+

WO1995034647A1 - Compositions et procedes d'amelioration de l'apport d'acides nucleiques a des cellules - Google Patents

Compositions et procedes d'amelioration de l'apport d'acides nucleiques a des cellules Download PDF

Info

Publication number
WO1995034647A1
WO1995034647A1 PCT/US1995/007543 US9507543W WO9534647A1 WO 1995034647 A1 WO1995034647 A1 WO 1995034647A1 US 9507543 W US9507543 W US 9507543W WO 9534647 A1 WO9534647 A1 WO 9534647A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
nucleic acid
complex
peptide
enhancing
Prior art date
Application number
PCT/US1995/007543
Other languages
English (en)
Inventor
Jon T. Conary
Kenneth L. Brigham
Original Assignee
Vanderbilt University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vanderbilt University filed Critical Vanderbilt University
Priority to AU27736/95A priority Critical patent/AU2773695A/en
Publication of WO1995034647A1 publication Critical patent/WO1995034647A1/fr

Links

Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • 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

Definitions

  • This present invention relates to delivery of exogenous nucleic acids into cells.
  • this invention provides a means to achieve enhanced transport of transfected nucleic acids into intracellular compartments, such as the nucleus or the mitochondria.
  • DNA charge associate with cationic substances has been documented for a number of substances including liposomes (Brigham, K.L. et al, Amer. J. Respir. Cell andMole. Biol. 8:209-213 (1993)) and glycoprotein complexes containing poly-L-lysine (Wilson, J.M. et al, J. Biol Chem. 267: 11483- 11489 (1992)). DNA charge interactions have also been employed to purify DNA (Richards, E.J., in Current Protocols in Molecular Biology, Ausubel, F.M. et al, eds. pp. 2.13.1-2.13.3 (1993)).
  • the successful delivery of an exogenous gene either to eukaryotic cells in culture (in vitro gene transfer) or to cells in a living organism (in vivo gene transfer, gene therapy) can be viewed as a two-step process.
  • the delivered gene must traverse the cell membrane (go from outside to inside the cell) and second, the delivered gene must cross the nuclear or mitochondrial membrane.
  • viral based vectors e.g., retroviruses, adenoviruses, and adeno-associated viruses
  • the present invention provides a very different, simple, highly efficient approach to transferring nucleic acids across intracellular membranes to overcome the problems in the art.
  • the present invention provides a purified complex comprising a nucleic acid bound to a nuclear localization peptide.
  • the complex can further comprise a liposome and or asialorosomucoid.
  • the present invention further provides a purified complex comprising a nucleic acid bound to a mitochondrial localization peptide.
  • the instant invention also provides a purified complex comprising a liposome, a nucleic acid and a nuclear or mitochondrial localization peptide.
  • the instant invention also provides a method of enhancing expression of a transfected nucleic acid in a eukaryotic cell comprising administering to the cell a purified complex comprising a nucleic acid bound to a nuclear localization peptide, thereby enhancing expression of the transfected nucleic acid in the cell.
  • the instant invention also provides a method of enhancing delivery of a nucleic acid to the nucleus of a cell, comprising administering to the cell a purified complex comprising a nucleic acid bound to a nuclear localization peptide, thereby enhancing the delivery of the nucleic acid to the nucleus of the cell.
  • the instant invention also provides a method of enhancing expression of a transfected nucleic acid in a eukaryotic cell in a subject comprising administering to the subject a purified complex comprising a nucleic acid bound to a nuclear localization peptide, thereby enhancing expression of the transfected nucleic acid in the cell.
  • the present invention additionally provides a method of enhancing delivery of a nucleic acid to a mitochondrion of a cell, comprising administering to the cell a complex comprising the nucleic acid bound to a mitochondrial localization peptide, thereby enhancing the delivery of the nucleic acid to the mitochondrion of the cell.
  • Also provided is a method of enhancing expression of a transfected nucleic acid in a cell in a plant comprising administering to the plant a complex comprising the nucleic acid bound to a plant nuclear localization peptide, thereby enhancing expression of the nucleic acid in the cell in the plant.
  • a synthetic intracellular localization peptide is used herein as part of a complex that consists of: 1) the synthetic intracellular localization peptide (also herein referred to as a "transport peptide”); and 2) a nucleic acid.
  • the complex can also include a liposome.
  • the general purpose of this invention is to enhance effective delivery of transfected genes into eukaryotic cells by providing a mechanism for those genes to reach and enter the cell nucleus or mitochondria. For example, increasing the percentage of transfected nucleic acids that enters the nucleus can increase expression of the transfected gene.
  • This invention includes the use of a protein-derived, nuclear or mitochondrial localization sequence as a delivery mechanism for a nucleic acid and the use of this delivery mechanism to increase the effectiveness of gene transfer by increasing the amount of a transfected gene that reaches the cell nucleus or mitochondria.
  • nucleic acid enhancing or expression of a nucleic acid is meant that the amount of the nucleic acid delivered or the amount of expression is increased relative to the amount of delivery or expression achieved by the same method without utilizing the inventive complex that includes an intracellular localization peptide.
  • nuclear localization peptide includes any peptide having a function of enhancing delivery of a cotransfected nucleic acid, bound to the peptide, to the nucleus of the transfected cell, as compared to the amount delivered to the nucleus when the same cell delivery method is used without the nuclear localization peptide.
  • nuclear localization peptides as well as a general description of characteristics of these nuclear localization peptides are provided herein.
  • these or any other potential peptides can be readily assayed by, for example, the methods described herein for assaying nuclear localization of labelled substances. Such assay is used to determine whether, when bound to a nucleic acid and administered to cells, a potential nuclear localization peptide enhances localization of the nucleic acid to the nucleus of the cells, as compared to the same type cells to which the nucleic acid without the peptide is administered by the same method.
  • This invention provides that, typically, useful nuclear localization peptides can provide higher amounts of nucleus-localized transfected nucleic acid than other known methods of enhancing transfection, such as liposomes, asialorosomucoid or adenoviruses. Thus, useful nuclear localization peptides can typically cause higher levels of expression of transfected DNA than other known methods of enhancing transfection such as liposomes.
  • bound means that the nucleic acid is complexed with the peptide, for example by charge-association between the negatively charged nucleic acid and positively charged amino acids in the peptide.
  • a polylysine region in the peptide can be particularly useful for this charge association, for both nuclear localization peptides and mitochondrial localization peptides.
  • standard chemical ligation methods can be employed to link the nucleic acid to the peptide, and covalent (e.g., thioester) bonds can be formed between the nucleic acid and the protein. Such methods are standard in the art.
  • the inventive complex can be used to enhance effectiveness of known methods of nucleic acid delivery in cells, thus enhancing the amount of nucleic acid that is expressed by increasing the percentage of the delivered DNA that enters the nucleus.
  • such method also enhances the delivery by known nucleic acid delivery methods of, for example, antisense RNAs, to the nucleus.
  • the present complex can be used in conjunction with any nucleic acid delivery method to enhance delivery of a nucleic acid to the nucleus of a cell. Therefore, the complex can comprise, in addition to the nucleic acid and the nuclear localization peptide, a liposome, such as cationic or anionic liposomes. This complex can then be used in conjunction with liposome delivery methods (e.g., Brigham, et al.
  • the complex can further comprise asialorosomucoid, another known transfection mediator.
  • the asialorosomucoid can bind to the nuclear localization signal via the polylysine region, and this complex can then be used in conjunction with asialorosomucoid delivery methods (Wilson, et al. (1992)).
  • the present complex can be used, for example, in methods of transfection utilizing adenoviruses (Rosenfeld, et al. (1992)).
  • the complex can be used in conjunction with poly-L-lysine antibody complex methods which target delivery of a transgene to a cell surface receptor (Trubetskoy, et al (1993)).
  • poly-L-lysine antibody complex methods which target delivery of a transgene to a cell surface receptor (Trubetskoy, et al (1993)).
  • the present complex can be utilized in cell type- targeted delivery methods, as will be elaborated upon below.
  • the nuclear localization peptide utilized herein can be derived from a naturally occurring nuclear localization sequence found in cells in proteins that are to be localized to the nucleus to function (e.g., Dingwall, C. and Laskey, R.A., Annu. Rev. Cell Biol 2:367-390 (1986); Goldfarb, D. S., et al, Nature 322:641-644 (1986)).
  • any peptide can be tested as described herein for usefulness in enhancing delivery to the nucleus.
  • a typical peptide can be about 4 to about 60 amino acids in length.
  • a preferable size range is from about 4 to about 25 amino acids, and even more preferably from about 4 to about 20 amino acids. Even more preferable is about 4 to about 13 amino acids.
  • a nuclear localization peptide typically contains a high concentration of positively charged amino acids residues; often it contains a high concentration specifically of lysine and/or arginine and usually it contains proline.
  • the amino acid residues can be mutated and/or modified so long as the modifications do not affect the nuclear localization function of the peptide.
  • the work "peptide” includes mimetics and the word “amino acid” includes modified amino acids, unusual amino acids, and D-form amino acids. All nuclear localization peptides encompassed by this invention have the function of localizing to the nucleus. Additionally, both straight- and branched-chain peptides can be utilized, as long as they retain this function.
  • An example of a minimal nuclear localization peptide comprises K R/K X R/K (listed herein as SEQ ID NO:6).
  • a nuclear localization peptide can be determined by observing its effect on the intracellular sorting of other proteins when they are attached to them by recombinant DNA methods (see, e.g., Lanford, RE. et al, Cell 37:801-813 (1984)).
  • SEQ ID NO: 1 provides a typical nuclear localization peptide similar to the SV40 T-antigen nuclear localization sequence. Any nuclear localization peptide can include a polylysine tail. Therefore, SEQ ID NO: 2 (termed peptide 1 herein) is the sequence of SEQ ID NO: 1 with a polylysine tail.
  • SEQ ID NO:2 for example, provides a 5-lysine polylysine tail; however, fewer or more lysines can be utilized. Typically, about 3 to about 10 lysines can be used. A preferable length is about 4-5 lysines.
  • SEQ ID NO:3 provides yet another example of a nuclear localization peptide. It contains the amino acid residues of peptide 1, but it has a leucine residue inserted between the nuclear localization region and the polylysine tail, causing the peptide to be less linear.
  • SEQ ID NO:4 is a sequence utilized by plants to localize proteins to the nucleus.
  • a complex including this peptide will be particularly useful in plants.
  • nuclear localization regions of proteins will be useful in the present invention. Additionally, these nuclear localization regions can be modified as desired, as long as they retain the characteristic of enhancing nucleic acid delivery to the nucleus.
  • the present invention also provides a purified complex comprising a nucleic acid bound to a mitochondrial localization peptide.
  • a complex is useful for enhancing the delivery of the bound nucleic acid to the mitochondria of a transfected cell.
  • this complex can also comprise a liposome, including cationic or anionic liposomes.
  • mitochondrial localization sequences are also known in the art, and thus, useful mitochondrial localization peptides, as determined by following the teachings herein, can be derived from, for example, proteins that are localized to the mitochondria by the cell.
  • Mitochondrial localization peptide refers to peptides having the function of directing delivery across the mitochondrial membrane into the mitochondria, as can be tested by the methods taught herein.
  • a mitochondrial localization peptide can be determined by detecting enhanced delivery of a co-transfected nucleic acid, bound to the peptide, to the mitochondria as compared to when the same delivery method is used to transfect the nucleic acid without the mitochondrial localization peptide.
  • Mitochondrial signals are known in the art to be characterized by being about 12-80 amino acid residues in length and to form amphipathic ⁇ -helical structures in the cytoplasm in which positively charged residues line up on one side of the helix while uncharged hydrophobic residues line up toward the opposite side. Therefore, positively charged amino acid residues are typically found periodically throughout the mitochondrial localization peptide. As described for nuclear localization signals, modified, unusual, and D-form animo acids can be utilized, and mimetics can therefore also be utilized, as long as they retain the function of directing delivery across the mitochondrial membrane into the mitochondria.
  • a mitochondrial localization signal is listed herein as SEQ ID NO.5.
  • a polylysine tail of a desired length for example, from about 3 to about 10 lysine residues, can be included, for example, at the carboxy terminus of the peptide.
  • a polylysine tail can be useful for allowing charge-association between the mitochondrial localization peptide and the nucleic acid to be delivered.
  • the present invention also provides a purified complex comprising a liposome, a nucleic acid and either a nuclear or a mitochondrial localization peptide.
  • the liposome can be cationic or anionic and, if desired, the liposome can contain components to target the complex to a selected type of cell in the body, as is known in the art (Cristiano, et al, Proc. Natl. Acad. Sci. USA 90:2122-2126 (1993); Nabel, G. J., Proc. Natl. Acad. Sci. USA 90:11307-11311 (1993)).
  • the present invention additionally provides a method of enhancing delivery of a nucleic acid to the cell nucleus and enhancing expression of a transfected nucleic acid in a cell comprising administering a purified complex comprising a nucleic acid bound to a nuclear localization peptide, thereby enhancing expression of the transfected nucleic acid in the cell.
  • Any cell can be transfected using techniques known in the art to transfect a selected cell type or to target a specific organ, regardless of whether a nuclear or a mitochondrial localization peptide is used.
  • the present method can enhance the expression obtainable by the known transfection method because it increases the percentage of nucleic acid that, once it is transfected into the cell, is then localized to the nucleus. Increasing the amount of DNA that enters the nucleus can increase the number of templates available for transcription into RNA, and thus increase the number of mRNA molecules available for protein synthesis. Therefore, whether the goal of the increased expression is to produce antisense RNA molecules or to produce a protein product, this method can enhance expression of these products.
  • lipofection with either cationic liposomes (such as Lipofectin, LipofectAce, etc., (BRL) (Brigham, K.L. et al, Amer. J. Respir. Cell and Mol Biol. 8:209-213 (1993)) or anionic liposomes (Nicolau et al, (1987)).
  • cationic liposomes such as Lipofectin, LipofectAce, etc., (BRL) (Brigham, K.L. et al, Amer. J. Respir. Cell and Mol Biol. 8:209-213 (1993)
  • anionic liposomes Nicolau et al, (1987)
  • Another useful delivery method includes the use of asialorosomucoid in a complex with poly-L-lysine and nucleic acid.
  • This method can be used in conjunction with the present method simply by complexing asialorosomucoid with the herein described complex comprising a nucleic acid bound to a nuclear localization peptide having a polylysine tail and following the known asialorosomucoid method.
  • adenovirus- or adenoassociated virus mediated transfection Rosenfeld, N.A. etal. (1992); Muzyczka, N. (1992)
  • can also be enhanced by the present method by using in the known virus method the present complex comprising a nucleic acid bound to a nuclear or mitochondrial localization peptide.
  • the present method can also be used to enhance delivery/expression with known delivery methods utilizing poly-L-lysine-antibody complexes (Trubetskoy, V.S. et al. (1993)). This method can be achieved simply by replacing the poly-L-lysine with an intracellular localization peptide, or by adding an intracellular localization peptide to the poly-L-lysine antibody complex. Additionally, liposomes can be used in this method.
  • any selected nuclear localization peptide can be utilized in this method.
  • the nuclear localization peptides listed as SEQ ED NOS: 1, 2, 3, 4, 6 and 7 can be utilized. Any other selected peptide can be determined for usefulness as described herein.
  • the nuclear localization peptide can be chosen according to the cell type to be transfected; i.e., for mammalian cells, one can select a mammalian-derived peptide amino acid sequence and for plant cells, one can select a plant-derived peptide amino acid sequence.
  • the present invention also provides a method of enhancing delivery of a nucleic acid to the nucleus of a cell and thus enhancing expression of the nucleic acid in a subject, comprising administering to the subject a purified complex comprising a nucleic acid bound to a nuclear localization peptide, thereby enhancing delivery of the nucleic acid to the nucleus of the cell of the subject.
  • a purified complex comprising a nucleic acid bound to a nuclear localization peptide, thereby enhancing delivery of the nucleic acid to the nucleus of the cell of the subject.
  • liposomes and preferably cationic liposomes, can be administered with the complex.
  • means known in the art for targeting a specific organ and/or cell type, such as cancer cells can be utilized.
  • the present invention additionally provides a method of enhancing delivery of a nucleic acid to mitochondria of a cell, comprising administering to the cell a complex comprising the nucleic acid bound to a mitochondrial localization peptide, thereby enhancing the delivery of the nucleic acid to the mitochondrion of the cell.
  • This method as also described herein for nuclear localization, can be performed in conjunction with known delivery methods, with cationic liposomes being particularly useful.
  • Administration of the complex can be achieved identically to that for nuclear localization.
  • An example of a mitochondrial localization peptide that can be useful in this method is that set forth in SEQ ID NO: 5.
  • Means of administration can be comparable to that already described for known in vivo methods for delivery of nucleic acids.
  • the complex and any additional components, as described herein
  • parenterally e.g., intravenously
  • intramuscular injection e.g., intraperitoneal injection
  • inhalation e.g., intravenous
  • the amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician.
  • dosage will approximate that which is typical for the administration of nucleic acids, particularly those that remain extrachromosomal, and can preferably be in the range of about 270 ⁇ g DNA 9 ⁇ g peptide and 900 ⁇ g liposome/kg about once every two to three weeks.
  • the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include, as noted above, an effective amount of the selected complex in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • Parenteral administration if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795, which is incorporated by reference herein.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected complex without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the present invention further provides a method of enhancing expression of a transfected nucleic acid in a cell in a plant comprising administering to the plant a complex comprising the nucleic acid bound to a plant nuclear localization peptide, thereby enhancing expression of the nucleic acid in the cell in the plant.
  • Plant nuclear localization sequences are known in the art, have the same characteristics as other nuclear localization sequences, and can be utilized herein as a plant nuclear localization peptide so long as it, by definition herein, directs delivery to the nucleus in plant cells. Therefore, plant nuclear localization peptides can be selected as described herein for any nuclear localization peptide.
  • a minimum plant localization peptide is therefore the peptide listed herein as SEQ ID NO:6.
  • Another example of a useful plant nuclear localization peptide is listed herein as SEQ ED NO:4, which is derived from Argobacterium tumefaciens.
  • Administration and dosages can be as already utilized for delivery/transfection of nucleic acids to cells of plants. Dosage will depend upon the purpose of the delivered nucleic acid and will typically range from about 3 ⁇ g/10 5 cells to about 40 ⁇ g/10 5 cells.
  • the present invention which enhances the delivery of nucleic acids to the nucleus or mitochondria can be very useful for delivery of nucleic acids.
  • the invention can be useful, for example, for any method or treatment wherein delivery of nucleic acids is desired, as in gene therapy.
  • one can transport a desirable gene into the nucleus of a target cell, thus, ultimately providing, for example, a protein or peptide encoded by that gene.
  • An example of useful gene therapy for which the present invention would be useful includes delivery and expression of the human cystic fibrosis transmembrane conductance regulator gene for treatment of cystic fibrosis.
  • the present invention can be utilized to deliver antisense RNAs, to block the ultimate production of a particular protein.
  • the present invention provides for delivery of nucleic acids to the mitochondria, which can be useful for, for example, correcting defects in mitochondrial DNA.
  • the present invention can be used for delivery of nucleic acids ex vivo for numerous purposes, such as transfecting desirable genes into cells prior to implantation of the cells in a body, or for producing and harvesting large quantities of an RNA or a protein encoded by a transfected gene from in vitro cell cultures.
  • this invention can be utilized to enhance antisense RNA delivery, for example, to block production of a particular protein or to enhance recovery of a specific RNA in single-stranded isolates from cells by binding to other RNAs.
  • the present method can be utilized to deliver an exogenous gene to cells of the plant for production of beneficial proteins in the plant, for example, growth enhancers or proteins to retard spoilage of any edible portions of the plant.
  • NLS nuclear localization
  • Table 2 shows the peptide 1 -mediated uptake of acridine orange-labeled DNA by nuclei isolated from BPAECs.
  • DNA alone was incubated with nuclei under the above optimum conditions.
  • Experiment 2 shows uptake when DNA is incubated with 2 ⁇ g peptide as described above (i.e., with ATP);
  • Experiment 3 shows uptake with 1 ⁇ g DNA plus 2 ⁇ g peptide, but without ATP added to the uptake incubation.
  • excess non-labeled DNA was added to the uptake incubation.
  • this method is functional in more than one cell type.
  • BPAECs as follows: Cells were grown to near confluence in a P-100 tissue culture dish. Plasmid DNA (30 ⁇ g) (pCMV4-CAT) containing the gene for chloramphenicol acetyltransferase (CAT) driven by a cytomegalovirus promoter was combined with 1 ⁇ g of NLS peptide. Following incubation for 15 minutes at room temperature, 0.01, 0.1, 0.25, 0.5, or 1.0 ⁇ g of the NLS peptide/plasmid DNA complex was combined with 90 ⁇ g of LipofectinTM (Bethesda Research Laboratories) and added per plate (100 mm) of BPAEC and the cells were incubated overnight at 37° C, in 5% CO 2 . Following this incubation the medium was changed and the cells were incubated for an additional 36-40 hours and then harvested for CAT assay.
  • pCMV4-CAT Plasmid DNA (30 ⁇ g) (pCMV4-CAT) containing the gene for chlor
  • Cells were harvested by scraping into 3 ml of phosphate buffered 0.1% NaCl and collected by centrifugation. The cell pellet was resuspended in 40 mM Tris- HC1 buffer, pH 7.5, containing 10 mM EDTA and 150 mM NaCl and frozen in an acetone dry ice bath for 5 minutes. The frozen pellet was thawed in a 37°C water bath and the freeze-thaw process was repeated two times. An aliquot (50 ⁇ l) of cell suspension was removed and assayed for CAT activity using standard methods (Berger, S. and Kimmel, A.R., eds., Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152:717-718 (1987)). The CAT assay was quantified as cpm of radiolabeled acetyl transferred from coenzyme A to chloramphenicol.
  • Table 3 shows the results of administering to BPAECs 30 ⁇ g pCMV4- CAT with varying concentrations of peptide 1.
  • One control as no lipofectin or peptide in the reaction, while another shows the reaction with 90 ⁇ g lipofectin and no peptide.
  • the experimental reactions show the results with 1.0, 0.5, 0.25, 0.1, and 0.01 ⁇ g peptide 1 to determine successful ratios of DNA to peptide.
  • a nuclear localization peptide comprising the complete histone protein (Boenringer Mannheim) was utilized in a liposome transfer experiment as described above. 1 ⁇ g of this nuclear localization peptide was administered with 90 ⁇ g lipofectin and with 30 ⁇ g of pCMV4-CAT DNA to BPAECs.
  • Table 5 provides the results from these experiments using complete histone protein.
  • a nuclear localization peptide comprising polylysine (listed herein as SEQ ID NO:7) was utilized in a liposome transfer experiment as described above. 1 ⁇ g of polylysine was complexed with 30 ⁇ g of pCMV4-CAT and 90 ⁇ g lipofectin and administered to BPAECs. The results of these polylysine experiments are shown in Table 6 below. These results demonstrate that polylysine alone does not detectably enhance liposome- mediated transfection of nucleic acids.
  • a mitochondrial localization peptide comprising the amino acid sequence set forth in SEQ ID NO: 5 was utilized in a liposome transfer experiment as described above for nuclear localization experiments. 1 ⁇ g of this mitochondrial localization peptide was administered with 90 ⁇ g lipofectin and 30 ⁇ g pCMV4-CAT DNA to BPAECs. CAT activity was then measured, and the results are shown below in Table 7.
  • DNA/liposome complex with either a nuclear or a mitochondrial localization peptide, at a 1:30 peptide:DNA ratio on a 37°C microscope stage and images of the chosen field were recorded every 2 minutes.
  • the nuclear localization peptide peptide 1 listed herein as SEQ ID NO: 2
  • the DNA fluorescence was limited to abundant, small, well defined regions of the cell periphery. After 24 minutes the fluorescence appeared to be exclusively nuclear and the nucleus of the cell was very well defined against the much darker cytoplasm.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions et des procédés destinés à améliorer l'apport d'acides nucléiques à des compartiments intracellulaires. L'invention concerne notamment un procédé d'amélioration de l'apport et de l'expression d'acides nucléiques au noyau, ainsi qu'un procédé d'amélioration de l'apport d'acides nucléiques aux mitochondries, utilisant un complexe de localisation nucléaire ou mitochondrial, respectivement.
PCT/US1995/007543 1994-06-13 1995-06-13 Compositions et procedes d'amelioration de l'apport d'acides nucleiques a des cellules WO1995034647A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU27736/95A AU2773695A (en) 1994-06-13 1995-06-13 Compositions for and methods of enhancing delivery of nucleic acids to cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US25885394A 1994-06-13 1994-06-13
US08/258,853 1994-06-13

Publications (1)

Publication Number Publication Date
WO1995034647A1 true WO1995034647A1 (fr) 1995-12-21

Family

ID=22982405

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/007543 WO1995034647A1 (fr) 1994-06-13 1995-06-13 Compositions et procedes d'amelioration de l'apport d'acides nucleiques a des cellules

Country Status (2)

Country Link
AU (1) AU2773695A (fr)
WO (1) WO1995034647A1 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997035873A2 (fr) * 1996-03-26 1997-10-02 Radulescu Razvan T Peptide presentant des proprietes antiproliferatives
EP0902088A2 (fr) * 1996-03-26 1999-03-17 Razvan T. Radulescu Peptides avec propriétés antiprolifératives
WO1999013096A1 (fr) * 1997-09-08 1999-03-18 University Of Florida Materiels et methodes d'apport intracellulaire de molecules actives biologiquement
EP0908521A1 (fr) * 1997-10-10 1999-04-14 Hoechst Marion Roussel Deutschland GmbH Système de transfection pour le transfert d'ADN dans des cellules
WO1999019502A1 (fr) * 1997-10-10 1999-04-22 Aventis Pharma Deutschland Gmbh Systeme de transfection pour transferer des acides nucleiques dans des cellules
EP0967288A1 (fr) * 1998-06-16 1999-12-29 Hoechst Marion Roussel Deutschland GmbH Système pour la transfection génique de celules
WO2000003683A2 (fr) * 1998-07-20 2000-01-27 Inex Pharmaceuticals Corporation Complexes d'acides nucleiques encapsules dans des liposomes
US6312956B1 (en) * 1999-10-01 2001-11-06 Vanderbilt University Nuclear targeted peptide nucleic acid oligomer
US6372720B1 (en) 1998-02-05 2002-04-16 Kenneth J. Longmuir Liposome fusion and delivery vehicle
US6498011B2 (en) 1998-07-24 2002-12-24 Novartis Ag Method for transformation of animal cells
US6752987B1 (en) 1995-02-28 2004-06-22 The Regents Of The University Of California Adenovirus encoding human adenylylcyclase (AC) VI
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US7235236B2 (en) 1995-02-28 2007-06-26 The Regents Of The University Of California Polynucleotide encoding human adenylylcyclase VI and uses thereof for enhancing cardiac function
WO2007095264A1 (fr) 2006-02-13 2007-08-23 Cardiac Pacemakers, Inc. Procédé et appareil permettant de contrôler l'expression génique par voie électromagnétique ou par la chaleur
FR2944806A1 (fr) * 2009-04-27 2010-10-29 Centre Nat Rech Scient Adressage d'acides nucleiques dans les mitochondries.
JP2013519662A (ja) * 2010-02-12 2013-05-30 プロカルタ バイオシステムズ リミテッド 核酸複合体
JP2013523111A (ja) * 2010-04-01 2013-06-17 プロカルタ バイオシステムズ リミテッド 転写因子デコイ
US8708948B2 (en) 2005-06-29 2014-04-29 Advanced Cardiovascular Systems, Inc. Intracoronary device and method of use thereof
JP2014522662A (ja) * 2011-08-04 2014-09-08 カナダ国 オルガネラターゲッティングナノキャリア
WO2016086227A2 (fr) 2014-11-26 2016-06-02 The Regents Of The University Of California Compositions thérapeutiques comprenant des facteurs de transcription et leurs procédés de préparation et d'utilisation
US20160222415A1 (en) * 2007-02-28 2016-08-04 Yeda Research And Development Co. Ltd. Nuclear targeting sequences
US9702012B2 (en) 2007-10-03 2017-07-11 Procarta Biosystems Ltd Transcription factor decoys, compositions and methods
US10240133B2 (en) 2013-09-17 2019-03-26 Yeda Research And Development Co. Ltd. ERK-derived peptides and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019768A1 (fr) * 1992-04-03 1993-10-14 The Regents Of The University Of California Systeme de livraison d'un polynucleotide a assemblage autonome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019768A1 (fr) * 1992-04-03 1993-10-14 The Regents Of The University Of California Systeme de livraison d'un polynucleotide a assemblage autonome

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ACCOUNTS OF CHEMICAL RESEARCH, Volume 26, Number 5, issued 1993, BEHR, "Synthetic Gene-Transfer Vectors", pages 274-278. *
BIOTECHNOLOGY, Volume 13, issued 13 March 1995, HODGSON, "The Vector Void in Gene Therapy", pages 222-225. *
BRITISH MEDICAL BULLETIN, Volume 51, Number 1, issued 1995, SCHOFIELD et al., "Non-Viral Approaches to Gene Therapy", pages 56-71. *
CURRENT OPINION IN BIOTECHNOLOGY, Volume 5, issued 1994, LEDLEY, "Non-Viral Gene Therapy", pages 626-636. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 262, Number 10, issued 05 April 1987, WU et al., "Receptor-Mediated in Vitro Gene Transformation by a Soluble DNA Carrier System", pages 4429-4432. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 264, Number 21, issued 25 July 1989, KANEDA et al., "Introduction and Expression of the Human Insulin Gene in Adult Rat Liver", pages 12126-12129. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 266, Number 22, issued 05 August 1991, WU et al., "Receptor-Mediated Gene Delivery in Vivo", pages 14338-14342. *
MOLECULAR AND GENERAL GENETICS, Volume 239, issued 1993, ROSSI et al., "The VirD2 Protein of Agrobacterium Tumefaciens Carries Nuclear Localization Signals Important for Transfer of T-DNA to Plants", pages 345-353. *
NATURE GENETICS, Volume 5, issued October 1993, ALTON et al., "Non-Invasive Liposome-Mediated Gene Delivery can Correct the Ion Transport Defect in Cystic Fibrosis Mutant Mice", pages 135-142. *
NUCLEIC ACIDS RESEARCH, Volume 23, Number 1, issued January 1995, SEIBEL et al., "Transfection of Mitochondria: Strategy Towards a Gene Therapy of Mitochondrial DNA Diseases", pages 10-17. *
TRENDS IN BIOCHEMICAL SCIENCES, Volume 16, issued December 1991, DINGWALL et al., "Nuclear Targeting Sequences - a Consensus?", pages 478-481. *

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7235236B2 (en) 1995-02-28 2007-06-26 The Regents Of The University Of California Polynucleotide encoding human adenylylcyclase VI and uses thereof for enhancing cardiac function
US6752987B1 (en) 1995-02-28 2004-06-22 The Regents Of The University Of California Adenovirus encoding human adenylylcyclase (AC) VI
WO1997035873A2 (fr) * 1996-03-26 1997-10-02 Radulescu Razvan T Peptide presentant des proprietes antiproliferatives
EP0902088A3 (fr) * 1996-03-26 1999-09-15 Razvan T. Radulescu Peptides avec propriétés antiprolifératives
EP0902088A2 (fr) * 1996-03-26 1999-03-17 Razvan T. Radulescu Peptides avec propriétés antiprolifératives
WO1997035873A3 (fr) * 1996-03-26 1997-12-31 Razvan T Radulescu Peptide presentant des proprietes antiproliferatives
WO1999013096A1 (fr) * 1997-09-08 1999-03-18 University Of Florida Materiels et methodes d'apport intracellulaire de molecules actives biologiquement
US6627618B2 (en) 1997-09-08 2003-09-30 University Of Florida Research Foundation, Inc. Materials and methods for intracellular delivery of biologically active molecules
US6090619A (en) * 1997-09-08 2000-07-18 University Of Florida Materials and methods for intracellular delivery of biologically active molecules
US6171863B1 (en) 1997-09-08 2001-01-09 University Of Florida Materials and methods for intracellular delivery of biologically active molecules
EP0908521A1 (fr) * 1997-10-10 1999-04-14 Hoechst Marion Roussel Deutschland GmbH Système de transfection pour le transfert d'ADN dans des cellules
WO1999019502A1 (fr) * 1997-10-10 1999-04-22 Aventis Pharma Deutschland Gmbh Systeme de transfection pour transferer des acides nucleiques dans des cellules
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US6372720B1 (en) 1998-02-05 2002-04-16 Kenneth J. Longmuir Liposome fusion and delivery vehicle
EP0967288A1 (fr) * 1998-06-16 1999-12-29 Hoechst Marion Roussel Deutschland GmbH Système pour la transfection génique de celules
WO2000003683A3 (fr) * 1998-07-20 2000-04-27 Inex Pharmaceuticals Corp Complexes d'acides nucleiques encapsules dans des liposomes
WO2000003683A2 (fr) * 1998-07-20 2000-01-27 Inex Pharmaceuticals Corporation Complexes d'acides nucleiques encapsules dans des liposomes
US6498011B2 (en) 1998-07-24 2002-12-24 Novartis Ag Method for transformation of animal cells
US6623966B1 (en) 1999-10-01 2003-09-23 Vanderbilt University Nuclear targeted peptide nucleic acid oligomer
US6312956B1 (en) * 1999-10-01 2001-11-06 Vanderbilt University Nuclear targeted peptide nucleic acid oligomer
US8708948B2 (en) 2005-06-29 2014-04-29 Advanced Cardiovascular Systems, Inc. Intracoronary device and method of use thereof
WO2007095264A1 (fr) 2006-02-13 2007-08-23 Cardiac Pacemakers, Inc. Procédé et appareil permettant de contrôler l'expression génique par voie électromagnétique ou par la chaleur
US10000771B2 (en) * 2007-02-28 2018-06-19 Yeda Research And Development Co. Ltd. Nuclear targeting sequences
US20160222415A1 (en) * 2007-02-28 2016-08-04 Yeda Research And Development Co. Ltd. Nuclear targeting sequences
US9702012B2 (en) 2007-10-03 2017-07-11 Procarta Biosystems Ltd Transcription factor decoys, compositions and methods
FR2944806A1 (fr) * 2009-04-27 2010-10-29 Centre Nat Rech Scient Adressage d'acides nucleiques dans les mitochondries.
WO2010125293A1 (fr) * 2009-04-27 2010-11-04 Centre National De La Recherche Scientifique (C.N.R.S) Adressage d'acides nucléiques dans les mitochondries
JP2013519662A (ja) * 2010-02-12 2013-05-30 プロカルタ バイオシステムズ リミテッド 核酸複合体
US10350299B2 (en) 2010-02-12 2019-07-16 Procarta Biosystems Ltd. Nucleic acid complexes
US9024005B2 (en) 2010-02-12 2015-05-05 Procarta Biosystems Ltd Nucleic acid complexes
US9669101B2 (en) 2010-02-12 2017-06-06 Procarta Biosystems Ltd Nucleic acid complexes
US9550991B2 (en) 2010-04-01 2017-01-24 Procarta Biosystems Ltd. Transcription factor decoys
JP2013523111A (ja) * 2010-04-01 2013-06-17 プロカルタ バイオシステムズ リミテッド 転写因子デコイ
AU2012289698B2 (en) * 2011-08-04 2017-04-20 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Organelle targeting nanocarriers
EP2739741A4 (fr) * 2011-08-04 2015-05-13 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri Food Nanovecteurs ciblant des organelles
JP2014522662A (ja) * 2011-08-04 2014-09-08 カナダ国 オルガネラターゲッティングナノキャリア
US10240133B2 (en) 2013-09-17 2019-03-26 Yeda Research And Development Co. Ltd. ERK-derived peptides and uses thereof
WO2016086227A2 (fr) 2014-11-26 2016-06-02 The Regents Of The University Of California Compositions thérapeutiques comprenant des facteurs de transcription et leurs procédés de préparation et d'utilisation

Also Published As

Publication number Publication date
AU2773695A (en) 1996-01-05

Similar Documents

Publication Publication Date Title
WO1995034647A1 (fr) Compositions et procedes d'amelioration de l'apport d'acides nucleiques a des cellules
US5843643A (en) Site-specific transfection of eukaryotic cells using polypeptide-linked recombinant nucleic acid
EP3971216A1 (fr) Fragment peptidique de support et son utilisation
SK281543B6 (sk) Farmaceutická kompozícia na transfekciu nukleovej kyseliny
US6372720B1 (en) Liposome fusion and delivery vehicle
US6387700B1 (en) Cationic peptides, Cys-Trp-(LYS)n, for gene delivery
EP0693939A1 (fr) Peptides de tranfert d'acides nucleiques et leur utilisation pour injecter des acides nucleiques dans des cellules eucaryotes
JPH10504709A (ja) ポリヌクレオチドの細胞への標的を定めた送達を増大させるための細菌成分の利用
US20040192627A1 (en) Mitochondrial genome replenishment
US20220195401A1 (en) Modified piggybac transposase polypeptide, polynucleotide encoding them, introducing carrier, kit, method of incorporating target sequence into cell genome, and method of producing cell
EP1644496A2 (fr) Vecteurs modifies pour la transfection d'organite
JPH07508419A (ja) アミノ酸輸送体，プラスミド，細菌，酵母菌および輸送体を有する植物のｄｎａ配列およびその使用
US20040102606A1 (en) Histone H2A -derived peptides useful in gene delivery
Déas et al. In vivo-targeted gene delivery using antibody-based nonviral vector
EP1007549B1 (fr) Compositions et procedes pour une transfection tres efficace
US6479464B1 (en) Compositions and methods for highly efficient transfection
WO1998050078A1 (fr) Peptides lipophiles et/ou lytiques permettant l'apport specifique d'acides nucleiques a des cellules
JP2002338503A (ja) Ｄｎａトランスフェクション用組成物
JP3808938B2 (ja) 核酸運搬体
KR20010074456A (ko) 핵산 전달 벡터, 이를 함유한 조성물 및 용도
EP2329023B1 (fr) Compositions et procédés pour la délivrance d arn codant pour des protéines pour corriger un dysfonctionnement mitochondrial
US20080194540A1 (en) Use of a Tricyclic Antidepressant Drug For Promoting Endocytosis
JPWO2005054486A1 (ja) 遺伝子導入試薬調製法
Anwer et al. Synthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes
US20030100496A1 (en) Compositions and methods for highly efficient transfection

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载