WO2006090697A1 - Agent thérapeutique pour maladie dégénérative apoptotique dans le tissu cellulaire de l’oeil contenant pedf et fgf2 - Google Patents

Agent thérapeutique pour maladie dégénérative apoptotique dans le tissu cellulaire de l’oeil contenant pedf et fgf2 Download PDF

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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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English (en)
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/zh
Priority to JP2007504722A priority patent/JP4971974B2/ja
Publication of WO2006090697A1 publication Critical patent/WO2006090697A1/fr
Priority to HK08111109.7A priority patent/HK1115324A1/xx

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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

L’invention entend proposer un nouveau procédé thérapeutique pour une maladie dégénérative apoptotique dans les tissus cellulaires de l’œil comme la dégénération du pigment de la rétine. Les efforts se sont portés sur l’administration concomitante de deux facteurs neurotrophiques : facteur dérivé de l’épithélium du pigment (PEDF) et facteur 2 de croissance de fibroblaste (FGF2). Les sites des actions sont considérés comme différents entre PEDF et FGF2. Un vecteur SIV-PEDF et un vecteur SIV-FGF2 ont été construits et administrés à l’espace sub-rétinien d’un rat RCS, qui est un modèle de maladie dégénérative du pigment de la rétine, avant d’en évaluer les effets. Au bout de 4 semaines, de 8 semaines et de 12 semaines après l’administration des vecteurs, on a pu observer un effet protecteur visuel des cellules beaucoup plus élevé dans un groupe avec administration concomitante que dans un groupe avec une seule administration. De plus, l’évaluation fonctionnelle de la rétine au moyen d’un électrorétinogramme a révélé que l’effet dans le groupe avec administration concomitante était notablement plus élevé que dans le groupe avec une seule administration. Il ressort des résultats ci-dessus pour la première fois que l’administration concomitante de PEDF et FGF2 a des effets supérieurs dans le traitement de la dégénération du pigment de la rétine que les procédés conventionnels.
PCT/JP2006/303052 2005-02-23 2006-02-21 Agent thérapeutique pour maladie dégénérative apoptotique dans le tissu cellulaire de l’oeil contenant pedf et fgf2 WO2006090697A1 (fr)

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CN2006800128827A CN101160139B (zh) 2005-02-23 2006-02-21 含有pedf以及fgf2的伴随眼组织细胞凋亡变性的疾患的治疗药物
JP2007504722A JP4971974B2 (ja) 2005-02-23 2006-02-21 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
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