WO2006090697A1 - Therapeutic agent for disease with apoptotic degeneration in eye tissue cell containing pedf and fgf2 - Google Patents

Therapeutic agent for disease with apoptotic degeneration in eye tissue cell containing pedf and fgf2 Download PDF

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Publication number
WO2006090697A1
WO2006090697A1 PCT/JP2006/303052 JP2006303052W WO2006090697A1 WO 2006090697 A1 WO2006090697 A1 WO 2006090697A1 JP 2006303052 W JP2006303052 W JP 2006303052W WO 2006090697 A1 WO2006090697 A1 WO 2006090697A1
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gene
vector
fgf2
pedf
immunodeficiency virus
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PCT/JP2006/303052
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French (fr)
Japanese (ja)
Inventor
Masanori Miyazaki
Yoshikazu Yonemitsu
Yasuhiro Ikeda
Katsuo Sueishi
Toshiaki Tabata
Akihiro Iida
Yasuji Ueda
Mamoru Hasegawa
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Dnavec Corporation
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Priority to CN2006800128827A priority Critical patent/CN101160139B/en
Priority to JP2007504722A priority patent/JP4971974B2/en
Publication of WO2006090697A1 publication Critical patent/WO2006090697A1/en
Priority to HK08111109.7A priority patent/HK1115324A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • Drugs for treating diseases with apoptotic degeneration in ocular tissue cells including PEDF and FGF2
  • the present invention relates to a pharmaceutical product for treating a disease associated with apoptotic degeneration in ocular tissue cells using a lentiviral vector loaded with a neurotrophic factor.
  • Retinitis pigmentosa is an intractable genetic disease in which the photoreceptor cell layer and pigment epithelial layer of the retina are extensively brought into apoptosis.
  • photoreceptor cells There are two types of photoreceptor cells: rods and cones.
  • the rods are mainly distributed in areas where the central force of the retina is slightly shifted, and are related to the way objects are seen in the dark and the field of view.
  • the cones are widely distributed in the macula, the central part of the retina, and are mainly related to the central visual acuity and color vision.
  • retinal pigmentosa In retinitis pigmentosa, the photoreceptor cells are damaged, so there are many cases of night blindness, visual field stenosis, and visual acuity symptom, and blindness occurs as it progresses. Part of retinal pigment degeneration is thought to be caused by abnormalities in genes that specifically act on photoreceptors and retinal pigment epithelial cells, but the cause of most retinal pigment degeneration is still unknown.
  • rod cGMP-phosphodiesterase a and b subunits rod cyclic nucleotide-sensitive cation channels, retinal guanyl cyclase, RPE65
  • the genes for cellular retinyl aldehyde binding protein and arrestin are known.
  • the genes for rhodopsin, peripherin 'RDS (retinal degeneration slow), lom-1, and X-linked retinal pigment degeneration are known. ing.
  • Diagnosis is based on fundus findings (in typical cases, retinal vascular narrowing, crude sesame salt-like retina, bone-like pigmentation, colorless in atypical cases Identities, white spots, etc.), visual field (stenosis matching the lesion site such as centripetal, ring-shaped, map-like, centrality), dark adaptation (rising of the second curve of the dark adaptation curve), visual acuity Decrease))
  • Physiological findings (reduction in electroretinogram (ERG) amplitude / disappearance), fluorescence fundus angiography findings (retinal pigment epithelial atrophy and retina choroidal atrophy).
  • FGF2 Fibroblast growth factor 2
  • a neurotrophic factor is a retinal photodamage model (Non-patent document 1), a retinal pigment degeneration model (Non-patent document 2-6), and retinal ganglion cell damage.
  • Ischemia reperfusion, optic nerve transection Models (Non-Patent Document 7) and other animal experiments have been examined for application to gene therapy in the ophthalmic field.
  • Non-patent Document 8 pigment epithelium derived factor
  • the present inventors constructed a SIV-PEDF vector in which PEDF was inserted into a SIV vector having the backbone of the simian immunodeficiency virus (SIV), a retrovirus, and examined it in model animals!
  • SIV simian immunodeficiency virus
  • good results have been obtained (Non-patent Document 8).
  • retinitis pigmentosa which is a serious disease that eventually leads to blindness
  • the development of a treatment that exhibits a higher effect is required.
  • Patent Document 1 International Application Number PCT / JP2002 / 005225 International Publication Number WO2002 / 101057
  • Patent Document 2 International Application Number PCT / JP00 / 03955 International Publication Number WO00 / 078987
  • Tokubori 1 Lau D, Fiannery J. iral-mediated FuF ⁇ 2 treatment of the constant 1 ight damage model of replica degeneration.Doc Ophthalmol. 2003 Jan; 106 (
  • Non-Patent Document 2 Akimoto M, Miyatake S, Kogishi J, Hangai M, Okazaki K, Takahashi JC, Saiki M, Iwaki M, Honda Y. Adenovirally expressed basic fibroblast growth factor rescues containing cells in RCS rats. 1999 Feb; 40 (
  • Non-Patent Document 3 Uteza Y, Rouillot JS, Kobetz A, Marchant D, Pecqueur S, Arnaud E, Prats H, Honiger J, Dufier JL, Abitbol M, Neuner— Jehle M. Intravitreous transplantation of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delay s dye cell degeneration in Royal College of Surgeons rats.Proc Natl Aca d Sci US A. 1999 Mar 16; 96 (6): 3126- 31.
  • Non-Patent Document 4 Neuner- Jehle M, Berghe LV, Bonnel S, Uteza Y, Benmeziane F, Roui Hot JS, Marchant D, Kobetz A, Dufier JL, Menasche M, Abitbol M. Ocular cell trans fection with the human basic fibroblast growth factor gene delays reservoir cell degeneration in RCS rats. Hum Gene Ther. 2000 Sep 1; 11 (13): 1875— 90.
  • Non-Patent Document 5 Lau D, McGee LH, Zhou S, Rendahl KG, Manning WC, Escobedo JA, Flannery JG. Retinal degeneration is slowed in transgenic rats by AAV— mediated de livery of FGF- 2. Invest Ophthalmol Vis Sci. 2000 Oct; 41 (ll): 3622-33.
  • Non-patent literature b Spencer B, Agarwala S, Gentry L, Brandt CR.HSV- 1 vector- delivere d FGF2 to the retina is neuroprotective but does not preserve functional responses.
  • Non-Patent Document 7 Sapieha PS, Peltier M, Rendahl KG, Manning WC, Di Polo A. Fibrobl ast growth factor— 2 gene delivery stimulates axon growth by adult retinal ganglion c ells after acute optic nerve injury. Mol Cell Neurosci. 2003 Nov ; 24 (3): 656-72.
  • Non-Patent Document 8 Miyazaki M, Ikeda Y, Yonemitsu Y, Goto Y, Sakamoto T, Tabata T, Ueda Y, Hasegawa M, Tobimatsu S, Ishibashi T, Sueishi K. Simian lentiviral vector-mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats.Gene Ther. 200 3 Aug; 10 (17): 1503-l l.
  • Non-Patent Document 9 Wahlin KJ, Campochiaro PA, Zack DJ, Adler R. Neurotrophic factors cause activation of intracellular signaling pathways in Muller cells and other cells of the inner retina, but not peptidess.Invest Ophthalmol Vis bci. 2000; 41 (3 ): 92 7-36.
  • Non-Patent Document 10 Valter K, van Driel D, Bisti S, Stone J. FGFRl expression and FGF Rl— FGF— 2 colocalisation in rat retina: sites of FGF— 2 action on rat derivatives. Growth Factors. 2002; 20 (4): 177- 88.
  • the present invention has been made in view of such circumstances, and the problem to be solved by the present invention is to find a new treatment method for a disease accompanied by apoptotic degeneration in ocular tissue cells.
  • PEDF and FGF2 are thought to have different sites of action.
  • the site of action of PEDF is the visual site of retinal photoreceptors and ganglion cells.
  • the site of action of FGF2 suggests that the FGF2 receptor is in the inner granular layer of the retina (Non-Patent Document 9).
  • Non-patent Document 10 There is a report (Non-patent Document 10) suggesting that it exists in cells. If PEDF and FGF2 are administered simultaneously, there is a possibility that a higher effect than before can be obtained due to the synergistic effect of both.
  • the present inventors constructed a SIV-PEDF vector and a SIV-FGF2 vector.
  • the SIV vector is a vector with simian immunodeficiency virus as the backbone, and since the introduced foreign gene can be continuously expressed in the host, it should provide drug delivery particularly suitable for the treatment of chronic diseases. Can do.
  • the above vector was administered into the subretinal space of RCS rats, a retinitis pigmentosa disease model, and the effect was examined. Four weeks after administration of the vector, the rat rear eye was collected and the expression levels of PEDF and FGF2 were measured. As a result, hPEDF and hFGF2 gene expression was confirmed by administration of SIV-hPEDF and SIV-hFGF2 vectors.
  • Simultaneous administration of PEDF and FGF2 is considered to be effective in treating not only retinal pigment degeneration but also diseases associated with apoptotic degeneration in ocular tissue cells. That is, the present invention relates to the treatment of diseases associated with apoptotic degeneration in ocular tissue cells by simultaneous administration of FGF2 and PEDF, and more specifically, the following inventions are provided.
  • a pharmaceutical product for treating a disease associated with apoptotic degeneration in an ocular tissue cell comprising any of the following (a) Kas et al. (D) together with a pharmaceutically acceptable medium:
  • PEDF Pigment epithelium derived factor
  • FGF2 fibroblast growth factor 2
  • PEDF Pigment epithelium derived factor
  • FGF2 fibroblast growth factor 2
  • PEDF Pigment epithelium derived factor
  • FGF2 fibroblast growth factor 2
  • a pharmaceutical product according to (2) above comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene,
  • simian immunodeficiency virus vector comprises a cPPT sequence and a Z or WPRE sequence
  • a simian immunodeficiency virus vector is pseudotyped with VSV-G! /, A pharmaceutical product according to any one of (2) to (5) above, (7) Monkey immunodeficiency virus vector force The pharmaceutical product according to any one of (2) to (6) above, which is derived from a gm strain,
  • a composition comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene, and a composition comprising a recombinant simian immunodeficiency virus vector carrying an FGF2 gene.
  • kits for treating a disease associated with apoptotic degeneration in ocular tissue cells comprising a composition comprising a recombinant simian immunodeficiency virus vector having a PEDF gene and an FGF2 gene,
  • a method for treating a disease associated with apoptotic degeneration in ocular tissue cells comprising administering PEDF and FGF2 or a gene encoding them,
  • An ocular tissue comprising a step of preparing a recombinant simian immunodeficiency virus vector retaining a PEDF gene using a gene transfer vector comprising a nucleotide sequence in which a PEDF gene is inserted into the nucleotide sequence of SEQ ID NO: 1.
  • An ocular tissue comprising a step of preparing a recombinant simian immunodeficiency virus vector retaining the FGF2 gene using a gene transfer vector comprising a nucleotide sequence in which the FGF2 gene is inserted into the nucleotide sequence of SEQ ID NO: 1.
  • a recombinant simian immunodeficiency virus vector carrying the PEDF gene and the FGF2 gene is prepared using a gene transfer vector containing the base sequence of the PEDF gene and the FGF2 gene inserted into the base sequence described in SEQ ID NO: 1.
  • a method for producing a pharmaceutical product for treating a disease associated with apoptotic degeneration in an ocular tissue cell comprising:
  • FIG. 1 shows the structures of an improved gene transfer vector, an improved packaging vector, a rev expression vector, and a VSV-G expression vector.
  • FIG. 2A is a diagram illustrating a process of constructing an improved gene transfer vector from a conventional gene transfer vector.
  • ( a ) shows the continuation of the process in Figure 2B.
  • FIG. 2B is a diagram showing a continuation of FIG. 2A. (a) shows the continuation of the process capability in Fig. 2A.
  • FIG. 3 is a diagram illustrating a process for constructing an improved packaging vector from a conventional packaging vector.
  • FIG. 4A is a diagram illustrating the construction process of a rev expression vector. ( ⁇ ) indicates the continuation of step 4 in Fig. 4.
  • FIG. 4 ⁇ This is a continuation of Fig. 4 ⁇ . ( ⁇ ) shows the continuation of the process power shown in Fig. 4.
  • FIG.5 (a) Conventional gene transfer vectors include cPPT alone, WPRE alone, cPPT and It is a figure explaining the structure of the vector carried simultaneously with WPRE. (B) This is a photograph observing the productivity of SIV vectors when using gene transfer vectors with cPPT alone, WPRE alone, cPPT and WPRE simultaneously in infection with MOI: 15. Upper left: Conventional cPPT, vector without WPRE (control) (_cPPT, -WPRE), upper right: cPPT alone (+ cPPT, -WPRE), lower left: WPRE alone (-cPPT, + WPRE), lower right : CPPT and WPRE installed simultaneously (+ cPPT and + WPRE).
  • FIG. 6 Results of examination of the productivity of SIV vectors by the percentage of foreign gene (EGFP) positive cells when using gene transfer vectors equipped with cPPT alone, WPRE alone, cPPT and WPRE simultaneously.
  • the MOI in the table represents the number of vector particles infected with one cell, and 0.3, 1.5, 7.5, and 15 are the numbers of ⁇ (vector particle number / cell number) in the actual infection experiment.
  • a (+) after the cPPT or WPRE indicates that the vector contains cPPT or WPRE, and (1) indicates that the vector does not contain cPPT or WPRE.
  • the numbers in the table are the percentage of EGFP positive cells (percentage:%).
  • the graph is a graph of the values in the table of (a). The vertical axis of the graph represents the percentage of EGFP positive cells (percentage:%).
  • FIG.7 Comparison of protein expression levels per cell in transgenic cells using MOPP: 15, gene transfer vectors with cPPT alone, WPRE alone, cPPT and WPRE simultaneously It is the result.
  • the numerical value represents the relative value of fluorescence intensity (comparison standard of protein expression level).
  • FIG. 8 Expression level of hPEDF protein when SIV-hPEDF vector is administered alone, SIV-hFGF2 vector is administered alone, SIV-hPEDF and SIV-hFGF2 vectors are administered simultaneously, and SIV-EGFP is administered to RCS rats 2 is a graph showing the expression level of hFGF2 protein.
  • FIG. 9 Four weeks after administration of RCS rats when SIV-hPEDF vector is administered alone, SIV-hFGF2 vector is administered alone, SIV-hPEDF and SIV-hFGF2 vector are administered simultaneously, and SIV-EGFP is administered It is a graph which shows the number of photoreceptor cells remaining.
  • FIG. 10 RCS rats were administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector were co-administered, and SIV-EGFP was administered. Is a graph showing the remaining number of photoreceptor cells in 8 weeks after administration.
  • FIG. 11 Post-administration of RCS rats administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector administered simultaneously, and SIV-EGFP administered It is a graph which shows the number of photoreceptor cells remaining for 12 weeks.
  • FIG. 12 Electroretinogram when RCS rats were administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector were co-administered, and SIV-EGFP was administered. It is a figure which shows the result.
  • FIG. 13 Electroretinogram when RCS rats were administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector were co-administered, and SIV-EGFP was administered. It is a figure which shows the result.
  • the present invention is directed to treatment of diseases associated with apoptosis degeneration in ocular and woven cells by pigment epithelium derived factor (PEDF) and fibroblast growth factor 2 (FGF2).
  • PEDF pigment epithelium derived factor
  • FGF2 fibroblast growth factor 2
  • the present inventors focused on the fact that the target cells for the apoptosis-suppressing action of PEDF and FGF2 are different, and for the disease accompanied by apoptotic degeneration in ocular tissue cells, co-administration of PEDF or FGF2 is one of these alone It was shown that a significantly higher effect than the administration can be obtained.
  • PEDF and FGF2 contained in the pharmaceutical product of the present invention may be a protein or a gene.
  • the amino acid sequence of human PEDF (hPEDF) protein is shown in SEQ ID NO: 5, and the amino acid sequence of human FGF 2 (hFGF2) protein is shown in SEQ ID NO: 6.
  • the cDNA sequence of hPEDF is shown in SEQ ID NO: 7, and the cDNA sequence of hFGF2 protein is shown in SEQ ID NO: 8.
  • the PEDF gene and the FGF2 gene contained in the pharmaceutical product of the present invention can be prepared by methods well known to those skilled in the art.
  • a cDNA library of human retinal pigment epithelial cells can be prepared by using a part or all of the base sequence described in SEQ ID NO: 7 or SEQ ID NO: 8 as a probe.
  • it can be prepared by performing a known nucleic acid amplification method using a part of the base sequence described in SEQ ID NO: 7 or SEQ ID NO: 8 as a primer and human retinal pigment epithelial cell mRNA as a saddle type.
  • the pharmaceutical product of the present invention is prepared from the above gene, it may be in the DNA state, but is preferably inserted into a vector.
  • the PEDF gene and the FGF2 gene may be held in separate vectors or simultaneously in one vector.
  • the type of the vector is not limited as long as it is a safe vector suitable for pharmaceutical use, but is preferably a lentiviral vector, most preferably a simian immunodeficiency virus vector.
  • a virus vector is characterized in that a gene is efficiently introduced into a host cell using a virus infection system.
  • many viral vectors have taken measures to eliminate the proliferation system and lack the self-replicating ability to prevent propagation in the introduced cells.
  • the vector particle has a protein outer shell called force psid.
  • Force psid also constitutes the structural protein power of the gag gene product.
  • the envelope has the function of determining the type of cells that are infected.
  • vector genomic RNA reverse transcriptase, which is the product of the pol gene.
  • a viral vector can be prepared by a packaging vector and a gene transfer vector.
  • the knocking vector carries the viral DNA from which the packaging signal has been removed.
  • Viral DNA contains viral protein sequences.
  • a knocking vector is introduced into the host, empty virus particles are created in the host cell (packaging cell) because there is no knocking signal.
  • One gene transfer vector carries a viral gene sequence necessary for integration into host chromosomal DNA and a foreign gene to be introduced. When this gene transfer vector is introduced into a packaging cell, the vector genomic DNA supplied with the gene transfer vector is integrated into the host chromosome, and then the vector genomic RNA is produced by transcription.
  • viral vector refers to a viral particle that lacks self-replicating ability and has the ability to introduce a nucleic acid molecule into a host.
  • a “recombinant” viral vector refers to a viral vector constructed by genetic recombination techniques. Viral vectors constructed using DNA encoding the viral genome and packaging cells are included in the recombinant virus vector.
  • the "monkey immunodeficiency virus (SIV) vector” refers to a vector in which a sequence essential for the function as a viral vector is a sequence based on the SIV genome among the nucleic acid molecules in the virus particle.
  • sequence essential for function as a viral vector means 5 ′ LTR (R 3 ⁇ 4, U5 region, packaging signal ( ⁇ ), RR RR, 3 ′ LTR in this order from the 5th side.
  • SIV vector of the present invention may be modified as long as it meets the above definition.
  • essential sequence for SIV is derived from SIV, it may include other sequences derived from SIV or sequences derived from other than SIV! /.
  • sequences that can be suitably included include, for example, cPPT ( central polypurine tract), inner ⁇
  • CMV central polypurine tract
  • WPRE woodchuck hepatitis virus posttranscriptional regul atory element
  • simian immunodeficiency virus includes all strains and subtypes of SIV.
  • SIV isolates include, but are not limited to, SIVagm, SIVcpz, SIVmac, SIVmnd, SIVsm, SIVsnm, and SIVsyk.
  • Simian immunodeficiency virus was found as an HIV-like virus in monkeys and together with HIV formed the Primates Lentivirus group (Eiji Ido, Masanori Hayami, simian immunodeficiency virus gene and infection, Pathogenicity.Protein Nucleic Acid Enzyme: Vol..39, No.8. 1994) o This group is further divided into 4 groups: 1) Acquired immune deficiency in humans!
  • HIV-1 group including SIVcpz isolated from HIV-1 and chimpanzee, which causes acquired immune deficiency syndrome (A IDS), 2) SIVsmm and lizard macaque isolated from Certicebus atys SIVmac isolated from aca mulatta and HIV-2 (Jaffar, S. et al., J. Acquir. Immune Defic. Syndr. Hum. Retroviral., 16 ( 5), 327-32, 1997) HIV-2 gnolepe, 3) SIVagm group represented by SIVagm isolated from African green monkey Cercopithecus aethiops, 4) SIVmnd group represented by SIVmnd isolated from mandrill Papio sphinx It is made up of.
  • a IDS acquired immune deficiency syndrome
  • SIVsmm and lizard macaque isolated from Certicebus atys SIVmac isolated from aca mulatta and HIV-2 Jaffar, S. et al., J. Acquir. Immun
  • SIVagm and SIVmnd have not been reported to be pathogenic in natural hosts (Ohta, Y. et al., Int. J. Cancer, 15, 41 (1), 115—22, 1988; Miura, T et al., J. Med. Primatol., 18 (3-4), 255-9, 1989; Masanori Hayami, Japanese Clinical, 47, 1, 1989), especially a kind of SIVagm used in this example
  • the TYO-1 strain has been reported to show no pathogenicity in natural hosts or in experimental infections with the power-quick macaque Macaca facicularis, or the power-giving monkey Macaca mulatta M.
  • virus vector prepared as is considered to be safer than vectors based on HIV-1 and other lentiviruses, and can be preferably used in the present invention.
  • the sequence is shown in SEQ ID NO: 12.
  • the simian immunodeficiency virus vector of the present invention may have a part of a genomic RNA sequence of another retrovirus.
  • human immunodeficiency virus Human Immunodeficiency Vi rus; HIV
  • feline immunodeficiency virus FMV
  • FMV feline immunodeficiency virus
  • CAEV Caprine Arthritis Encephalitis Virus
  • a vector having a chimeric sequence in which a part of the genome sequence is replaced with a part of the genome of the simian immunodeficiency virus is also included in the simian immunodeficiency virus vector of the present invention.
  • Pigment epithelium derived factor (PEDF) gene of the present invention The retained recombinant simian immunodeficiency virus vector (SIV-PEDF vector) refers to a recombinant SIV vector carrying a PEDF gene.
  • the recombinant simian immunodeficiency virus vector (SIV-FGF2 vector) retaining the FGF2 gene of the present invention refers to a recombinant SIV vector carrying the FGF2 gene.
  • the recombinant simian immunodeficiency virus vector carrying the PEDF gene and FGF2 gene of the present invention refers to a recombinant SIV vector carrying both the PEDF gene and the FGF2 gene.
  • the SIV-PEDF vector of the present invention may be of any kind as long as it falls within the above definition, and a preferable example includes a base sequence in which a PEDF gene is inserted into the base sequence described in SEQ ID NO: 1.
  • An SIV vector produced using a gene transfer vector can be mentioned, and a more preferred example is an SIV vector produced using a gene transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 2. Can do.
  • the SIV-FGF2 vector of the present invention may be of any kind as long as it falls within the above definition, but as a preferred example, the FGF2 gene is inserted into the base sequence described in SEQ ID NO: 1.
  • An SIV vector produced using a gene transfer vector containing the nucleotide sequence can be mentioned, and a more preferred example is an SIV produced using the gene transfer vector containing the nucleotide sequence shown in SEQ ID NO: 3.
  • a vector can be mentioned.
  • the recombinant simian immunodeficiency virus vector carrying the PEDF gene and the FGF2 gene of the present invention may be of any kind as long as it falls within the above definition, but a preferred example is SEQ ID NO:
  • An SIV vector produced using a gene transfer vector comprising a base sequence in which a PEDF gene and an FGF2 gene are inserted into the base sequence described in 1 can be mentioned.
  • VSV-G pseudotyping means that the vector envelope contains VSV-G protein, which is a surface glycoprotein of vesicular stomatitis virus (VSV).
  • VSV-G protein may be derived from any VSV strain. For example, the ability to use a VSV-G protein derived from an Indiana serotype strain (J. Virology 39: 519-528 (1981)) is not limited thereto.
  • VSV-G protein is modified from a naturally-occurring protein by substitution, deletion and / or addition of one or more amino acids. Also good.
  • a VSV-G pseudotyped vector can be produced by allowing VSV-G protein to coexist during virus production. For example, the expression of VSV-G in a knocking cell by transfection of the VSV-G expression vector or induction of expression from the VSV-G gene integrated into the host chromosomal DNA will generate this cellular force. The particles are pseudotyped with VSV-G.
  • the VSV-G protein forms a stable trimer of a single glycoprotein and is present on the membrane, making it difficult for vector particles to be destroyed during the purification process and allowing high concentration by centrifugation. (Yang, Y. et al., Hum Gene Ther: Sep, 6 (9), 1203-13. 1995).
  • the SIV vector carrying the PEDF gene of the present invention can further contain an envelope protein derived from another virus.
  • an envelope protein derived from a virus that infects human cells is suitable as such a protein.
  • Such a protein is not particularly limited, and examples thereof include a retroviral unphotopick envelope protein.
  • an envelope protein derived from the mouse leukemia virus (MuLV) 4070A strain is obtained as a retrovirus unphoto-mouth pick envelope protein.
  • An envelope protein derived from MuMLV 10A1 can also be used (for example, pCL-10Al (Im genex) (Naviaux, RK et al, J.
  • examples of such proteins include simple herpesvirus gB, gD, gH and gp85 proteins, EB virus gp350 and gp220 proteins, etc.
  • Hepadnaviridae proteins include hepatitis B virus S protein. Etc.
  • the recombinant simian immunodeficiency virus vector of the present invention can also be modified to LTR (long terminal repeat).
  • LTR is a sequence characteristic of retroviruses and exists at both ends of the viral genome.
  • the 5 'LTR acts as a promoter and promotes transcription of mRNA from proviruses. Therefore, if the gene transfer vector encoding the viral RNA genome packaged in the viral particle is replaced with another strong promoter, the gene transfer vector mRNA transcripts May increase packaging efficiency and vector titer.
  • the 5 'LTR is transcribed by the viral protein tat.
  • the activity is enhanced, and it is possible to remove tat from the knocking vector by replacing the 5 ′ LTR with a promoter independent of the tat protein.
  • viral RNA that infects cells and invades cells is reverse transcribed, then becomes a circular structure that combines the LTRs at both ends, and the binding site and viral integrase are coupled and integrated into the cell's chromosome.
  • the mRNA transcribed from the provirus is downstream from the transcription start point in the 5 ′ LTR, up to the polyA sequence of the 3 ′ LTR, and the promoter portion of the 5 ′ LTR is not packaged in the virus.
  • the 3 'LTR sequence is partially deleted to create a self-inactivating vector (SIN vector) that prevents transcription of the full-length vector mRNA of the target cell. It can also be raised.
  • a lentiviral provirus that has entered the chromosome of the target cell has a 3 'LTR U3 moiety bound to the 5' end. Therefore, the gene transfer vector transcript has a structure similar to that of the gene transfer vector from the gene transfer vector, when U3 is located at the 5 'end after reverse transcription and integrated into the chromosome of the target cell. RNA will be transcribed. If a lentivirus or similar protein is present in the target cell, the transcribed RNA can be repackaged and reinfected to other cells.
  • the 3 'LTR promoter allows expression of a host-derived gene located on the 3' side of the viral genome.
  • P-chome 'Gender 2 ' O Rosenberg, N., Jolicoeur, P., Retoroviral Pathogenesis. Cold Spling Harbor Laboratory Press, 475-585, 1997) This phenomenon has already been a problem in retroviral vectors, and SIN vectors have been developed as a way to avoid it (Yu, S. F. et al, Proc Natl. Acad. Sci.
  • the LTR is methylated by the host-side mechanism and the expression of the transgene is suppressed (Challita, PM and Kohn, D. B "Proc. Natl. Acad. Sci. USA 91: 2567, 1994).
  • Self-inactivating (SIN) vectors lose most of their LTR sequences when integrated into the host genome.
  • the self-inactive type produced by the present inventors by replacing the 3'LTR U3 region of the gene transfer vector with another promoter sequence.
  • the vector has been found to maintain stable expression for more than 2 months after introduction into primate ES cells (Patent Document 1).
  • SIN vectors designed to In particular, vectors in which one or more bases in the U3 region of the 3′LTR have been modified by substitution, deletion, and / or addition are included in the present invention.
  • the U3 region may be simply deleted, or another promoter may be inserted into this region, such as the CMV promoter, EF1 promoter, or CAG promoter. Can do.
  • the PEDF gene and the FGF2 gene encoded by the vector of the present invention are preferably designed to be transcribed by a promoter other than LTR.
  • a promoter other than LTR For example, when the LTR U3 region is replaced with a non-LTR promoter as described above, it is preferable to drive the expression of the PED F gene or the FGF2 gene by this modified LTR.
  • a non-LTR promoter is placed at a position different from the LTR region, and a PEDF gene or FGF2 gene is linked downstream thereof, so that the PE DF gene is independent of the LTR.
  • expression of the FGF2 gene can be induced.
  • An SIV vector constructed by the present inventors so that expression of a foreign gene is driven by a non-LTR promoter First, it was shown that this foreign gene is stably expressed in ES cells for a long period of time (Patent Document 1). Similarly, a vector in which a non-LTR promoter is linked upstream of the PEDF gene or FGF2 gene, and the promoter power of the PEDF gene or FGF2 gene is also transcribed is particularly suitable for the present invention! / ,I can.
  • the non-LTR promoter include CMV promoter, EF1 promoter, and CAG promoter, and CMV promoter is particularly preferable.
  • the nucleotide sequence of the CMV promoter used in this example is shown in SEQ ID NO: 13. Such a vector is particularly effective when constructed in the above-described self-inactivating (SIN) type vector.
  • Lentiviral vectors such as HIV vectors
  • HIV vectors when the host genome already carries HIV virus, recombination occurs between the foreign vector and the endogenous provirus, resulting in a replicable virus.
  • the SIV vector used in this study is a non-replicatable virus that has almost no homologous sequence with HIV and more than 80% of the virus-derived sequences, and this risk is safer than other lentiviral vectors. High nature.
  • the SIV-PEDF vector and SIV-F GF2 vector of the present invention are vectors from which SIV genomic sequences other than the above-described “sequence essential for function as a viral vector” have been removed, and preferably this vector is 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, and most preferably 80% or more of the genome sequence of the SIV from which it is derived.
  • a gene transfer vector DNA having a packaging signal is transcribed in a host cell, and a virus particle is formed in the presence of gag, pol protein and envelope protein.
  • the gag and pol proteins in the knocking cells can be supplied using a packaging vector.
  • the envelope protein may be supplied by a packaging vector or may be supplied by another vector. For example, as in the examples, it may be supplied by a VSV-G expression vector.
  • the gene transfer vector of the present invention basically comprises a 5, LTR, a packaging signal sequence, a PEDF gene and a Z or FGF2 gene, and a 3 'LTR sequence.
  • the LTR sequence may be subjected to the LTR modification described above as a modification of the SIV vector.
  • the above-mentioned cPPT sequence, CMV sequence, RRE sequence and the like may be incorporated.
  • the packaging signal sequence encoded by the gene transfer vector DNA is preferably incorporated as long as possible so that the structure formed by this sequence can be retained, while the packaging signal on the vector DNA and gag Therefore, in order to suppress the appearance frequency of wild-type virus due to recombination with the knocking vector supplying the pol protein, it is necessary to minimize sequence duplication between these vectors. Therefore, in the construction of gene transfer vector DNA, it is preferable to use as short a sequence as possible including sequences necessary for knocking in order to satisfy both packaging efficiency and safety.
  • the packaging vector is derived from SIVagm
  • the HIV-derived gene transfer vector is not packaged, and therefore only the SIV is derived from the packaging signal used for the gene transfer vector DNA. It is considered to be limited to
  • SIV-derived gene transfer vectors are also packaged. Therefore, in order to reduce the appearance frequency of recombinant viruses, different lentivirus-derived gene transfer vectors and packages are used. It is thought that vector particles can be formed by combining with a caging vector.
  • the SIV vector thus produced is also included in the vector of the present invention. In this case, a combination between primate lentiviruses (eg HIV and SIV) is preferred.
  • the gene transfer vector DNA is preferably modified so that the gag protein is not expressed.
  • Viral gag protein is recognized as a foreign substance by the living body, and may have antigenicity. It may also affect cell function. In order to avoid expressing the gag protein !, it can be modified so that it undergoes a frame shift by adding or deleting a base downstream of the start codon of gag. It is also preferable to delete a part of the coding region of gag protein. Generally, viral packaging requires the 5 'side of the coding region of the gag protein. Therefore, in the gene transfer vector 1, it is preferable that the C-terminal coding region of the gag protein is deleted. Pack It is preferable to delete the gag coding region as wide as possible without greatly affecting the caging efficiency.
  • a viral vector can be produced by introducing the gene transfer vector DNA having a packaging signal thus constructed into an appropriate packaging cell.
  • the produced viral vector can be recovered from, for example, the culture supernatant of packaging cells.
  • the gene transfer vector DNA has been modified to enhance the introduction efficiency and expression efficiency of the PEDF gene and the FGF2 gene.
  • An example of a modification that increases the introduction efficiency is the introduction of a cPPT sequence.
  • cPPT is a sequence originally present in the SIV genome.
  • the HIV virus has been reported for a long time (P. Charneau et al .: J. Virol 65: 2415-2431, 1991).
  • the introduction of cPPT improves the transfer of the vector genome to the nucleus, and the gene Increased efficiency has been reported (A. Sirven et al .: Blood 96: 4103-4110, 2000).
  • the base sequence of cPPT used in this example is shown in SEQ ID NO: 14.
  • An example of a modification that increases expression efficiency is the introduction of a WPRE sequence.
  • WPRE is a factor having a function of enhancing gene expression efficiency (US Patent 6284469: RNA export element and methods of use).
  • simultaneous introduction of two factors, cPPT and WPRE has been reported to further enhance individual effects (SC. Barry et al .: Hum. Gene Ther. 12: 1103 -1108, 2001). ).
  • the nucleotide sequence of WPRE used in this example is shown in SEQ ID NO: 15.
  • cPPT can take the same arrangement as that in a general lentiviral vector.
  • cPPT can be placed between the promoter and the external gene, or can be placed upstream of the RRE sequence.
  • 1S Preferably, upstream of the above non-LTR promoter that drives transcription of PEDF or FGF2.
  • WPRE can be placed downstream of the PEDF or FGF2 gene.
  • a gene transfer vector comprising a base sequence in which a PEDF gene is inserted into the base sequence described in SEQ ID NO: 1.
  • An SIV vector produced using a gene transfer vector containing a base sequence having a PEDF gene and an FGF2 gene inserted into the base sequence can be mentioned. A more preferred example is the base sequence described in SEQ ID NO: 2.
  • the packaging vector can be used after removing the sequence which is necessary for the introduction of the PEDF gene and the Z or FGF2 gene.
  • sequences that are not necessary include vif and vpr called modified genes and tat and rev of regulatory genes. It has been reported that modified gene products are not required in vectors (V. Kim et al: J. Virol 72: 811-816, 1998), and in recent years, vectors with modified genes deleted to increase safety. Is used.
  • a vector called the third generation has been developed in which tat has been deleted and rev is transferred to another plasmid to further increase safety.
  • a rev expression vector When rev is excluded from the packaging vector, a rev expression vector is constructed separately, and the re V expression vector holds the PEDF gene of the present invention such as SIV-PEDF vector and SIV-FGF2 vector and Z or FGF2 gene. It can be used in the production of SIV vectors.
  • the nucleotide sequence of rev of SIVagm TYO-1 strain is shown in SEQ ID NO: 16.
  • the packaging vector constructed as described above can be configured to include, for example, a promoter sequence, a viral core protein sequence (gag), a reverse transcriptase sequence (pol), and a polyA sequence, and are shown in the Examples.
  • an RRE sequence may be further included in the above configuration.
  • the ev expression vector can have a configuration in which a promoter for controlling the sequence is arranged upstream of the rev sequence and a polyA sequence is arranged downstream of the rev sequence.
  • the cell used for the nosing cell is not limited as long as it is a cell line generally used for virus production. Considering use for human gene therapy, humans or monkeys are considered appropriate for cell origin. Examples of human cell lines that can be used as knocking cells include 293 cells, 293T cells, 293EBNA cells, and SW480 cells. Cell, u87MG cell, HOS cell, C8166 cell, MT-4 cell, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell and the like. Examples of monkey-derived cell lines include COS1 cells, COS7 cells, CV-1 cells, BMT10 cells, and the like.
  • SIV vectors carrying the PEDF gene of the present invention can be purified to be substantially pure.
  • the purification method can be performed by a known purification method including filtration, centrifugation, column purification, and the like.
  • the vector solution can be precipitated and concentrated by filtering the vector solution through a 0.45 m filter and centrifuging at 42500 Xg for 90 minutes at 4 ° C.
  • the pharmaceutical product of the present invention may be prepared using PEDF protein and FGF2 protein.
  • PEDF protein and FGF2 protein may be prepared by a method well known to those skilled in the art.
  • the cDNA can be inserted into an appropriate expression vector and introduced into a host cell for expression. These cDNAs may be held in separate expression vectors or simultaneously in one expression vector.
  • the preparation of cDNA is as described above.
  • the expression vector can be appropriately selected according to the host cell.
  • the pharmaceutical agent of the present invention can be used for treatment and prevention of diseases associated with apoptotic degeneration in ocular tissue cells.
  • it can be used for the treatment and prevention of retinitis pigmentosa, glaucoma, retinal detachment, and retinal ischemic disease, and particularly preferably for the treatment and prevention of retinitis pigmentosa.
  • the SIV vector carrying the PEDF gene and / or FGF2 gene such as the SIV-PEDF vector and SIV-FGF2 vector described above, is appropriately combined with a desired pharmaceutically acceptable carrier or vehicle as necessary. It can be a medicine.
  • a “pharmaceutically acceptable carrier” is a material that can be administered together with a vector and does not significantly inhibit gene transfer by the vector. Specifically, for example, appropriate combinations with sterilized water, physiological saline, culture solution, serum, phosphate buffered saline (PBS) and the like can be considered. In addition, stabilizers, biocides and the like may be included.
  • the form of the pharmaceutical product of the present invention can be made into a single pharmaceutical product by making PEDF protein and FGF2 protein or SIV-PEDF vector and SIV-FGF2 vector present in the same medium.
  • SIV-PEDF vector When the SIV-FGF2 vector and the SIV-FGF2 vector are used as a single composition, they can be blended within the range in which the doses shown below can be secured. Alternatively, the SIV-PEDF vector and the SIV-FGF2 vector may be prepared as separate compositions and used as a therapeutic kit containing both compositions. The same kit can be used for the PEDF and FGF2 genes and proteins. In the case of using PEDF protein and FGF2 protein as the pharmaceutical of the present invention, the carrier and form are the same as in the case of the vector.
  • the administration route is not particularly limited as long as the effect of suppressing retinal apoptosis degeneration is obtained. Intravitreal administration and intra-anterior administration are preferred, and subretinal administration is more preferred.
  • the dose (per human eyeball) of the pharmaceutical agent containing SIV-PEDF and SIV-FGF2 of the present invention is, for example, 2.5 X 10 5 TU-2.5 X 10 8 TU, preferably 5.0 X 10 5 TU-5.0 X 10 7 Use TU as a guide.
  • FIG. 1 Four types of plasmids (gene transfer vector, packaging vector, rev expression vector, VSV-G expression vector) shown in FIG. 1 were used for the construction of the vector. Three gene transfer vectors, packaging vectors, and rev expression vectors were prepared by modifying the conventional vector plasmid (PCT / JP00 / 03955). The conventional VSV-G expression vector was used as it was.
  • the conventional gene transfer vector used is based on SIVagm, a non-pathogenic African green monkey immunodeficiency virus clone. 5, LTR region, RRE, CMV (cytomegalovirus) promoter, EGFP (enhanced green fluorescent protein) gene, 3, vector with LTR in that order.
  • the conventional gene transfer vector has been constructed by the present inventors, and the construction method and the like have already been reported (Patent Document 2).
  • the base sequence of the conventional gene transfer vector is shown in SEQ ID NO: 17.
  • a specific method for improving a vector is as follows. First, the conventional gene transfer vector was cleaved with the restriction enzyme Sac II, the sample was electrophoresed to remove the CMV promoter and EGFP gene, and self-ligation was performed. Next, in order to eliminate the Not I site of the plasmid, the vector was cut with Not I, the cut ends were smoothed with T4 DNA polymerase, and self-ligation was performed.
  • the vector was cleaved with the restriction enzyme Sac II, and the cleaved ends were dephosphorylated using BAP treatment.
  • a fragment in which a Sac II site was added to was prepared.
  • the CMV promoter fragment was incorporated into the Sac II site of the above-mentioned vector treated with BAP.
  • the vector was cleaved in order with Not I and BamH I, and an adapter prepared by annealing two synthetic oligos DNA2 F (SEQ ID NO: 20) and 2R (SEQ ID NO: 21) at the cleavage site was ligated.
  • the restriction enzyme site was modified.
  • the vector was digested with the restriction enzyme Sac II, and the digested end was dephosphorylated using BAP treatment.
  • a cPTT fragment (SEQ ID NO: 14) for introduction, the SIVagmTYOl genome (SEQ ID NO: Using the plasmid pSA212 incorporating 12) as a template, PCR was performed with primers 3F (SEQ ID NO: 22) and 3R (SEQ ID NO: 23). The end of the PCR amplified fragment was cleaved with SAC ⁇ to prepare a fragment in which SAC II sites were added to both ends of c PPT. The cPPT fragment was ligated to the Sac II site of the above vector treated with BAP.
  • the vector was digested with BamHI, and the digested end was dephosphorylated using BAP treatment.
  • a plasmid containing WPRE cDNA (SEQ ID NO: 15) was used as a template, and PCR was performed with primers 4F (SEQ ID NO: 24) and 4R (SEQ ID NO: 25).
  • the ends of the obtained PCR amplification products were cleaved with BamH I and Bgl II to prepare a fragment in which a restriction enzyme site was added to the end of WPRE.
  • the above WPRE fragment was ligated to the BamHI site of the vector to complete an improved gene transfer vector (SEQ ID NO: 1) without any onboard gene.
  • a mounted gene fragment was prepared and incorporated into the above-mentioned improved gene transfer vector Not I site.
  • the EGFP fragment was prepared by using a plasmid containing EGFP cDNA (SEQ ID NO: 26) as a template, PCR with primers 5F (SEQ ID NO: 27) and 5R (SEQ ID NO: 28), and cleaving with Not I.
  • the FGF2 fragment was prepared by using the plasmid containing hFGF2 cDNA (SEQ ID NO: 8) as a template, PCR with primers 6F (SEQ ID NO: 29) and 6R (SEQ ID NO: 30), and cleaving with Not I. did.
  • the PEDF fragment was templated with a plasmid containing hPEDF cDNA (SEQ ID NO: 7), PCR was performed with primers 7F (SEQ ID NO: 31) and 7R (SEQ ID NO: 32), and transferred to pGEM-T Easy vector (Promega). TA clawed and cut out with Not I.
  • conventional packaging vectors include vif and vpr called modified genes, and tat and rev regulatory genes.
  • modified genes are not required in vectors (V. Kim et al .: J. Virol 72: 811-816, 1998), and recently, modified genes have been deleted to increase safety. Vector is used. Also, the tat was deleted, and the rev was transferred to another plasmid for further safety. Vectors have been developed, and now the third generation of vectors is essential
  • the conventional packaging vector SEQ ID NO: 33
  • force assisting gene vif, vpr, tat
  • FIG. 3 The method has basically been reported previously with HIV vectors (T. Dull. Et al .: J. Virol 72: 8463-8471, 1998).
  • the plasmid of the knocking vector was cleaved with the restriction enzyme Not I, and then cleaved with Ecot22I.
  • the sample was electrophoresed to remove the EcoT22 to Not I fragment, and a large vector fragment and a portion of the EcoT22 to EcoT22I fragment of the pol gene were recovered.
  • An adapter prepared by annealing two types of synthetic oligos DNA1F (SEQ ID NO: 34) and 1R (SEQ ID NO: 35) was ligated to the EcoT22 to Not I site of the above vector. Subsequently, the vector was cleaved with EcoT22I, treated with BAP, the cleaved end was dephosphorylated, and the EcoT22I fragment of the pol gene was incorporated by recovery at the EcoT22I site treated with BAP.
  • the above vector was cleaved with Not I, subjected to BAP treatment, and the cleaved ends were dephosphorylated.
  • PCR was performed with primers 8F (SEQ ID NO: 36) and 8R (SEQ ID NO: 37) using a conventional packaging vector (SEQ ID NO: 33) as a template, and pGEM-T Easy vector. TA closed to (Promega).
  • the RRE fragment was cut out with Not I.
  • the RRE fragment was ligated to the Not I site of the dephosphorylated vector to complete the improved packaging vector (SEQ ID NO: 4).
  • the rev protein has been supplied from conventional packaging vectors. However, with the above improvement of the knocking vector, the rev protein will be supplied in the form of a separate expression plasmid. Built. Although rev is divided into two introns on the genome, it was decided to bind to one and incorporate it into the expression plasmid (Fig. 4A, B).
  • PCR fragments Two types were collected and mixed to form a PCR template, which was amplified using primers 1F and 2R to obtain the desired rev gene fragment (SEQ ID NO: 16) connecting the two fragments.
  • PCR The rev fragment amplified in step 1 was TA cloned into pGEM-T Easy vector. Subsequently, the vector was cleaved with EcoR I, and the rev fragment with the EcoR I site added was recovered.
  • the pCI vector for protein expression Promega was cleaved with EcoRI and the cleavage site was BAP-treated. The recovered rev fragment and the pCI expression vector were ligated to obtain a rev expression vector.
  • the DNA complex was added dropwise to a 15 cm petri dish, gently shaken and mixed, and incubated in a 37 ° C, 5% CO incubator for 3 hours. Petri dish after incubation
  • D-MEM medium containing 13 ml of 20% urine fetal serum was added and cultured.
  • the medium was replaced with 30 ml of D-MEM medium containing fresh 10% urine fetal serum and cultured.
  • the supernatant was collected and filtered through a 0.45 ⁇ m filter to obtain a vector solution.
  • the titer of the SIV vector includes a functional titer (Functional titer: TU / ml) calculated from the number of cells expressing the mounted gene protein and a value (Particle titer: particles / ml) calculated from the number of vector particles.
  • a functional titer Frctional titer: TU / ml
  • Particle titer particles / ml
  • the probe was prepared using DIG-labeled NTP, and DIG Easy Hyb, DIG Wash and Block Buffer Set (Roche) was used for the subsequent operations after hybridization.
  • Anti-DIG AP conjugate antibody (Roche) and CSPD (Roche) were used for chemiluminescence, and the signal was detected and quantified using a lumino image analyzer (Fuji Photo Film: LAS-1000).
  • the modified gene transfer vector, packaging vector, rev expression vector, and VSV-G expression vector, SIV vectors were prepared as follows. Vectors loaded with PEDF and FGF2 therapeutic genes were produced in units of 20 15 cm dishes.
  • 293T cells were seeded at about 1 X 10 7 (70-80% density the next day) per 15 cm plastic petri dish, and cultured for 24 hours in 20 ml of D-MEM medium containing 10% ushi fetal serum. . After culturing for 24 hours, the medium was replaced with 10 ml of OPTI-MEM medium and used for transfer. 40 ⁇ L of gene transfer vector after 10 ⁇ g of gene transfer vector, 5 ⁇ g of packaging vector, 2 ⁇ g of rev expression vector, and 2 ⁇ g of VSV-G expression vector in 1.5 ml of MEM-MEM medium Reagent (Invitrogen) was stirred and stirred for 15 minutes at room temperature .
  • MEM-MEM medium Reagent Invitrogen
  • the medium was replaced with 30 ml of D-MEM medium containing fresh! /, 10% ushi fetal serum, and cultured.
  • the supernatant was collected and 20 ml of fresh medium was added.
  • the collected supernatant was filtered through a 0.45 ⁇ m filter and stored at 4 ° C.
  • the supernatant was collected 3 days after the transfectate, filtered through a 0.45 ⁇ m filter, combined with the collection vector from the previous day, and concentrated using a high-speed centrifuge.
  • the collected vector solution was dispensed into a sterilized tube and centrifuged at 42500 G at 4 ° C for 1 hour.
  • This centrifugation operation was repeated twice to concentrate the vector solution 500 times to 1000 times.
  • Ability to precipitate the vector as a pellet The pellet was dissolved in PBS containing 5% urine fetal serum.
  • the concentrated vector was dispensed in small portions and stored at -80 ° C, and a portion of the vector was measured for particle titer. Particle titer was measured in the same manner as described above.
  • Example 4 Examination of inhibitory effect on photoreceptor cell degeneration by simultaneous administration of SIV-hPEDF and SIV-hFGF2 Retinitis pigmentosa is an intractable genetic disease, and there is no effective treatment at present.
  • the present inventors have introduced characteristics of SIV vectors into the retina in small animals' long-term safety test, and further, an effect determination test using SIV-PEDF in disease model animals (RCS rats) (Gene th erapy 10, 1503- 1511, 2003) with good results.
  • RCS rats disease model animals
  • Neurotrophic factors PEDF and FGF2 are thought to have different target cells. We examined the neuroprotective effects of simultaneous administration of these two types of neurotrophic factors.
  • Each vector solution of SIV-hPEDF, SIV-hFGF2, and SIV-EGFP (control) was prepared at a vector concentration: total 2.5 ⁇ 10 7 TU / ml.
  • RCS rats 3 weeks old are divided into 4 groups: SIV-hPEDF single administration group, SIV-hFGF2 single administration group, SIV-hPEDF and SIV-hFGF2 simultaneous administration group, SIV-EG FP administration group, and it is administered into the subretinal space did.
  • PEDF and FGF2 genes are introduced into retinal pigment epithelial cells, It is secreted and acts on nerve cells.
  • hFGF2 expression tended to be high in the SIV-hFGF2 single administration group and the co-administration group, and hPEDF and hF GF2 gene expression was confirmed by SIV-hPEDF and SIV-hFGF2 vector administration (FIG. 8).
  • ERG electrophysiological study
  • the electroretinogram is a test method that records changes in the retinal potential in response to light stimulation. A high amplitude is obtained when the retina is functioning.
  • the a wave is derived from photoreceptor cells, and the b wave is derived mainly from Müller cells and bipolar cells.
  • SIV-hPEDF and SIV-hFGF2 single administration groups significantly higher amplitude results were obtained for both a and b waves than the SIV-EGFP group.
  • a higher amplitude was obtained for both the a and b waves, and it was confirmed that a higher neuroprotective effect was obtained in the co-administration group and V (FIGS. 12 and 13).
  • a novel method for treating retinal pigment degeneration has been provided.
  • the simultaneous administration of PEDF and FGF2 of the present invention can be expected to have a significantly higher effect than conventional methods for treating retinal pigment degeneration.
  • the administration method using the SIV-PEDF vector and the SIV-FGF2 vector makes it possible to provide PEDF and FGF2 continuously in the patient's cells, and in terms of the frequency of invasiveness to the patient and the economic cost. It proved to be a very good treatment.

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Abstract

It is intended to provide a novel therapeutic method for a disease with apoptotic degeneration in eye tissue cells such as retinal pigment degeneration. Attention was paid on the concomitant administration of two neurotrophic factors: pigment epithelium-derived factor (PEDF) and fibroblast growth factor 2 (FGF2). The sites of actions are considered to be different between PEDF and FGF2. An SIV-PEDF vector and an SIV-FGF2 vector were constructed and administered to the subretinal space of an RCS rat, which is a disease model of retinal pigment degeneration, and their effects were evaluated. At 4 weeks, 8 weeks and 12 weeks after the administration of the vectors, a significantly higher visual cell protection effect was obtained in a group with concomitant administration than in a group with sole administration. Further, the functional evaluation of retina was carried out by electroretinogram and an effect in the group with concomitant administration was significantly higher than in the group with sole administration. From the above results, it was found for the first time that the concomitant administration of PEDF and FGF2 has higher effects in the treatment of retinal pigment degeneration than conventional methods.

Description

明 細 書  Specification
PEDFおよび FGF2を含む眼組織細胞におけるアポトーシス変性を伴う疾 患の治療薬  Drugs for treating diseases with apoptotic degeneration in ocular tissue cells, including PEDF and FGF2
技術分野  Technical field
[0001] 本発明は、神経栄養因子を搭載したレンチウィルスベクターを用いた、眼組織細胞 におけるアポトーシス変性を伴う疾患の治療用医薬品に関する。  [0001] The present invention relates to a pharmaceutical product for treating a disease associated with apoptotic degeneration in ocular tissue cells using a lentiviral vector loaded with a neurotrophic factor.
背景技術  Background art
[0002] 眼科領域の疾患には、適切な治療を施さないと失明に至る重篤なものがある。決定 的な治療法がなぐ対症的療法による治療に頼らざるを得ない疾患も少なくない。最 近、眼科領域の重篤な疾患の一部の発生 '進行に、アポトーシスが関与していること がわかってきた。  [0002] Some diseases in the ophthalmological field are serious enough to lead to blindness unless appropriate treatment is performed. Many diseases have to rely on treatment with symptomatic therapies without definitive treatment. Recently, it has been found that apoptosis is involved in the development and progression of some serious diseases in the ophthalmic field.
[0003] 網膜色素変性は、網膜の視細胞層及び色素上皮層が広範にお力されてアポトー シスに至る、難治性の遺伝性疾患である。視細胞には、大きく分けて杆体と錐体との 2種類の細胞がある。杆体は、主に網膜の中心力 少しずれた部分に多く分布し、暗 いところでの物の見え方や視野の広さなどに関係している。錐体は網膜の中心部で ある黄斑に多くに分布し、主に中心の視力や色覚などに関係する。網膜色素変性で は、視細胞が傷害されるために、夜盲、視野狭窄、視力低下の症状を呈し、進行にし たがい失明する例も多い。網膜色素変性の一部は、視細胞や網膜色素上皮細胞に 特異的に働く遺伝子の異常によって起こるとされているが、大部分の網膜色素変性 においては、いまだ原因不明である。現在までにわ力つている原因遺伝子としては、 常染色体劣性網膜色素変性では、杆体 cGMP-フォスフォジエステラーゼ aおよび b サブユニット、杆体サイクリックヌクレオチド感受性陽イオンチャンネル、網膜グァ -ル シクラーゼ、 RPE65、細胞性レチニルアルデヒド結合蛋白質、アレスチンの各遺伝子 が知られている。また常染色体優性網膜色素変性では、ロドプシン、ペリフェリン ' RD S (retinal degeneration slowの略）、ロム- 1、 X連鎖性網膜色素変性では網膜色素変 性 GTPase調節因子 (RPGR)の各遺伝子が知られている。診断は、眼底所見（定型例 では網膜血管狭小、粗造な胡麻塩状網膜、骨小体様色素沈着。非定型例では無色 素性、白点状、等)、視野 (求心性、輪状、地図状、中心性など病変の部位に一致し た狭窄)、暗順応 (暗順応曲線の第 2次曲線の閾値の上昇)、視力低下、）電気生理 学的所見 (網膜電図 (ERG)の振幅低下 ·消失)、蛍光眼底造影所見 (網膜色素上皮 萎縮および網脈絡膜萎縮による過蛍光）により判断される。 [0003] Retinitis pigmentosa is an intractable genetic disease in which the photoreceptor cell layer and pigment epithelial layer of the retina are extensively brought into apoptosis. There are two types of photoreceptor cells: rods and cones. The rods are mainly distributed in areas where the central force of the retina is slightly shifted, and are related to the way objects are seen in the dark and the field of view. The cones are widely distributed in the macula, the central part of the retina, and are mainly related to the central visual acuity and color vision. In retinitis pigmentosa, the photoreceptor cells are damaged, so there are many cases of night blindness, visual field stenosis, and visual acuity symptom, and blindness occurs as it progresses. Part of retinal pigment degeneration is thought to be caused by abnormalities in genes that specifically act on photoreceptors and retinal pigment epithelial cells, but the cause of most retinal pigment degeneration is still unknown. Among the causative genes that have been used to date, in autosomal recessive retinal pigment degeneration, rod cGMP-phosphodiesterase a and b subunits, rod cyclic nucleotide-sensitive cation channels, retinal guanyl cyclase, RPE65, The genes for cellular retinyl aldehyde binding protein and arrestin are known. For autosomal dominant retinal pigment degeneration, the genes for rhodopsin, peripherin 'RDS (retinal degeneration slow), lom-1, and X-linked retinal pigment degeneration are known. ing. Diagnosis is based on fundus findings (in typical cases, retinal vascular narrowing, crude sesame salt-like retina, bone-like pigmentation, colorless in atypical cases Identities, white spots, etc.), visual field (stenosis matching the lesion site such as centripetal, ring-shaped, map-like, centrality), dark adaptation (rising of the second curve of the dark adaptation curve), visual acuity Decrease)) Physiological findings (reduction in electroretinogram (ERG) amplitude / disappearance), fluorescence fundus angiography findings (retinal pigment epithelial atrophy and retina choroidal atrophy).
現在のところ、網膜色素変性の有効な治療法はなぐ対症療法が行われているに 過ぎないが、遺伝子治療、網膜移植、人工網膜などによる治療法が研究されている。 神経栄養因子を用いた遺伝子治療もそのひとつである。神経栄養因子は、アポトー シス抑制に効果があるといわれている。神経栄養因子である線維芽細胞成長因子 2 ( fibroblast growth factor 2： FGF2)は、網膜光傷害モデル (非特許文献 1)、網膜色素 変性モデル (非特許文献 2— 6)、網膜神経節細胞障害 (虚血再還流、？見神経切断） モデル (非特許文献 7)などを用いた動物実験によって、眼科領域における遺伝子治 療への応用が検討されている。また本発明者らは、神経栄養因子の一つである色素 上皮由来因子（Pigment epithelium derived factor : PEDF)を網膜色素変性の治療に 応用することを検討している（非特許文献 8)。本発明者らは、レトロウイルスであるサ ノレ免疫不全ウイノレス (SIV: simian immunodeficiency virus)をバックボーンにもつ SIV ベクターに PEDFを挿入した SIV-PEDFベクターを構築し、モデル動物にお!/、て検討 し、良好な結果を得ている（非特許文献 8)。しかし、最終的に失明に至る重篤な疾患 である網膜色素変性については、より高い効果を発揮する治療法の開発が求められ る。  At present, there are only symptomatic treatments that are effective treatments for retinitis pigmentosa, but gene therapy, retinal transplantation, artificial retina treatment, etc. are being studied. One example is gene therapy using neurotrophic factors. Neurotrophic factors are said to be effective in suppressing apoptosis. Fibroblast growth factor 2 (FGF2), a neurotrophic factor, is a retinal photodamage model (Non-patent document 1), a retinal pigment degeneration model (Non-patent document 2-6), and retinal ganglion cell damage. (Ischemia reperfusion, optic nerve transection) Models (Non-Patent Document 7) and other animal experiments have been examined for application to gene therapy in the ophthalmic field. In addition, the present inventors are examining the application of pigment epithelium derived factor (PEDF), which is one of neurotrophic factors, to the treatment of retinal pigment degeneration (Non-patent Document 8). The present inventors constructed a SIV-PEDF vector in which PEDF was inserted into a SIV vector having the backbone of the simian immunodeficiency virus (SIV), a retrovirus, and examined it in model animals! However, good results have been obtained (Non-patent Document 8). However, for retinitis pigmentosa, which is a serious disease that eventually leads to blindness, the development of a treatment that exhibits a higher effect is required.
特許文献 1：国際出願番号 PCT/JP2002/005225 国際公開番号 WO2002/101057 特許文献 2：国際出願番号 PCT/JP00/03955 国際公開番号 WO00/078987 Patent Document 1: International Application Number PCT / JP2002 / 005225 International Publication Number WO2002 / 101057 Patent Document 2: International Application Number PCT / JP00 / 03955 International Publication Number WO00 / 078987
特干文献 1 : Lau D, Fiannery J. iral-mediated FuF~2 treatment of the constant 1 ight damage model of photoreceptor degeneration. Doc Ophthalmol. 2003 Jan;106( Tokubori 1: Lau D, Fiannery J. iral-mediated FuF ~ 2 treatment of the constant 1 ight damage model of replica degeneration.Doc Ophthalmol. 2003 Jan; 106 (
1) :89- 98. 1): 89-98.
非特許文献 2 :Akimoto M, Miyatake S, Kogishi J, Hangai M, Okazaki K, Takahashi J C, Saiki M, Iwaki M, Honda Y. Adenovirally expressed basic fibroblast growth factor rescues photoreceptor cells in RCS rats. Invest Ophthalmol Vis Sci. 1999 Feb;40(Non-Patent Document 2: Akimoto M, Miyatake S, Kogishi J, Hangai M, Okazaki K, Takahashi JC, Saiki M, Iwaki M, Honda Y. Adenovirally expressed basic fibroblast growth factor rescues containing cells in RCS rats. 1999 Feb; 40 (
2) :273-9. 非特許文献 3 : Uteza Y, Rouillot JS, Kobetz A, Marchant D, Pecqueur S, Arnaud E, Prats H, Honiger J, Dufier JL, Abitbol M, Neuner— Jehle M. Intravitreous transplanta tion of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delay s photoreceptor cell degeneration in Royal College of Surgeons rats. Proc Natl Aca d Sci U S A. 1999 Mar 16;96(6):3126- 31. 2): 273-9. Non-Patent Document 3: Uteza Y, Rouillot JS, Kobetz A, Marchant D, Pecqueur S, Arnaud E, Prats H, Honiger J, Dufier JL, Abitbol M, Neuner— Jehle M. Intravitreous transplantation of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delay s dye cell degeneration in Royal College of Surgeons rats.Proc Natl Aca d Sci US A. 1999 Mar 16; 96 (6): 3126- 31.
非特許文献 4 : Neuner- Jehle M, Berghe LV, Bonnel S, Uteza Y, Benmeziane F, Roui Hot JS, Marchant D, Kobetz A, Dufier JL, Menasche M, Abitbol M. Ocular cell trans fection with the human basic fibroblast growth factor gene delays photoreceptor cell degeneration in RCS rats. Hum Gene Ther. 2000 Sep 1;11(13): 1875— 90. Non-Patent Document 4: Neuner- Jehle M, Berghe LV, Bonnel S, Uteza Y, Benmeziane F, Roui Hot JS, Marchant D, Kobetz A, Dufier JL, Menasche M, Abitbol M. Ocular cell trans fection with the human basic fibroblast growth factor gene delays reservoir cell degeneration in RCS rats. Hum Gene Ther. 2000 Sep 1; 11 (13): 1875— 90.
非特許文献 5 : Lau D, McGee LH, Zhou S, Rendahl KG, Manning WC, Escobedo JA, Flannery JG. Retinal degeneration is slowed in transgenic rats by AAV— mediated de livery of FGF- 2. Invest Ophthalmol Vis Sci. 2000 Oct;41(l l):3622- 33. Non-Patent Document 5: Lau D, McGee LH, Zhou S, Rendahl KG, Manning WC, Escobedo JA, Flannery JG. Retinal degeneration is slowed in transgenic rats by AAV— mediated de livery of FGF- 2. Invest Ophthalmol Vis Sci. 2000 Oct; 41 (ll): 3622-33.
非特許文献 b : Spencer B, Agarwala S, Gentry L, Brandt CR. HSV- 1 vector- delivere d FGF2 to the retina is neuroprotective but does not preserve functional responses.Non-patent literature b: Spencer B, Agarwala S, Gentry L, Brandt CR.HSV- 1 vector- delivere d FGF2 to the retina is neuroprotective but does not preserve functional responses.
Mol Ther. 2001 May;3(5 Pt 1):746- 56. Mol Ther. 2001 May; 3 (5 Pt 1): 746-56.
非特許文献 7 : Sapieha PS, Peltier M, Rendahl KG, Manning WC, Di Polo A. Fibrobl ast growth factor— 2 gene delivery stimulates axon growth by adult retinal ganglion c ells after acute optic nerve injury. Mol Cell Neurosci. 2003 Nov;24(3):656-72. 非特許文献 8 : Miyazaki M, Ikeda Y, Yonemitsu Y, Goto Y, Sakamoto T, Tabata T, Ueda Y, Hasegawa M, Tobimatsu S, Ishibashi T, Sueishi K. Simian lentiviral vector- mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats. Gene Ther. 200 3 Aug;10(17):1503-l l. Non-Patent Document 7: Sapieha PS, Peltier M, Rendahl KG, Manning WC, Di Polo A. Fibrobl ast growth factor— 2 gene delivery stimulates axon growth by adult retinal ganglion c ells after acute optic nerve injury. Mol Cell Neurosci. 2003 Nov ; 24 (3): 656-72. Non-Patent Document 8: Miyazaki M, Ikeda Y, Yonemitsu Y, Goto Y, Sakamoto T, Tabata T, Ueda Y, Hasegawa M, Tobimatsu S, Ishibashi T, Sueishi K. Simian lentiviral vector-mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats.Gene Ther. 200 3 Aug; 10 (17): 1503-l l.
非特許文献 9 :Wahlin KJ, Campochiaro PA, Zack DJ, Adler R. Neurotrophic factors cause activation of intracellular signaling pathways in Muller cells and other cells of the inner retina, but not photoreceptors. Invest Ophthalmol Vis bci. 2000 ;41(3):92 7-36. Non-Patent Document 9: Wahlin KJ, Campochiaro PA, Zack DJ, Adler R. Neurotrophic factors cause activation of intracellular signaling pathways in Muller cells and other cells of the inner retina, but not peptidess.Invest Ophthalmol Vis bci. 2000; 41 (3 ): 92 7-36.
非特許文献 10 :Valter K, van Driel D, Bisti S, Stone J. FGFRl expression and FGF Rl— FGF— 2 colocalisation in rat retina: sites of FGF— 2 action on rat photoreceptors. Growth Factors. 2002 ;20(4): 177- 88. Non-Patent Document 10: Valter K, van Driel D, Bisti S, Stone J. FGFRl expression and FGF Rl— FGF— 2 colocalisation in rat retina: sites of FGF— 2 action on rat derivatives. Growth Factors. 2002; 20 (4): 177- 88.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0005] 本発明はこのような状況を鑑みてなされたものであり、本発明が解決しょうとする課 題は、眼組織細胞におけるアポトーシス変性を伴う疾患の新たな治療方法を見出す ことである。 [0005] The present invention has been made in view of such circumstances, and the problem to be solved by the present invention is to find a new treatment method for a disease accompanied by apoptotic degeneration in ocular tissue cells.
課題を解決するための手段  Means for solving the problem
[0006] 本発明者らは、上記課題を解決すべく鋭意研究を行 ヽ、 2つの神経栄養因子： PED Fと FGF2の同時投与に思い至った。 PEDFと FGF2は作用部位が異なると考えられて いる。 PEDFの作用部位は網膜の視細胞および神経節細胞である力 FGF2の作用 部位にっ 、ては、 FGF2受容体が網膜の内顆粒層にあることを示唆する報告 (非特 許文献 9)と視細胞にあることを示唆する報告 (非特許文献 10)がある。 PEDFと FGF2 を同時投与すれば、両者の相乗効果によって、従来よりも高い効果を得られる可能 性がある。本発明者らは、 SIV-PEDFベクターおよび SIV-FGF2ベクターを構築した。 [0006] The present inventors have conducted intensive research to solve the above problems and have come up with the simultaneous administration of two neurotrophic factors: PED F and FGF2. PEDF and FGF2 are thought to have different sites of action. The site of action of PEDF is the visual site of retinal photoreceptors and ganglion cells. The site of action of FGF2 suggests that the FGF2 receptor is in the inner granular layer of the retina (Non-Patent Document 9). There is a report (Non-patent Document 10) suggesting that it exists in cells. If PEDF and FGF2 are administered simultaneously, there is a possibility that a higher effect than before can be obtained due to the synergistic effect of both. The present inventors constructed a SIV-PEDF vector and a SIV-FGF2 vector.
SIVベクターはサル免疫不全ウィルスをバックボーンとするベクターであり、導入外来 遺伝子を宿主内において継続的に発現させることが可能なため、慢性疾患の治療に おいては特に適したドラッグデリバリーを提供することができる。網膜色素変性疾患モ デルである RCSラットの網膜下腔に上記ベクターを投与し、その効果を検討した。ベ クタ一投与 4週間後において、ラット後眼部を採取し、 PEDFおよび FGF2の発現量を 測定したところ、 SIV-hPEDF、 SIV-hFGF2ベクター投与による hPEDFおよび hFGF2遺 伝子発現が確認された。また、視細胞の残存数をカウントしたところ、単独投与群に おいても視細胞保護効果が観察されたが、同時投与群は単独投与群よりも著しく高 ぃ視細胞保護効果が得られた。さらに、網膜電図により網膜の機能的評価を行った ところ、単独投与群、同時投与群の両方で機能維持が認められたが、同時投与群に おける効果は単独投与群よりも顕著に高い効果であった。さらに、ベクター投与 8週 間後および 12週間後においても、 SIV- hPEDFおよび SIV- hFGF2ベクターの同時投 与による顕著な神経保護効果が認められた。本発明者らは上記結果から、 PEDFお よび FGF2の同時投与は網膜色素変性の効果に高い効果があるということを、初めて 見出した。 PEDFおよび FGF2の同時投与は、網膜色素変性のみならず、眼組織細胞 におけるアポトーシス変性を伴う疾患の治療に有効と考えられる。すなわち、本発明 は、 FGF2および PEDFの同時投与による、眼組織細胞におけるアポトーシス変性を 伴う疾患の治療に関し、より具体的には以下の発明を提供するものである。 The SIV vector is a vector with simian immunodeficiency virus as the backbone, and since the introduced foreign gene can be continuously expressed in the host, it should provide drug delivery particularly suitable for the treatment of chronic diseases. Can do. The above vector was administered into the subretinal space of RCS rats, a retinitis pigmentosa disease model, and the effect was examined. Four weeks after administration of the vector, the rat rear eye was collected and the expression levels of PEDF and FGF2 were measured. As a result, hPEDF and hFGF2 gene expression was confirmed by administration of SIV-hPEDF and SIV-hFGF2 vectors. Further, when the number of remaining photoreceptor cells was counted, a photoreceptor protection effect was observed in the single administration group, but the photoreceptor administration effect was significantly higher in the simultaneous administration group than in the single administration group. Furthermore, functional evaluation of the retina using electroretinogram showed that functional maintenance was observed in both the single administration group and the simultaneous administration group, but the effect in the simultaneous administration group was significantly higher than that in the single administration group. Met. Furthermore, a remarkable neuroprotective effect was also observed by simultaneous administration of SIV-hPEDF and SIV-hFGF2 vectors at 8 and 12 weeks after vector administration. From the above results, the present inventors And FGF2 co-administration was found for the first time to be highly effective in the effects of retinal pigment degeneration. Simultaneous administration of PEDF and FGF2 is considered to be effective in treating not only retinal pigment degeneration but also diseases associated with apoptotic degeneration in ocular tissue cells. That is, the present invention relates to the treatment of diseases associated with apoptotic degeneration in ocular tissue cells by simultaneous administration of FGF2 and PEDF, and more specifically, the following inventions are provided.
(1)下記 (a)カゝら (d)の ヽずれかを薬学的に許容される媒体とともに含む、眼組織細 胞におけるアポトーシス変性を伴う疾患の治療用医薬品  (1) A pharmaceutical product for treating a disease associated with apoptotic degeneration in an ocular tissue cell, comprising any of the following (a) Kas et al. (D) together with a pharmaceutically acceptable medium:
(a)色素上皮由来因子（Pigment epithelium derived factor: PEDF)遺伝子および線 維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)遺伝子  (a) Pigment epithelium derived factor (PEDF) gene and fibroblast growth factor 2 (FGF2) gene
(b)色素上皮由来因子（Pigment epithelium derived factor : PEDF)タンパク質および 線維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)タンパク質  (b) Pigment epithelium derived factor (PEDF) protein and fibroblast growth factor 2 (FGF2) protein
(c)色素上皮由来因子（Pigment epithelium derived factor: PEDF)遺伝子および線 維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)タンパク質  (c) Pigment epithelium derived factor (PEDF) gene and fibroblast growth factor 2 (FGF2) protein
(d)色素上皮由来因子（Pigment epithelium derived factor : PEDF)タンパク質および 線維芽細胞成長因子 2 (fibroblast growth factor 2 : FGF2)遺伝子、  (d) Pigment epithelium derived factor (PEDF) protein and fibroblast growth factor 2 (FGF2) gene,
(2) PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクタ 一を含む医薬品であって、該 PEDF遺伝子および FGF2遺伝子力 別々の組換えサ ル免疫不全ウィルスベクターに保持されるカゝ、または 1つの組換えサル免疫不全ウイ ルスベクターに保持される、上記（1)に記載の医薬品、  (2) a recombinant monkey immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene, wherein the PEDF gene and the FGF2 gene have a capacity to be retained in separate recombinant sal immunodeficiency virus vectors; Or the pharmaceutical according to (1) above, which is retained in one recombinant simian immunodeficiency virus vector,
(3) PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターおよび FGF2遺伝 子を保持する組換えサル免疫不全ウィルスベクターを含む、上記（2)に記載の医薬 (3) The pharmaceutical according to (2) above, comprising a recombinant simian immunodeficiency virus vector retaining a PEDF gene and a recombinant simian immunodeficiency virus vector retaining an FGF2 gene
PP
TO、 TO,
(4) PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクタ 一を含む、上記（2)に記載の医薬品、  (4) A pharmaceutical product according to (2) above, comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene,
(5)サル免疫不全ウィルスベクターが cPPT配列および Zまたは WPRE配列を含む、 上記（2)から (4)の 、ずれかに記載の医薬品、  (5) The pharmaceutical product according to any one of (2) to (4) above, wherein the simian immunodeficiency virus vector comprises a cPPT sequence and a Z or WPRE sequence,
(6)サル免疫不全ウィルスベクターが VSV-Gでシユードタイプ化されて!/、る、上記（2 )から（5)の!、ずれかに記載の医薬品、 (7)サル免疫不全ウィルスベクター力 gm株由来である、上記（2)から（6)の 、ずれ かに記載の医薬品、 (6) A simian immunodeficiency virus vector is pseudotyped with VSV-G! /, A pharmaceutical product according to any one of (2) to (5) above, (7) Monkey immunodeficiency virus vector force The pharmaceutical product according to any one of (2) to (6) above, which is derived from a gm strain,
(8) PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターを含む組成物、 および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを含む組成物 を含む、眼組織細胞におけるアポトーシス変性を伴う疾患の治療用キット、  (8) A composition comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene, and a composition comprising a recombinant simian immunodeficiency virus vector carrying an FGF2 gene. Treatment kit,
(9) PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクタ 一を含む組成物を含む、眼組織細胞におけるアポトーシス変性を伴う疾患の治療用 キット、  (9) A kit for treating a disease associated with apoptotic degeneration in ocular tissue cells, comprising a composition comprising a recombinant simian immunodeficiency virus vector having a PEDF gene and an FGF2 gene,
(10)眼組織細胞におけるアポトーシス変性を伴う疾患が、網膜色素変性、緑内障、 網膜剥離、網膜虚血性疾患のいずれかである、上記（1)から（7)のいずれかに記載 の医薬品または上記（8)もしくは（9)に記載のキット、  (10) The medicine according to any one of (1) to (7) above, wherein the disease associated with apoptotic degeneration in ocular tissue cells is any one of retinal pigment degeneration, glaucoma, retinal detachment, and retinal ischemic disease (8) or the kit according to (9),
(11) PEDFおよび FGF2あるいはそれらをコードする遺伝子を投与する、眼組織細胞 におけるアポトーシス変性を伴う疾患の治療方法、  (11) A method for treating a disease associated with apoptotic degeneration in ocular tissue cells, comprising administering PEDF and FGF2 or a gene encoding them,
( 12) PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターおよび FGF2遺 伝子を保持する組換えサル免疫不全ウィルスベクターを投与する、上記（11)に記載 の治療方法、  (12) The therapeutic method according to (11) above, wherein a recombinant simian immunodeficiency virus vector retaining a PEDF gene and a recombinant simian immunodeficiency virus vector retaining an FGF2 gene are administered,
(13) PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターおよび FGF2遺 伝子を保持する組換えサル免疫不全ウィルスベクターを網膜下腔に投与する、上記 (12)に記載の治療方法、  (13) The therapeutic method according to (12) above, wherein a recombinant simian immunodeficiency virus vector retaining a PEDF gene and a recombinant simian immunodeficiency virus vector retaining an FGF2 gene are administered to the subretinal space,
(14) PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスべク ターを投与する、上記（11)に記載の治療方法、  (14) The treatment method according to (11) above, wherein a recombinant simian immunodeficiency virus vector carrying the PEDF gene and the FGF2 gene is administered,
(15) PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスべク ターを網膜下腔に投与する、上記（14)に記載の治療方法、  (15) The therapeutic method according to (14) above, wherein a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene is administered to the subretinal space,
( 16)配列番号： 1に記載の塩基配列に PEDF遺伝子が挿入された塩基配列を含む ジーントランスファーベクターを用いて PEDF遺伝子を保持する組換えサル免疫不全 ウィルスベクターを調製する工程を含む、眼組織細胞におけるアポトーシス変性を伴 う疾患の治療用医薬品の製造方法、  (16) An ocular tissue comprising a step of preparing a recombinant simian immunodeficiency virus vector retaining a PEDF gene using a gene transfer vector comprising a nucleotide sequence in which a PEDF gene is inserted into the nucleotide sequence of SEQ ID NO: 1. A method for producing a medicament for the treatment of diseases associated with apoptotic degeneration in cells,
(17)配列番号： 2に記載の塩基配列を含むジーントランスファーベクターを用いて PE DF遺伝子を保持する組換えサル免疫不全ウィルスベクターを調製する工程を含む、 上記（16)に記載の方法、 (17) PE using a gene transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 2 Including the step of preparing a recombinant simian immunodeficiency virus vector carrying the DF gene,
( 18)配列番号： 1に記載の塩基配列に FGF2遺伝子が挿入された塩基配列を含む ジーントランスファーベクターを用いて FGF2遺伝子を保持する組換えサル免疫不全 ウィルスベクターを調製する工程を含む、眼組織細胞におけるアポトーシス変性を伴 う疾患の治療用医薬品の製造方法、  (18) An ocular tissue comprising a step of preparing a recombinant simian immunodeficiency virus vector retaining the FGF2 gene using a gene transfer vector comprising a nucleotide sequence in which the FGF2 gene is inserted into the nucleotide sequence of SEQ ID NO: 1. A method for producing a medicament for the treatment of diseases associated with apoptotic degeneration in cells,
( 19)配列番号： 3に記載の塩基配列を含むジーントランスファーベクターを用 、て F GF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを調製する工程を含む 、上記（18)に記載の方法、  (19) The method according to (18) above, comprising the step of preparing a recombinant simian immunodeficiency virus vector retaining the FGF2 gene using a gene transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 3.
(20)配列番号： 1に記載の塩基配列に PEDF遺伝子および FGF2遺伝子が挿入され た塩基配列を含むジーントランスファーベクターを用いて PEDF遺伝子および FGF2 遺伝子を保持する組換えサル免疫不全ウィルスベクターを調製する工程を含む、眼 組織細胞におけるアポトーシス変性を伴う疾患の治療用医薬品の製造方法、 (20) A recombinant simian immunodeficiency virus vector carrying the PEDF gene and the FGF2 gene is prepared using a gene transfer vector containing the base sequence of the PEDF gene and the FGF2 gene inserted into the base sequence described in SEQ ID NO: 1. A method for producing a pharmaceutical product for treating a disease associated with apoptotic degeneration in an ocular tissue cell, comprising:
( 21 )配列番号: 4に記載の塩基配列を含むパッケージングベクターが導入されたパ ッケージング細胞に、該ジーントランスファーベクターを導入する工程を含む、上記（ 16)から（20)の!、ずれかに記載の方法。 (21) From (16) to (20) above, which includes a step of introducing the gene transfer vector into a packaging cell into which a packaging vector containing the nucleotide sequence of SEQ ID NO: 4 has been introduced. The method described in 1.
図面の簡単な説明 Brief Description of Drawings
[図 1]改良型ジーントランスファーベクター、改良型パッケージングベクター、 rev発現 ベクター、 VSV-G発現ベクターの構造を示す図である。 FIG. 1 shows the structures of an improved gene transfer vector, an improved packaging vector, a rev expression vector, and a VSV-G expression vector.
[図 2A]従来型ジーントランスファーベクターから改良型ジーントランスファーベクター を構築する工程を説明する図である。 ( a )は図 2B工程への続きを示す。 FIG. 2A is a diagram illustrating a process of constructing an improved gene transfer vector from a conventional gene transfer vector. ( a ) shows the continuation of the process in Figure 2B.
[図 2B]図 2Aの続きを示す図である。 ( a )は図 2A工程力もの続きを示す。  FIG. 2B is a diagram showing a continuation of FIG. 2A. (a) shows the continuation of the process capability in Fig. 2A.
[図 3]従来型パッケージングベクターから改良型パッケージングベクターを構築する 工程を説明する図である。  FIG. 3 is a diagram illustrating a process for constructing an improved packaging vector from a conventional packaging vector.
[図 4A]rev発現ベクターの構築工程を説明する図である。 ( β )は図 4Β工程への続き を示す。  FIG. 4A is a diagram illustrating the construction process of a rev expression vector. (β) indicates the continuation of step 4 in Fig. 4.
[図 4Β]図 4Αの続きを示す図である。 ( β )は図 4Α工程力もの続きを示す。  [Fig. 4 Β] This is a continuation of Fig. 4Α. (β) shows the continuation of the process power shown in Fig. 4.
[図 5] (a)従来型ジーントランスファーベクターに、 cPPT単独、 WPRE単独、 cPPTおよ び WPRE同時搭載させたベクターの構造を説明する図である。 （b) MOI:15での感染 において、 cPPT単独、 WPRE単独、 cPPTおよび WPRE同時搭載の各ジーントランスフ ァーベクターを用いたときの、 SIVベクターの生産性を観察した写真である。左上:従 来型の cPPT,WPRE無しのベクター（コントロール）（_cPPT,-WPRE)、右上： cPPT単独 搭載（+cPPT，- WPRE)、左下： WPRE単独搭載（- cPPT,+WPRE)、右下： cPPT,WPRE 同時搭載（+cPPT,+WPRE)。 [Fig.5] (a) Conventional gene transfer vectors include cPPT alone, WPRE alone, cPPT and It is a figure explaining the structure of the vector carried simultaneously with WPRE. (B) This is a photograph observing the productivity of SIV vectors when using gene transfer vectors with cPPT alone, WPRE alone, cPPT and WPRE simultaneously in infection with MOI: 15. Upper left: Conventional cPPT, vector without WPRE (control) (_cPPT, -WPRE), upper right: cPPT alone (+ cPPT, -WPRE), lower left: WPRE alone (-cPPT, + WPRE), lower right : CPPT and WPRE installed simultaneously (+ cPPT and + WPRE).
[図 6]cPPT単独、 WPRE単独、 cPPTおよび WPRE同時搭載の各ジーントランスファー ベクターを用いたときの、 SIVベクターの生産性を、外来遺伝子 (EGFP)陽性細胞数 の割合で検討した結果である。（a)表中の MOIとは 1個の細胞に感染させたベクター 粒子数を表し、 0.3, 1.5, 7.5, 15は実際に行った感染実験の ΜΟΙ (ベクター粒子数/ 細胞数）の数値を表す。 cPPTまたは WPREの後ろに記載された（+ )は、ベクターに c PPTまたは WPREが含まれることを示し、（一）は、ベクターに cPPTまたは WPREが含ま れないことを示す。表の数字は EGFP陽性細胞の割合（百分率：％)。（b)グラフは、 (a )の表の数値をグラフ化したもの。グラフの縦軸は、 EGFP陽性細胞の割合（百分率： %)を示す。  [Fig. 6] Results of examination of the productivity of SIV vectors by the percentage of foreign gene (EGFP) positive cells when using gene transfer vectors equipped with cPPT alone, WPRE alone, cPPT and WPRE simultaneously. (A) The MOI in the table represents the number of vector particles infected with one cell, and 0.3, 1.5, 7.5, and 15 are the numbers of ΜΟΙ (vector particle number / cell number) in the actual infection experiment. To express. A (+) after the cPPT or WPRE indicates that the vector contains cPPT or WPRE, and (1) indicates that the vector does not contain cPPT or WPRE. The numbers in the table are the percentage of EGFP positive cells (percentage:%). (B) The graph is a graph of the values in the table of (a). The vertical axis of the graph represents the percentage of EGFP positive cells (percentage:%).
[図 7]MOI:15での感染において、 cPPT単独、 WPRE単独、 cPPTおよび WPRE同時搭 載の各ジーントランスファーベクターを用いたときの、遺伝子導入細胞での、細胞当 たりの蛋白発現量を比較した結果である。数値は蛍光強度の相対値 (蛋白発現量の 比較目安)を表す。  [Fig.7] Comparison of protein expression levels per cell in transgenic cells using MOPP: 15, gene transfer vectors with cPPT alone, WPRE alone, cPPT and WPRE simultaneously It is the result. The numerical value represents the relative value of fluorescence intensity (comparison standard of protein expression level).
[図 8]RCSラットに、 SIV-hPEDFベクターを単独投与、 SIV-hFGF2ベクターを単独投与 、 SIV-hPEDFおよび SIV-hFGF2ベクターを同時投与、ならびに SIV- EGFPを投与した 場合の hPEDFタンパク質の発現量および hFGF2タンパク質の発現量を示すグラフで ある。  [Fig. 8] Expression level of hPEDF protein when SIV-hPEDF vector is administered alone, SIV-hFGF2 vector is administered alone, SIV-hPEDF and SIV-hFGF2 vectors are administered simultaneously, and SIV-EGFP is administered to RCS rats 2 is a graph showing the expression level of hFGF2 protein.
[図 9]RCSラットに、 SIV-hPEDFベクターを単独投与、 SIV-hFGF2ベクターを単独投与 、 SIV-hPEDFおよび SIV-hFGF2ベクターを同時投与、ならびに SIV- EGFPを投与した 場合の、投与後 4週間の視細胞残存数を示すグラフである。  [Fig. 9] Four weeks after administration of RCS rats when SIV-hPEDF vector is administered alone, SIV-hFGF2 vector is administered alone, SIV-hPEDF and SIV-hFGF2 vector are administered simultaneously, and SIV-EGFP is administered It is a graph which shows the number of photoreceptor cells remaining.
[図 10]RCSラットに、 SIV-hPEDFベクターを単独投与、 SIV-hFGF2ベクターを単独投 与、 SIV-hPEDFおよび SIV-hFGF2ベクターを同時投与、ならびに SIV- EGFPを投与し た場合の、投与後 8週間の視細胞残存数を示すグラフである。 [Fig. 10] RCS rats were administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector were co-administered, and SIV-EGFP was administered. Is a graph showing the remaining number of photoreceptor cells in 8 weeks after administration.
[図 11]RCSラットに、 SIV-hPEDFベクターを単独投与、 SIV-hFGF2ベクターを単独投 与、 SIV-hPEDFおよび SIV-hFGF2ベクターを同時投与、ならびに SIV- EGFPを投与し た場合の、投与後 12週間の視細胞残存数を示すグラフである。  [Fig. 11] Post-administration of RCS rats administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector administered simultaneously, and SIV-EGFP administered It is a graph which shows the number of photoreceptor cells remaining for 12 weeks.
[図 12]RCSラットに、 SIV-hPEDFベクターを単独投与、 SIV-hFGF2ベクターを単独投 与、 SIV-hPEDFおよび SIV-hFGF2ベクターを同時投与、ならびに SIV- EGFPを投与し た場合の網膜電図の結果を示す図である。  [Fig. 12] Electroretinogram when RCS rats were administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector were co-administered, and SIV-EGFP was administered. It is a figure which shows the result.
[図 13]RCSラットに、 SIV-hPEDFベクターを単独投与、 SIV-hFGF2ベクターを単独投 与、 SIV-hPEDFおよび SIV-hFGF2ベクターを同時投与、ならびに SIV- EGFPを投与し た場合の網膜電図の結果を示す図である。  [Fig. 13] Electroretinogram when RCS rats were administered SIV-hPEDF vector alone, SIV-hFGF2 vector alone, SIV-hPEDF and SIV-hFGF2 vector were co-administered, and SIV-EGFP was administered. It is a figure which shows the result.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0008] 本発明は、色素上皮由来因子（Pigment epithelium derived factor： PEDF)および線 維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)による眼糸且織細胞におけるァ ポトーシス変性を伴う疾患の治療用医薬品に関する。本発明者らは、 PEDFと FGF2の アポトーシス抑制作用の標的細胞が異なることに着目し、眼組織細胞におけるアポト 一シス変性を伴う疾患に対し、 PEDFまたは FGF2の同時投与は、これらいずれかの 単独投与よりも顕著に高い効果を得られることを示した。  [0008] The present invention is directed to treatment of diseases associated with apoptosis degeneration in ocular and woven cells by pigment epithelium derived factor (PEDF) and fibroblast growth factor 2 (FGF2). Related to pharmaceutical products. The present inventors focused on the fact that the target cells for the apoptosis-suppressing action of PEDF and FGF2 are different, and for the disease accompanied by apoptotic degeneration in ocular tissue cells, co-administration of PEDF or FGF2 is one of these alone It was shown that a significantly higher effect than the administration can be obtained.
[0009] 本発明の医薬品に含まれる PEDFと FGF2は、タンパク質であってもよぐ遺伝子で あってもよい。ヒト PEDF (hPEDF)タンパク質のアミノ酸配列を配列番号： 5に、ヒト FGF 2 (hFGF2)タンパク質のアミノ酸配列を配列番号： 6に示す。また、 hPEDFの cDNA配 列を配列番号： 7に、 hFGF2タンパク質の cDNA配列を配列番号： 8に示す。  [0009] PEDF and FGF2 contained in the pharmaceutical product of the present invention may be a protein or a gene. The amino acid sequence of human PEDF (hPEDF) protein is shown in SEQ ID NO: 5, and the amino acid sequence of human FGF 2 (hFGF2) protein is shown in SEQ ID NO: 6. In addition, the cDNA sequence of hPEDF is shown in SEQ ID NO: 7, and the cDNA sequence of hFGF2 protein is shown in SEQ ID NO: 8.
[0010] 本発明の医薬品に含まれる PEDF遺伝子と FGF2遺伝子は、当業者に周知の方法 で調製することができる。例えば、配列番号: 7または配列番号 : 8に記載された塩基 配列の一部または全部をプローブとしてヒト網膜色素上皮細胞の cDNAライブラリー 力 調製することができる。あるいは、配列番号： 7または配列番号: 8に記載された塩 基配列の一部をプライマーとして、ヒト網膜色素上皮細胞の mRNAを铸型として、公 知の核酸増幅法を行って調製可能である。上記遺伝子から本発明の医薬品を調製 する場合、 DNAの状態でもよいが、好ましくはベクターに挿入した状態であることが望 ましい。 PEDF遺伝子および FGF2遺伝子は、別々のベクターに保持されてもよぐ 1つ のベクターに同時に保持されていてもよい。ベクターの種類は、医薬品用途としてふ さわしい安全なベクターである限り制限はないが、好ましくは、レンチウィルスベクタ 一、最も好ましくは、サル免疫不全ウィルスベクターである。 [0010] The PEDF gene and the FGF2 gene contained in the pharmaceutical product of the present invention can be prepared by methods well known to those skilled in the art. For example, a cDNA library of human retinal pigment epithelial cells can be prepared by using a part or all of the base sequence described in SEQ ID NO: 7 or SEQ ID NO: 8 as a probe. Alternatively, it can be prepared by performing a known nucleic acid amplification method using a part of the base sequence described in SEQ ID NO: 7 or SEQ ID NO: 8 as a primer and human retinal pigment epithelial cell mRNA as a saddle type. . When the pharmaceutical product of the present invention is prepared from the above gene, it may be in the DNA state, but is preferably inserted into a vector. Good. The PEDF gene and the FGF2 gene may be held in separate vectors or simultaneously in one vector. The type of the vector is not limited as long as it is a safe vector suitable for pharmaceutical use, but is preferably a lentiviral vector, most preferably a simian immunodeficiency virus vector.
[0011] ウィルスの生活環は大きく感染と増殖の段階に分けられる。一般にウィルスベクタ 一は、ウィルスの感染システムを利用して、遺伝子を効率よく宿主の細胞内に導入出 来ることが特徴である。多くのウィルスベクターは、安全性確保のため、増殖システム を排除して自己複製能を欠如させ、導入された細胞内での増殖を防止する措置がと られている。 [0011] The life cycle of a virus is roughly divided into the stages of infection and propagation. In general, a virus vector is characterized in that a gene is efficiently introduced into a host cell using a virus infection system. In order to ensure safety, many viral vectors have taken measures to eliminate the proliferation system and lack the self-replicating ability to prevent propagation in the introduced cells.
[0012] ベクター粒子の構造を簡単に説明すると、ベクター粒子には、力プシドと呼ばれる 蛋白質の外殻がある。力プシドは gag遺伝子産物の構造蛋白質力も構成されている。 その力プシドの外側にエンベロープと呼ばれる膜構造がある。エンベロープは、感染 する細胞の種類を決定する機能を有する。力プシドの中には、 2コピーのベクターゲ ノム RNA、 pol遺伝子産物である逆転写酵素が存在している。ウィルスベクターは宿主 細胞に感染すると、ベクターゲノム RNAは自らの上記逆転写酵素により逆転写された 後、宿主染色体に組み込まれてプロウィルス DNAとなり、感染を成立させる。  [0012] Briefly explaining the structure of the vector particle, the vector particle has a protein outer shell called force psid. Force psid also constitutes the structural protein power of the gag gene product. There is a membrane structure called an envelope outside the force psid. The envelope has the function of determining the type of cells that are infected. Within the force psid, there are two copies of vector genomic RNA, reverse transcriptase, which is the product of the pol gene. When a viral vector infects a host cell, the vector genomic RNA is reverse transcribed by its own reverse transcriptase and then integrated into the host chromosome to become proviral DNA, thus establishing infection.
[0013] 一般にウィルスベクターは、パッケージングベクターとジーントランスファーベクター によって調製することができる。ノ ッケージングベクターは、パッケージングシグナル が除去されたウィルス DNAを搭載している。ウィルス DNAには、ウィルスタンパク質配 列が含まれている。ノ ッケージングベクターを宿主に導入すると、宿主細胞 (パッケー ジング細胞）では、ノ ッケージングシグナルがないために、空のウィルス粒子が作ら れる。一方のジーントランスファーベクターは、宿主染色体 DNAに組み込まれるため に必要なウィルス由来の遺伝子配列と、導入する外来遺伝子を搭載している。このジ ーントランスファーベクターをパッケージング細胞に導入すると、ジーントランスファー ベクター力 供給されるベクターゲノム DNAが宿主染色体に組み込まれた後、転写 によってベクターゲノム RNAが作られる。このベクターゲノム RNA力 パッケージング 細胞によって作られるウィルス粒子に取り込まれ、宿主内に核酸分子を導入する能 力を有するウィルス粒子が生成される。 [0014] 本発明において「ウィルスベクター」とは、自己複製能を欠如し、宿主内に核酸分子 を導入する能力を有するウィルス粒子を指す。「組換え」ウィルスベクターとは、遺伝 子組換え技術により構築されたウィルスベクターを言う。ウィルスゲノムをコードする D NAとパッケージング細胞を用いて構築したウィルスベクターは、組換えウィルスべク ターに含まれる。 [0013] Generally, a viral vector can be prepared by a packaging vector and a gene transfer vector. The knocking vector carries the viral DNA from which the packaging signal has been removed. Viral DNA contains viral protein sequences. When a knocking vector is introduced into the host, empty virus particles are created in the host cell (packaging cell) because there is no knocking signal. One gene transfer vector carries a viral gene sequence necessary for integration into host chromosomal DNA and a foreign gene to be introduced. When this gene transfer vector is introduced into a packaging cell, the vector genomic DNA supplied with the gene transfer vector is integrated into the host chromosome, and then the vector genomic RNA is produced by transcription. This vector genome RNA force Packaging It is taken up by virus particles produced by cells, and virus particles having the ability to introduce nucleic acid molecules into the host are generated. In the present invention, the “viral vector” refers to a viral particle that lacks self-replicating ability and has the ability to introduce a nucleic acid molecule into a host. A “recombinant” viral vector refers to a viral vector constructed by genetic recombination techniques. Viral vectors constructed using DNA encoding the viral genome and packaging cells are included in the recombinant virus vector.
[0015] 本発明において「サル免疫不全ウィルス（SIV)ベクター」とは、ウィルス粒子中の核 酸分子のうち、ウィルスベクターとしての機能に必須な配列が SIVゲノムに基づく配列 であるベクターを指す。本発明にお 、て「ウィルスベクターとしての機能に必須な配 列」とは、 5，側から順に、 5' LTR( R ¾, U5領域、パッケージングシグナル（ φ )、 RR Ε、 3 ' LTRのプロモーター領域以外の U3領域、 R領域の配列である。 5' LTR領域から パッケージングシグナルまでの塩基配列を配列番号：9に、 RRE配列を配列番号： 10 に、 3' LTRのプロモーター領域を欠如した U3領域力 R領域までの塩基配列を配列 番号： 11に示す。本発明の SIVベクターは、上述の定義に当てはまる限り、改変が施 されていてもよぐ例えば、「ウィルスベクターとしての機能に必須な配列」が SIV由来 である限り、他に SIV由来の配列または SIV以外の由来の配列を含んで 、てもよ!/、。 好適に含まれ得る配列として、例えば、後述する cPPT (central polypurine tract),内 咅 |5プロモ ~~タ' ~~ (CMV)、 WPRE (woodchuck hepatitis virus posttranscriptional regul atory element)を挙げることができる。  [0015] In the present invention, the "monkey immunodeficiency virus (SIV) vector" refers to a vector in which a sequence essential for the function as a viral vector is a sequence based on the SIV genome among the nucleic acid molecules in the virus particle. In the present invention, “sequence essential for function as a viral vector” means 5 ′ LTR (R ¾, U5 region, packaging signal (φ), RR RR, 3 ′ LTR in this order from the 5th side. U3 region and R region sequences other than the promoter region of 5 'LTR region to the packaging signal in SEQ ID NO: 9, RRE sequence in SEQ ID NO: 10 and 3' LTR promoter region The nucleotide sequence up to the lacking U3 region force R region is shown in SEQ ID NO: 11. The SIV vector of the present invention may be modified as long as it meets the above definition. As long as the “essential sequence for SIV” is derived from SIV, it may include other sequences derived from SIV or sequences derived from other than SIV! /. Examples of sequences that can be suitably included include, for example, cPPT ( central polypurine tract), inner 咅 | 5 promo ~~ ta '~~ (CMV) Mention may be made of the WPRE (woodchuck hepatitis virus posttranscriptional regul atory element).
[0016] 本発明にお 、てサル免疫不全ウィルス (simian immunodeficiency virus; SIV)には、 SIVの全ての株およびサブタイプが含まれる。 SIV単離株としては、 SIVagm、 SIVcpz、 S IVmac、 SIVmnd、 SIVsm、 SIVsnm、 SIVsyk等が例示できるがこれらに制限されない。  In the present invention, simian immunodeficiency virus (SIV) includes all strains and subtypes of SIV. Examples of SIV isolates include, but are not limited to, SIVagm, SIVcpz, SIVmac, SIVmnd, SIVsm, SIVsnm, and SIVsyk.
[0017] サル免疫不全ウィルス (Simian Immunodeficiency Virus, SIV)はサルにおける HIV 類似ウィルスとして発見され、 HIVとともに Primates Lentivirusグループを形成してい る（井戸栄治，速水正憲，サル免疫不全ウィルスの遺伝子と感染，病原性.蛋白質核 酸酵素： Vol..39, No.8. 1994) oこのグループはさらに大きく 4つのグループに分類さ れ、 1)ヒトにお ヽて後天'性免疫不全；！正候群 (acquired immune deficiency syndrome, A IDS)の原因となる HIV-1とチンパンジーより分離された SIVcpzを含む HIV-1グループ 、 2)スーティーマンガべィ Cercocebus atysより分離された SIVsmmとァカゲザル Mac aca mulattaより分離された SIVmac、およびヒトに対し低頻度ではあるが病原性を示 す HIV- 2 (Jaffar, S. et al., J. Acquir. Immune Defic. Syndr. Hum. Retroviral., 16(5), 327-32, 1997)よりなる HIV- 2グノレープ、 3)アフリカミドリザル Cercopithecus aethiops から分離された SIVagmに代表される SIVagmグループ、 4)マンドリル Papio sphinxから 分離された SIVmndに代表される SIVmndグループからなっている。 [0017] Simian immunodeficiency virus (SIV) was found as an HIV-like virus in monkeys and together with HIV formed the Primates Lentivirus group (Eiji Ido, Masanori Hayami, simian immunodeficiency virus gene and infection, Pathogenicity.Protein Nucleic Acid Enzyme: Vol..39, No.8. 1994) o This group is further divided into 4 groups: 1) Acquired immune deficiency in humans! HIV-1 group, including SIVcpz isolated from HIV-1 and chimpanzee, which causes acquired immune deficiency syndrome (A IDS), 2) SIVsmm and lizard macaque isolated from Certicebus atys SIVmac isolated from aca mulatta and HIV-2 (Jaffar, S. et al., J. Acquir. Immune Defic. Syndr. Hum. Retroviral., 16 ( 5), 327-32, 1997) HIV-2 gnolepe, 3) SIVagm group represented by SIVagm isolated from African green monkey Cercopithecus aethiops, 4) SIVmnd group represented by SIVmnd isolated from mandrill Papio sphinx It is made up of.
[0018] このうち、 SIVagmおよび SIVmndでは自然宿主における病原性の報告はなく（Ohta, Y. et al., Int. J. Cancer, 15, 41(1), 115—22, 1988; Miura, T. et al., J. Med. Primatol. , 18(3-4), 255-9, 1989;速水正憲， 日本臨床， 47, 1, 1989)、特に本実施例で用いた SIVagmの一種である TYO-1株は自然宿主でも、力-クイザル Macaca facicularis、ァ 力ゲザル Macaca mulattaに対する実験感染でも病原性を示さないことが報告されて いる M. et al, Gene Therapy, 1(6), :367-84, 1994; Honjo, S. et al., J. Med. Pri matol, 19(1), 9-20, 1990)。 SIVagmのヒトに対する感染、発症については報告がなく 、ヒトに対する病原性は知られていないが、一般に霊長類におけるレンチウィルスは 種特異性が高ぐ自然宿主から他種に感染、発症した例は少なぐその発症も低頻 度あるいは進行が遅いという傾向がある（Novembre, F. J. et al" J. Virol, 71(5), 408 6-91, 1997) o従って、 SIVagm、特に SIVagm TYO-1株をベースとして作製したウィル スベクターは、 HIV-1や他のレンチウィルスをベースとしたベクターと比べて安全性が 高いと考えられ、本発明において好適に用いられ得る。 SIVagm TYO-1株のゲノム塩 基配列を配列番号： 12に示す。  [0018] Of these, SIVagm and SIVmnd have not been reported to be pathogenic in natural hosts (Ohta, Y. et al., Int. J. Cancer, 15, 41 (1), 115—22, 1988; Miura, T et al., J. Med. Primatol., 18 (3-4), 255-9, 1989; Masanori Hayami, Japanese Clinical, 47, 1, 1989), especially a kind of SIVagm used in this example The TYO-1 strain has been reported to show no pathogenicity in natural hosts or in experimental infections with the power-quick macaque Macaca facicularis, or the power-giving monkey Macaca mulatta M. et al, Gene Therapy, 1 (6),: 367 -84, 1994; Honjo, S. et al., J. Med. Pri matol, 19 (1), 9-20, 1990). Although there are no reports of human infection or development of SIVagm, pathogenicity to humans is not known, but in general, lentiviruses in primates are rarely infected or developed from natural hosts with high species specificity to other species. The incidence of gulf also tends to be infrequent or slow (Novembre, FJ et al "J. Virol, 71 (5), 408 6-91, 1997) o Therefore, based on SIVagm, especially SIVagm TYO-1 The virus vector prepared as is considered to be safer than vectors based on HIV-1 and other lentiviruses, and can be preferably used in the present invention. The sequence is shown in SEQ ID NO: 12.
[0019] 本発明のサル免疫不全ウィルスベクターは、他のレトロウイルスのゲノム RNA配列の 一部を有していてもよい。例えば、ヒト免疫不全ウィルス（Human Immunodeficiency Vi rus ; HIV)、ネコ免疫不全ウイノレス（Feline Immunodeficiency Virus： FIV) (Poeschla, E . M. et al., Nature Medicine, 4(3), 354-7, 1998)、ャギ関節炎脳炎ウィルス（Caprine Arthritis Encephalitis Virus : CAEV) (Mselli— Lakhal, L. et al., Arch. Virol., 143(4), 6 81-95, 1998)などの他のレンチウィルスのゲノム配列の一部をサル免疫不全ウィルス のゲノムの一部と置換したキメラ配列を有するベクターも、本発明のサル免疫不全ゥ ィルスベクターに含まれる。  [0019] The simian immunodeficiency virus vector of the present invention may have a part of a genomic RNA sequence of another retrovirus. For example, human immunodeficiency virus (Human Immunodeficiency Vi rus; HIV), feline immunodeficiency virus (FIV) (Poeschla, E.M. et al., Nature Medicine, 4 (3), 354-7, 1998. ), Caprine Arthritis Encephalitis Virus (CAEV) (Mselli—Lakhal, L. et al., Arch. Virol., 143 (4), 6 81-95, 1998) A vector having a chimeric sequence in which a part of the genome sequence is replaced with a part of the genome of the simian immunodeficiency virus is also included in the simian immunodeficiency virus vector of the present invention.
[0020] 本発明の色素上皮由来因子（Pigment epithelium derived factor : PEDF)遺伝子を 保持する組換えサル免疫不全ウィルスベクター（SIV-PEDFベクター）とは、 PEDF遺 伝子を搭載して ヽる組換え SIVベクターを指す。また本発明の FGF2遺伝子を保持す る組換えサル免疫不全ウィルスベクター (SIV-FGF2ベクター)とは、 FGF2遺伝子を搭 載して 、る組換え SIVベクターを指す。また本発明の PEDF遺伝子および FGF2遺伝 子を保持する組換えサル免疫不全ウィルスベクターとは、 PEDF遺伝子と FGF2遺伝 子の両方を搭載して 、る組換え SIVベクターを指す。本発明の SIV-PEDFベクターは 、上述の定義に該当する限り、種類'構造を問わないが、好ましい例としては、配列 番号： 1に記載の塩基配列に PEDF遺伝子が挿入された塩基配列を含むジーントラン スファーベクターを用いて製造される SIVベクターを挙げることができ、より好ましい例 としては、配列番号： 2に記載の塩基配列を含むジーントランスファーベクターを用い て製造される SIVベクターを挙げることができる。同様に、本発明の SIV— FGF2ベクタ 一は、上述の定義に該当する限り、種類'構造を問わないが、好ましい例としては、 配列番号： 1に記載の塩基配列に FGF2遺伝子が挿入された塩基配列を含むジーン トランスファーベクターを用いて製造される SIVベクターを挙げることができ、より好まし い例としては、配列番号： 3に記載の塩基配列を含むジーントランスファーベクターを 用いて製造される SIVベクターを挙げることができる。また同様に、本発明の PEDF遺 伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターは、上述 の定義に該当する限り、種類'構造を問わないが、好ましい例としては、配列番号： 1 に記載の塩基配列に PEDF遺伝子および FGF2遺伝子が挿入された塩基配列を含む ジーントランスファーベクターを用いて製造される SIVベクターを挙げることができる。 [0020] Pigment epithelium derived factor (PEDF) gene of the present invention The retained recombinant simian immunodeficiency virus vector (SIV-PEDF vector) refers to a recombinant SIV vector carrying a PEDF gene. The recombinant simian immunodeficiency virus vector (SIV-FGF2 vector) retaining the FGF2 gene of the present invention refers to a recombinant SIV vector carrying the FGF2 gene. The recombinant simian immunodeficiency virus vector carrying the PEDF gene and FGF2 gene of the present invention refers to a recombinant SIV vector carrying both the PEDF gene and the FGF2 gene. The SIV-PEDF vector of the present invention may be of any kind as long as it falls within the above definition, and a preferable example includes a base sequence in which a PEDF gene is inserted into the base sequence described in SEQ ID NO: 1. An SIV vector produced using a gene transfer vector can be mentioned, and a more preferred example is an SIV vector produced using a gene transfer vector comprising the nucleotide sequence set forth in SEQ ID NO: 2. Can do. Similarly, the SIV-FGF2 vector of the present invention may be of any kind as long as it falls within the above definition, but as a preferred example, the FGF2 gene is inserted into the base sequence described in SEQ ID NO: 1. An SIV vector produced using a gene transfer vector containing the nucleotide sequence can be mentioned, and a more preferred example is an SIV produced using the gene transfer vector containing the nucleotide sequence shown in SEQ ID NO: 3. A vector can be mentioned. Similarly, the recombinant simian immunodeficiency virus vector carrying the PEDF gene and the FGF2 gene of the present invention may be of any kind as long as it falls within the above definition, but a preferred example is SEQ ID NO: An SIV vector produced using a gene transfer vector comprising a base sequence in which a PEDF gene and an FGF2 gene are inserted into the base sequence described in 1 can be mentioned.
SIV- PEDFベクターおよび SIV-FGF2ベクターなどの本発明の PEDF遺伝子および Zまたは FGF2遺伝子を保持する SIVベクターは、 VSV-Gシユードタイプ化されて!/、て もよい。 VSV- Gシユードタイプ化とは、ベクターのエンベロープに水疱性口内炎ウイ ルス (Vesicular stomatitis virus; VSV)の表面糖蛋白質である VSV-G蛋白質を含ま せることを言う。 VSV-G蛋白質は、任意の VSV株に由来するものであってよい。例え ば Indiana血清型株（J. Virology 39: 519-528 (1981))由来の VSV- G蛋白を用いるこ とができる力 これに限定されない。また、 VSV-G蛋白質は、天然由来の蛋白質から 1または複数のアミノ酸の置換、欠失、および/または付加などにより改変されていて もよい。 VSV-Gシユードタイプ化ベクターは、ウィルスの産生時に VSV-G蛋白質を共 存させること〖こより製造することができる。例えば、 VSV-G発現ベクターのトランスフエ クシヨンや、宿主染色体 DNAに組み込んだ VSV-G遺伝子からの発現誘導により、ノ ッケージング細胞内で VSV-Gを発現させることにより、この細胞力 産生されるウィル ス粒子が VSV-Gでシユードタイプ化される。 VSV-G蛋白質は 1種類の糖蛋白が安定 な 3量体を形成して膜上に存在するため、精製過程でのベクター粒子の破壊が起こ りにくく、遠心による高濃度の濃縮が可能となる（Yang, Y. et al.， Hum Gene Ther: Se p, 6(9), 1203-13. 1995)。 SIV vectors carrying the PEDF gene of the present invention, such as the SIV-PEDF vector and the SIV-FGF2 vector, and the Z or FGF2 gene may be VSV-G pseudotyped! /. VSV-G pseudotyping means that the vector envelope contains VSV-G protein, which is a surface glycoprotein of vesicular stomatitis virus (VSV). The VSV-G protein may be derived from any VSV strain. For example, the ability to use a VSV-G protein derived from an Indiana serotype strain (J. Virology 39: 519-528 (1981)) is not limited thereto. In addition, the VSV-G protein is modified from a naturally-occurring protein by substitution, deletion and / or addition of one or more amino acids. Also good. A VSV-G pseudotyped vector can be produced by allowing VSV-G protein to coexist during virus production. For example, the expression of VSV-G in a knocking cell by transfection of the VSV-G expression vector or induction of expression from the VSV-G gene integrated into the host chromosomal DNA will generate this cellular force. The particles are pseudotyped with VSV-G. The VSV-G protein forms a stable trimer of a single glycoprotein and is present on the membrane, making it difficult for vector particles to be destroyed during the purification process and allowing high concentration by centrifugation. (Yang, Y. et al., Hum Gene Ther: Sep, 6 (9), 1203-13. 1995).
[0022] SIV- PEDFベクターおよび SIV- FGF2ベクターなどの本発明の PEDF遺伝子および Zまたは FGF2遺伝子を保持する SIVベクターは、他のウィルス由来のエンベロープ 蛋白質をさらに含むことができる。例えば、このような蛋白質として、ヒト細胞に感染す るウィルスに由来するエンベロープ蛋白質が好適である。このような蛋白質としては、 特に制限はないが、レトロウイルスのアンフォト口ピックエンベロープ蛋白質などが挙 げられる。レトロウイルスのアンフォト口ピックエンベロープ蛋白質としては、例えばマ ウス白血病ウィルス (MuLV) 4070A株由来のエンベロープ蛋白質を用 、得る。また、 MuMLV 10A1由来のエンベロープ蛋白質を用いることもできる（例えば pCL-10Al(Im genex) (Naviaux, R. K. et al, J. Virol. 70: 5701-5705 (1996))。また、ヘルぺスウィル ス科の蛋白質としては、例えば単純へルぺスウィルスの gB、 gD、 gH、 gp85蛋白質、 E Bウィルスの gp350、 gp220蛋白質などが挙げられる。へパドナウィルス科の蛋白質とし ては、 B型肝炎ウィルスの S蛋白質などが挙げられる。  [0022] The SIV vector carrying the PEDF gene of the present invention, such as the SIV-PEDF vector and the SIV-FGF2 vector, and the Z or FGF2 gene can further contain an envelope protein derived from another virus. For example, an envelope protein derived from a virus that infects human cells is suitable as such a protein. Such a protein is not particularly limited, and examples thereof include a retroviral unphotopick envelope protein. For example, an envelope protein derived from the mouse leukemia virus (MuLV) 4070A strain is obtained as a retrovirus unphoto-mouth pick envelope protein. An envelope protein derived from MuMLV 10A1 can also be used (for example, pCL-10Al (Im genex) (Naviaux, RK et al, J. Virol. 70: 5701-5705 (1996)). Examples of such proteins include simple herpesvirus gB, gD, gH and gp85 proteins, EB virus gp350 and gp220 proteins, etc. Hepadnaviridae proteins include hepatitis B virus S protein. Etc.
[0023] 本発明の組換えサル免疫不全ウィルスベクターとしては、 LTR (long terminal repeat )に改変をカ卩えることもできる。 LTRはレトロウイルスに特徴的な配列であり、ウィルス ゲノムの両端に存在している。 5' LTRはプロモーターとして働き、プロウィルスからの mRNAの転写を促す。したがって、ウィルス粒子内にパッケージングされるウィルス RN Aゲノムをコードするジーントランスファーベクターの 5' LTRのプロモーター活性をも つ部分を別の強力なプロモーターと置換すれば、ジーントランスファーベクターの mR NA転写量が増大し、パッケージング効率が上昇、ベクター力価を上昇させる可能性 がある。さらに、例えばレンチウィルスの場合、 5' LTRはウィルス蛋白質 tatによる転写 活性の増強を受けることが知られており、 5' LTRを tat蛋白質に依存しないプロモータ 一に置換することで、ノ ッケージングベクターから tatを削除することが可能となる。ま た、細胞に感染し細胞内に侵入したウィルス RNAは逆転写された後、両端の LTRを 結合させた環状構造となり、結合部位とウィルスのインテグラーゼが共役して細胞の 染色体内にインテグレートされる。プロウィルスから転写される mRNAは 5' LTR内の転 写開始点より下流、 3' LTRの polyA配列までであり、 5' LTRのプロモーター部分はゥ ィルス内にパッケージングされない。したがって、プロモーターを置換したとしても標 的細胞の染色体に挿入される部分には変化が無い。以上のことから、 5' LTRのプロ モーターの置換は、より高力価で安全性の高いベクターを作製することにつながると 考えられる。従って、ジーントランスファーベクターの 5'側プロモーターの置換を行い 、パッケージングされるベクターの力価を上昇させることができる。 [0023] The recombinant simian immunodeficiency virus vector of the present invention can also be modified to LTR (long terminal repeat). LTR is a sequence characteristic of retroviruses and exists at both ends of the viral genome. The 5 'LTR acts as a promoter and promotes transcription of mRNA from proviruses. Therefore, if the gene transfer vector encoding the viral RNA genome packaged in the viral particle is replaced with another strong promoter, the gene transfer vector mRNA transcripts May increase packaging efficiency and vector titer. In addition, for example, in the case of lentivirus, the 5 'LTR is transcribed by the viral protein tat. It is known that the activity is enhanced, and it is possible to remove tat from the knocking vector by replacing the 5 ′ LTR with a promoter independent of the tat protein. In addition, viral RNA that infects cells and invades cells is reverse transcribed, then becomes a circular structure that combines the LTRs at both ends, and the binding site and viral integrase are coupled and integrated into the cell's chromosome. The The mRNA transcribed from the provirus is downstream from the transcription start point in the 5 ′ LTR, up to the polyA sequence of the 3 ′ LTR, and the promoter portion of the 5 ′ LTR is not packaged in the virus. Therefore, even if the promoter is replaced, there is no change in the portion inserted into the chromosome of the target cell. Based on the above, replacement of the 5 'LTR promoter is thought to lead to the production of higher-titer and safer vectors. Therefore, substitution of the 5 ′ promoter of the gene transfer vector can increase the potency of the packaged vector.
また、 3' LTRの配列を部分的に削除し、標的細胞力 全長のベクター mRNAが転写 されることを防止する自己不活性化型ベクター (Self Inactivating Vector： SINベクター )の作製により、安全性を上昇させることも可能である。標的細胞の染色体に侵入した レンチウィルスのプロウィルスは、 3' LTRの U3部分を 5'端に結合した形となる。した がってジーントランスファーベクターの転写産物は、逆転写後、標的細胞の染色体に 組み込まれた状態では 5'端に U3が配置され、そこ力もジーントランスファーベクター からの転写産物と同様の構造を持つ RNAが転写されることになる。仮に、標的細胞内 にレンチウィルスあるいはその類似の蛋白質が存在した場合、転写された RNAが再 びパッケージングされ、他の細胞に再感染する可能性がある。また 3' LTRのプロモー ターにより、ウィルスゲノムの 3'側に位置する宿主由来の遺伝子が発現されてしまう P丁目 '性力 2 "め O (Rosenberg, N., Jolicoeur, P., Retoroviral Pathogenesis. Retroviruses. Cold Spling Harbor Laboratory Press, 475—585, 1997)。この現象はレトロウイルスべ クタ一においてすでに問題とされ、回避の方法として SINベクターが開発された (Yu, S . F. et al, Proc. Natl. Acad. Sci. U S A, 83(10), 3194—8, 1986)。ジーントランスファ 一ベクター上の 3' LTRの U3部分を欠失させることにより、標的細胞内では 5' LTRや 3 ' LTRのプロモーターが無くなるため、全長の RNAや宿主遺伝子の転写が起こらない 。そして、内部プロモーター力もの目的遺伝子の転写のみが行われることになり、安 全性が高ぐ高発現のベクターとなることが期待される。このようなベクターは、本発明 において好適である。 SINベクターの構築は、公知の方法または本発明者らによる特 許出願：国際公開番号 WO2002/101057 (特許文献 1)の実施例 1-4に記載の方法な どに従えばよい。 In addition, the 3 'LTR sequence is partially deleted to create a self-inactivating vector (SIN vector) that prevents transcription of the full-length vector mRNA of the target cell. It can also be raised. A lentiviral provirus that has entered the chromosome of the target cell has a 3 'LTR U3 moiety bound to the 5' end. Therefore, the gene transfer vector transcript has a structure similar to that of the gene transfer vector from the gene transfer vector, when U3 is located at the 5 'end after reverse transcription and integrated into the chromosome of the target cell. RNA will be transcribed. If a lentivirus or similar protein is present in the target cell, the transcribed RNA can be repackaged and reinfected to other cells. In addition, the 3 'LTR promoter allows expression of a host-derived gene located on the 3' side of the viral genome. P-chome 'Gender 2 ' O (Rosenberg, N., Jolicoeur, P., Retoroviral Pathogenesis. Cold Spling Harbor Laboratory Press, 475-585, 1997) This phenomenon has already been a problem in retroviral vectors, and SIN vectors have been developed as a way to avoid it (Yu, S. F. et al, Proc Natl. Acad. Sci. USA, 83 (10), 3194-8, 1986) Gene transfer By deleting the U3 portion of the 3 'LTR on the same vector, 5' LTR and 3 ' Since the LTR promoter is lost, transcription of full-length RNA and host genes does not occur, and only the target gene with internal promoter strength is transferred. It is expected to be a highly expressed vector with high integrity. Such a vector is suitable in the present invention. The construction of the SIN vector may be performed by a known method or the method described in Example 1-4 of the patent application by the present inventors: International Publication No. WO2002 / 101057 (Patent Document 1).
[0025] レトロウイルスベクターなどそのゲノムに LTR配列を含むウィルスベクターを用いた 遺伝子治療の問題点の一つに導入遺伝子の発現が次第に低下することがある。こ れらのベクターは宿主ゲノムに組み込まれると、宿主側の機序によりその LTRがメチ ル化され、導入遺伝子の発現が抑制されてしまうことが原因の一つと考えられて 、る (Challita, P. M. and Kohn, D. B" Proc. Natl. Acad. Sci. USA 91:2567, 1994)。 自己 不活性化 (SIN)型ベクターでは、宿主ゲノムに組み込まれると LTR配列の大部分を失 うため、 LTRのメチルイ匕による遺伝子発現の減弱を受けにくい利点がある。本発明者 らによって、ジーントランスファーベクターの 3'LTRの U3領域を他のプロモーター配列 に置換して製造した自己不活性ィ匕型ベクターは、霊長類 ES細胞に導入後、 2力月以 上にわたって安定した発現を維持することが判明している（特許文献 1)。このように、 LTR U3領域の改変により自己不活性ィ匕するように設計された SINベクターは、本発 明において特に好適である。具体的には、 3'LTRの U3領域の 1または複数の塩基が 置換、欠失、および/または付カ卩により改変されたベクターは、本発明に含まれる。こ の U3領域は、単に欠失させてもよいし、あるいはこの領域に他のプロモーターを挿入 してもよい。このようなプロモーターとしては、例えば CMVプロモーター、 EF1プロモー ター、または CAGプロモーターなどを挙げることができる。  [0025] One of the problems of gene therapy using a viral vector containing an LTR sequence in its genome, such as a retroviral vector, is that the expression of the transgene gradually decreases. When these vectors are integrated into the host genome, the LTR is methylated by the host-side mechanism and the expression of the transgene is suppressed (Challita, PM and Kohn, D. B "Proc. Natl. Acad. Sci. USA 91: 2567, 1994). Self-inactivating (SIN) vectors lose most of their LTR sequences when integrated into the host genome. This is an advantage that the gene expression of the LTR is less susceptible to the attenuation of the gene expression caused by the MTR.The self-inactive type produced by the present inventors by replacing the 3'LTR U3 region of the gene transfer vector with another promoter sequence. The vector has been found to maintain stable expression for more than 2 months after introduction into primate ES cells (Patent Document 1). SIN vectors designed to In particular, vectors in which one or more bases in the U3 region of the 3′LTR have been modified by substitution, deletion, and / or addition are included in the present invention. The U3 region may be simply deleted, or another promoter may be inserted into this region, such as the CMV promoter, EF1 promoter, or CAG promoter. Can do.
[0026] また、本発明のベクターにコードされる PEDF遺伝子および FGF2遺伝子は、 LTR以 外のプロモーターにより転写されるように設計することが好ましい。例えば、上記のよ うに LTR U3領域を非 LTRプロモーターと置換した場合には、この改変 LTRにより PED F遺伝子または FGF2遺伝子の発現を駆動することが好ましい。あるいは、実施例に おいて示されるように、 LTR領域とは別の位置に非 LTRプロモーターを配置し、その 下流に PEDF遺伝子または FGF2遺伝子を連結することにより、 LTRに依存せずに PE DF遺伝子または FGF2遺伝子の発現を誘導することができる。本発明者らによって、 外来遺伝子の発現を非 LTRプロモーターにより駆動するように構築された SIVベクタ 一は、 ES細胞においてこの外来遺伝子を長期間安定に発現することが示された (特 許文献 1)。同様に、 PEDF遺伝子または FGF2遺伝子の上流に非 LTRプロモーター が連結されており、このプロモーター力も PEDF遺伝子または FGF2遺伝子が転写さ れるようなベクターは本発明にお 、て特に適して 、ると!/、うことができる。非 LTRプロ モーターとしては、例えば CMVプロモーター、 EF1プロモーター、または CAGプロモ 一ターが挙げられ、特に CMVプロモーターが好適である。本実施例で使用した CMV プロモーターの塩基配列を配列番号：13に示す。このようなベクターは、特に上記の 自己不活性化 (SIN)型ベクターにお 、て構築することで高 、効果を発揮する。 [0026] The PEDF gene and the FGF2 gene encoded by the vector of the present invention are preferably designed to be transcribed by a promoter other than LTR. For example, when the LTR U3 region is replaced with a non-LTR promoter as described above, it is preferable to drive the expression of the PED F gene or the FGF2 gene by this modified LTR. Alternatively, as shown in the Examples, a non-LTR promoter is placed at a position different from the LTR region, and a PEDF gene or FGF2 gene is linked downstream thereof, so that the PE DF gene is independent of the LTR. Alternatively, expression of the FGF2 gene can be induced. An SIV vector constructed by the present inventors so that expression of a foreign gene is driven by a non-LTR promoter First, it was shown that this foreign gene is stably expressed in ES cells for a long period of time (Patent Document 1). Similarly, a vector in which a non-LTR promoter is linked upstream of the PEDF gene or FGF2 gene, and the promoter power of the PEDF gene or FGF2 gene is also transcribed is particularly suitable for the present invention! / ,I can. Examples of the non-LTR promoter include CMV promoter, EF1 promoter, and CAG promoter, and CMV promoter is particularly preferable. The nucleotide sequence of the CMV promoter used in this example is shown in SEQ ID NO: 13. Such a vector is particularly effective when constructed in the above-described self-inactivating (SIN) type vector.
[0027] HIVベクターをはじめとするレンチウィルスベクターは、宿主ゲノムが HIVプロウィル スをすでに保持して 、る場合、外来ベクターと内因性プロウィルスの間で組換えが生 じ、複製可能ウィルスが発生しないかという危惧が指摘されている。これは、将来実 際に HIV感染患者に HIVベクターを用いる時には確かに大きな問題になろう。今回使 用した SIVベクターは、 HIVとの相同配列はほとんどない上、ウィルス由来の配列を 80 %以上取り除いた複製不能ウィルスであり、この危険性は極めて小さぐ他のレンチ ウィルスベクターに比べて安全性が高い。本発明の SIV-PEDFベクターおよび SIV-F GF2ベクターは、上述の「ウィルスベクターとしての機能に必須な配列」以外の SIVゲ ノム配列が一定以上除去されたベクターであり、好ましくは、このベクターが由来する SIVのゲノム配列の 40%以上、より好ましくは 50%以上、さらに好ましくは 60%以上、さ らに好ましくは 70%以上、最も好ましくは 80%以上が取り除かれた複製不能ウィルス である。 [0027] Lentiviral vectors such as HIV vectors, when the host genome already carries HIV virus, recombination occurs between the foreign vector and the endogenous provirus, resulting in a replicable virus. There are concerns about whether or not to do so. This will certainly be a major problem when HIV vectors are used in the future for HIV-infected patients. The SIV vector used in this study is a non-replicatable virus that has almost no homologous sequence with HIV and more than 80% of the virus-derived sequences, and this risk is safer than other lentiviral vectors. High nature. The SIV-PEDF vector and SIV-F GF2 vector of the present invention are vectors from which SIV genomic sequences other than the above-described “sequence essential for function as a viral vector” have been removed, and preferably this vector is 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, and most preferably 80% or more of the genome sequence of the SIV from which it is derived.
[0028] レトロウイルスの産生には、宿主細胞でパッケージングシグナルを有するジーントラ ンスファーベクター DNAを転写させ、 gag, pol蛋白質およびエンベロープ蛋白質の存 在下でウィルス粒子を形成させる。ノ ッケージング細胞内の gag, pol蛋白質はパッケ 一ジングベクターを用いて供給することができる。エンベロープ蛋白質は、パッケージ ングベクターによって供給されてもよいが、別のベクターによって供給されてもよい。 例えば実施例のように、 VSV-G発現ベクターによって供給されてもよい。  [0028] For the production of a retrovirus, a gene transfer vector DNA having a packaging signal is transcribed in a host cell, and a virus particle is formed in the presence of gag, pol protein and envelope protein. The gag and pol proteins in the knocking cells can be supplied using a packaging vector. The envelope protein may be supplied by a packaging vector or may be supplied by another vector. For example, as in the examples, it may be supplied by a VSV-G expression vector.
[0029] 本発明のジーントランスファーベクターは、最も基本的には、 5，LTR、パッケージン グシグナル配列、 PEDF遺伝子および Zまたは FGF2遺伝子、ならびに 3' LTR配列を 有する。 LTR配列は、 SIVベクターの改変として上述した LTRの改変が施されていても よい。また、上述の cPPT配列、 CMV配列、 RRE配列等が組み込まれていてもよい。ジ ーントランスファーベクター DNAにコードされるパッケージングシグナル配列は、この 配列により形成される構造を保持できるように可能な限り長く組み込むことが好ましい 一方で、該ベクター DNA上のパッケージングシグナルと、 gag,pol蛋白質を供給する ノ ッケージングベクターとの間で起こる組換えによる野生型ウィルスの出現頻度を抑 制するためにはこれらベクター間の配列の重複を最小限にする必要がある。従って、 ジーントランスファーベクター DNAの構築においては、パッケージング効率および安 全性の両者を満足させるために、ノ ッケージングに必要な配列を含むできる限り短 ヽ 配列を用いることが好まし 、。 [0029] The gene transfer vector of the present invention basically comprises a 5, LTR, a packaging signal sequence, a PEDF gene and a Z or FGF2 gene, and a 3 'LTR sequence. Have. The LTR sequence may be subjected to the LTR modification described above as a modification of the SIV vector. Moreover, the above-mentioned cPPT sequence, CMV sequence, RRE sequence and the like may be incorporated. The packaging signal sequence encoded by the gene transfer vector DNA is preferably incorporated as long as possible so that the structure formed by this sequence can be retained, while the packaging signal on the vector DNA and gag Therefore, in order to suppress the appearance frequency of wild-type virus due to recombination with the knocking vector supplying the pol protein, it is necessary to minimize sequence duplication between these vectors. Therefore, in the construction of gene transfer vector DNA, it is preferable to use as short a sequence as possible including sequences necessary for knocking in order to satisfy both packaging efficiency and safety.
[0030] 例えば、パッケージングベクターを SIVagm由来のものにした場合は、 HIV由来のジ ーントランスファーベクターはパッケージングされないため、ジーントランスファーべク ター DNAに用いられるパッケージングシグナルの由来としては SIVのみに制限される と考えられる。但し、 HIV由来のパッケージングベクターを用いた場合、 SIV由来のジ ーントランスファーベクターもパッケージングされるので、組換えウィルスの出現頻度 を低下させるために、異なるレンチウィルス由来のジーントランスファーベクターとパッ ケージングベクターとを組み合わせてベクター粒子を形成させることが可能であると 考えられる。このようにして製造された SIVベクターも、本発明のベクターに含まれる。 この場合、霊長類のレンチウィルスの間の組み合わせ（例えば、 HIVと SIV)であること が好ましい。 [0030] For example, when the packaging vector is derived from SIVagm, the HIV-derived gene transfer vector is not packaged, and therefore only the SIV is derived from the packaging signal used for the gene transfer vector DNA. It is considered to be limited to However, when HIV-derived packaging vectors are used, SIV-derived gene transfer vectors are also packaged. Therefore, in order to reduce the appearance frequency of recombinant viruses, different lentivirus-derived gene transfer vectors and packages are used. It is thought that vector particles can be formed by combining with a caging vector. The SIV vector thus produced is also included in the vector of the present invention. In this case, a combination between primate lentiviruses (eg HIV and SIV) is preferred.
[0031] ジーントランスファーベクター DNAでは、 gag蛋白質が発現しないように改変されて いることが好ましい。ウィルス gag蛋白質は、生体にとって異物として認識され、抗原 性が現れる可能性がある。また、細胞の機能に影響を及ぼす可能性もある。 gag蛋白 質を発現しな!、ようにするためには、 gagの開始コドンの下流に塩基の付加や欠失等 によりフレームシフトするように改変することができる。また、 gag蛋白質のコード領域 の一部を欠失させることが好ましい。一般にウィルスのパッケージングには、 gag蛋白 質のコード領域の 5'側が必要であるとされている。従って、ジーントランスファーベクタ 一においては、 gag蛋白質の C末側のコード領域を欠失していることが好ましい。パッ ケージング効率に大きな影響を与えな 、範囲でできるだけ広 ヽ gagコード領域を欠失 させることが好ましい。また、 gag蛋白質の開始コドン (ATG)を ATG以外のコドンに置 換することも好ましい。置換するコドンは、ノ ッケージング効率に対する影響が少ない ものを適宜選択する。これにより構築されたパッケージングシグナルを有するジーント ランスファーベクター DNAを、適当なパッケージング細胞に導入することにより、ウイ ルスベクターを生産させることができる。生産させたウィルスベクターは、例えばパッ ケージング細胞の培養上清から回収することができる。 [0031] The gene transfer vector DNA is preferably modified so that the gag protein is not expressed. Viral gag protein is recognized as a foreign substance by the living body, and may have antigenicity. It may also affect cell function. In order to avoid expressing the gag protein !, it can be modified so that it undergoes a frame shift by adding or deleting a base downstream of the start codon of gag. It is also preferable to delete a part of the coding region of gag protein. Generally, viral packaging requires the 5 'side of the coding region of the gag protein. Therefore, in the gene transfer vector 1, it is preferable that the C-terminal coding region of the gag protein is deleted. Pack It is preferable to delete the gag coding region as wide as possible without greatly affecting the caging efficiency. It is also preferable to replace the start codon (ATG) of the gag protein with a codon other than ATG. The codon to be replaced is appropriately selected so that it has little effect on knocking efficiency. A viral vector can be produced by introducing the gene transfer vector DNA having a packaging signal thus constructed into an appropriate packaging cell. The produced viral vector can be recovered from, for example, the culture supernatant of packaging cells.
さらにジーントランスファーベクター DNAでは、 PEDF遺伝子および FGF2遺伝子の 導入効率および発現効率を高める改変がなされて 、ることが好ま 、。導入効率を 上昇させる改変の例として、 cPPT配列の導入を挙げることができる。 cPPTは、もともと SIVゲノムに存在する配列である。 HIVウィルスにおいてはかなり以前から報告があり (P.Charneau et al.： J.Virol 65: 2415-2431, 1991)、 HIVベクターにおいては cPPTを 導入するとベクターゲノムの核への移行が良くなり、遺伝子導入効率が上昇すること が報告されている (A.Sirven et al.： Blood 96:4103-4110, 2000)。本実施例で使用し た cPPTの塩基配列を配列番号： 14に示す。また発現効率を高める改変の例としては 、 WPRE配列の導入を挙げることができる。 WPREは遺伝子発現効率を高める機能を 有する因子である（US Patent 6284469： RNA export element and methods of use)。 他のレンチウィルスベクターでは cPPTと WPREの 2つの因子の同時導入は個々の効 果を更に高める結果が報告されている (SC.Barry et al.： Hum. Gene Ther. 12: 1103 -1108, 2001)。本実施例で使用した WPREの塩基配列を配列番号： 15に示す。 SIV- PEDFベクターおよび SIV- FGF2ベクターなどの本発明の PEDF遺伝子および Zまた は FGF2遺伝子を保持する SIVベクターにおいて、 cPPTは、一般的なレンチウィルス ベクターにおける配置と同様の配置をとることができる。例えば、 cPPTをプロモーター と外部遺伝子との間に配置してもよぐまたは RRE配列の上流に配置することもできる 1S 好ましくは、 PEDFまたは FGF2の転写を駆動する上述の非 LTRプロモーターの上 流に配置する。 WPREは、 PEDFまたは FGF2遺伝子の下流に配置することができる。 このようなジーントランスファーベクターの好ましい具体例として、配列番号： 1に記載 の塩基配列に PEDF遺伝子が挿入された塩基配列を含むジーントランスファーベクタ 一を用いて製造される SIVベクター、配列番号： 1に記載の塩基配列に FGF2遺伝子 が挿入された塩基配列を含むジーントランスファーベクターを用いて製造される SIV ベクター、ならびに配列番号： 1に記載の塩基配列に PEDF遺伝子および FGF2遺伝 子が挿入された塩基配列を含むジーントランスファーベクターを用いて製造される SI Vベクターを挙げることができ、より好ましい例としては、配列番号： 2に記載の塩基配 列を含むジーントランスファーベクターを用いて製造される SIVベクター、配列番号： 3 に記載の塩基配列を含むジーントランスファーベクターを用いて製造される SIVベクタ 一を挙げることができる。 Furthermore, it is preferable that the gene transfer vector DNA has been modified to enhance the introduction efficiency and expression efficiency of the PEDF gene and the FGF2 gene. An example of a modification that increases the introduction efficiency is the introduction of a cPPT sequence. cPPT is a sequence originally present in the SIV genome. The HIV virus has been reported for a long time (P. Charneau et al .: J. Virol 65: 2415-2431, 1991). In the case of HIV vectors, the introduction of cPPT improves the transfer of the vector genome to the nucleus, and the gene Increased efficiency has been reported (A. Sirven et al .: Blood 96: 4103-4110, 2000). The base sequence of cPPT used in this example is shown in SEQ ID NO: 14. An example of a modification that increases expression efficiency is the introduction of a WPRE sequence. WPRE is a factor having a function of enhancing gene expression efficiency (US Patent 6284469: RNA export element and methods of use). In other lentiviral vectors, simultaneous introduction of two factors, cPPT and WPRE, has been reported to further enhance individual effects (SC. Barry et al .: Hum. Gene Ther. 12: 1103 -1108, 2001). ). The nucleotide sequence of WPRE used in this example is shown in SEQ ID NO: 15. In SIV vectors carrying the PEDF gene of the present invention, such as SIV-PEDF vector and SIV-FGF2 vector, and Z or FGF2 gene, cPPT can take the same arrangement as that in a general lentiviral vector. For example, cPPT can be placed between the promoter and the external gene, or can be placed upstream of the RRE sequence. 1S Preferably, upstream of the above non-LTR promoter that drives transcription of PEDF or FGF2. Deploy. WPRE can be placed downstream of the PEDF or FGF2 gene. As a preferred specific example of such a gene transfer vector, a gene transfer vector comprising a base sequence in which a PEDF gene is inserted into the base sequence described in SEQ ID NO: 1. SIV vector produced using one, SIV vector produced using a gene transfer vector comprising a nucleotide sequence in which the FGF2 gene is inserted into the nucleotide sequence described in SEQ ID NO: 1, and SEQ ID NO: 1 An SIV vector produced using a gene transfer vector containing a base sequence having a PEDF gene and an FGF2 gene inserted into the base sequence can be mentioned. A more preferred example is the base sequence described in SEQ ID NO: 2. An SIV vector produced using a gene transfer vector containing a sequence, and an SIV vector produced using a gene transfer vector containing the nucleotide sequence set forth in SEQ ID NO: 3.
[0033] 本発明にお!/、て、パッケージングベクターは、 PEDF遺伝子および Zまたは FGF2遺 伝子の導入に必要でな 、配列を取り除 、たものを用いることができる。必要でな 、配 列として、修飾遺伝子と呼ばれる vif,vprと制御遺伝子の tat,revを例示することができ る。修飾遺伝子産物はベクターにおいて必要でないことが報告されており (V.Kim et al: J.Virol 72:811-816, 1998)、近年は、安全性を高めるため、修飾遺伝子が削除さ れたベクターが使用されている。また、 tatも削除され、 revは別のプラスミドに移すこと により更に安全性が高められた、第三世代と呼ばれるベクターが開発されている。パ ッケージングベクターから revを除いた場合は、別に、 rev発現ベクターを構築し、該 re V発現ベクターを SIV- PEDFベクターおよび SIV- FGF2ベクターなどの本発明の PEDF 遺伝子および Zまたは FGF2遺伝子を保持する SIVベクターの製造において使用す ることができる。 SIVagm TYO-1株の revの塩基配列を配列番号： 16に示す。上記のよ うにして構築されたパッケージングベクターは、例えば、プロモーター配列、ウィルス コアタンパク質配列（gag)、逆転写酵素配列 (pol)、 polyA配列を含む構成とすること ができ、実施例に示すように上記構成にさらに RRE配列が含まれていてもよい。また ev発現ベクターは、 rev配列の上流に該配列を制御するためのプロモーター、 rev配 列の下流に polyA配列を配置した構成とすることができる。  [0033] In the present invention, the packaging vector can be used after removing the sequence which is necessary for the introduction of the PEDF gene and the Z or FGF2 gene. Examples of sequences that are not necessary include vif and vpr called modified genes and tat and rev of regulatory genes. It has been reported that modified gene products are not required in vectors (V. Kim et al: J. Virol 72: 811-816, 1998), and in recent years, vectors with modified genes deleted to increase safety. Is used. In addition, a vector called the third generation has been developed in which tat has been deleted and rev is transferred to another plasmid to further increase safety. When rev is excluded from the packaging vector, a rev expression vector is constructed separately, and the re V expression vector holds the PEDF gene of the present invention such as SIV-PEDF vector and SIV-FGF2 vector and Z or FGF2 gene. It can be used in the production of SIV vectors. The nucleotide sequence of rev of SIVagm TYO-1 strain is shown in SEQ ID NO: 16. The packaging vector constructed as described above can be configured to include, for example, a promoter sequence, a viral core protein sequence (gag), a reverse transcriptase sequence (pol), and a polyA sequence, and are shown in the Examples. As described above, an RRE sequence may be further included in the above configuration. In addition, the ev expression vector can have a configuration in which a promoter for controlling the sequence is arranged upstream of the rev sequence and a polyA sequence is arranged downstream of the rev sequence.
[0034] ノ^ケ一ジング細胞に使われる細胞としては、一般的にウィルスの産生に使用され る細胞株であれば制限はない。ヒトの遺伝子治療用に用いることを考えると、細胞の 由来としてはヒトまたはサルが適当であると考えられる。ノ ッケージング細胞として使 用されうるヒト細胞株としては、例えば 293細胞、 293T細胞、 293EBNA細胞、 SW480細 胞、 u87MG細胞、 HOS細胞、 C8166細胞、 MT- 4細胞、 Molt- 4細胞、 HeLa細胞、 HT1 080細胞、 TE671細胞などが挙げられる。サル由来細胞株としては、例えば、 COS1細 胞、 COS7細胞、 CV-1細胞、 BMT10細胞などが挙げられる。 [0034] The cell used for the nosing cell is not limited as long as it is a cell line generally used for virus production. Considering use for human gene therapy, humans or monkeys are considered appropriate for cell origin. Examples of human cell lines that can be used as knocking cells include 293 cells, 293T cells, 293EBNA cells, and SW480 cells. Cell, u87MG cell, HOS cell, C8166 cell, MT-4 cell, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell and the like. Examples of monkey-derived cell lines include COS1 cells, COS7 cells, CV-1 cells, BMT10 cells, and the like.
[0035] SIV- PEDFベクターおよび SIV- FGF2ベクターなどの本発明の PEDF遺伝子および Zまたは FGF2遺伝子を保持する SIVベクターは、実質的に純粋になるよう精製するこ とができる。精製方法はフィルター濾過、遠心分離、およびカラム精製等を含む公知 の精製'分離方法により行うことができる。例えば、ベクター溶液を 0.45 mのフィルタ 一にて濾過後、 42500 X g、 90分、 4°Cで遠心を行うことで、ベクターを沈殿'濃縮する ことができる。 [0035] SIV vectors carrying the PEDF gene of the present invention, such as the SIV-PEDF vector and the SIV-FGF2 vector, and the Z or FGF2 gene can be purified to be substantially pure. The purification method can be performed by a known purification method including filtration, centrifugation, column purification, and the like. For example, the vector solution can be precipitated and concentrated by filtering the vector solution through a 0.45 m filter and centrifuging at 42500 Xg for 90 minutes at 4 ° C.
[0036] また上述のとおり本発明の医薬品は PEDFタンパク質と FGF2タンパク質を用 、て調 製してもよぐ PEDFタンパク質と FGF2タンパク質は、これらの cDNAを当業者に周知 の方法で調製し、該 cDNAを適当な発現ベクターに挿入して宿主細胞に導入して発 現させることができる。これらの cDNAは、別々の発現ベクターに保持されてもよぐ 1 つの発現ベクターに同時に保持されていてもよい。 cDNAの調製は、上述したとおり である。発現ベクターは、宿主細胞に合わせて適宜選択することができる。  [0036] As described above, the pharmaceutical product of the present invention may be prepared using PEDF protein and FGF2 protein. PEDF protein and FGF2 protein may be prepared by a method well known to those skilled in the art. The cDNA can be inserted into an appropriate expression vector and introduced into a host cell for expression. These cDNAs may be held in separate expression vectors or simultaneously in one expression vector. The preparation of cDNA is as described above. The expression vector can be appropriately selected according to the host cell.
[0037] 本発明の医薬品は、眼組織細胞におけるアポトーシス変性を伴う疾患の治療およ び予防へ使用することができる。例えば、網膜色素変性、緑内障、網膜剥離、網膜虚 血性疾患の治療および予防に用いることができ、特に、網膜色素変性の治療および 予防には好適に用いることができる。上述した SIV-PEDFベクターおよび SIV-FGF2 ベクターなどの PEDF遺伝子および/または FGF2遺伝子を保持する SIVベクターは、 必要に応じて薬学的に許容される所望の担体または媒体と適宜組み合わせて上記 疾患用の医薬品とすることができる。「薬学的に許容される担体」とは、ベクターと共 に投与することが可能であり、ベクターによる遺伝子導入を有意に阻害しない材料で ある。具体的には、例えば滅菌水、生理食塩水、培養液、血清、リン酸緩衝生理食塩 水（PBS)などと適宜組み合わせることが考えられる。さらに、その他にも、安定剤、殺 生物剤等が含有されていてもよい。本発明の医薬品の形態は、 PEDFタンパク質およ び FGF2タンパク質または、 SIV- PEDFベクターおよび SIV- FGF2ベクターを同一の媒 体に存在させて組成物とし、一つの医薬品とすることができる。 SIV-PEDFベクターお よび SIV- FGF2ベクターを一つの組成物とする場合は、それぞれのベクターについて 下記に示す投与量を確保できる範囲で配合することができる。または、 SIV-PEDFベ クタ一および SIV-FGF2ベクターを別々の組成物として調製し、両組成物を含む治療 用キットとしてもよい。 PEDFおよび FGF2の遺伝子、タンパク質も同様のキットとするこ とができる。 PEDFタンパク質および FGF2タンパク質を本発明の医薬品とする場合に おける、担体および形態は、ベクターの場合と同様である。 [0037] The pharmaceutical agent of the present invention can be used for treatment and prevention of diseases associated with apoptotic degeneration in ocular tissue cells. For example, it can be used for the treatment and prevention of retinitis pigmentosa, glaucoma, retinal detachment, and retinal ischemic disease, and particularly preferably for the treatment and prevention of retinitis pigmentosa. The SIV vector carrying the PEDF gene and / or FGF2 gene, such as the SIV-PEDF vector and SIV-FGF2 vector described above, is appropriately combined with a desired pharmaceutically acceptable carrier or vehicle as necessary. It can be a medicine. A “pharmaceutically acceptable carrier” is a material that can be administered together with a vector and does not significantly inhibit gene transfer by the vector. Specifically, for example, appropriate combinations with sterilized water, physiological saline, culture solution, serum, phosphate buffered saline (PBS) and the like can be considered. In addition, stabilizers, biocides and the like may be included. The form of the pharmaceutical product of the present invention can be made into a single pharmaceutical product by making PEDF protein and FGF2 protein or SIV-PEDF vector and SIV-FGF2 vector present in the same medium. SIV-PEDF vector When the SIV-FGF2 vector and the SIV-FGF2 vector are used as a single composition, they can be blended within the range in which the doses shown below can be secured. Alternatively, the SIV-PEDF vector and the SIV-FGF2 vector may be prepared as separate compositions and used as a therapeutic kit containing both compositions. The same kit can be used for the PEDF and FGF2 genes and proteins. In the case of using PEDF protein and FGF2 protein as the pharmaceutical of the present invention, the carrier and form are the same as in the case of the vector.
[0038] 本発明の SIV-PEDFおよび SIV-FGF2を含む医薬品を投与する場合は、網膜アポト 一シス変性抑制効果を得られる限り、特に投与経路を問わないが、好ましくは、網膜 下腔投与、硝子体内投与、前房内投与であり、より好ましくは網膜下腔投与である。 本発明の SIV-PEDFおよび SIV-FGF2を含む医薬品の投与量（ヒト 1眼球あたり）は、 例えば、 2.5 X 105TU-2.5 X 108TU、好ましくは、 5.0 X 105TU-5.0 X 107 TUを目安とす ることがでさる。 [0038] In the case of administering a pharmaceutical comprising SIV-PEDF and SIV-FGF2 of the present invention, the administration route is not particularly limited as long as the effect of suppressing retinal apoptosis degeneration is obtained. Intravitreal administration and intra-anterior administration are preferred, and subretinal administration is more preferred. The dose (per human eyeball) of the pharmaceutical agent containing SIV-PEDF and SIV-FGF2 of the present invention is, for example, 2.5 X 10 5 TU-2.5 X 10 8 TU, preferably 5.0 X 10 5 TU-5.0 X 10 7 Use TU as a guide.
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に 組み入れられる。  All prior art documents cited in the present specification are incorporated herein by reference.
実施例  Example
[0039] 以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実 施例に限定されるものではない。  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[0040] [実施例 1]VSV- Gシユードタイプ SIVベクターの構築  [0040] [Example 1] Construction of VSV-G pseudo-type SIV vector
ベクターの構築には図 1に示す 4種のプラスミド（ジーントランスファーベクター、パッ ケージングベクター、 rev発現ベクター、 VSV- G発現ベクター）を用いた。 3種のジー ントランスファーベクター、パッケージングベクター、 rev発現ベクターについては、従 来型のベクタープラスミド（PCT/JP00/03955)を改良して作製した。 VSV-G発現べク ターにつ 、ては従来のものをそのまま用いた。  Four types of plasmids (gene transfer vector, packaging vector, rev expression vector, VSV-G expression vector) shown in FIG. 1 were used for the construction of the vector. Three gene transfer vectors, packaging vectors, and rev expression vectors were prepared by modifying the conventional vector plasmid (PCT / JP00 / 03955). The conventional VSV-G expression vector was used as it was.
[0041] プラスミド作製にあたっては、市販の各種キットを使用した。制限酵素は New Englan d Biolabs社製品を使用し、プラスミド DNAの抽出,精製,回収にはキアゲンのキット（QI Aquick PCR purification kit, QIAquick Nucleotide Removal kit, QIAquick Gel extrac tion kit, Plasmid Maxi kit)を使用した。 PCRには TaKaRaの EX Taq酵素を用い、使用 するプライマーは外部のメーカー（シグマジエノシス'ジャパン）に合成を依頼した。 D NA末端の脱リン酸化は TaKaRaの Alkalline Phosphatase (E.coli C75)を使用した。ライ ゲーシヨンには TaKaRaの DNA Ligation kit ver.2を用い、トランスフォーメーションに は TOYOBOの DH5 a COMPETENT highを利用した。 [0041] Various commercially available kits were used for plasmid preparation. Restriction enzymes are from New England Biolabs, and Qiagen kits (QIquick PCR purification kit, QIAquick Nucleotide Removal kit, QIAquick Gel extraction kit, Plasmid Maxi kit) are used for plasmid DNA extraction, purification, and recovery. did. The PCR used TaKaRa's EX Taq enzyme, and the primer used was commissioned to an external manufacturer (Sigma Dienosis' Japan). D For dephosphorylation of NA terminal, TaKaRa Alkalline Phosphatase (E. coli C75) was used. For ligation, TaKaRa DNA Ligation kit ver.2 was used, and for transformation, TOYOBO DH5 a COMPETENT high was used.
[0042] 1— 1.ジーントランスファーベクターの改良  [0042] 1— 1. Improvement of Gene Transfer Vector
従来型ジーントランスファーベクターに、 cPTT(central polypurine tract)および WPR E、woodcnucK hepatitis virus posttranscnptionai regulatory element)の 入を行 ヽ、 ジーントランスファーベクターの性能改良を図った（図 2A、 B)。使用した従来型のジ ーントランスファーベクターは、非病原性のアフリカミドリザル免疫不全ウィルスのクロ ーンである SIVagmを基本とし、 5，LTR領域、 RRE、 CMV (cytomegalovirus)プロモータ 一、 EGFP (enhanced green fluorescent protein)遺伝子、 3， LTRを順に有するベクタ 一である。該従来型ジーントランスファーベクターは、本発明者らによって構築された ものであり、構築方法等については、既に報告済みである (特許文献 2)。該従来型 ジーントランスファーベクターの塩基配列を、配列番号： 17に示す。  By adding cPTT (central polypurine tract), WPRE, and woodcnucK hepatitis virus posttranscnptionai regulatory element to the conventional gene transfer vector, we improved the performance of the gene transfer vector (Figs. 2A and B). The conventional gene transfer vector used is based on SIVagm, a non-pathogenic African green monkey immunodeficiency virus clone. 5, LTR region, RRE, CMV (cytomegalovirus) promoter, EGFP (enhanced green fluorescent protein) gene, 3, vector with LTR in that order. The conventional gene transfer vector has been constructed by the present inventors, and the construction method and the like have already been reported (Patent Document 2). The base sequence of the conventional gene transfer vector is shown in SEQ ID NO: 17.
[0043] 具体的なベクターの改良方法は以下のとおりである。まず、従来型のジーントランス ファーベクターを制限酵素 Sac IIで切断し、サンプルを泳動して CMVプロモーターと E GFP遺伝子を取り除き、セルフライゲーシヨンを行った。次に、プラスミドの Not I部位 をなくすために、上記ベクターを Not Iで切断し、切断末端を T4 DNAポリメラーゼで平 滑化し、セルフライゲーシヨンを行った。  [0043] A specific method for improving a vector is as follows. First, the conventional gene transfer vector was cleaved with the restriction enzyme Sac II, the sample was electrophoresed to remove the CMV promoter and EGFP gene, and self-ligation was performed. Next, in order to eliminate the Not I site of the plasmid, the vector was cut with Not I, the cut ends were smoothed with T4 DNA polymerase, and self-ligation was performed.
[0044] 続、て、上記ベクターを制限酵素 Sac IIで切断し、 BAP処理を用い、切断末端を脱 リン酸ィ匕した。従来型のジーントランスファーベクターをテンプレートにし、プライマー 1F (配列番号： 18)と 1R (配列番号： 19)で PCRを行い、 PCR産物を Sac IIで切断し、 CMVプロモーター（配列番号： 13)の末端に Sac II部位を付加した断片を作成した。 該 CMVプロモーター断片を、 BAP処理した上記ベクターの Sac II部位に組み込んだ。  [0044] Subsequently, the vector was cleaved with the restriction enzyme Sac II, and the cleaved ends were dephosphorylated using BAP treatment. Perform PCR with primers 1F (SEQ ID NO: 18) and 1R (SEQ ID NO: 19) using a conventional gene transfer vector as a template, cleave the PCR product with Sac II, and terminate the CMV promoter (SEQ ID NO: 13). A fragment in which a Sac II site was added to was prepared. The CMV promoter fragment was incorporated into the Sac II site of the above-mentioned vector treated with BAP.
[0045] ベクターを Not I、 BamH Iで順番に切断し、その切断部位に 2種の合成オリゴ DNA2 F (配列番号： 20)と 2R (配列番号： 21)をァニールして作成したアダプターをライゲー シヨンし、制限酵素部位を改変した。ベクターを制限酵素 Sac IIで切断し、 BAP処理を 用い、切断末端を脱リン酸化した。  [0045] The vector was cleaved in order with Not I and BamH I, and an adapter prepared by annealing two synthetic oligos DNA2 F (SEQ ID NO: 20) and 2R (SEQ ID NO: 21) at the cleavage site was ligated. The restriction enzyme site was modified. The vector was digested with the restriction enzyme Sac II, and the digested end was dephosphorylated using BAP treatment.
[0046] 導入用の cPTT断片（配列番号： 14)を得るために、 SIVagmTYOlゲノム（配列番号： 12)を組み込んだプラスミド pSA212をテンプレートとし、プライマー 3F (配列番号： 22 )と 3R (配列番号： 23)で PCRを行った。該 PCR増幅断片の末端を SAC Πで切断し、 c PPTの両端に SAC II部位を付加した断片を作成した。 cPPT断片を、 BAP処理した上 記ベクターの Sac II部位にライゲーシヨンした。 [0046] In order to obtain a cPTT fragment (SEQ ID NO: 14) for introduction, the SIVagmTYOl genome (SEQ ID NO: Using the plasmid pSA212 incorporating 12) as a template, PCR was performed with primers 3F (SEQ ID NO: 22) and 3R (SEQ ID NO: 23). The end of the PCR amplified fragment was cleaved with SACΠ to prepare a fragment in which SAC II sites were added to both ends of c PPT. The cPPT fragment was ligated to the Sac II site of the above vector treated with BAP.
[0047] ベクターを BamH Iで切断し、 BAP処理を用い、切断末端を脱リン酸ィ匕した。導入す る WPRE断片を得るために、 WPRE cDNA (配列番号： 15)の入ったプラスミドをテンプ レートとし、プライマー 4F (配列番号： 24)と 4R (配列番号： 25)で PCRを行った。得ら れた PCR増幅産物の末端を BamH Iと Bgl IIで切断し、 WPREの末端に制限酵素部位 を付加した断片を作成した。ベクターの BamH I部位に上記 WPRE断片をライゲーショ ンし、搭載遺伝子のない改良型ジーントランスファーベクター（配列番号： 1)を完成さ せた。 [0047] The vector was digested with BamHI, and the digested end was dephosphorylated using BAP treatment. In order to obtain a WPRE fragment to be introduced, a plasmid containing WPRE cDNA (SEQ ID NO: 15) was used as a template, and PCR was performed with primers 4F (SEQ ID NO: 24) and 4R (SEQ ID NO: 25). The ends of the obtained PCR amplification products were cleaved with BamH I and Bgl II to prepare a fragment in which a restriction enzyme site was added to the end of WPRE. The above WPRE fragment was ligated to the BamHI site of the vector to complete an improved gene transfer vector (SEQ ID NO: 1) without any onboard gene.
[0048] 搭載遺伝子断片を作成し、上記の改良型ジーントランスファーベクター Not I部位に 組み込んだ。 EGFP断片は、 EGFP cDNA (配列番号： 26)の入ったプラスミドをテンプ レートとし、プライマー 5F (配列番号： 27)と 5R (配列番号： 28)で PCRを行い、 Not Iで 切断し作成した。 FGF2断片は、 hFGF2 cDNA (配列番号: 8)の入ったプラスミドをテ ンプレートとし、プライマー 6F (配列番号： 29)と 6R (配列番号： 30)で PCRを行い、 No t Iで切断し作成した。 PEDF断片は hPEDF cDNA (配列番号： 7)の入ったプラスミド をテンプレートとし、プライマー 7F (配列番号： 31)と 7R (配列番号： 32)で PCRを行い 、 pGEM-T Easy vector (プロメガ社）へ TAクローユングし、 Not Iで切り出し作成した。  [0048] A mounted gene fragment was prepared and incorporated into the above-mentioned improved gene transfer vector Not I site. The EGFP fragment was prepared by using a plasmid containing EGFP cDNA (SEQ ID NO: 26) as a template, PCR with primers 5F (SEQ ID NO: 27) and 5R (SEQ ID NO: 28), and cleaving with Not I. The FGF2 fragment was prepared by using the plasmid containing hFGF2 cDNA (SEQ ID NO: 8) as a template, PCR with primers 6F (SEQ ID NO: 29) and 6R (SEQ ID NO: 30), and cleaving with Not I. did. The PEDF fragment was templated with a plasmid containing hPEDF cDNA (SEQ ID NO: 7), PCR was performed with primers 7F (SEQ ID NO: 31) and 7R (SEQ ID NO: 32), and transferred to pGEM-T Easy vector (Promega). TA clawed and cut out with Not I.
[0049] また、 cPPT,WPREを組み込んだプラスミドの構築と同時に、 cPPT,WPREの効果を 確認するために、 cPPTのみ、または WPREのみを組み込んだジーントランスファーべ クタ一もそれぞれ作成した。  [0049] At the same time as the construction of the plasmid incorporating cPPT and WPRE, in order to confirm the effect of cPPT and WPRE, a gene transfer vector incorporating only cPPT or only WPRE was also prepared.
[0050] 1 2.パッケージングベクターの改良  [0050] 1 2. Improvement of packaging vector
従来型パッケージングベクターには gag,polの他に、修飾遺伝子と呼ばれる vif,vprと 制御遺伝子の tat,revが含まれている。しかし、修飾遺伝子産物はベクターにおいて 必要でないことが分力り (V.Kim et al.: J.Virol 72:811-816, 1998)、近年は、安全性を 高めるため、修飾遺伝子が削除されたベクターが使用されている。また、 tatも削除さ れ、 revは別のプラスミドに移すことにより更に安全性が高められた、第三世代と呼ば れるベクターが開発されており、現在ではベクターの第三世代化は必須となっているIn addition to gag and pol, conventional packaging vectors include vif and vpr called modified genes, and tat and rev regulatory genes. However, it is a force that modified gene products are not required in vectors (V. Kim et al .: J. Virol 72: 811-816, 1998), and recently, modified genes have been deleted to increase safety. Vector is used. Also, the tat was deleted, and the rev was transferred to another plasmid for further safety. Vectors have been developed, and now the third generation of vectors is essential
。そこで本発明においても、従来型パッケージングベクター（配列番号： 33)力 補助 遺伝子 (vif,vpr,tat)を取り除き、 revを別のプラスミドに移し、安全性を高めることとした (図 3)。方法は、基本的には、 HIVベクターで以前に報告されている (T.Dull.et al.: J. Virol 72:8463-8471, 1998)。 . Therefore, in the present invention, the conventional packaging vector (SEQ ID NO: 33) force assisting gene (vif, vpr, tat) was removed, and rev was transferred to another plasmid to enhance safety (FIG. 3). The method has basically been reported previously with HIV vectors (T. Dull. Et al .: J. Virol 72: 8463-8471, 1998).
[0051] 具体的には、まず、ノ ッケージングベクターのプラスミドを制限酵素 Not Iで切断し、 続いて Ecot22Iで切断した。サンプルを泳動し、 EcoT22ト Not I断片を除去し、サイズ の大きいベクター断片と pol遺伝子の一部の EcoT22ト EcoT22I断片を回収した。  [0051] Specifically, first, the plasmid of the knocking vector was cleaved with the restriction enzyme Not I, and then cleaved with Ecot22I. The sample was electrophoresed to remove the EcoT22 to Not I fragment, and a large vector fragment and a portion of the EcoT22 to EcoT22I fragment of the pol gene were recovered.
[0052] 2種類の合成オリゴ DNA1F (配列番号： 34)と 1R (配列番号： 35)をァニールさせて 作製したアダプターを、上記ベクターの EcoT22ト Not I部位にライゲーシヨンした。続 いて、ベクターを EcoT22Iで切断し、 BAP処理を行い、切断末端を脱リン酸化し、 BAP 処理した EcoT22I部位に、回収して置 、た pol遺伝子の EcoT22I断片を組み込んだ。  [0052] An adapter prepared by annealing two types of synthetic oligos DNA1F (SEQ ID NO: 34) and 1R (SEQ ID NO: 35) was ligated to the EcoT22 to Not I site of the above vector. Subsequently, the vector was cleaved with EcoT22I, treated with BAP, the cleaved end was dephosphorylated, and the EcoT22I fragment of the pol gene was incorporated by recovery at the EcoT22I site treated with BAP.
[0053] 上記ベクターを Not Iで切断し、 BAP処理を行 、、切断末端を脱リン酸ィ匕した。 RRE 断片を得るために、従来型のパッケージングベクター（配列番号： 33)をテンプレート にし、プライマー 8F (配列番号： 36)と 8R (配列番号： 37)で PCRを行い、 pGEM- T Ea sy vector (プロメガ社）へ TAクローユングした。 Not Iで RRE断片を切り出した。脱リン 酸化したベクターの Not I部位へ RRE断片をライゲーシヨンし、改良型パッケージング ベクター (配列番号： 4)を完成させた。  [0053] The above vector was cleaved with Not I, subjected to BAP treatment, and the cleaved ends were dephosphorylated. To obtain an RRE fragment, PCR was performed with primers 8F (SEQ ID NO: 36) and 8R (SEQ ID NO: 37) using a conventional packaging vector (SEQ ID NO: 33) as a template, and pGEM-T Easy vector. TA closed to (Promega). The RRE fragment was cut out with Not I. The RRE fragment was ligated to the Not I site of the dephosphorylated vector to complete the improved packaging vector (SEQ ID NO: 4).
[0054] 1 3. rev発現ベクターの構築  [0054] 1 3. Construction of rev expression vector
rev蛋白はこれまで従来型のパッケージングベクターより供給されて 、たが、ノ ッケ 一ジングベクターの上記改良に伴い、 rev蛋白を別の発現プラスミドの形で供給する こととし、新たに発現ベクターを構築した。 revはゲノム上ではイントロンで 2つに分か れているが、一つに結合して発現プラスミドに組み込むこととした（図 4A、 B)。  The rev protein has been supplied from conventional packaging vectors. However, with the above improvement of the knocking vector, the rev protein will be supplied in the form of a separate expression plasmid. Built. Although rev is divided into two introns on the genome, it was decided to bind to one and incorporate it into the expression plasmid (Fig. 4A, B).
[0055] まず、従来のパッケージングベクターをテンプレートとし、 2つの断片を PCRにより作 製した。 5'側の断片はプライマー 1F (配列番号 : 38)と 1R (配列番号 : 39)を使い、 3' 側の断片はプライマー 2F (配列番号: 40)と 2R (配列番号: 41)を使用して増幅した。  [0055] First, two fragments were prepared by PCR using a conventional packaging vector as a template. The 5 'fragment uses primers 1F (SEQ ID NO: 38) and 1R (SEQ ID NO: 39), and the 3' fragment uses primers 2F (SEQ ID NO: 40) and 2R (SEQ ID NO: 41). Amplified.
2種の PCR断片を回収し、混合して PCRのテンプレートとし、プライマー 1Fと 2Rを用い て増幅し、 2つの断片をつなげた目的の rev遺伝子断片 (配列番号：16)を得た。 PCR で増幅した rev断片を、 pGEM- T Easy vectorへ TAクローユングした。続いて、該べク ターを EcoR Iで切断し、 EcoR I部位が付加された rev断片を回収した。一方、蛋白発 現用の pCIベクター（プロメガ社)を EcoR Iで切断し、切断部位を BAP処理した。回収 した rev断片と pCI発現ベクターをライゲーシヨンし、 rev発現ベクターとした。 Two types of PCR fragments were collected and mixed to form a PCR template, which was amplified using primers 1F and 2R to obtain the desired rev gene fragment (SEQ ID NO: 16) connecting the two fragments. PCR The rev fragment amplified in step 1 was TA cloned into pGEM-T Easy vector. Subsequently, the vector was cleaved with EcoR I, and the rev fragment with the EcoR I site added was recovered. On the other hand, the pCI vector for protein expression (Promega) was cleaved with EcoRI and the cleavage site was BAP-treated. The recovered rev fragment and the pCI expression vector were ligated to obtain a rev expression vector.
[0056] [実施例 2] cPPT,WPREを搭載した SIVベクターの機能評価 [0056] [Example 2] Functional evaluation of SIV vector loaded with cPPT and WPRE
cPPT,WPREの導入の効果を調べるために、 cPPT,WPRE同時搭載の他に、 cPPT単 独搭載、 WPRE単独搭載のベクターを生産し、従来型のコントロールと比較した。全 てのジーントランスファーベクターは EGFPを搭載して!/、るものを用いた。パッケージン グベクターは従来型 (配列番号： 33)を使用した。 In order to investigate the effects of the introduction of cPPT and WPRE, in addition to cPPT and WPRE being installed together, we produced vectors with cPPT alone and WPRE alone, and compared them with conventional controls. All gene transfer vectors loaded with EGFP! / Were used. The conventional packaging vector (SEQ ID NO: 33 ) was used.
[0057] 2— 1. SIVベクターの調製  [0057] 2— 1. Preparation of SIV vector
ヒト胎児腎細胞由来細胞株 293T細胞を 15cmプラスチックシャーレ 1枚あたり約 1 X 1 07 (翌日 70-80%密度）になるように播種し、 10%ゥシ胎児血清を含む D-MEM培地（G ibco BRL) 20mlで 24時間培養した。 24時間培養後、培地を 10mlの OPTI-MEM培地（ Gibco BRL)に置換して、細胞をトランスフエタトに用いた。 It was seeded so that the human fetal kidney cell-derived cell line 293T cells 15cm plastic Petri dish one per about 1 X 1 0 7 (day 70-80% density), D-MEM medium containing 10% © Shi calf serum ( (Gibco BRL) was cultured in 20 ml for 24 hours. After culturing for 24 hours, the medium was replaced with 10 ml of OPTI-MEM medium (Gibco BRL), and the cells were used for transfer.
[0058] シャーレ 1枚あたりジーントランスファーベクター 6 μ g、パッケージングベクター 3 μ g 、 VSV- G発現ベクター 1 μ gを 1.5mlの OPTI- MEM培地に溶解後、 40 μ 1の PLUS Reag ent (インビトロジェン社)を加えて撹拌し、 15分間室温で静置した。ジーントランスファ 一ベクターは、 cppT,WPRE同時搭載、 cPPT単独搭載、 WPRE単独搭載、または従来 型（cPPT,WPRE 、ずれも搭載して 、な 、）のものを使用した。これに 1.5mlの OPTI-M EM培地で希釈した 60 μ 1の LIPOFECTAMINE Reagent (インビトロジェン社）を加え撹 拌し、 15分間室温で静置した。 [0058] After 6 µg of gene transfer vector, 3 µg of packaging vector, and 1 µg of VSV-G expression vector were dissolved in 1.5 ml of OPTI-MEM medium per dish, 40 µ1 of PLUS Reag ent (Invitrogen) The mixture was stirred and allowed to stand at room temperature for 15 minutes. The gene transfer vector used was c pp T and WPRE mounted simultaneously, cPPT mounted alone, WPRE mounted alone, or a conventional type (cPPT, WPRE mounted with misalignment). To this, 60 μl of LIPOFECTAMINE Reagent (Invitrogen) diluted with 1.5 ml of OPTI-MEM medium was added, stirred and allowed to stand at room temperature for 15 minutes.
[0059] 上記 DNA複合体を 15cmシャーレの細胞に滴下し、穏やかに振とうさせて混ぜ、 37 °C,5%COインキュベーターで 3時間インキュベートした。インキュベート後、シャーレ  [0059] The DNA complex was added dropwise to a 15 cm petri dish, gently shaken and mixed, and incubated in a 37 ° C, 5% CO incubator for 3 hours. Petri dish after incubation
2  2
に 13mlの 20%ゥシ胎児血清を含む D- MEM培地をカ卩えて培養した。トランスフエタトの 翌日に、新しい 10%ゥシ胎児血清を含む D-MEM培地 30mlと交換し培養した。トラン スフエタトの 2日後に、上清を回収し、 0.45 μ mのフィルターで濾過し、ベクター液とし た。  Then, D-MEM medium containing 13 ml of 20% urine fetal serum was added and cultured. The next day after the transfectate, the medium was replaced with 30 ml of D-MEM medium containing fresh 10% urine fetal serum and cultured. Two days after transfer, the supernatant was collected and filtered through a 0.45 μm filter to obtain a vector solution.
[0060] 2- 2. SIVベクターの力価測定 SIVベクターの力価には、搭載遺伝子蛋白の発現細胞数から算出する機能力価 (F unctional titer： TU/ml)とベクター粒子数から算出する値（Particle titer： particles/ ml)がある。 cPPT,WPREの性能評価は Partilce titerをそろえて細胞に感染させて評 価するため、以下のようにドットブロッテイングによる方法で Particle titerを測定した。 [0060] 2- 2. Determination of titer of SIV vector The titer of the SIV vector includes a functional titer (Functional titer: TU / ml) calculated from the number of cells expressing the mounted gene protein and a value (Particle titer: particles / ml) calculated from the number of vector particles. In order to evaluate the performance of cPPT and WPRE by infecting cells with a partilce titer, the particle titer was measured by the dot blotting method as follows.
[0061] まず、上記のとおり生産したベクター液から、市販のキット（キアゲン社の QIAamp Vi ral RNA mini kit)を用いて RNA抽出を行った。次に、スロットブロッターを用いて Hybo ndN+メンブレン（アマシャム社）に RNAをブロッテイングした。同時に検量線用のモル 数を計算したプラスミド DNAもブロッテイングした。なお、 RNAの処理方法はメンブレン 付属のプロトコールに従った。 DNAは加熱急冷した。メンブレンをアルカリ固定した後 、ハイブリダィゼーシヨンを行った。ハイブリダィズに関してはロシュの DIGラベルでの 検出システムを使用した。プローブは DIGラベル NTPを用いて作製し、ハイブリダィゼ ーシヨン以降の操作は DIG Easy Hyb, DIG Wash and Block Buffer Set (ロシュ）を使 用した。 anti- DIG AP conjugate antibody (ロシュ）および CSPD (ロシュ）を用い、化学 発光させ、ルミノイメージアナライザー (富士写真フィルム： LAS-1000)を用いて、シグ ナルを検出'定量した。  [0061] First, RNA was extracted from the vector solution produced as described above using a commercially available kit (QIAamp Viral RNA mini kit manufactured by Qiagen). Next, RNA was blotted onto Hybond N + membrane (Amersham) using a slot blotter. At the same time, plasmid DNA for which the number of moles for the calibration curve was calculated was blotted. The RNA treatment method followed the protocol attached to the membrane. DNA was heated and cooled rapidly. After the membrane was fixed with alkali, hybridization was performed. For hybridization, we used a Roche DIG label detection system. The probe was prepared using DIG-labeled NTP, and DIG Easy Hyb, DIG Wash and Block Buffer Set (Roche) was used for the subsequent operations after hybridization. Anti-DIG AP conjugate antibody (Roche) and CSPD (Roche) were used for chemiluminescence, and the signal was detected and quantified using a lumino image analyzer (Fuji Photo Film: LAS-1000).
[0062] 2- 3. SIVベクターの細胞への遺伝子導入、評価  [0062] 2- 3. Introduction and evaluation of SIV vector into cells
Particle titerを測定した 4種類のベクターは、以下のように MOI(multiplicity of infect ion)を変えて細胞に感染させ、 FACS解析を行った。 293T細胞を 6ゥエルのプラスチッ クカルチャープレートに 1ゥエルあたり I X 106個で播き、 37°C、 5%COでー晚培養し Four types of vectors that measured particle titer were infected with cells by changing MOI (multiplicity of infect ion) as follows, and FACS analysis was performed. 293T cells are seeded on a 6-well plastic culture plate at 6 IX 10 per well and cultured at 37 ° C, 5% CO.
2  2
た。翌日、プレート 1ゥエルの細胞数を血球計数板で計算し、プレートの培地を除き、 新しい 10%ゥシ胎児血清を含む D- MEM培地 2mlで希釈したベクターを MOI(Particle s/cell)が 0.3、 1.5、 7.5、 15になるようにカ卩えた。感染 1日後、細胞の培地を 2mlの新し いものと交換した。感染 2日後、ベクターにより遺伝子導入された EGFPを蛍光顕微鏡 で観察し、 EGFP陽性細胞の割合を測定し、更に蛍光強度 (EGFP蛋白量の目安とな る値)も測定した。  It was. On the next day, count the number of cells in plate 1 well with a hemocytometer, remove the plate medium, and dilute the vector diluted with 2 ml of D-MEM medium containing fresh 10% urine fetal serum with a MOI (Particles / cell) of 0.3. , 1.5, 7.5, 15 One day after infection, the cell culture medium was replaced with 2 ml fresh. Two days after infection, EGFP gene-transfected with the vector was observed with a fluorescence microscope, the proportion of EGFP positive cells was measured, and the fluorescence intensity (a value that is a measure of EGFP protein amount) was also measured.
[0063] 2-4.ベクター機能評価の結果 [0063] 2-4. Results of vector function evaluation
従来型のジーントランスファーベクターをコントロールとして、 cPPT単独搭載、 WPRE 単独搭載、 cPPT, WPRE同時搭載の 4種類のベクターを生産した。ベクターデザイン の模式図を図 5-(a)に示す。 Using the conventional gene transfer vector as a control, we produced four types of vectors: cPPT alone, WPRE alone, cPPT, and WPRE simultaneously. Vector design A schematic diagram of is shown in Fig. 5- (a).
[0064] 生産したベクターの Particle titerを測定したところ、 4種ともベクター粒子の生産性 には違いが見られな力つた。ベクター粒子数で ΜΟΙ (1個の細胞に感染させたベクタ 一粒子数)を統一して 293T細胞に遺伝子導入し、蛍光顕微鏡で観察したところ、 MO 1:15では図 5- (b)に示すように、 cPPTと WPREのな!/、従来型のコントロール（-cPPT,-W PRE)では EGFP陽性細胞の数が少なく蛍光も弱カゝつた。 cPPT単独搭載 (+cPPT,-WP RE)では EGFP陽性細胞の数が増加した。 WPRE単独搭載（_cPPT,+WPRE)では EGF P陽性細胞の数はコントロールに比べわずかな増加しか認められなかったものの、 EG FP蛋白の蛍光は増強された。 cPPT,WPRE同時搭載（+cPPT,+WPRE)では 2つの因 子が相乗的に効果を発揮し、細胞数，蛍光強度の両方とも cPPT,WPRE単独搭載の ものより大幅に増加し、期待以上の結果となった。  [0064] When the particle titer of the produced vector was measured, all four types showed no difference in vector particle productivity. The number of vector particles was the same as the number of vector particles (the number of particles per vector infected with one cell), and the gene was introduced into 293T cells and observed with a fluorescence microscope. MO 1:15 shows that in Figure 5- (b) As shown in the figure, cPPT and WPRE! /, And conventional controls (-cPPT, -WPRE) had few EGFP positive cells and weak fluorescence. The number of EGFP positive cells increased with cPPT alone (+ cPPT, -WP RE). With WPRE alone (_cPPT, + WPRE), the number of EGF P-positive cells was only slightly increased compared to the control, but the fluorescence of EG FP protein was enhanced. When cPPT and WPRE are installed at the same time (+ cPPT and + WPRE), the two factors have a synergistic effect, and both the number of cells and fluorescence intensity are significantly increased compared to those equipped with cPPT and WPRE alone. As a result.
[0065] EGFP陽性細胞の割合を FACSで調べたところ（図 6)、 V、ずれも MOI依存的に遺伝 子挿入割合が増加した力 cPPT, WPRE同時搭載のものはコントロールに比べて約 1 0倍も導入効率が上昇した。つまり実質的な機能力価 (生産性)が 10倍上昇した。  [0065] When the percentage of EGFP positive cells was examined by FACS (Fig. 6), V, the difference was the MOI-dependent force that increased the gene insertion rate. The one with cPPT and WPRE was about 10 compared to the control. The introduction efficiency increased twice as much. In other words, the actual functional titer (productivity) increased 10 times.
[0066] また、 EGFP陽性細胞の平均蛍光強度を調べたところ（図 7)、 cPPT,WPRE同時搭 載のものは WPRE単独搭載のものよりかなり高くなつており、細胞当たりの蛋白発現量 も大幅に増大している事が明らかとなった。  [0066] In addition, when the average fluorescence intensity of EGFP positive cells was examined (Fig. 7), those with cPPT and WPRE were significantly higher than those with WPRE alone, and the protein expression level per cell was also significantly higher. It became clear that it was increasing.
[0067] [実施例 3]治療用遺伝子搭載 SIVベクターの大量調製および濃縮  [0067] [Example 3] Mass preparation and concentration of therapeutic gene-loaded SIV vectors
図 1に示す改良型のジーントランスファーベクター、パッケージングベクター、 rev発 現ベクター、および VSV-G発現ベクターの 4種類のプラスミドをもとに以下のように SIV ベクターを調製した。 PEDFおよび FGF2の治療遺伝子を搭載したベクターは、それぞ れ 15cmシャーレ 20枚単位で生産を行った。  Based on the four types of plasmids shown in Fig. 1, the modified gene transfer vector, packaging vector, rev expression vector, and VSV-G expression vector, SIV vectors were prepared as follows. Vectors loaded with PEDF and FGF2 therapeutic genes were produced in units of 20 15 cm dishes.
[0068] 293T細胞を 15cmプラスチックシャーレ 1枚あたり約 1 X 107 (翌日 70- 80%密度）にな るように播き、 10%ゥシ胎児血清を含む D-MEM培地 20mlで 24時間培養した。 24時間 培養後、培地を 10mlの OPTI-MEM培地に置換してトランスフエタトに用いた。シヤー レ 1枚あたりジーントランスファーベクター 10 μ g、パッケージングベクター 5 μ g、 rev 発現ベクター 2 μ g、 VSV- G発現ベクター 2 μ gを 1.5mlの ΟΡΤΙ- MEM培地に溶解後、 40 1の PLUS Reagent (インビトロジェン社）をカ卩えて撹拌し、 15分間室温で静置した 。これに 1.5mlの OPTI- MEM培地で希釈した 60 1の LIPOFECTAMINE Reagentをカロ え撹拌し、 15分間室温で静置した。この DNA複合体を、上記の 15cmシャーレの細胞 に滴下し、穏やかに振とうさせて混ぜ、 37°C,5%COインキュベーターで 3時間インキ [0068] 293T cells were seeded at about 1 X 10 7 (70-80% density the next day) per 15 cm plastic petri dish, and cultured for 24 hours in 20 ml of D-MEM medium containing 10% ushi fetal serum. . After culturing for 24 hours, the medium was replaced with 10 ml of OPTI-MEM medium and used for transfer. 40 μL of gene transfer vector after 10 μg of gene transfer vector, 5 μg of packaging vector, 2 μg of rev expression vector, and 2 μg of VSV-G expression vector in 1.5 ml of MEM-MEM medium Reagent (Invitrogen) was stirred and stirred for 15 minutes at room temperature . To this, 60 1 LIPOFECTAMINE Reagent diluted with 1.5 ml of OPTI-MEM medium was stirred and stirred for 15 minutes at room temperature. This DNA complex is dropped into the above 15cm Petri dish cells, gently shaken and mixed, and inked in a 37 ° C, 5% CO incubator for 3 hours.
2  2
ュペートした。上記シャーレに 13mlの 20%ゥシ胎児血清を含む D- MEM培地を加えて 口 しプ 。  I'm sorry. Add 13 ml of D-MEM medium containing 20% urine fetal serum to the petri dish.
[0069] トランスフエタトの翌日に、新し!/、10%ゥシ胎児血清を含む D- MEM培地 30mlと交換 し培養した。トランスフエタトの 2日後に、上清を回収し、新しい培地を 20mlカ卩えた。回 収した上清は 0.45 μ mのフィルターで濾過し、 4°Cで保存した。トランスフエタトの 3日 後に上清を回収し、 0.45 μ mのフィルターで濾過し、前日の回収ベクターと合わせ、 高速遠心機を利用して濃縮操作を行った。回収したベクター液を滅菌処理したチュ ーブに分注し、 42500G,4°C,1時間遠心した。この遠心操作を 2回繰り返し、ベクター 液を 500倍 1000倍に濃縮した。ベクターはペレットとして沈殿する力 ペレットは 5% ゥシ胎児血清を含む PBSに溶解した。濃縮したベクターは少量ずつ分注したのち- 80 °Cで保存し、一部を用いて Particle titerを測定した。 Particle titerの測定は前述の方 法と同様に行った。  [0069] On the day after the transfectate, the medium was replaced with 30 ml of D-MEM medium containing fresh! /, 10% ushi fetal serum, and cultured. Two days after transfer, the supernatant was collected and 20 ml of fresh medium was added. The collected supernatant was filtered through a 0.45 μm filter and stored at 4 ° C. The supernatant was collected 3 days after the transfectate, filtered through a 0.45 μm filter, combined with the collection vector from the previous day, and concentrated using a high-speed centrifuge. The collected vector solution was dispensed into a sterilized tube and centrifuged at 42500 G at 4 ° C for 1 hour. This centrifugation operation was repeated twice to concentrate the vector solution 500 times to 1000 times. Ability to precipitate the vector as a pellet. The pellet was dissolved in PBS containing 5% urine fetal serum. The concentrated vector was dispensed in small portions and stored at -80 ° C, and a portion of the vector was measured for particle titer. Particle titer was measured in the same manner as described above.
[0070] [実施例 4]SIV-hPEDF, SIV-hFGF2同時投与における視細胞変性抑制効果の検討 網膜色素変性は難治性遺伝性疾患で、現在有効な治療法が存在しない。本発明 者らは SIVベクターの小動物における網膜への導入特性'長期安全性試験、さらに疾 患モデル動物（RCSラット）を対象とした SIV-PEDF投与による効果判定試験（Gene th erapy 10, 1503-1511, 2003)を行い良好な結果を得ている。神経栄養因子 PEDF, F GF2は、標的細胞が異なると考えられている。これら 2種類の神経栄養因子の同時投 与の神経保護効果について、検討した。  [Example 4] Examination of inhibitory effect on photoreceptor cell degeneration by simultaneous administration of SIV-hPEDF and SIV-hFGF2 Retinitis pigmentosa is an intractable genetic disease, and there is no effective treatment at present. The present inventors have introduced characteristics of SIV vectors into the retina in small animals' long-term safety test, and further, an effect determination test using SIV-PEDF in disease model animals (RCS rats) (Gene th erapy 10, 1503- 1511, 2003) with good results. Neurotrophic factors PEDF and FGF2 are thought to have different target cells. We examined the neuroprotective effects of simultaneous administration of these two types of neurotrophic factors.
[0071] SIV- hFGF2および SIV- hPEDFは、上述のとおり構築したものを用いた。  [0071] SIV-hFGF2 and SIV-hPEDF constructed as described above were used.
SIV- hPEDF、 SIV- hFGF2、 SIV- EGFP (コントロール）の各ベクター溶液を、ベクター 濃度: total 2.5 X 107TU/mlとして作製した。 RCSラット 3週齢を、 SIV-hPEDF単独投 与群、 SIV- hFGF2単独投与群、 SIV- hPEDFおよび SIV- hFGF2同時投与群、 SIV- EG FP投与群の 4群に分け、網膜下腔に投与した。上記ベクターの網膜下投与により、 P EDFおよび FGF2遺伝子は網膜色素上皮細胞に導入され、そこ力 神経保護因子が 分泌されて神経細胞に作用する。 Each vector solution of SIV-hPEDF, SIV-hFGF2, and SIV-EGFP (control) was prepared at a vector concentration: total 2.5 × 10 7 TU / ml. RCS rats 3 weeks old are divided into 4 groups: SIV-hPEDF single administration group, SIV-hFGF2 single administration group, SIV-hPEDF and SIV-hFGF2 simultaneous administration group, SIV-EG FP administration group, and it is administered into the subretinal space did. By subretinal administration of the above vector, PEDF and FGF2 genes are introduced into retinal pigment epithelial cells, It is secreted and acts on nerve cells.
[0072] ベクター導入 4週間後、各動物群を観察し、ベクター投与の影響を検討した。 PEDF および FGF2が作用する視細胞、内顆粒層等の神経細胞の層および網膜色素上皮 細胞は網膜内に存在する。しかし、網膜のみを採取するのは困難である。そこで、ラ ット後眼部を採取した。後眼部には、網膜'脈絡膜'強膜の 3つの層が含まれる。採取 した後眼部の hPEDF, hFGF2蛋白量を、各々 ELISA法により測定した。ベクター導入 4週間後の眼球後眼部において、 hPEDFの発現は SIV-hPEDF投与群で高い傾向と なることが示された。また、 hFGF2の発現は SIV-hFGF2単独投与群および同時投与 群で高い傾向を示し、 SIV-hPEDF, SIV- hFGF2ベクター投与による hPEDFおよび hF GF2遺伝子発現が確認された（図 8)。  [0072] Four weeks after introduction of the vector, each group of animals was observed to examine the effects of vector administration. The photoreceptor cells on which PEDF and FGF2 act, the layer of nerve cells such as the inner granule layer, and the retinal pigment epithelial cells exist in the retina. However, it is difficult to collect only the retina. Therefore, the eye part was collected after the rat. The posterior segment contains three layers: the retina 'choroid' sclera. After collection, the amounts of hPEDF and hFGF2 proteins in the eye were measured by ELISA. In the posterior segment of the eyeball 4 weeks after introduction of the vector, hPEDF expression was shown to be higher in the SIV-hPEDF administration group. In addition, hFGF2 expression tended to be high in the SIV-hFGF2 single administration group and the co-administration group, and hPEDF and hF GF2 gene expression was confirmed by SIV-hPEDF and SIV-hFGF2 vector administration (FIG. 8).
[0073] 次に、ベクター導入 4週間後、 8週間後および 12週間後において、最大割面標本を 作製し、網膜 10ケ所 (A1- A10)における 100 m当りの視細胞核数を計測した。 SIV- h PEDF, SIV-hFGF2各単独群、及び同時投与群では、ベクター注入部位に近い 3箇 所 (A1-A3)の網膜組織において、有意に視細胞が残存しており、同時投与群では、 単独群より高い神経保護効果が認められた (図 9)。また、ベクター導入 8週間後およ び 12週間後においても、 SIV-hPEDFおよび SIV-hFGF2の同時投与による顕著な神 経保護効果が認められた（図 10および図 11)。  [0073] Next, at the 4th, 8th, and 12th weeks after the introduction of the vector, the maximum cut surface specimens were prepared, and the number of photoreceptor nuclei per 100 m was measured at 10 retinas (A1-A10). In the SIV-h PEDF and SIV-hFGF2 single groups and the co-administered group, photoreceptor cells remained significantly in three retinal tissues (A1-A3) near the vector injection site. A higher neuroprotective effect was observed than the single group (Fig. 9). In addition, at 8 and 12 weeks after vector introduction, remarkable neuroprotective effects were observed by simultaneous administration of SIV-hPEDF and SIV-hFGF2 (Figs. 10 and 11).
[0074] さらに、機能的評価として電気生理学的検討 (網膜電図: ERG)を行った。網膜電図 は光刺激に対する網膜電位の変化を記録する検査法であり、網膜が機能している場 合には高い振幅が得られる。 a波は視細胞に由来し、 b波は主としてミュラー細胞と双 極細胞に由来する。 SIV-hPEDF, SIV-hFGF2の両単独投与群において、 SIV-EGFP 群に比較して、 a, b波ともに有意に高い振幅結果が得られた。さらに同時投与群では 、 a,b波共により高い振幅が得られ、同時投与群でより高い神経保護効果が得られて V、ることが確認できた（図 12および図 13)。  Furthermore, an electrophysiological study (electroretinogram: ERG) was performed as a functional evaluation. The electroretinogram is a test method that records changes in the retinal potential in response to light stimulation. A high amplitude is obtained when the retina is functioning. The a wave is derived from photoreceptor cells, and the b wave is derived mainly from Müller cells and bipolar cells. In the SIV-hPEDF and SIV-hFGF2 single administration groups, significantly higher amplitude results were obtained for both a and b waves than the SIV-EGFP group. Furthermore, in the co-administration group, a higher amplitude was obtained for both the a and b waves, and it was confirmed that a higher neuroprotective effect was obtained in the co-administration group and V (FIGS. 12 and 13).
[0075] なお、追加実験群にお!、て組織学的検討を行ったところ、 SIV-hPEDF, SIV- hFGF 2の同時投与による明らかな炎症、増殖、及び血管新生所見は認められなかった (デ ータ示さず)。  [0075] In addition, when the histological examination was conducted in the additional experimental group, no clear inflammation, proliferation, and angiogenesis were observed by the simultaneous administration of SIV-hPEDF and SIV-hFGF 2 ( (Data not shown).
産業上の利用可能性 本発明によって、網膜色素変性の新規治療方法が提供された。本発明の PEDFお よび FGF2の同時投与は従来の網膜色素変性治療方法よりも顕著に高い効果を期 待することができる。特に、 SIV-PEDFベクターおよび SIV-FGF2ベクターを用いた投 与方法は、患者の細胞内における PEDFおよび FGF2持続的提供を可能とし、患者に 対する侵襲頻度、経済的コストの点にぉ 、ても極めて優れた治療法であると 、うこと が証明された。 Industrial applicability According to the present invention, a novel method for treating retinal pigment degeneration has been provided. The simultaneous administration of PEDF and FGF2 of the present invention can be expected to have a significantly higher effect than conventional methods for treating retinal pigment degeneration. In particular, the administration method using the SIV-PEDF vector and the SIV-FGF2 vector makes it possible to provide PEDF and FGF2 continuously in the patient's cells, and in terms of the frequency of invasiveness to the patient and the economic cost. It proved to be a very good treatment.

Claims

請求の範囲 The scope of the claims
[1] 下記 (a)カゝら (d)の 、ずれかを薬学的に許容される媒体とともに含む、眼組織細胞に おけるアポトーシス変性を伴う疾患の治療用医薬品。  [1] A medicinal product for treating a disease associated with apoptotic degeneration in ocular tissue cells, comprising any of the following (a) and others (d) together with a pharmaceutically acceptable medium.
(a)色素上皮由来因子（Pigment epithelium derived factor : PEDF)遺伝子および線 維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)遺伝子  (a) Pigment epithelium derived factor (PEDF) gene and fibroblast growth factor 2 (FGF2) gene
(b)色素上皮由来因子（Pigment epithelium derived factor : PEDF)タンパク質および 線維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)タンパク質  (b) Pigment epithelium derived factor (PEDF) protein and fibroblast growth factor 2 (FGF2) protein
(c)色素上皮由来因子（Pigment epithelium derived factor: PEDF)遺伝子および線 維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)タンパク質  (c) Pigment epithelium derived factor (PEDF) gene and fibroblast growth factor 2 (FGF2) protein
(d)色素上皮由来因子（Pigment epithelium derived factor : PEDF)タンパク質および 線維芽細胞成長因子 2 (fibroblast growth factor 2： FGF2)遺伝子  (d) Pigment epithelium derived factor (PEDF) protein and fibroblast growth factor 2 (FGF2) gene
[2] PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを 含む医薬品であって、該 PEDF遺伝子および FGF2遺伝子力 別々の組換えサル免 疫不全ウィルスベクターに保持されるカゝ、または 1つの組換えサル免疫不全ウィルス ベクターに保持される、請求項 1に記載の医薬品。  [2] A pharmaceutical comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene, wherein the PEDF gene and FGF2 gene force are held in separate recombinant monkey immunodeficiency virus vectors, or The medicament according to claim 1, which is carried by one recombinant simian immunodeficiency virus vector.
[3] PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターおよび FGF2遺伝子を 保持する組換えサル免疫不全ウィルスベクターを含む、請求項 2に記載の医薬品。 [3] The medicament according to claim 2, comprising a recombinant simian immunodeficiency virus vector retaining a PEDF gene and a recombinant simian immunodeficiency virus vector retaining an FGF2 gene.
[4] PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを 含む、請求項 2に記載の医薬品。 [4] The pharmaceutical product according to claim 2, comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene.
[5] サル免疫不全ウィルスベクター力 ScPPT配列および Zまたは WPRE配列を含む、請求 項 2から 4のいずれかに記載の医薬品。 [5] The simian immunodeficiency virus vector force The pharmaceutical product according to any one of claims 2 to 4, comprising a ScPPT sequence and a Z or WPRE sequence.
[6] サル免疫不全ウィルスベクターが VSV-Gでシユードタイプ化されている、請求項 2か ら 5のいずれかに記載の医薬品。 [6] The pharmaceutical product according to any one of claims 2 to 5, wherein the simian immunodeficiency virus vector is pseudotyped with VSV-G.
[7] サル免疫不全ウィルスベクター力 gm株由来である、請求項 2から 6のいずれかに記 載の医薬品。 [7] The pharmaceutical product according to any one of claims 2 to 6, which is derived from a simian immunodeficiency virus vector gm strain.
[8] PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターを含む組成物、およ び FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを含む組成物を含 む、眼組織細胞におけるアポトーシス変性を伴う疾患の治療用キット。 [8] Reducing apoptotic degeneration in ocular tissue cells, including a composition comprising a recombinant simian immunodeficiency virus vector carrying the PEDF gene and a composition comprising a recombinant simian immunodeficiency virus vector carrying the FGF2 gene Kit for treatment of accompanying diseases.
[9] PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを 含む組成物を含む、眼組織細胞におけるアポトーシス変性を伴う疾患の治療用キット [9] A kit for treating diseases associated with apoptotic degeneration in ocular tissue cells, comprising a composition comprising a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene
[10] 眼組織細胞におけるアポトーシス変性を伴う疾患が、網膜色素変性、緑内障、網膜 剥離、網膜虚血性疾患のいずれかである、請求項 1から 7のいずれかに記載の医薬 品または請求項 8もしくは 9に記載のキット。 [10] The pharmaceutical product according to any one of claims 1 to 7, or the disease according to any one of claims 1 to 7, wherein the disease associated with apoptotic degeneration in ocular tissue cells is any one of retinal pigment degeneration, glaucoma, retinal detachment, and retinal ischemic disease. Or the kit according to 9.
[11] PEDFおよび FGF2あるいはそれらをコードする遺伝子を投与する、眼組織細胞にお けるアポトーシス変性を伴う疾患の治療方法。 [11] A method for treating a disease associated with apoptotic degeneration in ocular tissue cells, comprising administering PEDF and FGF2 or a gene encoding them.
[12] PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターおよび FGF2遺伝子を 保持する組換えサル免疫不全ウィルスベクターを投与する、請求項 11に記載の治 療方法。 [12] The treatment method according to claim 11, wherein a recombinant simian immunodeficiency virus vector retaining a PEDF gene and a recombinant simian immunodeficiency virus vector retaining an FGF2 gene are administered.
[13] PEDF遺伝子を保持する組換えサル免疫不全ウィルスベクターおよび FGF2遺伝子を 保持する組換えサル免疫不全ウィルスベクターを網膜下腔に投与する、請求項 12 に記載の治療方法。  [13] The method according to claim 12, wherein a recombinant simian immunodeficiency virus vector retaining a PEDF gene and a recombinant simian immunodeficiency virus vector retaining an FGF2 gene are administered into the subretinal space.
[14] PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを 投与する、請求項 11に記載の治療方法。  [14] The treatment method according to [11], wherein a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene is administered.
[15] PEDF遺伝子および FGF2遺伝子を保持する組換えサル免疫不全ウィルスベクターを 網膜下腔に投与する、請求項 14に記載の治療方法。 [15] The method according to claim 14, wherein a recombinant simian immunodeficiency virus vector carrying a PEDF gene and an FGF2 gene is administered into the subretinal space.
[16] 配列番号： 1に記載の塩基配列に PEDF遺伝子が挿入された塩基配列を含むジーン トランスファーベクターを用いて PEDF遺伝子を保持する組換えサル免疫不全ウィル スベクターを調製する工程を含む、眼組織細胞におけるアポトーシス変性を伴う疾患 の治療用医薬品の製造方法。 [16] An eye comprising a step of preparing a recombinant monkey immunodeficiency virus vector retaining a PEDF gene using a gene transfer vector comprising a nucleotide sequence in which a PEDF gene is inserted into the nucleotide sequence of SEQ ID NO: 1. A method for producing a pharmaceutical product for treating a disease associated with apoptotic degeneration in tissue cells.
[17] 配列番号： 2に記載の塩基配列を含むジーントランスファーベクターを用いて PEDF遺 伝子を保持する組換えサル免疫不全ウィルスベクターを調製する工程を含む、請求 項 16に記載の方法。 [17] The method according to claim 16, comprising the step of preparing a recombinant simian immunodeficiency virus vector carrying a PEDF gene using a gene transfer vector comprising the nucleotide sequence of SEQ ID NO: 2.
[18] 配列番号： 1に記載の塩基配列に FGF2遺伝子が挿入された塩基配列を含むジーン トランスファーベクターを用いて FGF2遺伝子を保持する組換えサル免疫不全ウィル スベクターを調製する工程を含む、眼組織細胞におけるアポトーシス変性を伴う疾患 の治療用医薬品の製造方法。 [18] An eye comprising a step of preparing a recombinant monkey immunodeficiency virus vector retaining the FGF2 gene using a gene transfer vector comprising a nucleotide sequence in which the FGF2 gene is inserted into the nucleotide sequence of SEQ ID NO: 1. Diseases with apoptotic degeneration in tissue cells Method for the preparation of therapeutic drugs.
[19] 配列番号： 3に記載の塩基配列を含むジーントランスファーベクターを用いて FGF2遺 伝子を保持する組換えサル免疫不全ウィルスベクターを調製する工程を含む、請求 項 18に記載の方法。  [19] The method according to claim 18, comprising the step of preparing a recombinant simian immunodeficiency virus vector carrying the FGF2 gene using a gene transfer vector comprising the nucleotide sequence of SEQ ID NO: 3.
[20] 配列番号： 1に記載の塩基配列に PEDF遺伝子および FGF2遺伝子が挿入された塩 基配列を含むジーントランスファーベクターを用いて PEDF遺伝子および FGF2遺伝 子を保持する組換えサル免疫不全ウィルスベクターを調製する工程を含む、眼組織 細胞におけるアポトーシス変性を伴う疾患の治療用医薬品の製造方法。  [20] A recombinant simian immunodeficiency virus vector carrying the PEDF gene and the FGF2 gene using a gene transfer vector containing a base sequence in which the PEDF gene and the FGF2 gene are inserted into the base sequence of SEQ ID NO: 1. A method for producing a pharmaceutical product for treating a disease associated with apoptotic degeneration in an ocular tissue cell, comprising a preparing step.
[21] 配列番号: 4に記載の塩基配列を含むパッケージングベクターが導入されたパッケ一 ジング細胞に、該ジーントランスファーベクターを導入する工程を含む、請求項 16か ら 20の!、ずれかに記載の方法。  [21] The method according to any one of claims 16 to 20, further comprising the step of introducing the gene transfer vector into a packaging cell into which the packaging vector comprising the base sequence of SEQ ID NO: 4 has been introduced. The method described.
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