino acid residue is removed from the alignment, and thus However, global alignment does not take into account that% homology can be significantly reduced. Thus, many sequence comparison methods are designed to optimize alignment without unduly degrading the overall homology score taking into account that insertions and deletions can occur. This is achieved by inserting “gaps” during sequence alignment to maximize local homology.

  However, in these relatively complex methods, a “gap penalty” is assigned to each gap that occurs in the alignment, so if the number of amino acids is the same, the fewer the gaps in the sequence alignment, the smaller the two comparison sequences. High relevance-higher scores will be obtained than if there are many gaps. “Affine gap costs” are typically used that require a relatively high cost for the existence of a gap and a relatively small penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, higher gap penalties optimize the alignment with fewer gaps. Many alignment programs can change the gap penalty. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

  Therefore, to calculate the maximum% homology, it is first necessary to optimize the alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit Package (University of Wisconsin, USA; Devereux et al., 1984, New Clayk, Inc.). Acids Research 12: p387). Examples of other software that can perform sequence comparison include the BLAST package (Ausubel et al., 1999, ibid.-Chapter 18), FASTA (Atschul et al.), 1990. Year, Journal of Molecular Biology (J. Mol. Biol.), P403-410) and GeneWorks (GENEWORKS) set of comparative tools. BLAST and FASTA can be used for offline and online searching (Ausubel et al., 1999 ibid, p7-58 to 7-60). However, for some applications, it is preferred to use the GCG bestfit program. A new tool called BLAST2 Sequences can also be used to compare protein and nucleotide sequences (FEMS Microbiol Letters, 1999, 174 (2): p247-50; FEMS microbio Rosie Letters, 1999, 177 (1): p187-8 and tatiana@ncbi.nlm.nih.gov).

  Although the final% homology can be measured in terms of identity, the alignment process itself is usually not based on an all-or-nothing pair comparison. Rather, a scaled similarity score matrix is generally used that assigns a score to each pair of comparisons based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix-BLAST program default matrix. In general, the GCG Wisconsin program uses public default values or, if provided, custom symbol comparison tables (see user manual for more details). For some applications, it is preferable to use public default values for the GCG package and a default matrix such as BLOSUM62 for other software.

  Once these software have optimized the alignment, it is possible to calculate% homology, preferably% sequence identity. Typically, such software does this as part of the sequence comparison and produces a numerical result.

  Also, deletions, insertions or substitutions of amino acid residues in these sequences can result in functionally equivalent substances by producing inconspicuous changes. As long as the secondary binding activity of this substance is retained, an amino acid can be intentionally substituted based on the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic similarity of the amino acid residue. it can. For example, aspartic acid and glutamic acid as negatively charged amino acids, lysine and arginine as positively charged amino acids, leucine, isoleucine, valine, glycine, alanine as uncharged polar head group-containing amino acids having similar hydrophilicity values , Asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

  The conservative substitution can be performed, for example, according to the following table. Amino acids in the same block of the second column, preferably in the same row of the third column, can be substituted for each other:

The present invention also provides homologous substitutions (in the text herein, both substitution and replacement are used in the sense that an existing amino acid residue is interchanged with another amino acid residue), In other words, it is possible to perform the same kind of substitution such as basic group and basic group, acidic group and acidic group, and polar group and polar group. In addition, non-homologous substitution, that is, substitution from one class of residues to another class of residues, ornithine (hereinafter referred to as Z), ornithine diaminobutyrate (hereinafter referred to as B), norleucine ornithine ( Substitutions involving the inclusion of unnatural amino acids such as pyrylalanine, thienylalanine, naphthylalanine and phenylglycine can also be made.

It can also be substituted by the following unnatural amino acids: alpha ^* and alpha-disubstituted ^* amino acids, N-alkyl amino acids ^* , lactic acid ^* , trifluorotyrosine ^* , p-Cl-phenylalanine ^* , p-Br-phenylalanine. ^* , Neutral derivatives of neutral amino acids such as p-I-phenylalanine ^* , L-allyl-glycine ^* , β-alanine ^* , L-α-aminobutyric acid ^* , L-γ-aminobutyric acid ^* , L-α-aminoiso Butyric acid ^* , L-ε-aminocaproic acid ^# , 7-aminoheptanoic acid ^* , L-methionine sulfone ^{# *} , L-norleucine ^* , L-norvaline ^* , p-nitro-L-phenylalanine ^* , L-hydroxyproline ^# , L- thioproline ^* 4-methyl--Phe ^*, phenylalanine of pentamethyl -Phe ^*, etc. Methyl derivatives of Phe), L-Phe (4- ^amino) #, L-Tyr ^(methyl) *, L-Phe (4- ^isopropyl) *, L-Tic (1,2,3,4- tetrahydroisoquinoline -3 -Carboxylic acid) ^* , L-diaminopropionic acid ^# and L-Phe (4-benzyl) ^* . In the above description (for homologous or non-homologous substitution), the symbol * indicates that the derivative is hydrophobic, # indicates that the derivative is hydrophilic, and ** indicates amphiphilicity Used.

  Variant amino acid sequences include amino acid spacers such as glycine or β-alanine residues as well as suitable spacer groups that can be inserted between any two amino acid residues of the sequence, eg, methyl, ethyl groups Alternatively, it can contain an alkyl group such as a propyl group. It will be appreciated by those skilled in the art that there may be other forms of variation, including the presence of one or more amino acids in the peptoid form. Although defined so as not to be uncertain, the “peptoid form” is used to mean a variant amino acid residue in which the α-carbon substituent is attached to the nitrogen atom of the residue rather than the α-carbon. Methods for producing peptoid-type peptides are known in the art. J. et al. Et al. (Simon RJ et al.), PNAS (1992) 89 (20): p9367-9371 and Howell D. et al. C. (Horwell DC), Trends Biotechnol. (1995) 13 (4): p132-134.

  The term “fragment” means that the polypeptide comprises a portion of the wild-type amino acid sequence. This can include one or more consecutive large array sections or multiple small sections. The polypeptide can also contain other sequence elements, for example, it can be a fusion protein with another protein. Preferably, the polypeptide comprises at least 50% wild type sequence, more preferably at least 65%, and most preferably at least 80%.

  In terms of function, said mutant, variant, homologue or fragment is transduced to at least part of the brain, motor nerves or cerebrospinal fluid (CSF0) when used to pseudotype an appropriate vector. Need to be able to.

  This mutant, variant, homologue or fragment should be able to confer retrograde transport capability to the vector system as an alternative or additionally.

  Vector delivery systems for use in the present invention can include nucleotide sequences that can hybridize to the nucleotide sequences presented herein (including complementary sequences of those presented herein). In a preferred embodiment, the present invention relates to nucleotide sequences of the present invention under stringent conditions (eg, 65 ° C. and 0.1 SSC) and nucleotide sequences presented herein (complementary sequences of the sequences presented herein). Nucleotide sequences that can hybridize to

NOI
In the present invention, the EOI is preferably one or more NOIs (nucleotide sequences of interest) that can be delivered to target cells in vitro or in vivo.

  When the vector system of the present invention is a viral vector system, it is possible to replace or complement a viral gene with one or more types of NOI, which may be heterologous NOI, by manipulating the genome of this virus.

  The term “heterologous” refers to a nucleic acid or protein sequence bound to a nucleic acid or protein sequence that does not bind in nature.

  In the present invention, the term NOI includes any nucleotide sequence that does not necessarily have to be a complete natural DNA or RNA sequence. Thus, the NOI may be, for example, a synthetic RNA / DNA sequence, a recombinant RNA / DNA sequence (made by recombinant DNA technology), a cDNA sequence or a partial genomic DNA sequence, or a combination thereof. This sequence need not be a coding region. Even if this is a coding region, it need not be a complete coding region. Furthermore, the RNA / DNA sequence may have a sense orientation or an antisense orientation. Preferably, the sense orientation is taken. This sequence is preferably cDNA, includes or is transcribed from it.

  Typically, the retroviral vector genome comprises an LTR at its 5 ′ and 3 ′ ends, an appropriate insertion site for inserting one or more NOIs, and / or the genome into a vector particle in the producer cell. Contains packaging signals that allow stuffing. In some cases, suitable primer binding sites and integration sites that allow reverse transcription of vector RNA into DNA and integration of proviral DNA into the genome of the target cell can also be included. In a preferred embodiment, the retroviral vector particle has a reverse transcription system (coexistable reverse transcription and primer binding sites) and an integration system (coexistable integrase and integration sites).

  The NOI can encode the protein of interest (“POI”). This would allow examining the effect of foreign gene expression on target cells (such as TH positive neurons) using this vector delivery system. For example, the retroviral delivery system could be used to screen a cDNA library for specific effects on the brain, motor nerves or CSF.

  For example, a novel survival / neuroprotective factor for dopaminergic neurons could be identified that could allow transfected TH + cells to persist in the presence of apoptosis-inducing factors.

  Suitable NOIs according to the present invention include those for therapeutic and / or diagnostic uses, such as cytokines, chemokines, hormones, antibodies, antioxidant molecules, genetically engineered immunoglobulin-like molecules, a single Chain antibody, fusion protein, enzyme, immune co-stimulatory molecule, immunoregulatory molecule, antisense RNA, transcription dominant negative mutant of the target protein, toxin, conditional toxin, antigen, tumor suppressor protein, growth Examples include, but are not limited to, factors, membrane proteins, vasoactive proteins / peptides, antiviral proteins and ribozymes, and sequences encoding these derivatives (including binding reporter groups). These NOIs can also encode prodrug activating enzymes.

  The expression product encoded by the NOI can be a protein secreted from the cell. Alternatively, the NOI expression product is not secreted and shows activity in the cell. In any case, it is preferred that the NOI expression product exhibit a spatial bystander effect or a distant bystander effect, which means that the expression product is produced in a cell. Means that nearby or distant (eg, metastasized) related cells with a common phenotype are killed.

  NOI or its expression product can act to modulate the biological activity of a compound or pathway. As used herein, the term “modulate” includes, for example, the meaning of enhancing or inhibiting biological activity. Such regulation may be direct (eg, involving cleavage of the protein or competitive binding of another substance to the protein) or indirect (eg, by blocking initiation of protein production). It can be.

  The NOI may be capable of blocking or suppressing the expression of the target cell gene. For example, NOI can be an antisense sequence. Inhibition of gene expression by antisense technology is known.

  The NOI or sequence derived therefrom may be capable of “knocking out” against expression of a specific gene in the target cell. Several “knockout” methods are known in the art. For example, the NOI can be capable of inhibiting the expression of a particular gene by integrating into the genome of the neuron. NOI affects, for example, the ability to fold the encoded protein by introducing an immature stop codon or by removing downstream coding sequences from frame synchronization (and thus affecting its function). ) To inhibit expression.

  Alternatively, the NOI may be capable of enhancing or inducing ectopic expression of the target cell gene. The NOI or sequence derived therefrom may be capable of being “knocked in” for expression of a particular gene.

  In one preferred embodiment, the NOI encodes a ribozyme. Ribozymes are RNA molecules that can act to catalyze specific intracellular chemical reactions without requiring the involvement of proteins. For example, Group I ribozymes take the form of introns that can mediate self excision from self-splicing precursor RNA. Other ribozymes are derived from self-cleaving RNA structures that are essential for the replication of viral RNA molecules. Like protein enzymes, ribozymes can be folded to form secondary and tertiary structures that serve as binding sites specific for substrates and cofactors such as metal ions. Examples of such structures include hammerheads, hairpins or stem loops, pseudoknots, and hepatitis delta antigenome ribozymes have been reported.

  Each ribozyme has a motif that recognizes and binds to the recognition site of the target RNA. This motif takes the form of one or more “binding arms”, but is generally two binding arms. The binding arms of the hammerhead ribozyme are flanking sequences helix I and helix III located at both ends of helix II. These lengths are variable, usually 6 to 10 nucleotides each, but can be shorter or longer. The length of these flanking sequences can affect the cutting rate. For example, reducing the total number of nucleotides in a flanking sequence from 20 to 12 increases the turnover rate of ribozymes that cleave HIV sequences by a factor of 10 (Goodchild, JVK, 1991, Archives of Bio). Chemistry and Biophysics (Arch Biochem Biophys) 284: p386-391). The catalytic motif in the ribozyme helix II of the hammerhead ribozyme cleaves the target RNA at a site called the cleavage site. Whether a ribozyme can cleave a given RNA depends on the presence or absence of a ribozyme recognition site that contains an appropriate cleavage site.

  Each type of ribozyme recognizes its own cleavage site. The cleavage site of the hammerhead ribozyme has a nucleotide base triplet GUX (where G is guanine, U is uracil, and X is any nucleotide base) immediately upstream. The hairpin ribozyme has a BCUGNYR cleavage site, 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 with a hairpin ribozyme is performed between G and N in the cleavage site.

  For more information on ribozymes, see “Molecular Biology and Biotechnology” (RA Meyers, Ed., 1995, VCH Publishers Inc, p831-8320 and “Retro “Retroviruses” (edited by JM Coffin et al., 1997, Cold Spring Harbor Laboratory Press, p683).

  Ribozyme expression can be induced in any cell, but it only has an effect in cells where a transcript of the target gene is present.

  Alternatively, the substance can reduce the bioavailable amount of a polypeptide of the invention, rather than directly hindering binding between components. This is considered to be due to, for example, suppressing the expression of this component at the level of transcription, transcript stability, translation or post-translational stability. Examples of such substances are antisense RNA or double stranded interfering RNA that suppresses the amount of mRNA biosynthesis.

  In another preferred embodiment, the NOI comprises siRNA. Post-transcriptional gene silencing (PTGS) mediated by double-stranded RNA (dsRNA) is a conserved cellular defense mechanism to control the expression of foreign genes. It is believed that random integration of elements such as transposons or viruses causes expression of homologous single-stranded mRNA or dsRNA that activates sequence-specific degradation of viral genomic RNA. This silencing effect is called RNA interference (RNAi). The RNAi mechanism processes long dsRNAs into 21-25 nucleotide (nt) RNA duplexes. These products, called small interfering RNAs or silencing RNAs (siRNAs), are sequence specific mediators of mRNA degradation. In differentiated mammalian cells,> 30 bp dsRNA has been shown to activate the interferon response resulting in termination of protein synthesis and nonspecific degradation of mRNA.

  However, this reaction can be avoided by using a 21 nt siRNA duplex, which makes it possible to analyze gene function in cultured mammalian cells.

  In one embodiment, siRNA expression can be regulated by using an RNA polymerase III promoter, such as U6, whose activity can be modulated by the addition of tetracycline.

  In another embodiment, the NOI comprises microRNA. MicroRNAs are a very large group of small RNAs that are normally produced in vivo, and at least some of these regulate the expression of target genes. The founding members of the microRNA family organization are let-7 and lin-4. The Let-7 gene encodes a small but highly conserved RNA species that regulates the expression of endogenous protein coding genes during the development of worms. This active RNA species is first transcribed into a precursor form of about 70 nt and then processed into a mature form of about 21 nt after transcription. Both let-7 and lin-4 are transcribed into a hairpin RNA precursor and then processed into a mature form by the Dicer enzyme.

  In another embodiment, the NOI comprises a hairpin-like double stranded interfering RNA. This short hairpin can be expressed from a single promoter, eg, U6. In another embodiment, RNAi can be effectively mediated by incorporating two promoters, eg, a U6 promoter that expresses a region of sense and a U6 promoter that expresses the reverse complement of the same sequence of this target. it can. In another embodiment, the effect of RNA, i.e., double-stranded interference, is 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. be able to.

  In another embodiment, the NOI may encode a short RNA that can act to alter the process of splicing (“exon-skipping”), alter the process of polyadenylation, or inhibit translation. it can.

NOI can also be an antibody. As used herein, an “antibody” includes an entire immunoglobulin molecule or a portion thereof, or a biobiostere or mimetic thereof, or a derivative thereof, or a combination thereof. Some examples of immunoglobulin molecules include Fab, F (ab) ′ ₂ and Fv. Examples of biological isotopes include single chain Fv (ScFv) fragments, chimeric antibodies, and bivalent antibodies.

  Transduced target cells that express specific genes or lack expression of specific genes have applications in drug discovery and target identification. The expression system could be used to determine which genes have the desired effect on the target cell, such as genes or proteins that can prevent or block the induction of apoptosis in cells. Similarly, if it is found that preventing or blocking the expression of a particular gene has an undesirable effect on the target cell, this is an opportunity to find a treatment that can ensure that the expression of that gene is not impaired. There is a case.

  Thus, the present invention relates to diseases such as experimental allergic encephalomyelitis, an animal model of multiple sclerosis, and experimental autoimmune neuritis, an animal model of acute and chronic inflammatory demyelinating polyneuropathy. Can be used in combination with a model. Other disease models are known to those skilled in the art.

  The NOI delivered by the vector delivery system can be capable of immortalizing target cells. Several immortalization techniques are known in the art (eg, Katakura Y et al (1998) Methods Cell Biol. 57: p69-). 91).

  The vector delivery system may be either a non-viral delivery system or a viral delivery system.

  In some preferred embodiments, the vector delivery system is a viral delivery vector system.

  In some other preferred embodiments, the vector delivery system is a retroviral vector delivery system.

  As used herein, the term “immortalized” is used for cells that can be cultured and expanded for more than 11 passages that can be maintained in continuous culture for more than about 2 months.

  Immortalized motor and sensory neurons and brain cells are useful in laboratory procedures, screening programs, and in therapeutic applications. For example, immortalized dopaminergic neurons can be used for transplantation for the treatment of Parkinson's disease and the like.

  The NOI delivered by the vector delivery system can be a selection gene or a marker gene. A variety of selectable labels have been successfully used for retroviral vectors. These are described in “Retroviruses” (Cold Spring Harbor Laboratory Press, 1997, J.M. Coffin, S.M.). SM Hughes, edited by HE Barmus (ed. P444), examples of which are bacterial neomycins that confer resistance to G418 and hygromycin, respectively. And hygromycin phosphotransferase gene; mutant mouse dihydrofolate reductase gene conferring resistance to methotrexate; allows cells to grow in media containing mycophenolic acid, xanthine and aminopterin A bacterial gpt gene; a bacterial hisD gene that allows cells to grow in medium without histidine but with histidinol; a multidrug resistance gene (mdr) that confers resistance to various drugs; and against puromycin or phleomycin Examples include, but are not limited to, bacterial genes that confer resistance. All of these labels are dominant selectable, and by using them, many of the cells expressing these genes can be chemically selected.

  The NOI delivered by the present vector delivery system can allow the therapeutic gene-gene itself to induce a therapeutic effect, or can encode a product that allows the gene to induce a therapeutic effect. In this sense, it can be-.

  The term “mimetic” means any chemical that can be a peptide, polypeptide, antibody, or other organic chemical with similar binding specificity as an antibody.

  As used herein, the term “derivative” includes chemical modification of an antibody. Examples of such modifications include hydrogen substitution with alkyl, acyl or amino groups.

Disease Generally speaking, the present invention is useful for improving the distribution of the expressed protein. For example, when this vector is administered to a certain site, the protein is released and this is released into the brain and nervous system. Can act on another part.

  The vector system used in the present invention is particularly useful for treating and / or preventing diseases associated with the death or dysfunction of cells of neural tissues such as brain cells including neurons, CSF, and / or glial cells. Therefore, this vector system is useful for the treatment and / or prevention of neurodegenerative diseases.

  In particular, the vector system used in the present invention can be used to treat and / or prevent diseases involving death or dysfunction of motor or sensory neurons.

  Treatable diseases include pain; Parkinson's disease, motor neuron diseases including amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) and motor diseases such as Huntington's disease; Alzheimer's disease; spinal muscle atrophy and lysosome accumulation The disease may be included, but is not limited to these.

  Amyotrophic lateral sclerosis (ALS) is a motor neuron degenerative disease with an annual incidence of 1 to 2 per 100,000 people. The disease is characterized by degeneration of spinal, brainstem and motor cortex motoneurons that lead to weakness of the lupus and extremities, medulla and respiratory muscles. About 5-10% of ALS is familial. Genes for which mutations or halotypes are thought to be associated with disease predisposition include SOD1, ALS2, and VEGF (Lambrechts et al., Nature Genetics 2003; 2003 June 6 online publication; Oosthuyse et al. Nature Genetics 2001; June; Vol. 28, p 131-138).

  In particular, the vector system used in the present invention is useful for the treatment and / or prevention of ALS. In this embodiment, the NOI can be capable of suppressing SOD1 expression.

  Another NOI (s) can encode a group of molecules that prevent apoptosis and thus prevent cell death. Suitable molecules include XIAP and NAIP. Alternatively, NOI (s) can encode a group of neurotrophic molecules that promote regeneration such as IGF-1, GDNF, VEGF and cardiotrophin (CT1).

  Lysosomal storage disease or glycolipid storage disease is a genetic disease that occurs when the rate of glycolipid synthesis is not balanced with the rate of intracellular degradation. For this reason, undegraded glycolipid accumulates in lysosomes. Such diseases include Fabry disease, Niemann-Pick disease, gangliosidosis, metachromatic cerebral white matter atrophy and many types of mucopolysaccharidosis.

  Spinal muscular atrophy (SMA) is an anterior horn cell disorder and is an autosomal recessive disease. Anterior horn cells are in the spinal cord. SMA affects voluntary muscles for activities such as remission, walking, head and neck control, and swallowing. SMA categories include type I SMA, also known as Weldnig Hoffmann disease, type II, type III, type IV (adult-onset) and adult-onset, often referred to as Kugelberg-Welander disease or juvenile spinal muscular atrophy X-linked SMA is included. This type is also called Kennedy syndrome or bulbar spinal muscular atrophy. SMA is a motor neuron disease common in humans, the most severe type of which causes death by the age of two. This disease is due to a mutation in the telomere motor neuron survival gene SMN1. In particular, the vector system used in the present invention is useful for the treatment and / or prevention of SMA. In this embodiment, the NOI can be capable of encoding a gene for replacement with a defective SMN1 gene. Another NOI (s) can encode a group of molecules that prevent cell death by blocking apoptosis. Suitable molecules include XIAP and NAIP. Alternatively, NOI (s) can encode neurotrophic molecules that promote regeneration such as IGF-1, GDNF, neurotrophin-3 (NT-3), VEGF and cardiotrophin (CT1). .

  In another embodiment, the vector system used in the present invention is useful for the treatment and / or prevention of Parkinson's disease. In this embodiment, the NOI can be capable of encoding a neuroprotective molecule. In particular, NOI (s) can encode a group of molecules that prevent the death of TH-positive neurons or promote the regeneration and functional recovery of damaged nigrostriatal system. In another embodiment, such NOI encodes an enzyme or group of enzymes involved in L-DOPA or dopamine synthesis, such as tyrosine hydroxylase, GTP cyclohydrase I, aromatic acid dopa decarboxylase and vesicular monoamine transporter 2. can do.

  The vector system of the present invention can also be used for the treatment and / or prevention of inflammatory neurological diseases including autoimmune nervous system diseases.

  Inflammatory responses have been developed to protect the body against trauma and infection. When trauma or infection occurs, blood leukocytes are transported to the site of injury through a complex cascade of events, killing potential pathogens and promoting tissue repair. However, this strong inflammatory response can damage normal tissue and failure to regulate the innate immune response results in a variety of lesions. Multiple sclerosis (MS) is known to be an inflammatory disease of the brain, but now inflammation is a disease of diseases such as stroke, traumatic brain injury, HIV-related dementia, Alzheimer's disease and prion disease. It has been suggested that it can contribute significantly.

  As mentioned above, MS is a chronic inflammatory disease of the CNS and its pathogenesis is presumed to be autoimmune. MS is thought to be caused by blood-derived T cells specific for CNS antigens. The T cells induce the production of non-antigen-specific mononuclear cells that can destroy oligodendrocytes directly and / or by releasing substances that are toxic to myelin in the CNS.

  Other autoimmune nervous system disorders include Guillain-Barre syndrome, myasthenia gravis, acute disseminated encephalomyelitis, stiff man syndrome, autoimmune neuritis, motor dysfunction, chronic inflammatory demyelinating polyneuropathy Disorders, multifocal motor neuropathy, paraproteinemia neuropathy, autoimmune diseases of the neuromuscular junction, other motor unit diseases, inflammatory myopathy, autoimmune myositis, paraneoplastic ) Neurological diseases, connective tissue disorders, and nervous system complications of vasculitis.

  As another embodiment related to the treatment and / or prevention of inflammatory diseases, the nucleotide of interest delivered by the vector system used in the present invention is an up-regulated anti-inflammatory molecule such as an anti-inflammatory cytokine or this anti-inflammatory molecule. Encode molecules that can be. Accordingly, one embodiment of the present invention relates to a method for the treatment of nervous system inflammatory diseases such as MS comprising delivering an anti-inflammatory molecule directly to the CNS.

  Cytokines that may be useful in the treatment of MS and possibly other diseases include IL-1β, IL-2, IL-4, IL-6, IL-1n, IFN-β, IFN-γ. , TNF-α, p55TNFR-Ig, p75dTNFR, TGF-β, PDGF-α and NGF. More generally speaking, it should be understood that anti-inflammatory cytokines can be effectively delivered by the present invention in the treatment and / or prevention of nervous system inflammatory diseases.

  Another method involves the delivery of nucleotides of interest that inhibit or encode inflammatory molecules such as inflammatory cytokines. That is, it is envisaged to use inhibitors such as those described above, such as ribozymes, siRNAs, antibodies and antisense sequences.

  Yet another method involves the delivery of myelin proteins and / or growth factors that restore and / or regenerate damaged nerve myelin sheaths.

  The table below lists some examples of diseases that can be treated using the methods and vectors of the invention, and proposed therapeutic mechanisms, as well as examples of genes that can be regulated to treat these diseases It is a summary.

Furthermore, by delivering the gene using the knowledge that retrograde transport to the brain occurs after delivery to the subretinal region, it affects the site of the optic nerve, optic nerve cross, optic nerve cord or lateral knee (LGN) region Any disease can be treated. Such diseases include (but are not limited to) glaucoma, other diseases secondary to elevated intraocular pressure, and neurodystrophy such as multiple sclerosis. Suitable expressed genes include growth or survival factors such as erythropoietin or VEGF for stroke treatment, and neuroprotective factors such as PEDF, GDNF or neurotrophin for the treatment of optic neuropathy (eg, Leber's congenital disease).

  In particular, as a preferred embodiment for the treatment of motor neuron disease, the vector system is a lentiviral vector system. This is because highly efficient retrograde transport and long-term expression are achieved using a lentiviral vector system that advantageously has the rabies virus G pseudotype. Adenovirus and HSV, and even though not so much, AAV are retrogradely transported, but in a lentiviral vector system with the rabies virus G pseudotype, high efficiency is achieved by selective transduction to neurons. Retrograde transport is achieved. Conveniently, a lentiviral vector pseudotyped with rabies virus G can specifically target motor neurons with high efficiency. Furthermore, the use of lentiviral vectors eliminates the toxicity problems that are common when using adenovirus and HSV, for example. Retrograde transport takes place via the intramuscular route with little or no transduction of mature muscle cells (Mazarakis et al., Supra), thus improving the efficiency of transduction of motor neurons To obtain the selectivity required for the phenotype is a further advantage of the lentiviral vector system pseudotyped with rabies virus glycoprotein G. On the other hand, when AAV is used, transduction of motoneurons is not Also produces a long-lasting expression in (Lu et al. Neuroscience Res. January 2003, 45 (1): p33-40). it is conceivable that.

Pharmaceutical Compositions The present invention also provides the use of a vector delivery system in the manufacture of a pharmaceutical composition. This pharmaceutical composition can be used to deliver an EOI such as NOI to target cells in need thereof.

  The vector delivery system may be either a non-viral delivery system or a viral delivery system.

  In some preferred embodiments, the vector delivery system is a viral delivery vector system.

  In some other preferred embodiments, the vector delivery system is a retroviral vector delivery system, preferably a lentiviral vector delivery system.

  The pharmaceutical composition can be used to treat an individual by gene therapy, in which case the composition comprises or can produce a therapeutically effective amount of a vector system according to the invention.

  The methods and pharmaceutical compositions of the present invention can be used for treatment of humans or animals. Preferably, the subject is a mammal. More preferably, the subject is a human. Usually, the actual dosage that is most appropriate for the subject will be determined by a physician, and the dosage will vary depending on the age, weight and response of the particular patient.

  The composition can optionally contain a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can be any suitable binder, lubricant, suspending agent, coating agent, solubilizer and (e.g., as a carrier, excipient or diluent) (e.g., Other carrier agents that can promote or increase viral entry into the target site (such as lipid delivery systems) can be included.

  Where appropriate, the pharmaceutical compositions may be administered by inhalation, administration as a suppository or pessary, topical administration as a lotion, solution, cream, ointment or dust, application of a skin patch, starch or Oral administration as tablets containing excipients such as lactose, capsules or vaginal suppositories alone or in admixture with excipients, or elixirs, solutions or suspensions containing flavoring agents or coloring agents Or the composition can be injected systemically, for example, intracavernous, intravenously, intramuscularly or subcutaneously. For systemic administration, the composition can be optimally 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. . For buccal or sublingual administration, the composition can be administered in the form of a tablet or lozenge that may be formulated by conventional methods.

  Conveniently, the vector system used in the present invention can be administered by direct injection into the patient. For the treatment of neurodegenerative diseases such as Parkinson's disease, the vector system can be injected into the brain. The vector system can be injected directly into any target area of the brain (eg, linear or substantia nigra). Alternatively, the vector system can be injected into a given region and transduced into the target region by retrograde transport of the vector system. Intramuscular injection is particularly preferred because it is the least invasive administration method.

  The table above outlines suitable sites for treatment by injection, including intraspinal, intrathecal, amygdala, DRG, skin, herpetic neuralgia, cerebral cortex, striatum , Substantia nigra, hypothalamus, pituitary, glioma bed, retina, vitreous, muscle, spinal cord and intracerebroventricular injection.

Transport The invention relates to the use of a vector system comprising at least part of a CVS coat protein or a mutant, fragment or homologue variant thereof for transducing a target site, the vector system being retrograde Uses that are intended to move to the site by sex transport.

  Viral particles can move in the same direction as the nerve impulse, i.e. from the cell body along the axon to the axon terminal. This is called antegrade transport.

  In the present invention, it was revealed that the protein-containing vector system of the present invention can be transported retrogradely, that is, can move in the opposite direction. Retrograde transport (or movement) of the vector means that it is taken up by the axon terminal and moves toward the cell body.

  This type of transport is often referred to as “Fast transport”, which translates to 50-200 mm per day in the synaptic direction of the membranous organelle (forward) or back to the original cell body (retrograde). (Hirokawa (1997) Curr Opin Neurobiol 7 (5): p605-614). However, the details of the retrograde transport mechanism are unclear. This probably transports the entire virus particle in conjunction with the internalized receptor. The fact that the vector system of the present invention can be specifically transported in this way (as shown herein) suggests that the env protein can be involved.

  HSV vectors, adenoviral vectors, and hybrid HSV / adeno-associated viral vectors have all been shown to be retrogradely transported in the brain (Hellorou, Mallet (1997) Molecular. • Neurobiology 15 (2): p241-256; Ridoux et al. (1994) Brain Res 648: p171-175; Constantini et al. (1999) Human Gene Therapy 10: p2481-2494). Infusion of an adenoviral vector system expressing glial cell line-derived neurotrophic factor (GDNF) into rat striatum enables expression in dopaminergic axon terminals and cell bodies via retrograde transport (Herreu) (Holloulou, Mallet (1997), supra; Bilan-Bleuel et al. Proceedings of the National Academy of Sciences USA 94. : P8818-8823).

  Retrograde transport can be detected by several mechanisms known in the art. In the example shown herein, when a vector system that expresses a heterologous gene is injected into the linear body, expression of this gene is detected in the substantia nigra. Obviously, retrograde transport along neurons from the striatum to the basal ganglia is involved in this phenomenon. Also known is a method of monitoring a labeled protein or virus and directly monitoring its retrograde movement by real-time confocal microscopy (Hirokawa (1997), supra).

  By retrograde transport, it can be expressed in both the axon terminal and cell body of transduced neurons. These two parts of the cell are thought to be located in different areas of the nervous system. Thus, a single administration (eg, injection) of the vector system of the invention can transduce many distal sites.

  The present invention also uses the vector system of the present invention for transducing a target site, comprising the step of administering the vector system to an administration site remote from the target site to allow good penetration and distribution throughout the CNS. I will provide a. For example, by administering to a region of the brain, the EOI can be distributed, different parts of the brain and / or different cell types.

  The target site can be any site of interest. This may or may not be anatomically coupled to the site of administration. The target site can be a site capable of receiving the vector from the administration site via axonal transport, eg, antegrade or (more preferably) retrograde transport.

  For a given site of administration, it is believed that there are several possible target sites that can be identified using tracers by methods known in the art (Ridoux et al. ( (1994) said literature).

  For example, when the CVS / EIAV vector is injected into the striatum, the pallidum bulb, cortex, various thalamic nuclei, amygdala, hypothalamus, supraoptic nucleus, deep mesencephalic nucleus, substantia nigra, lower inferior arm Transgene expression occurs in the caudal region of the brain stem, such as the nucleus, the parabrachial nucleus, the genetic nucleus, the paracerebellar nucleus, the ventral cochlear nucleus, and the facial nucleus.

  A target site is considered “away from the administration site” if it is located (or primarily located) in a different region than the administration site. These two sites can be distinguished by their spatial location, form and / or function.

  The basal ganglia of the brain consist of several pairs of nuclei, the two nuclei of each pair being located in the opposite cerebral hemisphere. The largest nucleus is a linear body consisting of a caudate nucleus and a lens nucleus. Further, each lens nucleus is divided into an outer part called a putamen and an inner part called a pallet ball. The substantia nigra and red nucleus of the midbrain and the subthalamic nucleus of the diencephalon are functionally linked to the basal ganglia. Axons from the substantia nigra end in the caudate nucleus or putamen. The subthalamic nucleus is associated with the pallidum. For the conductivity of the rat basal ganglia, see Oorschott (1996) Journal of Comparative Neurology 366: p580-599.

  In a preferred embodiment, the site of administration is a linear body of the brain, particularly the caudate putamen. When injected into this putamen, various distal sites of the brain can be labeled, for example, target sites located in the pallidum, amygdala, subthalamic nucleus or substantia nigra. Typically, transduction of pallidal cells results in retrograde labeling of thalamic cells. In a preferred embodiment, the target (s) sites (or one of these sites) are substantia nigra.

  Within a given target site, the vector system can transduce target cells. The target cell can be a cell that is present in a neural tissue such as a sensory or motor neuron, astrocyte, oligodendrocyte, microglia or ependymocyte.

  The vector system is preferably administered by direct injection. Methods for injection into the brain (particularly the striatum) are known in the art (Bilang-Bleuel et al (1997) Proceedings of the National Academy of Science. (Pro. Natl. Acad. Sci.) USA, 94: p8818-8823; Choi-Lundberg, et al. (1998), Experimental Neurology (Exp. Neuro. 154: p261-275; Choi-Lundberg, et al. (1997) Science 275: p838-841; Mandel, et al. (1997) Proceeding Of the -The National Academy of Sciences (Pro. Natl. Acad. Sci. USA, 94: p14083-14088). Stereotaxic injection can be performed.

As described above, in the case of transduction in tissues such as the brain, since it is necessary to use a very small volume, the virus preparation is concentrated by centrifugation. The resulting formulation is at least 10 ⁸ t. u. / Ml, preferably 10 ^{8 to} 10 ¹⁰ t. u. / Ml, more preferably at least 10 ⁹ t. u. Must have a titer of / ml. (This titer is expressed in transducing units per ml (tuu / ml) quantified using the standard D17 cell line). Variations in transgene expression can be improved by increasing the number of injection sites and decreasing the injection rate (Hellorou, Mallet (1997), supra). The number of injection sites is usually 1 to 10 sites, more generally 2 to 6 sites. 1 to 5 × 109 t. u. For doses containing / ml, the infusion rate is often 0.1 to 10 μl / min, usually about 1 μl / min.

  In addition, we can detect NOI expression in various parts of the brain and spinal cord, such as upper garment, buffy coat cells, hippocampus, corpus callosum and septum, after administration to CSF, for example, by intrathecal delivery Was revealed.

Transplantation The present invention also provides immortalized cells of the CNS, such as sensory or motor neurons or brain cells, and their use in transplantation methods.

  Transplant protocols using fetal dopaminergic neurons, equivalent cells from other species and neural progenitor cells are known (Dunnett, Bjorkund (1999) Nature 339). Volume, Appendix A, pages 32-39). Similar methods could be used to transplant the cells of the present invention.

Examples For details of the EIAV vector system used in the following examples, its production and viral transduction methods, see Mazarakis et al (2001), supra, And in WO 02/36170, in particular in the section “Materials and Methods” of Mazarakis et al (2001) and in the Examples section of WO 02/36170.

The rabies virus G mutant EIAV vector was pseudotyped using the wild type and two variants of the rabies virus G coat protein of the ERA strain. The sequence of the rabies virus strain ERA is shown in FIGS. 1 and 2 (SEQ ID NOs: 1 and 2). By substituting the 333rd amino acid arginine with glutamine, a single mutant of wild type ERA strain (ERAwt) was prepared. This mutant that exists in nature and is not pathogenic to mature mice was named ERAsm. Furthermore, by substituting the 330th amino acid from K to N, a double mutant of ERAwt named ERAdm was obtained. These two coat proteins were used to pseudotype an EIAV vector that expresses the marker gene LacZ.

More specifically, a partial PCR fragment of ERAwt in which two amino acids were changed above was amplified using the following primers:
(5 ′ to 3 ′) CTA CAA CTC AGT CAT GAC TTG GAA TGA GAT CCT CCC CTC AAA AGG GTG TTT AAG AGT TGG GGG GAG G
(5 'to 3') CCT TTT GAG GGG AGG ATC TCA TTC CAA GTC ATG ACT GAG TTG TAG TGA GCA TCG GCT TCC ATC AAG GTC
In addition, the full length fragment of ERAdm (with the two amino acids above changed) was amplified using the following primers:
(5 'to 3') ACC GTC CTT GAC ACG AAG CT
(5 'to 3') GGG GGA GGT GTG GGA GGT TT
The fragment obtained as described above was cloned into pSA91 using an appropriate restriction enzyme. Successful clones were sequenced and used to create EIAV vectors by transient transduction.

  The sequence of ERAADM is shown in FIG. 3 (SEQ ID NO: 3).

CVS
CVS (countervirus standard) rabies virus glycoprotein cDNA was obtained from ATCC (ATCC No. 40280 (designation) pKB3-JE-13). A fragment containing the complete coding sequence of this glycoprotein was excised using EcoRI, cloned into pSA91 and sequenced (Bk 1092 pg 75). The resulting sequence is shown in FIG. 4 (SEQ ID NO: 4).

The titers of various pseudotyped EIAV vectors determined by transduction efficiency on viral transduced D17 cells were as follows:
pONY8Z ERAwt 7x10 ⁸ TU / ml
pONY8Z ERAsm 9x10 ⁸ TU / ml
pONY8Z ERAAdm 1x10 ⁸ TU / ml
pONY8Z CVS 7x10 ⁸ TU / ml

Example 1-Injection of EIAV pseudotyped with rabies virus G or VSV-G coat protein into cerebrospinal fluid (CSF) and treatment of MS using intrathecal route for gene therapy Under a Hypnorm and Hypnovel anesthesia, a 5 μl Hamilton syringe with a rounded 33 gauge needle was used. A total of 8 rats were injected with 10 μl of viral vector solution at coordinates: AP = −0.92; L = 1.4; V = 3.3. Group 1 rats (n = 4) were injected with EIAV pseudotyped with VSV-G coat protein. In the second group of rats (n = 4), all viral vectors were pseudotyped with rabies virus G. The virus titer was 7 × 10 ⁸ TU / ml. This lentiviral solution was slowly injected at a rate of 0.2 μl / min using an injection pump (World Precision Instruments Inc. 5-0 after virus vector injection). The skin was closed with Vicryl continuous suture and the post-operative rat was kept warm until fully recovered All surgical procedures were approved by local veterinarians and ethics committees and regulated by the Home Office It carried out according to.

  After injection into CSF, it was found that the marker gene LacZ was expressed in various regions of the brain and spinal cord (FIG. 5). Vectors pseudotyped with rabies virus G were able to infect upper garments and buffy coat cells (FIGS. 5A to 5C). Strong bilateral transduction was observed in the hippocampus (mainly CA3), corpus callosum, and septum (FIGS. 5D to 5I). Moreover, this virus was able to spread to the spinal cord (FIGS. 6A to 6F).

  In contrast, pseudotyping with VSV-G showed no signs of transport or distribution.

  As is apparent from the above results, the present invention is considered to be an alternative treatment method for inflammatory neurological diseases. Delivering cytokine-encoding genes to CSF via the lentivirus according to the present invention has the following main advantages: i) being able to reach high concentrations of cytokines over a wide range within the CNS; ii) The exogenous gene incorporated into the host cell DNA is expressed persistently over a long period of time; and iii) no immune response to the viral particle occurs.

Example 2 Gene Transfer into Neonatal Mouse Muscle Using EIAV-Rabies Virus G and EIAV-CVS Whether EIAV Vectors Pseudotyped with Rabies Virus G or CVS Envelope Protein Are Retrogradely Transported to the Mouse Spinal Cord In order to clarify the above, P6 newborn mice were injected intramuscularly with pONY8Z rabies G virus stock solution (titer 5.7 × 10 ⁸ TU / ml). Seven mice were injected with pONY8Z rabies virus G (10 μl, n = 2; 20 μl, n = 2; 30 μl, n = 3). The other group of mice was injected with pONY8Z CVS (titer 7 × 10 ⁸ TU / ml, n = 3, injection volume = 30 μl).

  The results are shown in FIGS. 7 and 8, and this experiment showed that many motoneurons (MN) were retrogradely transduced after injection of the virus particles into the gastrocnemius muscle. In this study, 10-12 MN per section (about 50% of MN) were positive for X-gal in pONY8Z rabies virus G-injected mice (FIG. 7). In EIAV-CVS injected mice, 7-8 MN per section were x-gal positive (FIG. 8). The transduced cells were found to be localized only on one side of the anterior horn. Examination of the transduced cell morphology suggested that the cell was a motor neuron (large cell). Β-gal immunostaining was also performed. Myocytes were also transduced in EIAV-rabies virus G injected mice (FIG. 7).

Example 3 Gene Transfer to Rat Spinal Cord Using EIAV-CVS For intraspinal injection, anesthetized 2-month-old rats were placed in a stereotaxic frame and spinal cord adapter (Storting Co., IL) PONY8.0Z vector (n = 3) (7 × 10 ⁸ TU / ml) pseudotyped with CSV from one lumbar spinal cord where the laminectomy was performed after the spinal cord was fixed using ) Was injected. The infusion was controlled using an infusion pump (World Precision Instruments Inc., Sarasota, USA) at a rate of 0.1 μl per minute with a 10 μl Hamilton syringe with a 33 gauge needle. It was. After the injection, the injection needle was left for 5 minutes and then extracted. Four weeks after virus injection, rats were transcardially perfused with 4% w / v paraformaldehyde. Thereafter, the spinal cord and brain were dissected to examine the X-gal reaction.

  The results of this experiment are shown in FIG. Infusion of EIAV-Lac CVS into the spinal cord resulted in strong transduction at the site of injection and retrograde transport to the contralateral side of the spinal cord. Interestingly, brainstem and cortical motor neurons were transduced by retrograde transport (FIG. 9).

Example 4 Injection of EIAV vector pseudotyped with CVS coat protein into a linear body Approximately 2 × 10 ⁶ TU of each vector was placed in the linear body of a mature male Wistar rat (300 g), and the stereotaxic coordinates AP Slow infusion using 0 mm, ML 3.5 mm, DV 4.75 mm and left for 2-4 weeks. The rats were then sacrificed and transcardial perfusion was performed with 4% paraformaldehyde. After overnight incubation with 4% paraformaldehyde, the brain was cryoprotected with 30% sucrose for at least 3 days and then frozen and cut into 40 μm coronal sections. Using this, examination by X-gal staining and immunohistochemistry was performed.

  As is clear from FIG. 10, when the EIAV vector pseudotyped with the CVS coat protein was injected into the linear body, strong expression was observed in the pallidal bulb. Retrograde transport was observed in the cortex, various thalamic nuclei, amygdala, hypothalamus, supraoptic nucleus, deep midbrain nucleus and substantia nigra. In addition, retrograde transport to the caudal region of the brainstem was also observed. In this region, various nuclei such as the nucleus of the lower limb arm, the parachortic nucleus, the genetic nucleus, the paracerebellar peduncle nucleus, the ventral cochlear nucleus and the facial nucleus nucleus are positive for X-gal staining. Met.

Example 5 Retrograde transport to the brain after subretinal delivery of a lentiviral vector pseudotyped with rabies virus coat protein

Methods An EIAV viral vector based on the pONY8.0 CMV GFP genome pseudotyped with the rabies virus coat protein (pSA91ERAwt) was generated using a transient three-plasmid transfection method. As a result of biological measurement of the titer of this virus (batch number OBM039), it was calculated to be 1 × 10e10 TU / ml. A total of 4 μl (2 × 2 μl) was injected under the retina of C57 / bl-6J mice and tissues were collected at various time points for gene expression analysis.

  As described above, when the EIAV vector pseudotyped with this rabies virus is delivered under the retina, this vector extends along the optic nerve to the optic nerve cross in the base of the brain, and from here along the optic nerve cord, a part of the subcortical thalamus It was found to be retrogradely transported to the knee (LGN) region (FIG. 11).

  The optic nerve fibers from each eye cross over in a very unique manner at the optic nerve crossover-the fibers that originate in the nasal side of the retina cross over to the contralateral hemisphere, but the fibers that originate in the ear side retina Extends to the same side of the brain without crossover. Thus, delivery under the retina of a single eye can be retrogradely transported to both hemispheres of the brain. Alternatively, if the injection under the retina is limited to a specific region of the eye on the nasal side or ear side, only one hemisphere of the brain can be targeted.

Example 6-In vitro confirmation of SMA fibroblasts Construction of the Smart2SMN and pONY 8.7NCSMN vectors as shown in Figure 12 was reported in 2001 by Mazarakis et al. The SMN gene was provided by Dr. Arthur Burghes (Ohio State University, Ohio, USA). This Smart2SMN vector was pseudotyped with an ERA strain-derived rabies virus G coat protein.

  SMA fibroblasts become an in vitro model of SMA. It was removed from a type I SMA patient.

  Primary cultured fibroblasts were established from type I SMA patients by standard methods (DiDonato et al., 2003; Human Gene Therapy 14: p179).

  In this cell, the expression of SMN protein is very little or not recognized. The Smart2 SMN vector pseudotyped with the rabies virus G coat protein has been described in 50 and 100 according to the method described in Mazarakis et al., Human Molecular Genetics, 2001. Used to transduce SMA fibroblasts with MOI.

  As a negative control, Smart2LacZ ERAwt transduced and non-transduced cells were used. Immunohistochemistry was used to confirm SMN protein expression from pSMT2SMN ERAwt. Confocal microscopy showed strong positive SMN staining in the cytoplasm. Moreover, it was found from this experiment that gems in the SMA fibroblast nucleus were recovered by using EIAV (FIG. 13). The best results were obtained using MOI 100. Such staining was not observed in the negative control.

  Cell counts revealed an average of 8 gems per SMN transduced cell. According to Scordis et al. (PNAS, 100: p4114-4119) and Didonato et al. (Hum Gen Ther 14: p179-188) from SMA patients An average of 3 to 6 nuclear gems was found in the treated fibroblasts.

  To examine the efficiency of the SMN vector, the canine osteosarcoma cell line D17 was used. FIG. 14 shows a Western blot using an SMN antibody recognizing SMN (Transduction Laboratories, and an antibody against the HA tag demonstrating SMN expression of the cells transduced with the SMN vector. It is.

  D17 expresses some SMN protein, but when cells are transduced with the SMN vector, overexpression is observed compared to control cells transduced with the LacZ vector. Expression of the SMN transgene was confirmed using an HA tag antibody.

Example 7-SMN gene replacement in an SMA animal model The SMN-1 gene replacement method utilizing gene therapy can be used to prevent cell death against SMA animal models and motor neurons of SMA patients.

Type III SMA mouse model Type III mice had muscle weakness, motor neuron degeneration, and decreased SMN protein levels (versus an average of 9.8 nuclear gems per motor neuron in age-matched controls) , Calculated as an average of 4.5 nuclear gems for type III SMA mice).

  Smart2SMNHA was injected into the unilateral leg muscles of 4 type III mice. The mice were then perfused with 4% paraformaldehyde, the spinal cord was removed and stored at -80 ° C. Motor neuron SMN expression was monitored using HA and SMN antibodies. Confocal microscopy showed that motoneurons were efficiently transduced by retrograde transport, as shown by HA tag immunostaining (FIG. 15A). Another analysis showed that the SMN gene was successfully transferred into the muscles of these mice (FIG. 15B).

  The immune response elicited by SMN gene transfer into the type III mouse model was negligible, as is apparent from FIG.

Mouse model of type I SMA The mouse model of type I SMA is a model of severe SMA. The mouse exhibits motor neuron death, muscle weakness, and dies by 14 days after birth. The purpose of this experiment was to prolong the survival of mice by delivering to the muscle a LentiVector ™ that expresses the SMN gene.

  In this study, 1 to 2 day old neonatal SMN mice are used. Injection into newborns is performed as follows. Mice are briefly anaesthetized for a short time. Viral vectors are injected using a Hamilton microinjector with a 33 gauge needle. The following groups are used in this study.

Smart2-SMN group (n = 8)
Leg muscles: 20 μl each
Intraperitoneal: 10 μl
Diaphragm muscle: 10 μl
Facial muscle: 20μl
Tongue: 10 μl
Intracranial (brain stem): 5 μl
Chest trunk muscle: 10 μl
Smart2-GDNF group (n = 4)
Leg muscles: 20 μl each
Intraperitoneal: 10 μl
Diaphragm muscle: 10 μl
Facial muscle: 20μl
Tongue: 10 μl
Intracranial (brain stem): 5 μl
Chest trunk muscle: 10 μl
Smart2-SMN + Smart2-GDNF group (n = 6)
Leg muscles: 20 μl each
Intraperitoneal: 10 μl
Diaphragm muscle: 10 μl
Facial muscle: 20μl
Tongue: 10 μl
Intracranial (brain stem): 5 μl
Chest trunk muscle: 10 μl
Smart2-LacZ group (n = 6)
Leg muscles: 20 μl each
Intraperitoneal: 10 μl
Diaphragm muscle: 10 μl
Facial muscle: 20μl
Tongue: 10 μl
Intracranial (brain stem): 5 μl
Chest trunk muscle: 10 μl
All lentiviral vectors for these experiments are pseudotyped with rabies virus G so that retrograde transport of the virus and transduction to motor neurons can be achieved.

  When the EIAV gene is transferred to a mouse model of type I SMA, the transgene is expressed extensively and the survival time of the mouse is prolonged. Immunostaining of SMN revealed that the transgene was strongly expressed not only in spinal motor neurons but also in DRG neurons. Therefore, when Smart2SMN or pONY8.7NCSMN was injected into the muscle of type I mice, it was retrograde. It is suggested that transport results in transduction for motor neurons and DRG cells (Figure 17). Such staining was not observed in mice injected with Smart2LacZ (FIG. 17). Lentiviral vector-mediated expression of the SMN gene in type I SMA mice extends the survival time of these mice by 35% compared to LacZ injected control mice and 50% compared to non-injected mice.

Example 8-Prolonging Survival of SOD1 Transformed Mice by VEGF Gene Delivery To prevent or block the progression of motor neuron degeneration in ALS patients, an ALS mouse model was used to identify XIAP (Aegera Therapeutics ( The effect of lentivector (trademark) expressing anti-apoptotic molecules such as Aegera Therapeutics Inc.) and neuroprotective molecules such as VEGF, IGF-1, and GDNF and the siRNA method on the survival time of motor neurons will be examined.

Gene Therapy in SOD1 Transformed Mice To examine functional efficiency in SOD1 mice, Smart2LacZ, Smart2VEGF and Smart2XIAP were each injected intramuscularly (Table 1). Three groups were used for this experiment. Group 1 mice were injected with Smart2VEGF (n = 7). The second group was injected with a lentivector (trademark) expressing the anti-apoptotic protein XIAP (n = 6). The control group (n = 6) was injected with the Smart2LacZ vector. The targeted muscles were 3 groups of leg muscles, facial muscles and diaphragm muscles (Table 1).

Administration of Smart2hVEGF delays the onset of disease and prolongs survival of SOD1-transformed mice compared to LacZ control mice. Disease onset was delayed by an average of 30 days. hVEGF-injected mice survive at least 41 days longer than the LacZ group. However, Smart2XIAP has no effect on SOD1-transformed mice. In addition, VEGF administration improves the motor function of SOD1 mice compared to the LacZ group. This result was from a rotarod and footprint test.

  All publications mentioned in the above specification are herein incorporated by reference. It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed method and system of the present invention without departing from the scope or spirit of the invention. Although the invention has been described in terms of certain preferred embodiments, it will be understood that the claims of the invention should not be unduly limited to such embodiments. On the contrary, various modifications of the disclosed embodiments for carrying out the invention which are obvious to those skilled in biology or related fields are intended to be within the scope of the following claims.

EXAMPLES Next, the present invention will be further described with reference to examples. However, these examples are useful for the convenience of those skilled in the art in practicing the present invention, and do not limit the scope of the present invention. In these examples, reference is made to the following figures.
FIG. 1 (SEQ ID NO: 1) shows the ERA wild type polynucleotide and amino acid sequence. FIG. 2 (SEQ ID NO: 2) shows the ERA wild type polynucleotide and amino acid sequence. FIG. 3 (SEQ ID NO: 3) shows the polynucleotide sequence of ERAADM. FIG. 4 (SEQ ID NO: 4) shows the polynucleotide sequence of the CVS rabies virus glycoprotein. FIG. 5 shows the results of Example 1, illustrating the transduction efficiency of EIAV-LacZ in the brain after injection into CSF. FIG. 6 shows Example 1, which illustrates the expression of the marker gene LacZ in the spinal cord after EIAV-LacZ injection into the CSF. FIG. 7 shows the results of Example 2. FIG. 8 shows the results of Example 2. FIG. 9 shows the results of Example 3. FIG. 10 shows the results of Example 4 using CVS. FIG. 11 shows GFP fluorescence observed in the optic nerve crossing (A), optic nerve axon (B), and optic nerve cell body (C) after gene delivery of pONY8.0CMVGFP virus under the retina. It is. FIG. 12a shows a schematic representation of a replacement vector containing the SMN gene. FIG. 12b shows a schematic representation of pONY8.7NCSMN. FIG. 13 shows the results of confocal analysis of SMN immunolabeling after in vitro transduction with a Smart2 SMN vector pseudotyped with rabies virus G coat protein. A and B) Recovery of SMN protein in SMA fibroblasts transduced by lentiviral vector-mediated expression of SMN. C) Cells that were not transduced. D) b-gal immunostaining in SMA fibroblasts transduced with Smart2LacZ. Note the strong cytoplasmic and nuclear staining in A and B. FIG. 14 shows a Western blot supporting SMN expression in transduced D17 fibroblasts. D17 cells have been transduced with Smart2SMN, SMN-HA and LacZ vectors. FIG. 15 shows the results of SMN gene therapy in a mild SMA model. A) Transduction of spinal motoneurons by injecting SMN-HA expressing lentiviral vector ™ intramuscularly in a mouse model of type III SMA. B) SMN expression in muscle monitored with anti-HA tag antibody. FIG. 16 shows the results of studies on immune responses of type II mice injected with Smart2SMN intramuscularly. FIG. 17 shows the transfer of the SMN gene in a mouse model of type I SMA. DRG cells (A) and spinal motor neurons (B) were transduced by retrograde transport by intramuscular injection of SMN expression vector (C) control.

Claims (144)

Use of a vector system to transduce a target site, said vector system being transferred to said target site by diffusion, and said vector system comprising a rabies virus G coat protein or a mutant, variant thereof A homologue or fragment or a CVS coat protein or a mutant, variant, homologue or fragment thereof, or at least part thereof, and the target site is at least part of the central nervous system Use to. Use according to claim 1, wherein the target site is at least part of the brain, spinal cord and / or spinal nerve. Use according to claim 1 or 2, further comprising the step of administering the vector system to an administration site remote from the target site. 4. Use according to claim 3, wherein the administration site is cerebrospinal fluid. 4. Use according to claim 3, wherein the administration site is part of the brain. 4. Use according to claim 3, wherein the administration is carried out intramuscularly or via the peripheral nervous system. Use according to any of the preceding claims, wherein the rabies virus G coat protein mutant is a rabies virus G coat protein in which the 333rd amino acid arginine is replaced with glutamine. 7. Use according to any one of claims 1 to 6, wherein the rabies virus G coat protein mutant has the 333rd amino acid arginine replaced with glutamine and the 330th amino acid lysine. Use that is a rabies virus G coat protein substituted with asparagine, or a variant, fragment or homologue thereof. Use according to any preceding claim, wherein the vector system is a viral vector system. Use according to any preceding claim, wherein the vector system is a retroviral vector system. Use according to any of the preceding claims, wherein the vector system is used to deliver EOI. Use according to claim 11, wherein the EOI is NOI. Use according to claim 11 or 12, wherein the EOI can perform a substitution or up-regulation of expression on a gene in a target cell. Use according to claim 12 or 13, wherein the EOI is a NOI as a selection gene, marker gene or therapeutic gene. Use according to claim 11 or 12, wherein the EOI can block or suppress the expression of a gene in a target cell. 16. Use according to claim 15, wherein the EOI is an antisense RNA, antibody, siRNA, ribozyme, short hairpin RNA or microRNA. Use according to any one of claims 11 to 16, wherein the EOI is able to integrate into the genome of the target cell. 18. Use according to any one of claims 11 to 17, wherein the EOI has a therapeutic effect or encodes a protein having a therapeutic effect. Use according to any one of claims 11 to 18, wherein the EOI can encode the protein of interest ("POI"). Use according to any of the preceding claims, wherein the vector system can be derived from a lentivirus. 21. Use according to claim 20, wherein the vector system can be derived from EIAV or HIV. A method for treating and / or preventing a disease in a patient comprising the step of administering the vector system according to any one of claims 1 to 21 to a patient in need thereof. 23. A pharmaceutical composition comprising the vector system according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier, diluent or excipient. 23. A method for carrying out a method for treating and / or preventing a disease in a subject in need thereof, wherein the target cell is transduced using the vector system according to any one of claims 1 to 22. A method comprising the steps of: 25. A method according to claim 22 or 24 for treating and / or preventing movement disorders. 26. The method according to claim 25, wherein the movement disease is Parkinson's disease, motor neuron disease or Huntington's disease. 25. A method according to claim 22 or 24 for treating and / or preventing lysosomal storage diseases. 28. The method according to claim 27, wherein the lysosomal storage disease is Gaucher disease, Fabry disease, Niemann-Pick disease, gangliosidosis, mucopolysaccharidosis or metachromatic cerebral white matter atrophy. 25. A method according to claim 22 or 24 for treating and / or preventing Alzheimer's disease, ALS or pain. 25. A method according to claim 22 or 24 for treating and / or preventing SMA. 25. A method according to claim 22 or 24 for treating and / or preventing diseases of the optic nerve, optic nerve crossing, optic nerve cord or LGN region. 25. A method according to claim 22 or 24 for treating and / or preventing inflammatory neurological diseases. 40. The method according to claim 32, wherein the disease is an autoimmune nervous system disease. 34. The method according to claim 32 or 33, wherein the disease is multiple sclerosis, stroke, traumatic brain injury, HIV-related dementia, Alzheimer's disease, prion disease, Guillain-Barre syndrome, myasthenia gravis, encephalomyelitis , Stiff man syndrome, autoimmune neuritis, motor dysfunction, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, paraproteinemia neuropathy, neuromuscular junction autoimmunity A method that is a neurological complication of disease, inflammatory myopathy, autoimmune myositis, parameoplastic neurological disease, connective tissue or vasculitis. A method for analyzing the effect of POI in CNF, comprising the step of using the vector system according to any one of the preceding claims. Use of a vector system comprising said EOI for biodistribution of EOI, said vector system comprising a rabies virus G coat protein or a mutant, variant, homologue or fragment thereof or a CVS coat protein or a mutant thereof , A variant, homologue or use of at least part of or comprising a fragment. 40. The use according to claim 36, further comprising administering the vector system to an administration site remote from the target site. 38. Use according to claim 36 or 37, wherein the vector system is administered intramuscularly or via cerebrospinal fluid, brain or peripheral nervous system. 39. Use according to any one of claims 36 to 38, wherein the rabies virus G coat protein mutant is a rabies virus G coat protein in which the 333rd amino acid arginine is replaced with glutamine. 39. Use according to any one of claims 36 to 38, wherein the rabies virus G coat protein mutant has the 333rd amino acid arginine replaced with glutamine and the 330th amino acid lysine. A rabies virus G coat protein in which is substituted with asparagine, or a variant, fragment or homologue thereof. 41. Use according to any one of claims 36 to 40, wherein the vector system is a viral vector system. Use according to any one of claims 36 to 41, wherein the vector system is a retroviral vector system. Use according to any one of claims 36 to 42, wherein the EOI is NOI. 44. The use according to claim 43, wherein the EOI can perform substitution or up-regulation of expression to a gene in a target cell. 45. Use according to claim 43 or 44, wherein the EOI is a NOI as a selection gene, marker gene or therapeutic gene. 46. Use according to any one of claims 36 to 45, wherein the EOI can block or suppress the expression of a gene in a target cell. 47. Use according to claim 46, wherein said EOI is an antisense RNA, antibody, siRNA, ribozyme, short hairpin RNA or microRNA. 48. Use according to any one of claims 36 to 47, wherein the EOI can be integrated into the genome of the target cell. 49. Use according to any one of claims 36 to 48, wherein the EOI has a therapeutic effect or encodes a protein having a therapeutic effect. 50. Use according to any one of claims 36 to 49, wherein the EOI can encode the protein of interest ("POI"). 51. Use according to any one of claims 36 to 50, wherein the vector system can be derived from a lentivirus. 52. The use according to claim 51, wherein the vector system can be derived from EIAV or HIV. 53. A method for treating and / or preventing a disease in a patient comprising administering the vector system according to any one of claims 36 to 52 to a patient in need thereof. 54. A pharmaceutical composition comprising the vector system according to any one of claims 36 to 53 and a pharmaceutically acceptable carrier, diluent or excipient. 54. A method of performing a method for treating and / or preventing a disease in a subject in need thereof, wherein the target cell is transduced using the vector system according to any one of claims 36 to 53. A method comprising: 56. A method according to claim 53 or 55 for the treatment and / or prevention of movement disorders. 57. The method according to claim 56, wherein the movement disease is Parkinson's disease, motor neuron disease or Huntington's disease. 56. A method according to claim 53 or 55 for treating and / or preventing lysosomal storage diseases. 59. The method according to claim 58, wherein the lysosomal storage disease is Gaucher disease, Fabry disease, Niemann-Pick disease, gangliosidosis, mucopolysaccharidosis or metachromatic cerebral white matter atrophy. 56. A method according to claim 53 or 55 for treating and / or preventing Alzheimer's disease, ALS or pain. 56. A method according to claim 53 or 55 for treating and / or preventing SMA. 56. A method according to claim 53 or 55 for treating and / or preventing diseases of the optic nerve, optic nerve crossing, optic nerve cord or LGN region. 56. A method according to claim 53 or 55 for treating and / or preventing inflammatory neurological diseases. 64. The method according to claim 63, wherein the disease is an autoimmune nervous system disease. 64. The method according to claim 63 or 64, wherein the disease is multiple sclerosis, stroke, traumatic brain injury, HIV-related dementia, Alzheimer's disease, prion disease, Guillain-Barre syndrome, myasthenia gravis, encephalomyelitis , Stiff man syndrome, autoimmune neuritis, motor dysfunction, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, paraproteinemic neuropathy, neuromuscular junction autoimmune disease, inflammation A neurological complication of muscular dysfunction, autoimmune myositis, paraneoplastic neurological disease, connective tissue or vasculitis A method for analyzing the effect of POI in the CNS, comprising the step of using the vector system according to any one of claims 36 to 65. Use of a vector system to transduce a target site, said vector system being transferred to said target site by retrograde transport, and said vector system comprising CVS coat protein or a mutant, variant thereof , A homologue or fragment, or use thereof, wherein the target site is at least part of the central nervous system. 68. Use according to claim 67, wherein the target site is at least part of the brain, spinal cord and / or spinal nerve. 61. Use according to claim 59 or 60, further comprising administering the vector system to a site of administration remote from the target site. 70. The use according to claim 69, wherein the administration site is cerebrospinal fluid. 70. The use according to claim 69, wherein the administration site is part of the brain. 70. Use according to claim 69, wherein the administration is carried out intramuscularly or via the peripheral nervous system. 75. Use according to any one of claims 67 to 72, wherein the vector system is a viral vector system. 74. Use according to claim 73, wherein the vector system is a retroviral vector system. 75. Use according to any one of claims 67 to 74, wherein the vector system is used to deliver EOI. 76. The use according to claim 75, wherein the EOI is a NOI. 77. Use according to claim 75 or 76, wherein the EOI can perform a substitution or up-regulation of expression on a gene in a target cell. 78. Use according to any one of claims 75 to 77, wherein the EOI is a selection gene, a marker gene or a NOI as a therapeutic gene. 77. Use according to claim 75 or 76, wherein the EOI can block or suppress the expression of a gene in a target cell. 80. Use according to claim 79, wherein the EOI is an antisense RNA, antibody, siRNA, ribozyme, short hairpin RNA or microRNA. 81. Use according to any one of claims 75 to 80, wherein the EOI is able to integrate into the genome of the target cell. 82. Use according to any one of claims 75 to 81, wherein the EOI has a therapeutic effect or encodes a protein having a therapeutic effect. 83. Use according to any one of claims 75 to 82, wherein the EOI can encode a protein of interest ("POI"). 84. Use according to any one of claims 67 to 83, wherein the vector system can be derived from a lentivirus. 85. Use according to claim 84, wherein the vector system can be derived from EIAV or HIV. 84. A method for treating and / or preventing a disease in a patient comprising administering the vector system according to any one of claims 67 to 83 to a patient in need thereof. 87. A pharmaceutical composition comprising the vector system of any one of claims 67 to 86 and a pharmaceutically acceptable carrier, diluent or excipient. 87. A method for conducting a disease treatment and / or prevention method in a subject in need thereof, wherein the target cell is transduced using the vector system according to any one of claims 67 to 86. A method comprising: 90. A method according to claim 86 or 88 for treating and / or preventing movement disorders. 90. The method according to claim 89, wherein the movement disease is Parkinson's disease, motor neuron disease or Huntington's disease. 90. A method according to claim 86 or 88 for treating and / or preventing lysosomal storage diseases. 92. The method according to claim 91, wherein the lysosomal storage disease is Gaucher disease, Fabry disease, Niemann-Pick disease, gangliosidosis, mucopolysaccharidosis or metachromatic cerebral white matter atrophy. 90. A method according to claim 86 or 88 for treating and / or preventing Alzheimer's disease, ALS or pain. 90. A method according to claim 86 or 88 for treating and / or preventing SMA. 90. A method according to claim 86 or 88 for treating and / or preventing diseases of the optic nerve, optic nerve crossing, optic nerve cord or LGN region. 90. A method according to claim 86 or 88 for treating and / or preventing inflammatory neurological diseases. 99. The method according to claim 96, wherein the disease is an autoimmune nervous system disease. 98. The method according to claim 96 or 97, wherein the disease is multiple sclerosis, stroke, traumatic brain injury, HIV-related dementia, Alzheimer's disease, prion disease, Guillain-Barre syndrome, myasthenia gravis, encephalomyelitis , Stiff man syndrome, autoimmune neuritis, motor dysfunction, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, paraproteinemic neuropathy, neuromuscular junction autoimmune disease, inflammation A neurological complication of muscular dysfunction, autoimmune myositis, paraneoplastic neurological disease, connective tissue or vasculitis. 99. A method for analyzing the effect of POI in the CNS, comprising the step of using the vector system according to any one of claims 67 to 98. Use of a vector system for transducing an intrauterine target site or a neonatal target site, the vector system comprising a rabies virus G coat protein or a mutant, variant, homologue or fragment thereof or Use which is or comprises at least part of a CVS coat protein or a mutant, variant, homologue or fragment thereof. 101. Use according to claim 100, wherein the target site is at least part of the brain, spinal cord and / or spinal nerve. 102. Use according to claim 100 or 101, further comprising administering the vector system to an administration site remote from the target site. 102. Use according to claim 102, wherein said administration site is cerebrospinal fluid. 102. Use according to claim 102, wherein the administration site is part of the brain. Use according to claim 102, wherein the administration is carried out intramuscularly or via the peripheral nervous system. 110. Use according to any one of claims 100 to 105, wherein the rabies virus G coat protein mutant is a rabies virus G coat protein in which the 333rd amino acid arginine is replaced with glutamine. 106. Use according to any one of claims 100 to 105, wherein the rabies virus G coat protein mutant has the 333rd amino acid arginine replaced with glutamine and the 330th amino acid lysine. A rabies virus G coat protein in which is substituted with asparagine, or a variant, fragment or homologue thereof. 108. Use according to any one of claims 100 to 107, wherein the vector system is a viral vector system. Use according to claim 108, wherein the vector system is a retroviral vector system. 110. Use according to any one of claims 100 to 109, wherein the vector system is used to deliver EOI. Use according to claim 110, wherein the EOI is NOI. 112. Use according to claim 110 or 111, wherein the EOI is capable of replacing or up-regulating expression of a gene in a target cell. Use according to any one of claims 110 to 112, wherein the EOI is a selection gene, a marker gene or a NOI as a therapeutic gene. 112. Use according to claim 110 or 111, wherein the EOI can block or suppress the expression of a gene in the target cell. 115. Use according to claim 114, wherein the EOI is an antisense RNA, antibody, siRNA, ribozyme, short hairpin RNA or microRNA. 116. Use according to any one of claims 110 to 115, wherein the EOI is capable of integrating into the genome of the target cell. 117. Use according to any one of claims 110 to 116, wherein the EOI has a therapeutic effect or encodes a protein having a therapeutic effect. 118. Use according to any one of claims 110 to 117, wherein the EOI can encode a protein of interest ("POI"). Use according to any one of claims 110 to 118, wherein the vector system can be derived from a lentivirus. 120. Use according to claim 119, wherein the vector system can be derived from EIAV or HIV. 108. A method of treating and / or preventing a disease in a patient comprising the step of administering the vector system of any one of claims 100 to 107 to a patient in need thereof. 122. A pharmaceutical composition comprising the vector system of any one of claims 100 to 121 and a pharmaceutically acceptable carrier, diluent or excipient. 122. A method of performing a method for treating and / or preventing a disease in a subject in need thereof, wherein the target cell is transduced using the vector system according to any one of claims 100 to 121. A method comprising: 124. A method according to claim 121 or 123 for treating and / or preventing motor disease. 125. The method according to claim 124, wherein the motor disease is Parkinson's disease, motor neuron disease or Huntington's disease. 124. A method according to claim 121 or 123 for treating and / or preventing lysosomal storage diseases. 127. The method according to claim 126, wherein the lysosomal storage disease is Gaucher disease, Fabry disease, Niemann-Pick disease, gangliosidosis, mucopolysaccharidosis or metachromatic cerebral white matter atrophy. 124. A method according to claim 121 or 123 for treating and / or preventing Alzheimer's disease, ALS or pain. 124. A method according to claim 121 or 123 for treating and / or preventing SMA. 124. A method according to claim 121 or 123 for treating and / or preventing diseases of the optic nerve, optic nerve crossing, optic nerve cord or LGN region. 124. A method according to claim 121 or 123 for treating and / or preventing inflammatory neurological diseases. 132. The method according to claim 131, wherein the disease is an autoimmune nervous system disease. 132. The method according to claim 131 or 132, wherein the disease is multiple sclerosis, stroke, traumatic brain injury, HIV-related dementia, Alzheimer's disease, prion disease, Guillain-Barre syndrome, myasthenia gravis, encephalomyelitis , Stiff man syndrome, autoimmune neuritis, motor dysfunction, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, paraproteinemic neuropathy, neuromuscular junction autoimmune disease, inflammation A neurological complication of muscular dysfunction, autoimmune myositis, paraneoplastic neurological disease, connective tissue or vasculitis. 134. A method for analyzing the effect of POI in the CNS, comprising the step of using the vector system according to any one of claims 100 to 133. An EIAV vector comprising at least part of a CVS coat protein or a mutant, variant, homologue or fragment thereof. 136. Use of the vector of claim 135 in the manufacture of a pharmaceutical composition for treating and / or preventing a disease in a subject. 136. A pharmaceutical composition comprising the vector system of claim 135 and a pharmaceutically acceptable carrier, diluent or excipient. 136. A method of performing a method for treating and / or preventing a disease in a subject in need thereof, comprising transducing target cells using the vector system of claim 135. 122. Use of the vector according to claim 121 for use according to any one of claims 1 to 22, 36 to 53, 67 to 86 or 100 to 121. A method for analyzing a gene of a target cell or a function of a protein encoded by a gene, according to any one of claims 1 to 22, 36 to 53, 67 to 86, 100 to 121, or 135 A method comprising the step of suppressing or blocking expression of the gene using the vector or vector system described. A cell transduced with the vector or vector system according to any one of claims 1 to 22, 36 to 53, 67 to 86, 100 to 121, or 135. 142. Immortalized brain cells, spinal cord cells or motor or sensory neurons according to claim 141. 142. Use of an immortalized cell according to claim 141 in the manufacture of a therapeutic agent for transplantation. 145. A method of performing a method for treating and / or preventing a disease in a subject in need thereof, comprising the step of transplanting immortalized cells according to claim 141 into the subject.