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WO2007111333A1 - Procede de reduction des dommages des cellules lors de la transfection d'acide nucleique - Google Patents

Procede de reduction des dommages des cellules lors de la transfection d'acide nucleique Download PDF

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Publication number
WO2007111333A1
WO2007111333A1 PCT/JP2007/056393 JP2007056393W WO2007111333A1 WO 2007111333 A1 WO2007111333 A1 WO 2007111333A1 JP 2007056393 W JP2007056393 W JP 2007056393W WO 2007111333 A1 WO2007111333 A1 WO 2007111333A1
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WIPO (PCT)
Prior art keywords
nucleic acid
cell damage
cells
introduction
acidic polysaccharide
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Ceased
Application number
PCT/JP2007/056393
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English (en)
Japanese (ja)
Inventor
Kenji Masuda
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Toyobo Co Ltd
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Toyobo Co Ltd
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Filing date
Publication date
Application filed by Toyobo Co Ltd filed Critical Toyobo Co Ltd
Publication of WO2007111333A1 publication Critical patent/WO2007111333A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin

Definitions

  • the present invention relates to a method for reducing cell damage that does not hinder introduction into a cell when a nucleic acid is introduced. More specifically, the present invention relates to a method for achieving both efficient introduction of nucleic acid and reduction of cell damage by adding acidic polysaccharide at an appropriate time after introduction of nucleic acid.
  • nucleic acid introduction reagents are now commercially available and widely used. These methods are also considered to be suitable for in vivo use because they are highly safe, can be easily manufactured on a large scale, and can be easily used.
  • the disadvantages of these techniques include cell damage, cells that are difficult to introduce nucleic acids, and low reproducibility of experiments. Among these, the most prominent are It is a cellular disorder.
  • Cell damage is affected by factors such as the type and amount of nucleic acid introduction reagent, the mixing ratio of DNA and nucleic acid introduction reagent, the type of cells, the number of cells at the time of nucleic acid introduction, and the state of the cells.
  • the introduction rate of nucleic acids into cells is also affected by these factors, and when the above conditions are adjusted to reduce damage to cells, a reduction in introduction rate is often observed.
  • Non-patent document 1 Life Sciences 75, 2203-2216 (2004)
  • FIG. 2 A graph showing cell viability and gene transfer rate when examining the timing of addition of parin to lower molecular weight after transfection with Lipofectamine 2000 for COS-7 cells. The view of the figure is the same as in Figure 1.
  • an object of the present invention is to provide a method for reducing cell damage that hinders gene transfer when a nucleic acid is introduced into an animal cell.
  • the present inventors have also found that cell damage is reduced by using compounds other than heparin having a low molecular weight such as fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, etc. It was.
  • the present invention has the following constitutional power.
  • a method for reducing cell damage when a nucleic acid is introduced into an animal cell using a nucleic acid introduction (transfusion) reagent characterized in that an acidic polysaccharide component is added to the medium after the introduction of the nucleic acid. How to reduce obstacles.
  • the acidic polysaccharide is one or more selected from the group consisting of heno ⁇ phosphorus, heparan sulfate, chondroitin sulfate, fucoidan, dextran sulfate, and keratan sulfate.
  • a reagent or kit for reducing cell damage comprising an acidic polysaccharide used in the method according to claim 1 as a component.
  • nucleic acid it has become possible to reduce cell damage that appears at the time of nucleic acid introduction without hindering introduction of nucleic acid into cells.
  • nucleic acid can be introduced into animal cells simply, easily and with good reproducibility.
  • cytotoxicity means cell death caused by addition of a nucleic acid introduction reagent, or cell shape change.
  • This cell damage can be evaluated by the MTT method, LDH method, etc. after nucleic acid introduction.
  • MTT method tetrazolium salts such as MTT, XTT, MTS, and WST are used, and these substances are reduced by mitochondrial dehydrogenase in living cells to utilize formazan.
  • the number of viable cells can be calculated by measuring the absorbance of the generated formazan.
  • the LDH method evaluates cell damage by measuring lactate dehydrogenase released from dead cell force damaged by cell membranes.
  • the present invention is a method for reducing cell damage, which comprises adding an acidic polysaccharide component after introduction of a nucleic acid.
  • the acidic polysaccharide component is preferably dissolved in a koffer and added as an aqueous solution.
  • the buffer for dissolving the acidic polysaccharide component is not particularly limited, and examples thereof include a HEPES buffer and a phosphate buffer.
  • the so-called acidic polysaccharide having an acidic residue is used as the polysaccharide added after transfection.
  • Preferred are those having a sulfate group, such as heparin, heparan sulfate, chondroitin sulfate, fucoidan, dextran sulfate, and keratan sulfate. More preferably, it contains a plurality of sulfate groups per two sugar molecules, and is not particularly limited, and examples thereof include heparin, heparan sulfate, fucoidan, and dextran sulfate. More preferably, the acidic polysaccharide is derived from a living body, and examples thereof include heparin and hexanelan sulfate.
  • the acidic polysaccharide may be reduced in molecular weight.
  • low molecular weight heparin or low molecular weight fucoidan may be used.
  • the term “reducing molecular weight” as used herein means degrading acidic polysaccharides chemically or enzymatically.
  • the low molecular weight heparin and low molecular weight fucoidan used are preferably those having a molecular weight of 2,000 to 8, OOODa, and more preferably a molecular weight of 4,000 to 6, OOODa.
  • the acidic polysaccharide in the present invention is not limited in the type of force considered to be supplied as various salt compounds.
  • sodium salt, ammonium salt, lithium salt, calcium salt can be used.
  • the time until the addition of acidic polysaccharide is longer than 2 hours.
  • a more preferred lower limit is 3 hours, more preferably 4 hours, and a more preferred upper limit is 12 hours, more preferably 10 hours. This makes it possible to reduce cell damage without lowering the gene introduction rate.
  • the medium may be replaced after the introduction of the nucleic acid, and the nucleic acid introduction reagent may be removed, and then the acidic polysaccharide component may be added.
  • the medium replacement timing is preferably immediately before the addition of the acidic polysaccharide component. In this case, a further effect of reducing cell damage may be obtained as compared with the case of simply adding an acidic polysaccharide component.
  • the method for introducing a nucleic acid into a cell is not particularly limited! How to introduce nucleic acid Examples of the method include a calcium phosphate method, a DEAE-dextran method, and a ribofusion method.
  • a reagent for introducing nucleic acid a force using one kind of polymer, polyethyleneimine, and Lipofectamine 2000 mainly composed of lipids.
  • the scope of the present invention is not limited to these. Therefore, it is possible to use a reagent based on other lipids or other polymers, or a misaligned reagent based on calcium phosphate or DEAE-dextran! /.
  • Non-Patent Document 1 only discloses the result that cell damage can be reduced when plasmid DNA is introduced.
  • RNA and siRNA introduced by plasmid DNA alone are introduced, cell damage can be reduced by adding acidic polysaccharide.
  • the type of cells used is not limited.
  • COS-7 cells derived from the African green monkey kidney
  • HeLa cells derived from human eclampsia cancer
  • the present invention further extends to reagents and kits for reducing cell damage comprising an acidic polysaccharide as a component used in the method of the present invention. This is because it is expected that by supplying a reagent containing the method of the present invention, a number of users can easily perform nucleic acid introduction experiments with good reproducibility.
  • a form in which a parin solution is supplied to 0.1 to: LOmgZmL of heparin solution or a low molecular weight container is conceivable.
  • LOmgZmL of heparin solution or a low molecular weight container Use it in combination with a commercially available or separately prepared transfection reagent.
  • a nuclear acid is introduced using a transfection reagent, and after 2 hours (not including 2 hours), a non-heine solution or a low-molecular-weight non-heine solution is added to the medium. In this way, cell damage can be prevented.
  • set the above low molecular weight hetero solution and transfection reagent What was made into is also contained in the reagent or kit of this invention.
  • a set of nucleic acids to be introduced into cells is also included in the reagent or kit of the present invention.
  • Example 1 Effects of timing of addition of parin to low molecular weight silkworms on cell viability and gene transfer rate
  • COS-7 cells were seeded on a 12 well plate at 1.0 X 10 5 cells / well. Transfection was performed using polyethyleneimine (25 kDa, manufactured by Aldrich) or Lipofectamine 2000 (manufactured by Invitrogen).
  • transfection was performed according to the following procedure.
  • PQBI25 manufactured by Quantum Biotechnologies
  • polyethyleneimine 3. O ⁇ g were mixed in serum-free D-MEM and allowed to stand at room temperature for 20 minutes. The medium was replaced with serum-free D-MEM in advance, and the prepared plasmid DNA / polyethyleneimine complex solution was added to each well and incubated in a 37 ° C, 5% CO incubator.
  • Lipofectamine 2000 transfection was performed according to the following procedure. PQBI25 1. 0; z g and Lipofectamine2000 2.5 ⁇ L were mixed in Opti-MEM I Reduced Serum Medium (manufactured by Invitrogen) and allowed to stand at room temperature for 20 minutes (see Lipofectamine2000 instruction manual). To each well, add the prepared plasmid DNA and Lipofectamine 2000 complex solution, and incubate at 37 ° C and 5% CO.
  • FIGS. 1 and 2 show the results shown in FIGS. 1 and 2 .
  • Figures 1 and 2 show the cell viability and gene transfer rate when transfection was performed using polyethyleneimine and Lipofectamine 2000, respectively, and the timing of the addition of parin to low molecular weight was examined. Regardless of which nucleic acid transfection reagent is used, if the low molecular weight is added to the low molecular weight 2 hours after transfection, a sufficient effect of reducing cell damage can be seen! / A drop in the gene transfer rate was also observed. On the other hand, when 4 hours after transfection, the addition of low molecular weight nitrogen does not significantly reduce the rate of gene transfer, and this condition reduces cell damage and increases efficiency. It was found that both nucleic acid introductions were feasible.
  • Figure 1 focuses on the timing of the addition of parin to low molecular weight ⁇ , and its influence on cell viability and gene transfer rate.
  • Polyethyleneimine used in Non-Patent Document 1 25 The results of studies on COS-7 cells using kDa, Aldrich) as a nucleic acid transfer reagent are shown.
  • Fig. 2 shows a typical nucleic acid transfer reagent, Lipofectamine 2000 (Invitrogen), and COS-7 cells were subjected to transfection, and the cell viability of the addition of parin to low molecular weight was The results of examining the effect on the gene transfer rate are shown below.
  • nucleic acid introduction reagent to be used is not limited to polyethyleneimine, and even when Lipofectamine 2000 is used, cell damage can be reduced by reducing the molecular weight and adding paris.
  • Example 2 Effect of addition of various acidic polysaccharides on cell viability and gene transfer rate
  • the cells were seeded at 1.5 ⁇ 10 5 cells / well.
  • the transformation was performed using Lipofectamine 2000 (Invitrogen) according to the following procedure.
  • pQBI2 5 manufactured by Quantum Biotechnologies
  • pQBI2 5 manufactured by Quantum Biotechnologies
  • g and Lipofectamine2000 2.5 ⁇ L3 ⁇ 4 Opti-MEM I Reduced Serum Medium manufactured by Invitrogen
  • were mixed and allowed to stand at room temperature for 20 minutes see the Lipofectamine2000 instruction manual.
  • To each well add the prepared plasmid DNA and Lipofectamine 2000 complex solution and add 37 ° C, 5% CO
  • acidic polysaccharides low molecular weight ⁇ henoline, fucoidan, hemolin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, and hyaluronic acid.
  • tetrazolium salt WST-8 For cell damage after transformation, use a commercially available kit containing the tetrazolium salt WST-8. Used and evaluated. Specifically, add 100 L of viable cell count measurement reagent (SF strength tester) to each well, incubate at 37 ° C for 20 minutes, and then use a microplate reader and refer to the absorbance at 650 nm. Absorbance at 450 nm was measured. Based on the obtained absorbance, the survival rate of non-transfected cells was defined as 100%, and the cell survival rate after transfection was calculated. Next, the gene transfer rate was measured by FACS (fluorescence activated cell sorting) analysis. That is, cells 2 days after transfection were treated with trypsin, suspended in PBS, used as samples, and FACSCalibur (Betaton Dickinson) was used to calculate the gene transfer rate based on the presence or absence of GFP expression.
  • FACS fluorescence activated cell sorting
  • Table 1 shows the relationship between the characteristics of the examined acidic polysaccharides and the intensity of the effect of reducing cell damage.
  • Table 1 shows the results of studying COS-7 cells and HeLa cells using Lipofectamine 2000 and adding various compounds after 6 hours.
  • COS In both 7 cells and HeLa cells, acidic polysaccharides such as low molecular weight heparin, fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B reduce cell damage.
  • acidic polysaccharides such as low molecular weight heparin, fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B reduce cell damage.
  • hyaluronic acid was not able to confirm the reduction of cell damage.
  • 4 types of low molecular weight ⁇ henoline, fucoidan, heparin, and dextran sulfate containing multiple sulfate groups (preferably 1.5 to 4 molecules) per 2 sugar molecules can significantly reduce cell damage. It was possible.
  • the present inventors can reduce cell damage by using compounds other than heparin, such as fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, etc. I found. [0031] [Table 1]
  • Example 1 siRNA was used instead of plasmid DNA, and the effect of parin on lowering the molecular weight was examined.
  • nucleic acid of the present invention By the method for reducing cell damage at the time of introduction of nucleic acid of the present invention, it is possible to reduce damage to cells without preventing efficient introduction of nucleic acid. It can be used in basic fields such as gene product function analysis, protein expression, gene expression suppression, and transcriptional regulatory region analysis, and can be used in gene therapy and other application fields, making a significant contribution to industry. .

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Abstract

Le problème à résoudre dans le cadre de la présente invention consiste à proposer un procédé de réduction des dommages des cellules sans interférer la transfection de gène lors du transfert d'un acide nucléique dans une cellule animale. La solution proposée est un procédé de réduction des dommages des cellules lors du transfert d'un acide nucléique dans une cellule animale en utilisant un réactif de transfection d'acide nucléique, caractérisé par le fait de comprendre l'addition d'un composant polysaccharide acide dans le milieu juste après la transfection d'acide nucléique.
PCT/JP2007/056393 2006-03-29 2007-03-27 Procede de reduction des dommages des cellules lors de la transfection d'acide nucleique Ceased WO2007111333A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2006090308 2006-03-29
JP2006-090308 2006-03-29
JP2006-090307 2006-03-29
JP2006090307 2006-03-29

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WO2007111333A1 true WO2007111333A1 (fr) 2007-10-04

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120177594A1 (en) * 2009-07-22 2012-07-12 Kazunori Kataoka Polyion complex comprising phd2 expression inhibiting substance
CN116159145A (zh) * 2023-01-31 2023-05-26 四川大学 含七叶皂苷和/或其盐化合物的转染复合物在促转染的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074385A1 (fr) * 2000-04-03 2001-10-11 New York Blood Center, Inc. Proteines angiogeniques onchocerca volvulus et leurs utilisations
WO2004050078A1 (fr) * 2002-12-05 2004-06-17 Takara Bio Inc. Remede
JP2005533016A (ja) * 2002-05-03 2005-11-04 エフエムシー バイオポリマー エイエス 非ウイルス性遺伝子送達システム

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001074385A1 (fr) * 2000-04-03 2001-10-11 New York Blood Center, Inc. Proteines angiogeniques onchocerca volvulus et leurs utilisations
JP2005533016A (ja) * 2002-05-03 2005-11-04 エフエムシー バイオポリマー エイエス 非ウイルス性遺伝子送達システム
WO2004050078A1 (fr) * 2002-12-05 2004-06-17 Takara Bio Inc. Remede

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DRAGOMIR A. ET AL.: "Heparin can improve the viability of transfected cystic fibrosis cell lines in vitro", LIFE SCI., vol. 75, no. 18, 2004, pages 2203 - 2216, XP004533196 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120177594A1 (en) * 2009-07-22 2012-07-12 Kazunori Kataoka Polyion complex comprising phd2 expression inhibiting substance
US8791086B2 (en) * 2009-07-22 2014-07-29 The University Of Tokyo Polyion complex comprising PHD2 expression inhibiting substance
CN116159145A (zh) * 2023-01-31 2023-05-26 四川大学 含七叶皂苷和/或其盐化合物的转染复合物在促转染的应用

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