WO2016129656A1 - Agent pour améliorer l'efficacité du transfert de gène exogène à des cellules de mammifère - Google Patents

Agent pour améliorer l'efficacité du transfert de gène exogène à des cellules de mammifère Download PDF

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Publication number
WO2016129656A1
WO2016129656A1 PCT/JP2016/054042 JP2016054042W WO2016129656A1 WO 2016129656 A1 WO2016129656 A1 WO 2016129656A1 JP 2016054042 W JP2016054042 W JP 2016054042W WO 2016129656 A1 WO2016129656 A1 WO 2016129656A1
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peptide
tyrosine kinase
kinase activity
foreign gene
improving
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PCT/JP2016/054042
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English (en)
Japanese (ja)
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浩史 冨田
江里子 菅野
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国立大学法人岩手大学
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Priority to JP2016574847A priority Critical patent/JP6730705B2/ja
Publication of WO2016129656A1 publication Critical patent/WO2016129656A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to an agent for improving the efficiency of introducing a foreign gene into a mammalian cell.
  • AAV adeno-associated virus
  • the present inventors have energetically conducted research on therapeutic methods for visual impairment, and as a result, in gene therapy using AAV vectors, epithelium expressed in iris pigment epithelial cells as target cells.
  • EGFR growth factor receptor
  • tyrphostin which is a low molecular weight organic compound that inhibits kinase activity
  • Non-patent Document 1 tyrphostin is known to be cytotoxic at low concentrations (eg, 10 ⁇ M in in vitro experiments).
  • hydroxyurea used as an anticancer agent is also known to have an effect of improving the efficiency of introduction of a foreign gene when a foreign gene is introduced into a mammalian cell using an AAV vector. Its action is extremely weak.
  • an object of the present invention is to provide a novel agent for improving the efficiency of introducing a foreign gene, which is used when a foreign gene is introduced into a mammalian cell using an AAV vector.
  • a binding peptide of a peptide having membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR is applied to mammalian cells using an AAV vector. It has been found that when introducing a foreign gene, it is useful as an active ingredient of an agent for improving the efficiency of introducing a foreign gene.
  • An agent for improving the efficiency of introducing a foreign gene used when introducing a foreign gene into a mammalian cell using the AAV vector of the present invention made in view of the above points is, as described in claim 1, a membrane permeation agent A binding peptide of a peptide having sex and a peptide that inhibits the tyrosine kinase activity of EGFR is used as an active ingredient.
  • the foreign gene introduction efficiency improving agent according to claim 2 is the foreign gene introduction efficiency improving agent according to claim 1, wherein the mammalian cell is a retinal cell.
  • the foreign gene introduction efficiency improving agent according to claim 3 is the foreign gene introduction efficiency improving agent according to claim 1, wherein the foreign gene is a photosensitive cation selective channel gene.
  • the method for improving the efficiency of introducing a foreign gene into a mammalian cell comprises, as described in claim 4, a membrane permeability when the foreign gene is introduced into a mammalian cell using an AAV vector in vitro. And a peptide that inhibits the tyrosine kinase activity of EGFR.
  • the present invention also relates to a binding peptide comprising a peptide having membrane permeability and a peptide that inhibits tyrosine kinase activity of EGFR as described in claim 5.
  • the method for introducing a foreign gene into a mammalian cell using an AAV vector in vitro comprises a peptide having membrane permeability and a peptide inhibiting the tyrosine kinase activity of EGFR as described in claim 6. It is performed in the presence of a binding peptide.
  • a peptide having membrane permeability and an EGFR tyrosine kinase are used as active ingredients of an agent for improving the efficiency of introduction of a foreign gene used when a foreign gene is introduced into a mammalian cell using an AAV vector. Binding peptides of peptides that inhibit activity can be provided.
  • Example 2 is a graph showing the effect of various substances in Example 1 on the expression efficiency of the mCherry gene. It is a graph which shows the effect
  • FIG. 1 is a graph showing the effect of various substances in Example 1 on the expression efficiency of the mCherry gene. It is a graph which shows the effect
  • the agent for improving the efficiency of introducing a foreign gene used when a foreign gene is introduced into a mammalian cell using the AAV vector of the present invention is a peptide having a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR.
  • the binding peptide is an active ingredient.
  • the peptide having membrane permeability in the present invention is a peptide having a permeability to the tyrosine kinase activity of EGFR, and the peptide having membrane permeability is a known amino acid sequence having an action of passing through the cell membrane into the cytoplasm.
  • YGRKKRRRRR SEQ ID NO: 1 derived from HIV-1 Tat
  • RRRRNRTRRRNRRRVR SEQ ID NO: 2
  • TRQARNRRRRRWRRQR SEQ ID NO: 3
  • RAMVRRAAARNRNRTAR SEQ ID NO: 4
  • TRRQRTRRRARNR SEQ ID NO: 5
  • HTLV-II Rex HTLV-II Rex, etc. Mentioned are (if necessary e.g., Shiro Niki, proteins nucleic acid enzymes, see Vol.47 No.11 (2002) 1415-1419). These amino acid sequences may have 1 to 5 amino acids deleted, substituted or added as long as they have membrane permeability.
  • the peptide that inhibits EGFR tyrosine kinase activity may have a known amino acid sequence, for example, DEYLI (SEQ ID NO: 6) derived from Y992 which is an autophosphorylation site of EGFR, VPEYINQ (SEQ ID NO: 7) derived from Y1068, DYQQD (SEQ ID NO: 8) derived from Y1148, ENAEYLR derived from Y1173 (SEQ ID NO: 9) (See, for example, Mineo Abe, et al., British Journal of Pharmacology, 2006; 147: 402-411) if necessary.
  • These amino acid sequences may be those in which 1 to 5 amino acids are deleted, substituted or added as long as they inhibit the EGFR tyrosine kinase activity.
  • the peptide having a membrane permeability and the peptide that inhibits EGFR tyrosine kinase activity may be bound to the C-terminus of the peptide having membrane permeability by binding a peptide that inhibits EGFR tyrosine kinase activity.
  • a peptide having membrane permeability may be bound to the C-terminus of the peptide that inhibits the tyrosine kinase activity of EGFR.
  • Specific examples include peptides in which VPYINQ derived from Y1068 and DYQQD derived from Y1148 are bound to the C-terminus of YGRKKRRQRRR derived from HIV-1 Tat, and RENEYLR derived from Y1173 at the C-terminus of RRRRNRTRRRNRRRVR derived from FHV coat. Examples thereof include a bound peptide and a peptide in which YGRKKRRQRRR derived from HIV-1 Tat is bound to the C-terminus of VPEYINQ derived from Y1068.
  • binding peptide of the peptide having membrane permeability in the present invention and the peptide that inhibits the tyrosine kinase activity of EGFR can be chemically synthesized using a peptide synthesizer, but may be prepared by a genetic engineering technique.
  • the binding peptide of the peptide having membrane permeability in the present invention and the peptide that inhibits the tyrosine kinase activity of EGFR maintains the stability when a foreign gene is introduced into a mammalian cell using an AAV vector, etc.
  • the N-terminus may be acetylated or the C-terminus may be amidated.
  • the binding peptide of the peptide having membrane permeability and the peptide inhibiting the tyrosine kinase activity of EGFR is, for example, a water-soluble powder. Therefore, when introducing a foreign gene into a mammalian cell in vitro using an AAV vector, the cell is infected with the virus after being dissolved in the cell culture medium and incubated at 37 ° C. for 1 to 24 hours, for example. Thus, the efficiency of introducing foreign genes can be improved.
  • a peptide-binding peptide of the present invention and a peptide that inhibits the tyrosine kinase activity of EGFR are dissolved in an AAV vector solution incorporating a foreign gene and then administered into the body.
  • the efficiency of introducing a foreign gene can be improved.
  • the binding amount of the peptide having membrane permeability and the peptide that inhibits the tyrosine kinase activity of EGFR is dissolved in a cell culture solution or in a solution of an AAV vector incorporating a foreign gene, for example, 0. What is necessary is just to set suitably so that it may become the range of 1 micromol-10M.
  • the mammalian cells may be cells of various sites such as humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats and dogs.
  • the foreign gene may be a gene that exerts a therapeutic effect on a disease, for example, by being introduced into a target cell.
  • mammalian cells include mammalian retinal cells (such as neurons, glial cells, and pigment epithelial cells that constitute the retina).
  • foreign genes for photosensitive cation selective channels such as the photoreceptor channel rhodopsin that play an important role in visual function.
  • the binding peptide of the peptide possessing and the peptide that inhibits the tyrosine kinase activity of EGFR contributes to the restoration of more effective visual function by the AAV vector incorporating a smaller amount of the photosensitive cation selective channel gene.
  • Example 1 Cynomolgus monkey-derived fibroblast cell line CYNOM-K1 was seeded in a 96-well plate at 1 ⁇ 10 4 cells each, and incubated at 37 ° C. in MEM medium containing 10% FBS for 1 day.
  • the cells were washed twice with MEM medium containing 2% FBS, and then AAV-pmCherry (1 ⁇ 10 12 to 13 particles / particulate / 1 ⁇ 10 12-13 particle / mL) 47.5 ⁇ L of MEM medium containing 2.5 ⁇ L + 2% FBS was added as a virus solution and incubated for 2 hours at 37 ° C., and then 50 ⁇ L of MEM medium containing 18% FBS was added. After 1 day, the medium was changed and further cultured for 3 days.
  • AAV-pmCherry (1 ⁇ 10 12 to 13 particles / particulate / 1 ⁇ 10 12-13 particle / mL) 47.5 ⁇ L of MEM medium containing 2.5 ⁇ L + 2% FBS was added as a virus solution and incubated for 2 hours at 37 ° C., and then 50 ⁇ L of MEM medium containing 18% FBS was added. After 1 day, the medium was changed and further cultured for 3 days.
  • a Hoechst 33342 solution as a nuclear stain was added to 5 ⁇ g / mL, incubated at room temperature for 30 minutes, and a cell photograph was taken with a fluorescence microscope. From the photograph taken, the expression efficiency of the mCherry gene was calculated as the ratio of the number of cells stained red with mCherry to the total number of cells stained blue with Hoechst 33342. The results are shown in FIG. 1 (relative values using the untreated group as a control). As is apparent from FIG.
  • the binding peptide of the test substance which has a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR, increases the expression efficiency by improving the efficiency of introducing the mCherry gene in a concentration-dependent manner. Improved and its action was comparable to tyrphostin. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
  • Example 2 Similar to Example 1 except that various concentrations of Acetyl-YGRKKRRQRRRDYQQD-CONH 2 (SEQ ID NO: 11: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) were used as test substances.
  • SEQ ID NO: 11 water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8.
  • FIG. 2 the effect on the expression efficiency of the mCherry gene was evaluated. The results are shown in FIG. As apparent from FIG. 2, the effect of improving the expression efficiency of the mCherry gene of the test substance was comparable to that of tyrphostin. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
  • Example 3 Similar to Example 1 except that various concentrations of Acetyl-VPEYINQYGRKKRRRRR-CONH 2 (SEQ ID NO: 12: water-soluble white powder by chemical synthesis using Shimadzu's peptide synthesizer PSSM-8) were used as test substances. Then, when the effect on the expression efficiency of the mCherry gene was evaluated, the effect of improving the expression efficiency of the mCherry gene comparable to tyrphostin was shown. In addition, this test substance was less toxic than tyrphostin (for example, even 1 mM does not show cytotoxicity in in vitro experiments).
  • Example 4 Tomita Hiroshi, et sl. Mol Ther. , 2014; 22 (8): 1434-1440, and the following experiment was conducted.
  • a modified channelrhodopsin (mVChR1) gene having a basic structure of channel rhodopsin (VChR1) derived from green algae Volbox was incorporated into an AAV vector, and an AAV vector solution (2.5 ⁇ 10 11 particles / mL) containing mVChR1 gene was 5 ⁇ L.
  • Visual evoked potentials were measured 2 months after administration. Specifically, an LED light source is placed in front of the eyes of a rat that has undergone an implantation operation of a recording electrode in the brain visual cortex one week before the measurement, and 10 ms of light stimulation is performed 200 times at 1 second intervals. The associated changes in the potential of the visual cortex were recorded. The results are shown in FIG. As is clear from FIG. 3, the binding peptide of the test substance, which has a membrane permeability and a peptide that inhibits the tyrosine kinase activity of EGFR, improves the expression efficiency by improving the efficiency of mVChR1 gene introduction into retinal cells. Improved and increased the amplitude of the visual evoked potential (the main cells to be transfected are ganglion cells).
  • the present invention has an industrial applicability in that it can provide a novel improver of the efficiency of introducing a foreign gene used when a foreign gene is introduced into a mammalian cell using an AAV vector. Have.

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Abstract

Le problème que cherche à résoudre la présente invention est de proposer un nouvel agent pour améliorer l'efficacité d'un transfert de gène exogène, destiné à être utilisé lors du transfert de gènes exogènes à des cellules de mammifère au moyen d'un vecteur viral adéno-associé. Cet agent destiné à améliorer le transfert de gène exogène et ainsi fournir une solution au problème mentionné ci-dessus consiste en un principe actif sous la forme d'un peptide lié comprenant un peptide capable de traverser une membrane et un peptide capable d'inhiber l'activité tyrosine kinase des récepteurs du facteur de croissance épidermique. Le peptide lié comprenant un peptide capable de traverser une membrane et un peptide capable d'inhiber l'activité tyrosine kinase des récepteurs du facteur de croissance épidermique selon la présente invention contribue, par exemple, à une récupération plus efficace de la fonction visuelle grâce un vecteur viral adéno-associé contenant une plus petite quantité d'un gène d'un canal à cations sensible à la lumière.
PCT/JP2016/054042 2015-02-12 2016-02-11 Agent pour améliorer l'efficacité du transfert de gène exogène à des cellules de mammifère WO2016129656A1 (fr)

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Citations (1)

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JP7142369B2 (ja) 2022-09-27
JP2020191872A (ja) 2020-12-03
JP6730705B2 (ja) 2020-07-29
JPWO2016129656A1 (ja) 2017-11-24

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