WO2022068884A1 - 核酸递送方法及系统 - Google Patents

核酸递送方法及系统 Download PDF

Info

Publication number
WO2022068884A1
WO2022068884A1 PCT/CN2021/121765 CN2021121765W WO2022068884A1 WO 2022068884 A1 WO2022068884 A1 WO 2022068884A1 CN 2021121765 W CN2021121765 W CN 2021121765W WO 2022068884 A1 WO2022068884 A1 WO 2022068884A1
Authority
WO
WIPO (PCT)
Prior art keywords
stranded dna
cells
nucleotides
transfection
transfection reagent
Prior art date
Application number
PCT/CN2021/121765
Other languages
English (en)
French (fr)
Inventor
宋杰
程进
唐林林
Original Assignee
上海交通大学
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 上海交通大学 filed Critical 上海交通大学
Priority to EP21874539.6A priority Critical patent/EP4223881A4/en
Priority to CN202180066876.4A priority patent/CN116583606A/zh
Publication of WO2022068884A1 publication Critical patent/WO2022068884A1/zh
Priority to US18/192,674 priority patent/US20230272416A1/en

Links

Images

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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to nucleic acid delivery methods and systems.
  • the present application relates to methods and systems for expressing exogenous RNA and/or exogenous protein by nucleic acid delivery.
  • plasmid DNA plasmid DNA
  • RNA RNA
  • RNA molecules are not easy to obtain, have poor stability and are easily degraded, and are not ideal for the delivery and expression of foreign genes.
  • the present application provides a method and system for nucleic acid delivery.
  • the inventors of the present application have surprisingly found that utilizing the methods and systems of the present application to deliver exogenous nucleic acid molecules results in one or more of the following advantages (eg, as compared to traditional double-stranded plasmid DNA delivery systems):
  • the application provides a composition (eg, a transfection composition).
  • the composition may comprise: single-stranded DNA and at least one transfection reagent.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may be about 100 to about 0.1.
  • the mass ratio of the single-stranded DNA to the transfection reagent in the composition is from about 100:1 to about 1:10.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent in the composition is from about 1 to about 0.2.
  • the mass ratio of the single-stranded DNA to the transfection reagent in the composition is from about 2:1 to about 1:5.
  • the single-stranded DNA is circular single-stranded DNA.
  • the single-stranded DNA is linear single-stranded DNA.
  • the single-stranded DNA comprises nucleic acid sequences encoding one or more RNAs and/or proteins.
  • the single-stranded DNA comprises nucleic acid sequences encoding two or more RNAs and/or proteins.
  • the single-stranded DNA comprises a sense strand nucleic acid sequence encoding RNA and/or protein.
  • the single-stranded DNA comprises antisense nucleic acid sequences encoding RNA and/or proteins.
  • the single-stranded DNA comprises at least 40 nucleotides.
  • the single-stranded DNA comprises at least 1500 nucleotides.
  • the transfection reagent comprises cationic lipids and/or cationic polymers.
  • the transfection reagent comprises cationic liposomes.
  • the composition further comprises one or more cell culture media.
  • the single-stranded DNA is present in the composition in an amount of about 10 ng to about 1000 ng.
  • the concentration of the single-stranded DNA in the composition is from about 0.5 nM to about 3 nM.
  • the application provides a kit that can include a composition (eg, a transfection composition) as described herein.
  • a composition eg, a transfection composition
  • the present application provides a kit.
  • the kit may comprise: single-stranded DNA, at least one transfection reagent, and instructions for use (eg, instructions for use).
  • the instructions for use may describe administering the single-stranded DNA to the transfection reagent in a mass ratio of about 100 to about 0.1 to a target cell to deliver at least a portion of the nucleic acid contained in the single-stranded DNA to the target in cells.
  • the instructions for use describe administering the single-stranded DNA to the transfection reagent in a mass ratio of from about 5:1 to about 1:10 to target cells to convert the single-stranded DNA into the single-stranded DNA. At least a portion of the nucleic acid contained is delivered to the target cell.
  • the instructions for use describe administering the single-stranded DNA to the transfection reagent in a mass ratio of about 10 to about 0.1 to a target cell so that at least the single-stranded DNA contained in the A portion of the nucleic acid is delivered to the target cell.
  • the instructions for use describe administering the single-stranded DNA to the transfection reagent in a mass ratio of about 2:1 to about 1:5 to a target cell so that the single-stranded DNA is incorporated into the single-stranded DNA. At least a portion of the nucleic acid contained is delivered to the target cell.
  • the instructions for use describe that the single-stranded DNA and the transfection reagent are mixed according to the mass ratio or according to the mass ratio to prepare a transfection composition.
  • the single-stranded DNA and the at least one transfection reagent are included in the kit in a manner that they do not mix with each other.
  • the single-stranded DNA and the at least one transfection reagent are each independently present in individually packaged containers.
  • the content of the single-stranded DNA is about 10 ng to about 1000 ng.
  • the concentration of the single-stranded DNA in the kit is from about 0.5 nM to about 3 nM.
  • the single-stranded DNA is circular single-stranded DNA.
  • the single-stranded DNA is linear single-stranded DNA.
  • the single-stranded DNA comprises nucleic acid sequences encoding one or more RNAs and/or proteins.
  • the single-stranded DNA comprises nucleic acid sequences encoding two or more RNAs and/or proteins.
  • the single-stranded DNA comprises a sense strand nucleic acid sequence encoding RNA and/or protein.
  • the single-stranded DNA comprises antisense nucleic acid sequences encoding RNA and/or proteins.
  • the single-stranded DNA in the kit comprises at least 40 nucleotides.
  • the single-stranded DNA in the kit comprises at least 1500 nucleotides.
  • the transfection reagent in the kit comprises a cationic lipid and/or a cationic polymer.
  • the transfection reagent in the kit comprises cationic liposomes.
  • one or more cell culture media are also included in the kit.
  • the present application provides a method of delivering nucleic acid into a cell.
  • the method can include contacting the cell with single-stranded DNA comprising the nucleic acid and a transfection reagent.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may be about 5 to about 0.1.
  • the mass ratio of the single-stranded DNA to the transfection reagent is from about 5:1 to about 1:10.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent is from about 1 to about 0.2.
  • the mass ratio of the single-stranded DNA to the transfection reagent is from about 2:1 to about 1:5.
  • the present application provides a method of expressing exogenous RNA and/or exogenous protein in a cell.
  • the method may comprise introducing into the cell a single-stranded DNA encoding the exogenous RNA and/or the exogenous protein.
  • the method further comprises growing the cell under conditions that permit expression of the exogenous RNA and/or the exogenous protein.
  • the present application provides a method of reducing cytotoxicity associated with DNA transfection.
  • the method may comprise: upon transfection, introducing into the cell single-stranded DNA rather than the corresponding double-stranded DNA.
  • the present application provides a method of reducing cellular immunogenicity associated with DNA transfection.
  • the method may comprise: upon transfection, introducing into the cell single-stranded DNA rather than the corresponding double-stranded DNA.
  • the present application provides a method of increasing the DNA transfection efficiency of cells.
  • the method may comprise: upon transfection, introducing into the cell single-stranded DNA rather than the corresponding double-stranded DNA.
  • introducing the single-stranded DNA into the cell comprises contacting the cell with a composition described herein.
  • introducing the single-stranded DNA into the cell comprises contacting the cell with the single-stranded DNA and a transfection reagent, and the single-stranded DNA is linked to the single-stranded DNA.
  • the ratio of the mass ratio of the transfection reagent is about 5 to about 0.1.
  • introducing the single-stranded DNA into the cell comprises contacting the cell with the single-stranded DNA and a transfection reagent, and the single-stranded DNA is linked to the single-stranded DNA.
  • the mass ratio of the transfection reagent is about 5:1 to about 1:10.
  • introducing the single-stranded DNA into the cell comprises contacting the cell with the single-stranded DNA and a transfection reagent, and the single-stranded DNA is linked to the single-stranded DNA.
  • the ratio of the mass ratio of the transfection reagent is about 1 to about 0.2.
  • introducing the single-stranded DNA into the cell comprises contacting the cell with the single-stranded DNA and a transfection reagent, and the single-stranded DNA is linked to the single-stranded DNA.
  • the mass ratio of the transfection reagent is about 2:1 to about 1:5.
  • the contacting eg, contacting the cells with the single-stranded DNA and a transfection reagent, or contacting the cells with a composition described herein
  • Incubation at about 25°C to about 40°C is included.
  • the contacting comprises incubating at about 37°C.
  • the contacting comprises incubating for at least 4 hours.
  • the single-stranded DNA is circular single-stranded DNA.
  • the single-stranded DNA is linear single-stranded DNA.
  • the single-stranded DNA comprises nucleic acid sequences encoding one or more exogenous RNAs and/or exogenous proteins.
  • the single-stranded DNA comprises nucleic acid sequences encoding two or more exogenous RNAs and/or exogenous proteins.
  • the single-stranded DNA comprises a sense strand nucleic acid sequence encoding an exogenous RNA and/or an exogenous protein.
  • the single-stranded DNA comprises an antisense nucleic acid sequence encoding an exogenous RNA and/or an exogenous protein.
  • the single-stranded DNA comprises at least 40 nucleotides.
  • the single-stranded DNA comprises at least 1500 nucleotides.
  • the single-stranded DNA is present in an amount of about 10 ng to about 1000 ng.
  • the concentration of the single-stranded DNA is from about 0.5 nM to about 3 nM.
  • the transfection reagent comprises a cationic lipid and/or a cationic polymer.
  • the transfection reagent comprises cationic liposomes.
  • the methods described herein are in vivo methods, in vitro methods, or ex vivo methods.
  • the cells are somatic cells, tumor cells and/or immune cells.
  • the cells are selected from the group consisting of lung cancer cells, kidney cells, melanoma cells, liver cancer cells, cervical cancer cells, glioma cells, epithelial cells, colon cancer cells, fibroblasts cells, breast cancer cells, gastric cancer cells and immune effector cells.
  • the cells are selected from the group consisting of: A549 cells, C6 cells, MCF-7 cells, HepG2 cells, CT26 cells, Hela cells, Helf cells, MDCK cells, B16 cells, Hep3B cells , L929 cells, 4T1 cells, 293T cells, MGC803 cells and macrophages.
  • the application provides cells treated by the method of any of the aspects of the application.
  • the present application provides organisms (eg, non-human organisms), or components thereof, that can comprise cells described herein (eg, cells treated by the methods of the present application).
  • the moiety is selected from the group consisting of tissues, organs, and combinations thereof.
  • the application provides the use of single-stranded DNA for the preparation of a composition (eg, a transfection composition, such as a cell transfection composition, or a nucleic acid delivery composition).
  • a composition eg, a transfection composition, such as a cell transfection composition, or a nucleic acid delivery composition.
  • the composition can be used for one or more of the following: delivering at least a portion of the nucleic acid contained in the single-stranded DNA into a cell; expressing in the cell exogenous RNA or exogenous RNA encoded by the single-stranded DNA protein; increase DNA transfection efficiency of cells; reduce cytotoxicity associated with DNA transfection; and/or reduce cellular immunogenicity associated with DNA transfection.
  • the composition may be a composition as described in any aspect of the present application.
  • the cells are somatic cells, tumor cells and/or immune cells.
  • the cells are selected from the group consisting of lung cancer cells, kidney cells, melanoma cells, liver cancer cells, cervical cancer cells, glioma cells, epithelial cells, colon cancer cells, fibroblasts, breast cancer cells, Gastric cancer cells and immune effector cells.
  • the cells are selected from the group consisting of: A549 cells, C6 cells, MCF-7 cells, HepG2 cells, CT26 cells, Hela cells, Helf cells, MDCK cells, B16 cells, Hep3B cells, L929 cells, 4T1 cells , 293T cells, MGC803 cells and macrophages.
  • Figure 1 shows the structure and construction flow of the exemplary vectors pScaf-EGFP(+), pScaf-EGFP(-) and pScaf-EGFP-mCherry in the present application.
  • Figures 2A-2B show the transfection efficiency and protein expression of the exemplary single-stranded DNA of the present application in each cell line.
  • Figure 3 shows the protein expression of the exemplary single-stranded DNA of the present application in each cell line.
  • Figures 4A-4C show the protein expression after transfection of MDCK cells with the exemplary single-stranded DNA of the present application.
  • Figure 5 shows the protein expression of the exemplary single-stranded DNA of the present application compared with the corresponding double-stranded plasmid DNA after transfection of cells.
  • Figure 6 shows the protein expression of the exemplary single-stranded DNA of the present application compared with the corresponding double-stranded plasmid DNA after transfection of cells.
  • Figure 7 shows the protein expression of the exemplary single-stranded DNA of the present application compared with the corresponding double-stranded plasmid DNA after transfection of cells.
  • Figure 8 shows the cytotoxic effect of transfected cells with exemplary single-stranded DNA of the present application compared to the corresponding double-stranded plasmid DNA.
  • Figure 9 shows the results of electrophoresis validation of exemplary linear single-stranded DNA of the present application.
  • 1kb shows the 1kb DNA marker
  • the first lane is the corresponding double-stranded DNA
  • the second lane is the reverse product amplification result
  • the third lane is the forward product amplification result.
  • Figure 10 shows the expression of exemplary linear single-stranded DNA of the present application after transfection of various cell lines.
  • Figure 11 shows the results of DNase (S1 enzyme) verification of exemplary single-stranded DNA of the present application.
  • Figures 12A-12B show the effect of exemplary single-stranded DNA of the present application in eliciting an immune response compared to plasmid double-stranded DNA.
  • Figures 13A-13B show the effect of exemplary single-stranded DNA of the present application in eliciting an immune response compared to plasmid double-stranded DNA.
  • nucleic acid refers to a combination of at least two base-sugar-phosphate units.
  • polynucleotides differ from oligonucleotides in that polynucleotides generally contain more than 120 monomeric units.
  • nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • Nucleotides are the monomeric units of nucleic acid polymers.
  • Antisense nucleic acids are nucleic acids that interfere with DNA and/or RNA function.
  • Natural nucleic acids have a phosphate backbone, and artificial nucleic acids may contain other types of backbones but have the same bases as natural nucleic acids.
  • RNA can be in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA and/or ribozymes, among others.
  • DNA can be in the form of plasmid DNA, viral DNA, linear DNA, chromosomal DNA or derivatives thereof. In addition to these forms, DNA and RNA can also be in single-, double-, triple- or quadruple-stranded form.
  • the term may also include PNAs (peptide nucleic acids), phosphorothioates and other variants of the phosphate backbone of natural nucleic acids.
  • single-stranded DNA generally refers to a nucleic acid molecule consisting essentially of one DNA strand.
  • DNA can exist in double-stranded or single-stranded form.
  • double-stranded DNA dsDNA
  • single-stranded (ssDNA) DNA typically has only one DNA strand.
  • ssDNA can also include partially single-stranded DNA, wherein the DNA can have double-stranded and single-stranded segments.
  • ssDNA can have a portion that is not paired with another DNA strand and another portion that is paired with another DNA strand.
  • ssDNA can be formed by a variety of methods. For example, ssDNA can be formed by denaturing dsDNA.
  • denaturation refers to the process that can separate the two strands of dsDNA, thereby reducing the number of Watson-Crick pairs.
  • ssDNA can also be formed by nucleic acid amplification (eg, rolling circle amplification or asymmetric amplification, etc.).
  • the ssDNA can be circular or linear.
  • exogenous DNA sequence refers to a nucleic acid molecule that is not derived from the host in which it is found. It can be the same as the host's DNA or heterologous.
  • an exogenous DNA sequence can be a nucleic acid sequence inserted into an expression vector for expression of an RNA or protein of interest (eg, exogenous RNA or exogenous protein).
  • the exogenous DNA sequence can be derived from a variety of sources, including DNA, cDNA, synthetic DNA and RNA, and the like.
  • the exogenous DNA sequence may comprise genomic DNA, which may or may not include naturally occurring or artificial introns.
  • genomic DNA can be linked to a promoter region or a poly A signal sequence.
  • exogenous DNA sequences include, but are not limited to, DNA sequences that can be expressed to produce the gene product (eg, RNA of interest or protein of interest) desired for expression in the host cell.
  • the gene product can affect the viability or state of the host cell.
  • the application provides a composition (eg, a transfection composition).
  • the composition may comprise: single-stranded DNA and at least one transfection reagent.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 5 to about 0.1.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 4.5 to about 0.5, about 4 to about 1, about 3.5 to about 1.5, about 3 to about 0.5, about 2.5 to about 0.1, about 2 to about 0.1, about 1.5 to about 0.1, about 1 to about 0.1, about 1 to about 0.2, about 1.5 to about 0.2, about 1.5 to about 0.25, about 1.5 to about 0.3, about 1 to about 0.25, about 1.25 to about 0.2, about 0.75 to about 0.25, about 0.5 to about 0.3, etc.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 100 to about 0.1, about 50 to about 0.1, about 20 to about 0.1, about 10 to about 0.1, about 5 to about 0.1, about 100 to about 0.5, about 50 to about 0.5, about 20 to about 0.5, about 10 to about 0.5, about 5 to about 0.5, about 100 to about 1, about 50 to about 1, about 20 to about 1, about 10 to about 1, or about 5 to about 1, etc.
  • the mass ratio of the single-stranded DNA to the transfection reagent in the composition is from about 5:1 to about 1:10.
  • about 5:1 to about 1:10 means that the mass of the single-stranded DNA relative to the mass of the transfection reagent is gradually reduced from about 5:1 until the mass of the two is substantially the same (ie The mass ratio of the two is about 1:1), and then the relative mass of the transfection reagent continues to increase until the mass of the single-stranded DNA relative to the mass of the transfection reagent is about 1:10.
  • the mass ratio of the single-stranded DNA to the transfection reagent in the composition is about 4:1 to about 1:10, about 3:1 to about 1:10, about 2:1 to about 1:10, about 1:1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 2:1 To about 1:2, about 2:1 to about 1:3, about 2:1 to about 1:4, about 2:1 to about 1:5, etc.
  • the single-stranded DNA in the composition is circular single-stranded DNA
  • the mass ratio of the circular single-stranded DNA to the transfection reagent is about 5:1 to about 1 :10.
  • the single-stranded DNA in the composition is linear single-stranded DNA
  • the mass ratio of the linear single-stranded DNA to the transfection reagent is about 5:1 to about 1:10 .
  • the single-stranded DNA comprises nucleic acid sequences encoding one or more RNAs and/or proteins.
  • the single-stranded DNA can be transcribed/reverse transcribed into RNA molecules (eg, miRNA, mRNA, or other RNA molecules) as a template within a cell.
  • the single-stranded DNA can be converted to double-stranded DNA, which can exist either free in the cell or integrated into the genome of the cell (eg, in a random insertion manner, or integrated into a specific site). point).
  • the single-stranded DNA can be transcribed and translated to express the encoded target protein (eg, a therapeutic protein, a labeled protein such as a fluorescent protein or a light-emitting protein, etc.).
  • the single-stranded DNA comprises nucleic acid sequences encoding two or more RNAs and/or proteins.
  • the single-stranded DNA can comprise a first coding region, which can encode a first RNA or a first protein, and a second coding region, which can encode independently of the first coding region.
  • a second RNA or a second protein that is the same or different from the RNA or the first protein.
  • One or more non-coding regions may be included between the first coding region and the second coding region. In some cases, substantially no non-coding regions are included between the first coding region and the second coding region.
  • the single-stranded DNA may comprise a third coding region or more coding regions. Protein complexes or protein/nucleic acid complexes can be formed between the expression products of the respective coding regions.
  • the single-stranded DNA (eg, single-stranded circular DNA or single-stranded linear DNA) can comprise nucleic acid sequences encoding one or more RNAs and/or proteins (eg, encoding two or more RNAs and/or nucleic acid sequence of a protein), which may comprise at least about 40 nucleotides, such as at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, at least about 900 nucleotides, at least about 950 nucle
  • the single-stranded DNA comprises a sense strand (also referred to as coding strand, sense strand, plus strand or + strand) nucleic acid sequence encoding RNA and/or protein.
  • the nucleic acid sequence contained in the single-stranded DNA is substantially identical to the coding sequence of the protein of interest (e.g., the sequence of the mRNA encoding the protein of interest) that it expresses.
  • the single-stranded DNA comprises antisense nucleic acid sequences encoding RNA and/or proteins.
  • the antisense strand nucleic acid sequence is substantially complementary to the sense strand sequence (eg, the nucleic acid sequence encoding the protein of interest).
  • the antisense strand nucleic acid sequence is fully complementary to the sense strand sequence.
  • the single-stranded DNA described in this application may also contain other elements, such as promoter sequences (eg, CMV promoter), enhancer sequences and/or polyA sequences (eg, SV40 polyA), and the like.
  • promoter sequences eg, CMV promoter
  • enhancer sequences eg, enhancer sequences
  • polyA sequences eg, SV40 polyA
  • At least one transfection agent is also included.
  • transfection generally refers to the process or action of transferring nucleic acid (eg, exogenous nucleic acid) from outside the cell membrane into the cell membrane, and can also refer to the introduction of nucleic acid (eg, exogenous nucleic acid) into a cell (eg, exogenous nucleic acid) , processes in eukaryotic cells, such as mammalian cells).
  • transfection includes, for example, “stable transfection” and “transient transfection”.
  • Stable transfection generally refers to the introduction and integration of exogenous nucleic acid (eg, DNA) into the genome of a transfected cell.
  • Transient transfection generally refers to the introduction into a cell of exogenous nucleic acid (eg, DNA) that is not integrated into the genome of the transfected cell. Nucleic acid transfection can be performed by various means, such as electroporation, use of transfection reagents, and the like.
  • transfection reagent may refer to a substance (eg, a compound) used to mediate nucleic acid entry into a cell.
  • transfection reagents can include, but are not limited to, cationic liposomes, lipids, polyamines, calcium phosphates, polyethyleneimine, polylysine, and/or combinations thereof.
  • the transfection reagent may have a positive charge that binds to the negative charge of the nucleic acid.
  • cationic liposomes or polylysine complexes have a net positive charge that enables them to bind DNA or RNA.
  • the transfection reagent may comprise a polymer, such as a DNA polycation complex.
  • cationic proteins such as histones, protamines or synthetic polymers such as polylysine, polyarginine, DEAE dextran, polyphenylene ether, polyethyleneimine, etc. may be included.
  • the transfection reagent comprises a cationic lipid, eg, a cationic liposome, such as Lipofectamine 2000 or Lipofectamine 3000, and the like.
  • transfection reagents can include, for example, pH sensitive lipids, amphiphilic compounds, other liposomes, and the like.
  • the transfection reagent may include DOTMA (dioleoylpropyltrimethylammonium chloride), DOPE (dioleoylphosphatidylethanolamine), DOSPA (dimethyl-2,3-trifluoroacetate) Dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium), DOGS(N-(2-sperminecarboxyl)-N',N'-dioctadecylglycine amide) and/or other liposome-forming substances.
  • DOTMA dioleoylpropyltrimethylammonium chloride
  • DOPE dioleoylphosphatidylethanolamine
  • DOSPA dimethyl-2,3-trifluoroacetate
  • the composition further comprises one or more cell culture media.
  • it may comprise MEM medium (minimum essential medium, minimal essential medium, or low-quantity Eagle medium) and the like.
  • the amount of the medium can be, for example, at least about 20 ⁇ l, at least about 30 ⁇ l, at least about 40 ⁇ l, at least about 50 ⁇ l, at least about 60 ⁇ l, at least about 70 ⁇ l, at least about 80 ⁇ l, at least about 90 ⁇ l, at least about 100 ⁇ l, at least about 110 ⁇ l , at least about 120 ⁇ l, at least about 130 ⁇ l, at least about 140 ⁇ l, at least about 150 ⁇ l, at least about 160 ⁇ l, at least about 170 ⁇ l, at least about 180 ⁇ l, at least about 190 ⁇ l, at least about 200 ⁇ l, at least about 300 ⁇ l, at least about 400 ⁇ l, at least about 500 ⁇ l, at least about About 600 ⁇ l or more.
  • the single-stranded DNA (eg, circular single-stranded DNA, or linear single-stranded DNA) is present in the composition in an amount of about 10 ng to about 1500 ng.
  • the concentration of the single-stranded DNA (eg, circular single-stranded DNA, or linear single-stranded DNA) in the composition is from about 0.1 nM to about 5 nM.
  • the concentration of the single-stranded DNA is from about 0.1 nM to about 5 nM.
  • the application provides a kit that can include a composition (eg, a transfection composition) as described herein.
  • a composition eg, a transfection composition
  • the present application provides a kit.
  • the kit may comprise: single-stranded DNA (eg, single-stranded DNA described herein), at least one transfection reagent (eg, transfection reagent described herein), and instructions for use (eg, instructions for use) .
  • the instructions for use may describe administering the single-stranded DNA to the transfection reagent in a mass ratio of about 5 to about 0.1 to a target cell to deliver at least a portion of the nucleic acid contained in the single-stranded DNA to the target in cells.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 4.5 to about 0.5, about 4 to about 1, about 3.5 to about 1.5, about 3 to about 0.5, about 2.5 to about 0.1, about 2 to about 0.1, about 1.5 to about 0.1, about 1 to about 0.1, about 1 to about 0.2, about 1.5 to about 0.2, about 1.5 to about 0.25, about 1.5 to about 0.3, about 1 to about 0.25, about 1.25 to about 0.2, about 0.75 to about 0.25, about 0.5 to about 0.3, etc.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 100 to about 0.1, about 50 to about 0.1, about 20 to about 0.1, about 10 to about 0.1, about 5 to about 0.1, about 100 to about 0.5, about 50 to about 0.5, about 20 to about 0.5, about 10 to about 0.5, about 5 to about 0.5, about 100 to about 1, about 50 to about 1, about 20 to about 1, about 10 to about 1, or about 5 to about 1, etc.
  • the instructions for use describe administering the single-stranded DNA to the transfection reagent in a mass ratio of from about 5:1 to about 1:10 to target cells to convert the single-stranded DNA into the single-stranded DNA. At least a portion of the nucleic acid contained is delivered to the target cell.
  • the mass ratio of the single-stranded DNA to the transfection reagent may be about 4:1 to about 1:10, about 3:1 to about 1:10, about 2:1 to about 1:10, about 1 :1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1 :1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 2:1 to about 1:2, about 2 :1 to about 1:3, about 2:1 to about 1:4, about 2:1 to about 1:5, etc.
  • the instructions for use describe that the single-stranded DNA and the transfection reagent are mixed according to the mass ratio or according to the mass ratio to prepare a transfection composition. That is, the single-stranded DNA and the transfection reagent can be mixed according to the ratio described in this application to be a transfection composition (optionally, the transfection composition can also include a buffer or other solvents, such as cell culture media described in this application, etc.). The transfection composition can then be administered to cells or tissues.
  • the single-stranded DNA and the transfection reagent are directly or simultaneously applied to cells or tissues according to the mass ratio or the mass ratio described in this application, without pre-mixing and preparing into a transfection composition.
  • the single-stranded DNA and the at least one transfection reagent are included in the kit in a manner that they do not mix with each other.
  • the single-stranded DNA and the at least one transfection reagent may each be independently present in individually packaged containers.
  • the kit includes 2 or more containers packaged separately from each other, each of which contains the single-stranded DNA and the transfection reagent of the present application, and optionally other reagents (eg, the present invention). cell culture medium described in the application, etc.).
  • the content of the single-stranded DNA may be about 10 ng to about 1500 ng.
  • the concentration of the single-stranded DNA may be about 0.1 nM to about 5 nM.
  • the concentration of the single-stranded DNA may be about 0.1 nM to about 5 nM.
  • the single-stranded DNA may comprise nucleic acid sequences encoding one or more RNAs and/or proteins.
  • the single-stranded DNA can be transcribed/reverse transcribed into RNA molecules (eg, miRNA, mRNA, or other RNA molecules) as a template within a cell.
  • the single-stranded DNA can be converted to double-stranded DNA, which can exist either free in the cell or integrated into the genome of the cell (eg, in a random insertion manner, or integrated into a specific site). point).
  • the single-stranded DNA can be transcribed and translated to express the encoded target protein (eg, a therapeutic protein, a labeled protein such as a fluorescent protein or a light-emitting protein, etc.).
  • the single-stranded DNA may comprise nucleic acid sequences encoding two or more RNAs and/or proteins.
  • the single-stranded DNA can comprise a first coding region, which can encode a first RNA or a first protein, and a second coding region, which can encode independently of the first coding region.
  • a second RNA or a second protein that is the same or different from the RNA or the first protein.
  • One or more non-coding regions may be included between the first coding region and the second coding region. In some cases, substantially no non-coding regions are included between the first coding region and the second coding region.
  • the single-stranded DNA may comprise a third coding region or more coding regions. Protein complexes or protein/nucleic acid complexes can be formed between the expression products of the respective coding regions.
  • the single-stranded DNA (eg, single-stranded circular DNA or single-stranded linear DNA) can comprise nucleic acid sequences encoding one or more RNAs and/or proteins (eg, encoding two or more RNAs and/or nucleic acid sequence of a protein), which may comprise at least about 40 nucleotides, such as at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, at least about 900 nucleotides, at least about 950 nucle
  • the single-stranded DNA comprises a sense strand (also referred to as coding strand, sense strand, plus strand or + strand) nucleic acid sequence encoding RNA and/or protein.
  • a sense strand also referred to as coding strand, sense strand, plus strand or + strand
  • the nucleic acid sequence contained in the single-stranded DNA is substantially identical to the coding sequence of the protein of interest it expresses (eg, the sequence of the mRNA encoding the protein of interest).
  • the single-stranded DNA comprises antisense nucleic acid sequences encoding RNA and/or proteins.
  • the antisense strand nucleic acid sequence is substantially complementary to the sense strand sequence (eg, the nucleic acid sequence encoding the protein of interest).
  • the antisense strand nucleic acid sequence is fully complementary to the sense strand sequence.
  • the single-stranded DNA described in this application may also contain other elements, such as promoter sequences (eg, CMV promoter), enhancer sequences and/or polyA sequences (eg, SV40 polyA), and the like.
  • promoter sequences eg, CMV promoter
  • enhancer sequences eg, enhancer sequences
  • polyA sequences eg, SV40 polyA
  • One or more transfection reagents may be included in the kit.
  • transfection reagents can include, but are not limited to, cationic liposomes, lipids, polyamines, calcium phosphates, polyethyleneimine, polylysine, and/or combinations thereof.
  • the transfection reagent may have a positive charge that binds to the negative charge of the nucleic acid.
  • cationic liposomes or polylysine complexes have a net positive charge that enables them to bind DNA or RNA.
  • the transfection reagent may comprise a polymer, such as a DNA polycation complex.
  • cationic proteins such as histones, protamines or synthetic polymers such as polylysine, polyarginine, DEAE dextran, polyphenylene ether, polyethyleneimine, etc. may be included.
  • the transfection reagent comprises a cationic lipid, eg, a cationic liposome, such as Lipofectamine 2000 or Lipofectamine 3000, and the like.
  • transfection reagents can include, for example, pH sensitive lipids, amphiphilic compounds, other liposomes, and the like.
  • the transfection reagent may include DOTMA (dioleoylpropyltrimethylammonium chloride), DOPE (dioleoylphosphatidylethanolamine), DOSPA (dimethyl-2,3-trifluoroacetate) Dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium), DOGS(N-(2-sperminecarboxyl)-N',N'-dioctadecylglycine amide) and/or other liposome-forming substances.
  • DOTMA dioleoylpropyltrimethylammonium chloride
  • DOPE dioleoylphosphatidylethanolamine
  • DOSPA dimethyl-2,3-trifluoroacetate
  • One or more cell culture media may also be included in the kit.
  • it may comprise MEM medium (minimum essential medium, minimal essential medium, or low-quantity Eagle medium) and the like.
  • the amount of the medium can be, for example, at least about 20 ⁇ l, at least about 30 ⁇ l, at least about 40 ⁇ l, at least about 50 ⁇ l, at least about 60 ⁇ l, at least about 70 ⁇ l, at least about 80 ⁇ l, at least about 90 ⁇ l, at least about 100 ⁇ l, at least about 110 ⁇ l , at least about 120 ⁇ l, at least about 130 ⁇ l, at least about 140 ⁇ l, at least about 150 ⁇ l, at least about 160 ⁇ l, at least about 170 ⁇ l, at least about 180 ⁇ l, at least about 190 ⁇ l, at least about 200 ⁇ l, at least about 300 ⁇ l, at least about 400 ⁇ l, at least about 500 ⁇ l, at least about About 600 ⁇ l or more.
  • the present application provides a method of delivering nucleic acid into a cell.
  • the method can include: contaminating the cell with a single-stranded DNA described herein (eg, a circular single-stranded DNA of the present application or a linear single-stranded DNA of the present application) comprising the nucleic acid (eg, nucleic acid to be delivered). DNA) and the transfection reagent described in this application.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may be about 5 to about 0.1.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 4.5 to about 0.5, about 4 to about 1, about 3.5 to about 1.5, about 3 to about 0.5, about 2.5 to about 0.1, about 2 to about 0.1, about 1.5 to about 0.1, about 1 to about 0.1, about 1 to about 0.2, about 1.5 to about 0.2, about 1.5 to about 0.25, about 1.5 to about 0.3, about 1 to about 0.25, about 1.25 to about 0.2, about 0.75 to about 0.25, about 0.5 to about 0.3, etc.
  • the mass ratio of the single-stranded DNA to the transfection reagent is from about 5:1 to about 1:10.
  • the mass ratio of the single-stranded DNA to the transfection reagent may be about 4:1 to about 1:10, about 3:1 to about 1:10, about 2:1 to about 1:10, about 1 :1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1 :1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 2:1 to about 1:2, about 2 :1 to about 1:3, about 2:1 to about 1:4, about 2:1 to about 1:5, etc.
  • the present application provides a method of expressing exogenous RNA and/or exogenous protein in a cell.
  • the method may comprise introducing into the cell a single-stranded DNA encoding the exogenous RNA and/or the exogenous protein.
  • the single-stranded DNA eg, circular single-stranded DNA or linear single-stranded DNA
  • the method further comprises growing the cell under conditions that permit expression of the exogenous RNA and/or the exogenous protein.
  • the present application provides a method of reducing cytotoxicity associated with DNA transfection.
  • the method may include: upon transfection, introducing into the cell the single-stranded DNA described herein instead of the corresponding double-stranded DNA.
  • the present application provides a method of reducing cellular immunogenicity associated with DNA transfection.
  • the method may include: upon transfection, introducing into the cell the single-stranded DNA described herein instead of the corresponding double-stranded DNA.
  • the present application provides a method of increasing the DNA transfection efficiency of cells.
  • the method may include: upon transfection, introducing into the cell the single-stranded DNA described herein instead of the corresponding double-stranded DNA.
  • introducing the single-stranded DNA described in the present application instead of the corresponding double-stranded DNA generally means that substantially no (eg, in an amount that is difficult to accurately detect by conventional methods) to be introduced into the cell Introducing double-stranded DNA (eg, plasmid DNA) with identical or corresponding (eg, complementary or at least 80% homologous, at least 85% homologous, at least 90% homologous) nucleotide sequences, but only by adding the The single-stranded DNA described herein is introduced into a cell to allow expression of the RNA of interest or protein of interest in the cell.
  • double-stranded DNA eg, plasmid DNA
  • identical or corresponding nucleotide sequences eg, complementary or at least 80% homologous, at least 85% homologous, at least 90% homologous
  • introducing the single-stranded DNA into the cell comprises contacting the cell with a composition described herein.
  • introducing the single-stranded DNA eg, circular single-stranded DNA or linear single-stranded DNA
  • introducing the single-stranded DNA comprises: binding the cell to the single-stranded DNA contact with the transfection reagent, and the ratio of the mass ratio of the single-stranded DNA to the transfection reagent is about 5 to about 0.1.
  • the ratio of the mass ratio of the single-stranded DNA to the transfection reagent may range from about 4.5 to about 0.5, about 4 to about 1, about 3.5 to about 1.5, about 3 to about 0.5, about 2.5 to about 0.1, about 2 to about 0.1, about 1.5 to about 0.1, about 1 to about 0.1, about 1 to about 0.2, about 1.5 to about 0.2, about 1.5 to about 0.25, about 1.5 to about 0.3, about 1 to about 0.25, about 1.25 to about 0.2, about 0.75 to about 0.25, about 0.5 to about 0.3, etc.
  • introducing the single-stranded DNA eg, circular single-stranded DNA or linear single-stranded DNA
  • introducing the single-stranded DNA comprises: binding the cell to the single-stranded DNA contact with the transfection reagent, and the mass ratio of the single-stranded DNA to the transfection reagent is about 5:1 to about 1:10.
  • the mass ratio of the single-stranded DNA to the transfection reagent may be about 4:1 to about 1:10, about 3:1 to about 1:10, about 2:1 to about 1:10, about 1 :1 to about 1:10, about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1 :1 to about 1:5, about 1:1 to about 1:4, about 1:1 to about 1:3, about 1:1 to about 1:2, about 2:1 to about 1:2, about 2 :1 to about 1:3, about 2:1 to about 1:4, about 2:1 to about 1:5, etc.
  • the contacting eg, contacting the cells with the single-stranded DNA and a transfection reagent, or contacting the cells with a composition described herein
  • Incubation at about 20°C to about 45°C is included.
  • the incubation is carried out at about 29°C to about 35°C, at about 31°C to about 33°C, or at about 32°C to about 34°C, and the like.
  • the contacting comprises performing the incubation for at least 4 hours, eg, performing the incubation for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours hours, at least 28 hours, at least 29 hours, at least 30 hours, at least 32 hours, at least 34 hours, at least 36 hours, at least 38 hours, at least 40 hours, at least 42 hours, at least 44 hours, at least 45 hours, at least 46 hours, At least 47 hours, at least 48 hours, at least 49 hours, at least 50 hours, at least 51 hours, at least 52 hours, at least 53 hours, at least 54 hours or more, etc.
  • the single-stranded DNA may comprise nucleic acid sequences encoding one or more RNAs and/or proteins.
  • the single-stranded DNA can be transcribed/reverse transcribed into RNA molecules (eg, miRNA, mRNA, or other RNA molecules) as a template within a cell.
  • the single-stranded DNA can be converted to double-stranded DNA, which can exist either free in the cell or integrated into the genome of the cell (eg, in a random insertion manner, or integrated into a specific site). point).
  • the single-stranded DNA can be transcribed and translated to express the encoded target protein (eg, a therapeutic protein, a labeled protein such as a fluorescent protein or a light-emitting protein, etc.).
  • the single-stranded DNA may comprise nucleic acid sequences encoding two or more RNAs and/or proteins.
  • the single-stranded DNA can comprise a first coding region, which can encode a first RNA or a first protein, and a second coding region, which can encode independently of the first coding region.
  • a second RNA or a second protein that is the same or different from the RNA or the first protein.
  • One or more non-coding regions may be included between the first coding region and the second coding region. In some cases, substantially no non-coding regions are included between the first coding region and the second coding region.
  • the single-stranded DNA may comprise a third coding region or more coding regions. Protein complexes or protein/nucleic acid complexes can be formed between the expression products of the respective coding regions.
  • the single-stranded DNA (eg, single-stranded circular DNA or single-stranded linear DNA) can comprise nucleic acid sequences encoding one or more RNAs and/or proteins (eg, encoding two or more RNAs and/or nucleic acid sequence of a protein), which may comprise at least about 40 nucleotides, such as at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides, at least about 500 nucleotides, at least about 550 nucleotides, at least about 600 nucleotides, at least about 650 nucleotides, at least about 700 nucleotides, at least about 750 nucleotides, at least about 800 nucleotides, at least about 850 nucleotides, at least about 900 nucleotides, at least about 950 nucle
  • the single-stranded DNA may comprise a sense strand (also referred to as coding strand, sense strand, positive strand or + strand) nucleic acid sequence encoding RNA and/or protein.
  • a sense strand also referred to as coding strand, sense strand, positive strand or + strand
  • the nucleic acid sequence contained in the single-stranded DNA is substantially identical to the coding sequence of the protein of interest it expresses (eg, the sequence of the mRNA encoding the protein of interest).
  • the single-stranded DNA comprises antisense nucleic acid sequences encoding RNA and/or proteins.
  • the antisense strand nucleic acid sequence is substantially complementary to the sense strand sequence (eg, the nucleic acid sequence encoding the protein of interest).
  • the antisense strand nucleic acid sequence is fully complementary to the sense strand sequence.
  • the nucleotide sequence of the EGFP cssDNA antisense strand sequence (2Ambystoma lateralex x Ambystoma jeffersonianum) of the present application can be as shown in SEQ ID NO:7.
  • the single-stranded DNA described in this application may also contain other elements, such as promoter sequences (eg, CMV promoter), enhancer sequences and/or polyA sequences (eg, SV40 polyA), and the like.
  • promoter sequences eg, CMV promoter
  • enhancer sequences eg, enhancer sequences
  • polyA sequences eg, SV40 polyA
  • transfection reagent can refer to a substance (eg, a compound) used to mediate the entry of nucleic acid into a cell.
  • transfection reagents can include, but are not limited to, cationic liposomes, lipids, polyamines, calcium phosphates, polyethyleneimine, polylysine, and/or combinations thereof.
  • the transfection reagent may have a positive charge that binds to the negative charge of the nucleic acid.
  • cationic liposomes or polylysine complexes have a net positive charge that enables them to bind DNA or RNA.
  • the transfection reagent may comprise a polymer, such as a DNA polycation complex.
  • cationic proteins such as histones, protamines or synthetic polymers such as polylysine, polyarginine, DEAE dextran, polyphenylene ether, polyethyleneimine, etc. may be included.
  • the transfection reagent comprises a cationic lipid, eg, a cationic liposome, such as Lipofectamine 2000 or Lipofectamine 3000, and the like.
  • transfection reagents can include, for example, pH sensitive lipids, amphiphilic compounds, other liposomes, and the like.
  • the transfection reagent may include DOTMA (dioleoylpropyltrimethylammonium chloride), DOPE (dioleoylphosphatidylethanolamine), DOSPA (dimethyl-2,3-trifluoroacetate) Dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium), DOGS(N-(2-sperminecarboxyl)-N',N'-dioctadecylglycine amide) and/or other liposome-forming substances.
  • DOTMA dioleoylpropyltrimethylammonium chloride
  • DOPE dioleoylphosphatidylethanolamine
  • DOSPA dimethyl-2,3-trifluoroacetate
  • the method may also include administering one or more cell culture media.
  • it may involve administration of MEM medium (minimum essential medium, minimal essential medium, or low-quantity Eagle medium) and the like.
  • the amount of the medium can be, for example, at least about 20 ⁇ l, at least about 30 ⁇ l, at least about 40 ⁇ l, at least about 50 ⁇ l, at least about 60 ⁇ l, at least about 70 ⁇ l, at least about 80 ⁇ l, at least about 90 ⁇ l, at least about 100 ⁇ l, at least about 110 ⁇ l , at least about 120 ⁇ l, at least about 130 ⁇ l, at least about 140 ⁇ l, at least about 150 ⁇ l, at least about 160 ⁇ l, at least about 170 ⁇ l, at least about 180 ⁇ l, at least about 190 ⁇ l, at least about 200 ⁇ l, at least about 300 ⁇ l, at least about 400 ⁇ l, at least about 500 ⁇ l, at least about About 600 ⁇ l or more.
  • the culture medium can be incubated with the single-stranded DNA of the present application and/or the transfection reagent of the present application (eg, incubation for at least about 5 min, at least about 10 min, at least about 12 min, at least about 13 min, at least about 14 min, at least about 15 min, at least about 20 min, at least about 25 min, at least about 30 min, at least about 35 min or longer, etc.), for example, incubating at about 20°C to about 45°C.
  • the transfection reagent of the present application eg, incubation for at least about 5 min, at least about 10 min, at least about 12 min, at least about 13 min, at least about 14 min, at least about 15 min, at least about 20 min, at least about 25 min, at least about 30 min, at least about 35 min or longer, etc.
  • the incubation is carried out at about 29°C to about 35°C, at about 31°C to about 33°C, or at about 32°C to about 34°C, and the like.
  • the single-stranded DNA (eg, circular single-stranded DNA, or linear single-stranded DNA) is used in an amount of about 10 ng to about 1500 ng.
  • the single-stranded DNA (eg, circular single-stranded DNA, or linear single-stranded DNA) is used at a concentration of about 0.1 nM to about 5 nM.
  • a concentration of about 0.1 nM to about 5 nM is used at a concentration of about 0.1 nM to about 5 nM.
  • the amount of the transfection reagent used is from about 10 ng to about 4500 ng.
  • the methods described herein are in vivo methods, in vitro methods, or ex vivo methods.
  • the cells are somatic cells, tumor cells and/or immune cells.
  • the cells can be selected from the group consisting of lung cancer cells, kidney cells, melanoma cells, liver cancer cells, cervical cancer cells, glioma cells, epithelial cells, colon cancer cells, fibroblasts, breast cancer cells, gastric cancer cells and immune cells effector cells.
  • the cells can be selected from the group consisting of: A549 cells, C6 cells, MCF-7 cells, HepG2 cells, CT26 cells, Hela cells, Helf cells, MDCK cells, B16 cells, Hep3B cells, L929 cells, 4T1 cells , 293T cells, MGC803 cells and macrophages.
  • the application provides cells treated by the method of any of the aspects of the application.
  • the single-stranded DNA of the present application is introduced into the cell instead of the corresponding double-stranded DNA.
  • the cells are contacted with a composition of the present application.
  • the single-stranded DNA described herein is introduced into the cell, thereby expressing exogenous RNA and/or exogenous protein.
  • the present application provides organisms (eg, non-human organisms), or components thereof, that can comprise cells described herein (eg, cells treated by the methods of the present application).
  • the moiety is selected from the group consisting of tissues (eg, tumor tissue, etc.), organs, and combinations thereof.
  • the single-stranded DNA of the present application or the compositions described herein can be used to prepare a drug (eg, a nucleic acid drug), eg, as a gene therapy and/or a nucleic acid vaccine.
  • a drug eg, a nucleic acid drug
  • the cells are somatic cells, tumor cells and/or immune cells.
  • the cells are selected from the group consisting of lung cancer cells, kidney cells, melanoma cells, liver cancer cells, cervical cancer cells, glioma cells, epithelial cells, colon cancer cells, fibroblasts, breast cancer cells, Gastric cancer cells and immune effector cells.
  • the cells are selected from the group consisting of: A549 cells, C6 cells, MCF-7 cells, HepG2 cells, CT26 cells, Hela cells, Helf cells, MDCK cells, B16 cells, Hep3B cells, L929 cells, 4T1 cells , 293T cells, MGC803 cells and macrophages.
  • the invention of the present application provides the application of single-stranded DNA in gene transfection, specifically including circular single-stranded DNA generated based on traditional phage vectors and linear single-stranded DNA obtained by asymmetric PCR method in gene transfection.
  • the transfection efficiency of circular single-stranded DNA is proportional to the concentration of the transfection reagent, and the same concentration is easier to be delivered by the transfection reagent into cells that are difficult to transfect and express The higher the amount, the lower the cytotoxicity.
  • the circular single-stranded DNA can significantly reduce the cytotoxicity caused by the polymer, so that the transfection efficiency can be effectively improved, and it does not cause obvious cell toxicity.
  • linear single-stranded DNA also proved that In view of the feasibility of gene transfection, single-stranded DNA is used for gene transfection, and the expression of related genes can be seen, so it shows that single-stranded DNA can be used as a carrier for gene transfection for gene transfection.
  • the present application provides the use of single-stranded DNA in gene transfection.
  • the single-stranded DNA can include circular single-stranded DNA generated based on phage vector and linear single-stranded DNA generated by asymmetric PCR; the single-stranded DNA and transfection reagent can be incubated in a cell culture dish for 24-48 hours after incubation .
  • the nucleic acid number of the single-stranded DNA may be 1000-50000 nt.
  • the transfection reagent can be selected from cationic polymers.
  • the transfection reagent is one or more selected from Lipofectamine 2000 or polyetherimide.
  • the preparation method of the circular single-stranded DNA may include the following steps: (1), inserting a gene into a vector to obtain a gene vector; (2), co-transfecting the gene vector and the helper phage vector into competent cells (3) According to the length of the single-stranded DNA, the circular single-stranded DNA of a single band is obtained by culturing for different times.
  • the phage vector may be an M13-based plasmid vector.
  • the preparation method of the linear single-stranded DNA may include the following steps: (a), designing an upstream primer and a downstream primer based on genes; (b), performing a PCR reaction by controlling the ratio of the upstream primer and the downstream primer; (c), combining the The PCR products were separated and purified by agarose gel electrophoresis.
  • the upstream primer sequence can be shown as SEQ ID NO:3, and the downstream primer sequence can be shown as SEQ ID NO:4.
  • the molar ratio of the upstream primer to the upstream primer may be 50:1.
  • the invention of the present application provides an application of single-stranded DNA (ssDNA) in gene transfection, specifically, inserting a target gene into pScaf based on the circular single-stranded DNA of the pScaf vector
  • ssDNA single-stranded DNA
  • cssDNA circular single-stranded DNA
  • Linear single-stranded DNA (lssDNA) was generated by conventional asymmetric PCR methods.
  • the generated lssDNA and cssDNA contain sequences such as CMV enhancer and promoter, target gene, SV40polyA, etc., which reduces the introduction of other irrelevant sequences, thus minimizing the length of DNA fragments.
  • the present application provides the application of single-stranded DNA in gene transfection.
  • the single-stranded DNA can include circular single-stranded DNA generated based on a phage vector and linear single-stranded DNA generated by asymmetric PCR.
  • the single-stranded DNA and the transfection reagent are incubated in a cell culture dish for 24-48 hours. The obvious expression of the target protein can be seen.
  • the number of nucleic acids of the single-stranded DNA can be synthesized in a length between 1000-50000 nt.
  • transfection reagents can be selected from cationic polymers such as Lipofectamine 2000 (lip 2000) or polyetherimide (PEI).
  • the preparation method based on the cssDNA produced by the pScaf vector may include the following steps: (1), inserting the target gene into the pScaf vector through restriction enzyme cleavage site ligation to obtain the pScaf-target gene vector; (2), adding 10-20ng of pScaf - The target gene vector and 20-40ng of M13-based helper phage vector (helper plasmid pSB4423) were co-transfected into 100 ⁇ L XL1-Blue competent cells, and the culture temperature was 30 °C; (3), pick a single clone in 1 mL medium Activated in medium for 1h, then transferred to 5mL liquid medium, and expanded at 30°C for 4-5h; (4), the bacterial liquid obtained in step (3) was cultured in 200mL 2 ⁇ YT medium for 8-24h, according to the purpose Gene length, control culture time to obtain the target product of single band, no heteroband; (5), utilize nanodrop to measure the concentration of target product
  • the target gene may include EGFP gene and EGFP-mCherry gene.
  • the EGFP gene sequence can be shown as SEQ ID NO: 1.
  • the EGFP-mCherry gene sequence can be shown as SEQ ID NO:2.
  • the restriction sites can be KpnI (GGTACC) and BamHI (GGATCC).
  • the pScaf-target gene vector can be used to integrate the M13 replicon with the initiation site and termination site of the single-stranded DNA, thereby reducing the generation of by-products.
  • step (4) of the method the DNA obtained by controlling the incubation time can be confirmed as circular single-stranded DNA by S1 nuclease verification.
  • the preparation method of lssDNA based on traditional asymmetric PCR may include the following steps: (a), using the pEGFP-N1 plasmid as a template, using the PrimerSTAR (R045A, TAKARA) enzyme of Takara Company, the designed upstream primer and downstream primer Carry out PCR reaction; (b), carry out agarose gel electrophoresis on the obtained PCR product; (c), cut out the target band according to the DNA marker position; (d), recover target DNA; (e), utilize nanodrop to detect lssDNA (f), using the obtained lssDNA for gene transfection.
  • the EGFP gene sequence can be as shown in SEQ ID NO:1.
  • the upstream primer sequence can be shown as SEQ ID NO:3
  • the downstream primer sequence can be shown as SEQ ID NO:4.
  • the molar ratio of upstream primer to upstream primer can be 50:1.
  • the beneficial effects of the invention of the present application may include: the invention of the present application applies ssDNA in gene transfection, which is different from the traditional transfection of circular double-stranded plasmids, the transfection efficiency of cssDNA, the concentration of DNA and the concentration of transfection reagents In direct proportion, the same concentration of cssDNA is more easily delivered into more difficult-to-transfect cells by the transfection reagent and the expression is higher, resulting in lower cytotoxicity, and when transfected with commercial cationic polymer transfection reagents, cssDNA It can significantly reduce the cytotoxicity caused by the polymer, so that the transfection efficiency can be effectively improved, and it does not cause obvious cytotoxicity; in addition, lssDNA also proves the feasibility of gene transfection, so it is proved that ssDNA can be used as a gene Potential vectors for transfection and gene delivery.
  • the expression vector was constructed and circular single-stranded DNA was obtained by the following steps:
  • the pScaf vector (Addgene plasmid#111401) was subjected to restriction enzyme digestion by KpnI and BamHI restriction enzymes, and then the EGFP gene fragment and EGFP-mCherry gene fragment obtained by PCR amplification were respectively connected to the pScaf vector to obtain pScaf-EGFP vector and pScaf-mCherry-EGFP vector (as shown in Figure 1);
  • step 3) The culture in step 3) was inoculated into 5 mL of 2 ⁇ YT (containing 5 mM MgCl 2 , 100 ⁇ g/mL ampicillin sodium, 20 ⁇ g/mL chloramphenicol) medium, and cultured at 30° C. and 220 rpm for 4 hours ;
  • step 4) The culture of step 4) was inoculated into 200mL 2 ⁇ YT medium, and the culture was continued under the conditions of 30°C and 220rpm, wherein pScaf-EGFP was cultured for 8 hours, and pScaf-EGFP-mCherry was cultured for 12 hours;
  • step 6) Centrifuge the culture in step 5) at 8000 ⁇ rcf for 10 min at 4°C, and collect the supernatant;
  • step 6 Add 3.2g PEG-8000 and 2.4g NaCl to the supernatant obtained in step 6), mix well, and place in an ice-water bath for 30min;
  • step 8) Centrifuge the mixed solution of step 7) at 10,000 ⁇ rcf and 4°C for 30min to obtain a white precipitate;
  • step 10) Repeat step 9) three times to remove residual bacteria
  • step 11 Add 3 mL of lysis solution (0.2mol/L NaOH, 1% SDS) to the solution obtained in step 10), mix up and down, and lyse at room temperature for 5 min;
  • step 14 Add 2 times the volume of absolute ethanol to the supernatant obtained in step 13), and place the mixture at -20°C for precipitation overnight;
  • step 15 Centrifuge the solution obtained in step 14) at 10,000 ⁇ rcf at 4°C for 30 min to obtain EGFP circular single-stranded DNA (EGFP cssDNA) (its nucleic acid sequence is shown in SEQ ID NO: 5) and EGFP-mCherry ring Shaped single-stranded DNA (EGFP-mCherry cssDNA) (its nucleic acid sequence is shown in SEQ ID NO: 6);
  • S1 nuclease is extracted from Aspergill suoryzae and can specifically degrade single-stranded DNA.
  • the EGFP cssDNA and the P7560 positive control bacteria circular single-stranded DNA
  • the double-stranded plasmid plDNA cannot be degraded.
  • the EGFP cssDNA prepared in this example is single-stranded DNA.
  • the EGFP-mCherry cssDNA was also single-stranded DNA.
  • the first lane shows 5kb DNA marker
  • the second lane is EFGP CssDNA without S1 nuclease treatment
  • the third lane is EFGP CssDNA treated with S1 nuclease
  • the fourth lane is P7560 without S1 nuclease treatment
  • the 5th lane is P7560 treated with S1 nuclease
  • the 6th lane is EGFP plasmid double-stranded DNA without S1 nuclease treatment
  • the 7th lane is EGFP plasmid double-stranded DNA treated with S1 nuclease.
  • Example 2 Delivery of the single-stranded DNA of Example 1 into cells by electroporation and detection of its expression
  • the single-stranded DNA is delivered into cells by the following steps:
  • Example 2 20 ⁇ g of the EGFP cssDNA obtained in Example 1 was added to the electroporation cuvette.
  • the expression of the EGFP cssDNA in various cell lines was verified. Observation after 24 hours of culture revealed that EGFP cssDNA could be expressed in these cell lines (results are shown in Figures 2A-2B). Fluorescent expression images of EGFP cssDNA in each cell line are shown in Figure 2A. Figure 2B shows the electroporation transfection/expression efficiency of EGFP cssDNA in each cell line. It can be seen that the circular single-stranded DNA of the present application can effectively express the target protein in various cell lines.
  • Example 3 Delivery of the single-stranded DNA of Example 1 into cells using transfection reagents and detection of its expression
  • the single-stranded DNA is delivered into cells by the following steps:
  • Pre-seeded cells (1 ⁇ 10 5 cells) in a 24-well plate and cultured in an incubator for 12 hours.
  • EGFP cssDNA sense strand or antisense strand
  • EGFP-mCherry cssDNA were mixed with transfection reagents, respectively, in various cell lines (eg, A549 cells, C6 cells, MCF-7 cells, HepG2 cells, CT26 cells, Hela cells, Helf cells and MDCK cells).
  • Figure 4A shows the expression of EGFP protein after transfection of EGFP-mCherry cssDNA.
  • Figure 4B shows the expression of mCherry protein after transfection of EGFP-mCherry cssDNA.
  • Figure 4C shows the superposition of EGFP protein expression and mCherry protein expression, showing the co-expression of the two.
  • Nucleic acid transfection was performed by the following steps (different DNA:transfection reagent ratios were tested for differences in transfection efficiency):
  • Ratio 1 Add 0.5 ⁇ g of cationic polymer (Lipofectamine 2000) to 50 ⁇ L of MEM medium, mix well at room temperature for 5 min. Subsequently, 1.3 nM EGFP cssDNA (in 50 ⁇ L MEM medium, about 460 ng) and 1.3 nM of the corresponding double-stranded plasmid plDNA (in 50 ⁇ L MEM medium, about 460 ng) were cationically polymerized with the prepared 0.5 ⁇ g (Lipofectamine2000, which has been mixed in 50 ⁇ L MEM medium) to obtain a uniformly mixed 100 ⁇ L transfection composition, which was placed at room temperature for 15 min.
  • cationic polymer Lipofectamine 2000
  • Ratio 2 Add 1 ⁇ g of cationic polymer (Lipofectamine 2000) to 50 ⁇ L of MEM medium, mix well at room temperature and place for 5 minutes. Subsequently, 1.3 nM EGFP cssDNA (in 50 ⁇ L MEM medium, about 460 ng) and 1.3 nM of the corresponding double-stranded plasmid plDNA (in 50 ⁇ L MEM medium, about 460 ng) were mixed with the prepared 1 ⁇ g cationic polymer, respectively. (Lipofectamine2000, which has been mixed in 50 ⁇ L of MEM medium) and mixed to obtain 100 ⁇ L of the mixed transfection composition, which was placed at room temperature for 15 min.
  • Lipofectamine2000 which has been mixed in 50 ⁇ L of MEM medium
  • Ratio 3 Add 2 ⁇ g of cationic polymer (Lipofectamine 2000) to 50 ⁇ L of MEM medium, mix well at room temperature for 5 minutes. Subsequently, 1.3 nM EGFP cssDNA (in 50 ⁇ L MEM medium, about 460 ng) and 1.3 nM of the corresponding double-stranded plasmid plDNA (in 50 ⁇ L MEM medium, about 460 ng) were mixed with the prepared 2 ⁇ g cationic polymer, respectively. (Lipofectamine2000, which has been mixed in 50 ⁇ L of MEM medium) and mixed to obtain 100 ⁇ L of the mixed transfection composition, which was placed at room temperature for 15 min.
  • Lipofectamine2000 which has been mixed in 50 ⁇ L of MEM medium
  • the transfection efficiency and expression of the EGFP cssDNA in various cell lines were verified.
  • various cell lines eg, A549 cells, B16 cells, and HepG2 cells, including more difficult-to-transfect cell lines
  • the transfection efficiency of single-stranded DNA is significantly higher than that of double-stranded plasmid DNA.
  • the transfection efficiency of single-stranded DNA can be improved by using a higher ratio of transfection reagents (the results are shown in Figures 5-7).
  • Figure 5 shows the transfection effect in A549 cells
  • Figure 6 shows the transfection effect in the B16 cell line
  • Figure 7 shows the transfection effect in the HepG2 cell line.
  • MDCK cells into a 96-well plate Seeding MDCK cells into a 96-well plate: plating at a density of 5 ⁇ 10 3 cells/well, adding about 100 ⁇ L of cell suspension to each well, and repeating the same sample 5 times.
  • the cytotoxicity of the single-stranded DNA combination with a large number of transfection reagents when transfecting cells was verified and compared with the corresponding double-stranded plasmid DNA transfection composition.
  • pEGFP-N1 plasmid (Addgene plasmid#54767): 2 ⁇ L (10ng/ ⁇ L), upstream primer: 2 ⁇ L (0.2 ⁇ M), downstream primer: 2 ⁇ L (10 ⁇ M), PrimeSTAR Max Premix (25 ⁇ L, R045A, Takara ); ddH2O: 19 ⁇ L.
  • pEGFP-N1 plasmid 2 ⁇ L (10 ng/ ⁇ L), upstream primer: 2 ⁇ L (10 ⁇ M), downstream primer: 2 ⁇ L (0.2 ⁇ M), PrimeSTAR Max Premix (25 ⁇ L, R045A, Takara); ddH2O: 19 ⁇ L.
  • upstream primer sequence in forward product amplification and reverse product amplification is shown in SEQ ID NO: 3
  • downstream primer sequence is shown in SEQ ID NO: 4.
  • 95°C for 10 min 95°C for 30s, 60°C for 30s, 72°C for 1min (including 35 cycles of 95°C for 30s, 60°C for 30s, and 72°C for 1 min), and 72°C for 10 min.
  • linear single-stranded DNA can also express the target protein product in cells after transfection.
  • results of this example show that single-stranded DNA has lower immunostimulatory activity on macrophages at the cellular level than the corresponding double-stranded plasmid plDNA.
  • mice 20 female mice were randomly divided into 4 groups, 5 mice in each group, which were control group, Lipofectamine2000 administration group, EGFP cssDNA and Lipofectamine2000 complex administration group and double-stranded plasmid plDNA Lipofectamine2000 complex administration group. Group.
  • the control group was given 100 ⁇ L of normal saline
  • the liposome administration group was given 100 ⁇ L Lipofectamine2000 solution of equal mass
  • the single-stranded DNA administration group was given 9 ⁇ g EGFP cssDNA per tail vein.
  • the plDNA administration group was injected with 18 ⁇ g plDNA/tail vein.
  • results of this example show that single-stranded DNA is less immunostimulatory in vivo than the corresponding double-stranded plasmid plDNA, indicating lower side effects, better safety in clinical practice, and Greater clinical application value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种组合物，其包含单链DNA以及至少一种转染试剂，所述组合物中所述单链DNA与所述转染试剂质量比的比值为约100至约0.1。还提供了使用所述组合物递送核酸的方法和系统。

Description

核酸递送方法及系统 技术领域
本申请涉及核酸递送方法及系统。特别地，本申请涉及通过核酸递送来表达外源RNA和/或外源蛋白的方法及系统。
背景技术
传统基因递送是指利用递送载体或者特定技术，将质粒DNA(plDNA)或RNA等核酸分子导入到细胞或者机体中的过程。
然而，使用双链的plDNA来递送核酸常常引入来自宿主的序列，造成非目标蛋白表达。此外，外源基因表达效率往往不理想，对于比较长的核酸分子尤其如此。
另一方面，RNA分子不易获取，稳定性差且容易降解，对于递送和表达外源基因来说也并不理想。
因此，亟需更加安全和高效的外源核酸递送手段。
发明内容
本申请提供了一种核酸递送的方法和系统。本申请的发明人惊讶地发现，利用本申请的方法和系统来递送外源核酸分子，获得了以下的一种或多种优势(例如，如传统的双链质粒DNA递送系统相比)：
有更高的递送效率(例如，更高的细胞转染效率)；
有更高的表达量；
能够有效递送更长的核酸分子，从而表达例如，两个或更多个外源蛋白；
能够降低与DNA转染相关的细胞毒性；和/或
能够降低与DNA转染相关的机体免疫反应，进而增加安全性。
一方面，本申请提供了一种组合物(例如，转染组合物)。所述组合物可包含：单链DNA以及至少一种转染试剂。在所述组合物中，所述单链DNA与所述转染试剂质量比的比值可以为约100至约0.1。
在某些实施方式中，所述组合物中所述单链DNA与所述转染试剂的质量比为约100:1至约1:10。
在某些实施方式中，所述组合物中所述单链DNA与所述转染试剂质量比的比值为约1至约0.2。
在某些实施方式中，所述组合物中所述单链DNA与所述转染试剂的质量比为约2:1至约1:5。
在某些实施方式中，所述单链DNA为环状单链DNA。
在某些实施方式中，所述单链DNA为线性单链DNA。
在某些实施方式中，所述单链DNA包含编码一种或多种RNA和/或蛋白质的核酸序列。
在某些实施方式中，所述单链DNA包含编码两种或更多种RNA和/或蛋白质的核酸序列。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的正义链核酸序列。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。
在某些实施方式中，所述单链DNA包含至少40个核苷酸。
在某些实施方式中，所述单链DNA包含至少1500个核苷酸。
在某些实施方式中，所述转染试剂包含阳离子脂质和/或阳离子聚合物。
在某些实施方式中，所述转染试剂包含阳离子脂质体。
在某些实施方式中，所述组合物还包含一种或多种细胞培养基。
在某些实施方式中，所述组合物中所述单链DNA的含量为约10ng至约1000ng。
在某些实施方式中，所述组合物中所述单链DNA的浓度为约0.5nM至约3nM。
另一方面，本申请提供了一种试剂盒，其可包含本申请所述的组合物(例如，转染组合物)。
另一方面，本申请提供了一种试剂盒。所述试剂盒可包含：单链DNA，至少一种转染试剂，以及使用说明(例如使用说明书)。所述使用说明可记载将所述单链DNA与所述转染试剂按照约100至约0.1的质量比比值施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
在某些实施方式中，所述使用说明记载了将所述单链DNA与所述转染试剂按照约5:1至约1:10的质量比施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
在某些实施方式中，所述使用说明记载了将所述单链DNA与所述转染试剂按照约10至约0.1的质量比比值施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
在某些实施方式中，所述使用说明记载了将所述单链DNA与所述转染试剂按照约2:1至约1:5的质量比施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
在某些实施方式中，所述使用说明记载了将所述单链DNA与所述转染试剂按照所述质量比比值或者按照所述质量比混合配制为转染组合物。
在某些实施方式中，所述单链DNA与所述至少一种转染试剂以彼此不互相混合的方式被包含于所述试剂盒中。
在某些实施方式中，在所述试剂盒中，所述单链DNA与所述至少一种转染试剂各自独立地存在于单独包装的容器中。
在某些实施方式中，所述试剂盒中，所述单链DNA的含量为约10ng至约1000ng。
在某些实施方式中，所述试剂盒中，所述单链DNA的浓度为约0.5nM至约3nM。
在某些实施方式中，所述单链DNA为环状单链DNA。
在某些实施方式中，所述单链DNA为线性单链DNA。
在某些实施方式中，所述单链DNA包含编码一种或多种RNA和/或蛋白质的核酸序列。
在某些实施方式中，所述单链DNA包含编码两种或更多种RNA和/或蛋白质的核酸序列。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的正义链核酸序列。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。
在某些实施方式中，所述试剂盒中的所述单链DNA包含至少40个核苷酸。
在某些实施方式中，所述试剂盒中的所述单链DNA包含至少1500个核苷酸。
在某些实施方式中，所述试剂盒中的所述转染试剂包含阳离子脂质和/或阳离子聚合物。
在某些实施方式中，所述试剂盒中的所述转染试剂包含阳离子脂质体。
在某些实施方式中，所述试剂盒中还包含一种或多种细胞培养基。
另一方面，本申请提供了一种向细胞中递送核酸的方法。所述方法可包括：使所述细胞与包含所述核酸的单链DNA及转染试剂接触。所述单链DNA与所述转染试剂质量比的比值可以为约5至约0.1。
在该方法的某些实施方式中，所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。
在该方法的某些实施方式中，所述单链DNA与所述转染试剂质量比的比值为约1至约0.2。
在该方法的某些实施方式中，所述单链DNA与所述转染试剂的质量比为约2:1至约1:5。
另一方面，本申请提供了一种在细胞内表达外源RNA和/或外源蛋白质的方法。所述方法可包括：向所述细胞中引入编码所述外源RNA和/或所述外源蛋白质的单链DNA。
在该方法的某些实施方式中，所述方法还包括使所述细胞在允许所述外源RNA和/或所述外源蛋白质表达的条件下生长。
另一方面，本申请提供了一种降低与DNA转染有关的细胞毒性的方法。所述方法可包括：转染时，向所述细胞中引入单链DNA而非相应的双链DNA。
另一方面，本申请提供了一种降低与DNA转染有关的细胞免疫原性的方法。所述方法可包括：转染时，向所述细胞中引入单链DNA而非相应的双链DNA。
另一方面，本申请提供了一种提高细胞的DNA转染效率的方法。所述方法可包括：转染时，向所述细胞中引入单链DNA而非相应的双链DNA。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA包括：使所述细胞与本申请所述的组合物接触。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂质量比的比值为约5至约0.1。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂质量比的比值为约1至约0.2。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂的质量比为约2:1至约1:5。
在本申请所述方法的某些实施方式中，所述接触(例如，使所述细胞与所述单链DNA及转染试剂接触，或者使所述细胞与本申请所述的组合物接触)包括在约25℃至约40℃下进行孵育。
在本申请所述方法的某些实施方式中，所述接触包括在约37℃下进行孵育。
在本申请所述方法的某些实施方式中，所述接触包括孵育至少4小时。
在本申请所述方法的某些实施方式中，所述单链DNA为环状单链DNA。
在本申请所述方法的某些实施方式中，所述单链DNA为线性单链DNA。
在本申请所述方法的某些实施方式中，所述单链DNA包含编码一种或多种外源RNA和/或外源蛋白质的核酸序列。
在本申请所述方法的某些实施方式中，所述单链DNA包含编码两种或更多种外源RNA 和/或外源蛋白质的核酸序列。
在本申请所述方法的某些实施方式中，所述单链DNA包含编码外源RNA和/或外源蛋白质的正义链核酸序列。
在本申请所述方法的某些实施方式中，所述单链DNA包含编码外源RNA和/或外源蛋白质的反义链核酸序列。
在本申请所述方法的某些实施方式中，所述单链DNA包含至少40个核苷酸。
在本申请所述方法的某些实施方式中，所述单链DNA包含至少1500个核苷酸。
在本申请所述方法的某些实施方式中，所述单链DNA的含量为约10ng至约1000ng。
在本申请所述方法的某些实施方式中，所述单链DNA的浓度为约0.5nM至约3nM。
在本申请所述方法的某些实施方式中，所述转染试剂包含阳离子脂质和/或阳离子聚合物。
在本申请所述方法的某些实施方式中，所述转染试剂包含阳离子脂质体。
在某些实施方式中，本申请所述的方法为体内方法、体外方法或离体方法。
在本申请所述方法的某些实施方式中，所述细胞为体细胞，肿瘤细胞和/免疫细胞。
在本申请所述方法的某些实施方式中，所述细胞选自：肺癌细胞，肾细胞，黑色素瘤细胞，肝癌细胞，宫颈癌细胞，胶质瘤细胞，上皮细胞，结肠癌细胞，成纤维细胞，乳腺癌细胞，胃癌细胞和免疫效应细胞。
在本申请所述方法的某些实施方式中，所述细胞选自：A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞,MDCK细胞，B16细胞，Hep3B细胞，L929细胞，4T1细胞，293T细胞，MGC803细胞和巨噬细胞。
另一方面，本申请提供了经本申请中任一个方面的方法处理而得到的细胞。
另一方面，本申请提供了生物体(例如，非人生物体)或其组成部分，其可包含本申请所述的细胞(例如，经本申请的方法处理的细胞)。
在某些实施方式中，所述组成部分选自：组织、器官及其组合。
另一方面，本申请提供了单链DNA用于制备组合物(例如转染组合物，如细胞转染组合物，或者核酸递送组合物)的用途。所述组合物可用于下述的一项或多项：将所述单链DNA中包含的至少部分核酸递送至细胞内；在细胞内表达由所述单链DNA编码的外源RNA或外源蛋白；提高细胞的DNA转染效率；降低与DNA转染有关的细胞毒性；和/或降低与DNA转染有关的细胞免疫原性。所述组合物可以是本申请的任一方面所述的组合物。
在某些实施方式中，所述细胞为体细胞，肿瘤细胞和/免疫细胞。
在某些实施方式中，所述细胞选自：肺癌细胞，肾细胞，黑色素瘤细胞，肝癌细胞，宫颈癌细胞，胶质瘤细胞，上皮细胞，结肠癌细胞，成纤维细胞，乳腺癌细胞，胃癌细胞和免疫效应细胞。
在某些实施方式中，所述细胞选自：A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞,MDCK细胞，B16细胞，Hep3B细胞，L929细胞，4T1细胞，293T细胞，MGC803细胞和巨噬细胞。
本领域技术人员能够从下文的详细描述中容易地洞察到本公开的其它方面和优势。下文的详细描述中仅显示和描述了本公开的示例性实施方式。如本领域技术人员将认识到的，本公开的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示了本申请中示例性载体pScaf-EGFP(+)、pScaf-EGFP(-)及pScaf-EGFP-mCherry的结构和构建流程。
图2A-2B显示了本申请的示例性单链DNA在各细胞系中的转染效率和蛋白表达情况。
图3显示了本申请的示例性单链DNA在各细胞系中的蛋白表达情况。
图4A-4C显示了本申请的示例性单链DNA转染MDCK细胞后的蛋白表达情况。
图5显示了本申请的示例性单链DNA与相应的双链质粒DNA相比，转染细胞后的蛋白表达情况。
图6显示了本申请的示例性单链DNA与相应的双链质粒DNA相比，转染细胞后的蛋白表达情况。
图7显示了本申请的示例性单链DNA与相应的双链质粒DNA相比，转染细胞后的蛋白表达情况。
图8显示了本申请的示例性单链DNA与相应的双链质粒DNA相比，转染细胞后产生的细胞毒性影响。
图9显示了本申请的示例性线性单链DNA的电泳验证结果。1kb显示1kb DNA标记，第1泳道为相应的双链DNA,第2泳道为反向产物扩增结果，第3泳道为正向产物扩增结果。
图10显示了本申请的示例性线性单链DNA转染不同细胞系后的表达情况。
图11显示了本申请的示例性单链DNA的DNA酶(S1酶)验证结果。
图12A-12B显示了本申请的示例性单链DNA与质粒双链DNA相比，在引起免疫反应方面的效果。
图13A-13B显示了本申请的示例性单链DNA与质粒双链DNA相比，在引起免疫反应方面的效果。
具体实施方式
以下由特定的具体实施方式说明本申请涉及的发明，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“核酸”、“核酸分子”及“多核苷酸”可互换地使用，通常是指至少两个碱基-糖-磷酸单元的组合。在此，多核苷酸与寡核苷酸的区别在于多核苷酸通常包含超过120个单体单元。在本申请中，所述“核酸”包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。核苷酸是核酸聚合物的单体单元。反义核酸是干扰DNA和/或RNA功能的核酸。天然核酸具有磷酸骨架，人工核酸可能含有其他类型的骨架，但与天然核酸具有相同的碱基。RNA可以是tRNA(转移RNA)、snRNA(小核RNA)、rRNA(核糖体RNA)、mRNA(信使RNA)、反义RNA和/或核糖酶等形式。DNA可以是质粒DNA、病毒DNA、线性DNA、染色体DNA或它们的衍生物的形式。除了这些形式之外，DNA和RNA也可以是单链、双链、三链或四链的形式。该术语还可包括PNA(肽核酸)、硫代磷酸酯和其他天然核酸磷酸骨架的变体。
在本申请中，术语“单链DNA”也被称为“ssDNA”,通常是指主要由一条DNA链构成的核酸分子。DNA能够以双链或单链的形式存在。在双链DNA(dsDNA)中，有两条反向平行的DNA链通过，例如鸟嘌呤-胞嘧啶和腺嘌呤-胸苷对之间的Watson-Crick结合互补(鸟嘌呤-胞嘧啶或腺嘌呤-胸腺嘧啶配对)相互结合。反之，单链(ssDNA)DNA通常仅具有一条DNA链。在这一条DNA链中，有至少两个碱基-糖-磷酸酯的组合，它们彼此共价结合。在某些，情况下，单链DNA可以在自身内部形成环或二级结构，其中一条链的两个或更多个不同部分之间发生Watson-Crick配对。ssDNA也可以包括部分单链结构的DNA，其中DNA可具有双链和单链链段。例如，ssDNA可以具有未与另一条DNA链配对的部分和与另一条DNA链配对的另一部分。ssDNA可以通过多种方法形成。例如，可以通过使dsDNA变性而形成ssDNA。其中，变性是指可以将dsDNA的两条链分开从而减少Watson-Crick配对数量的过程。也可以通过核酸扩增(例如，滚环扩增或不对称扩增等)来形成ssDNA。所述ssDNA可以是 环状的，也可以是线性的。
在本申请中，术语“外源DNA序列”、“外源核酸”和“外源DNA”可互换地使用，通常是指不来源于其所处的宿主的核酸分子。其可以与宿主的DNA相同或者是异源的。例如，外源DNA序列可以是插入表达载体中用于表达目的RNA或蛋白(例如，外源RNA或外源蛋白质)的核酸序列。所述外源DNA序列可以源自各种来源，包括DNA、cDNA、合成DNA和RNA等。所述外源DNA序列可以包含基因组DNA，所述基因组DNA可以包括或不包括天然存在或人工的内含子。此外，所述基因组DNA可以与启动子区域或poly A信号序列相连。所述外源DNA序列包括但不限于下述DNA序列，其可表达产生希望在宿主细胞中表达的基因产物(例如，目的RNA或目的蛋白质)。所述基因产物可以影响宿主细胞的活力或状态。
术语“约”或“大约”通常表示在值的统计学上有意义的范围内。这样的范围可以在一个数量级内，例如可在给定值或范围的上下50％以内，如在上下20％以内，上下15％以内，上下10％以内，或者上下5％以内。术语“约”或“大约”所涵盖的允许偏差取决于所研究的具体系统，并且本领域技术人员可以根据本申请的内容理解。
组合物
一方面，本申请提供了一种组合物(例如，转染组合物)。所述组合物可包含：单链DNA以及至少一种转染试剂。在所述组合物中，所述单链DNA与所述转染试剂质量比的比值的范围可以为约5至约0.1。例如，所述单链DNA与所述转染试剂质量比的比值范围可以为约4.5至约0.5，约4至约1，约3.5至约1.5，约3至约0.5，约2.5至约0.1，约2至约0.1，约1.5至约0.1，约1至约0.1，约1至约0.2，约1.5至约0.2，约1.5至约0.25，约1.5至约0.3，约1至约0.25，约1.25至约0.2，约0.75至约0.25，约0.5至约0.3等。例如，所述单链DNA与所述转染试剂质量比的比值范围可以为约100至约0.1、约50至约0.1、约20至约0.1、约10至约0.1、约5至约0.1、约100至约0.5、约50至约0.5、约20至约0.5、约10至约0.5、约5至约0.5、约100至约1、约50至约1、约20至约1、约10至约1、或约5至约1等。
在某些实施方式中，所述组合物中所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。其中，约5:1至约1:10的意思是指，所述单链DNA的质量相对于所述转染试剂的质量由约5:1逐渐减少，直至二者的质量基本上相同(即二者质量比为约1:1)，而后所述转染试剂的相对质量继续增加，直至所述单链DNA的质量相对于所述转染试剂的质量为约1:10。在某些情形中，所述组合物中所述单链DNA与所述转染试剂的质量比为约4:1至约1:10，约 3:1至约1:10，约2:1至约1:10，约1:1至约1:10，约1:1至约1:9，约1:1至约1:8，约1:1至约1:7，约1:1至约1:6，约1:1至约1:5，约1:1至约1:4，约1:1至约1:3，约1:1至约1:2，约2:1至约1:2，约2:1至约1:3，约2:1至约1:4，约2:1至约1:5等。例如，约100:1至约1:10，约100:1至约1:10，约50:1至约1:10，约20:1至约1:10，约10:1至约1:10，约100:1至约1:5，约100:1至约1:5，约50:1至约1:5，约20:1至约1:5，约10:1至约1:5，约100:1至约1:1，约100:1至约1:1，约50:1至约1:1，约20:1至约1:1，或约10:1至约1:1等。
在某些实施方式中，所述组合物中的所述单链DNA为环状单链DNA，且所述环状单链DNA与所述转染试剂的质量比为约5:1至约1:10。例如，约4:1至约1:10，约3:1至约1:10，约2:1至约1:10，约1:1至约1:10，约1:1至约1:9，约1:1至约1:8，约1:1至约1:7，约1:1至约1:6，约1:1至约1:5，约1:1至约1:4，约1:1至约1:3，约1:1至约1:2，约2:1至约1:2，约2:1至约1:3，约2:1至约1:4，约2:1至约1:5等。例如，约100:1至约1:10，约100:1至约1:10，约50:1至约1:10，约20:1至约1:10，约10:1至约1:10，约100:1至约1:5，约100:1至约1:5，约50:1至约1:5，约20:1至约1:5，约10:1至约1:5，约100:1至约1:1，约100:1至约1:1，约50:1至约1:1，约20:1至约1:1，或约10:1至约1:1等。
在某些实施方式中，所述组合物中的所述单链DNA为线性单链DNA，且所述线性单链DNA与所述转染试剂的质量比为约5:1至约1:10。例如，约4:1至约1:10，约3:1至约1:10，约2:1至约1:10，约1:1至约1:10，约1:1至约1:9，约1:1至约1:8，约1:1至约1:7，约1:1至约1:6，约1:1至约1:5，约1:1至约1:4，约1:1至约1:3，约1:1至约1:2，约2:1至约1:2，约2:1至约1:3，约2:1至约1:4，约2:1至约1:5等。例如，约100:1至约1:10，约100:1至约1:10，约50:1至约1:10，约20:1至约1:10，约10:1至约1:10，约100:1至约1:5，约100:1至约1:5，约50:1至约1:5，约20:1至约1:5，约10:1至约1:5，约100:1至约1:1，约100:1至约1:1，约50:1至约1:1，约20:1至约1:1，或约10:1至约1:1等。
在某些实施方式中，所述单链DNA包含编码一种或多种RNA和/或蛋白质的核酸序列。例如，所述单链DNA可以在细胞内作为模板被转录/反转录为RNA分子(例如，miRNA，mRNA或其他RNA分子)。在某些情形中，所述单链DNA可以被转化为双链DNA，其可以游离存在于细胞中，也可以整合到细胞的基因组中(例如，以随机插入的方式，或者整合到特定的位点处)。所述单链DNA可以经转录和翻译后表达其所编码的目标蛋白质(例如，治疗性蛋白，标记性蛋白如荧光蛋白或发光蛋白等)。
在某些实施方式中，所述单链DNA包含编码两种或更多种RNA和/或蛋白质的核酸序列。例如，所述单链DNA可包含第一编码区和第二编码区，所述第一编码区可以编码第一RNA 或第一蛋白质，所述第二编码区可以独立地编码与所述第一RNA或第一蛋白质相同或不同的第二RNA或者第二蛋白质。所述第一编码区与所述第二编码区之间可以包含一个或多个非编码区。在某些情形中，所述第一编码区与所述第二编码区之间基本上不包含非编码区。在某些情形中，所述单链DNA可包含第三编码区或更多编码区。各个编码区的表达产物之间可以形成蛋白复合物或蛋白/核酸复合物。
例如，所述单链DNA(例如，单链环状DNA或者单链线性DNA)可包含编码一种或多种RNA和/或蛋白质的核酸序列(如编码两种或更多种RNA和/或蛋白质的核酸序列)，其可包含至少约40个核苷酸，例如至少约50个核苷酸，至少约100个核苷酸，至少约200个核苷酸，至少约300个核苷酸，至少约400个核苷酸，至少约450个核苷酸，至少约500个核苷酸，至少约550个核苷酸，至少约600个核苷酸，至少约650个核苷酸，至少约700个核苷酸，至少约750个核苷酸，至少约800个核苷酸，至少约850个核苷酸，至少约900个核苷酸，至少约950个核苷酸，至少约1000个核苷酸，至少约1050个核苷酸，至少约1100个核苷酸，至少约1150个核苷酸，至少约1200个核苷酸，至少约1250个核苷酸，至少约1300个核苷酸，至少约1350个核苷酸，至少约1400个核苷酸，至少约1450个核苷酸，至少约1500个核苷酸，至少约1550个核苷酸，至少约1600个核苷酸，至少约1650个核苷酸，至少约1700个核苷酸，至少约1750个核苷酸，至少约1800个核苷酸，至少约1850个核苷酸，至少约1900个核苷酸，至少约1950个核苷酸，至少约2000个核苷酸，至少约2100个核苷酸，至少约2200个核苷酸，至少约2300个核苷酸，至少约2400个核苷酸，至少约2500个核苷酸，至少约2600个核苷酸，至少约2700个核苷酸，至少约2800个核苷酸，至少约2900个核苷酸，至少约3000个核苷酸，至少约3100个核苷酸，至少约3200个核苷酸，至少约3300个核苷酸，至少约3400个核苷酸，至少约3500个核苷酸，至少约3600个核苷酸，至少约3700个核苷酸，至少约3800个核苷酸，至少约3900个核苷酸，至少约4000个核苷酸，至少约4100个核苷酸，至少约4200个核苷酸，至少约4300个核苷酸，至少约4400个核苷酸，至少约4500个核苷酸，至少约4600个核苷酸，至少约4700个核苷酸，至少约4800个核苷酸，至少约4900个核苷酸，至少约5000个核苷酸，至少约5100个核苷酸，至少约5200个核苷酸，至少约5300个核苷酸，至少约5400个核苷酸，至少约5500个核苷酸，至少约5600个核苷酸，至少约5700个核苷酸，至少约5800个核苷酸，至少约5900个核苷酸，至少约6000个核苷酸或更多。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的正义链(也称为编码链，有义链，正链或+链)核酸序列。例如，所述单链DNA中包含的核酸序列与其所表达的目标 蛋白质的编码序列(例如，编码目标蛋白的mRNA的序列)基本上一致。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。例如，所述反义链核酸序列与正义链序列(例如，目标蛋白质的编码核酸序列)基本上互补。在某些实施方式中，所述反义链核酸序列与正义链序列完全互补。
本申请所述的单链DNA还可以包含其他元件，例如启动子序列(如CMV启动子)，增强子序列和/或polyA序列(如SV40polyA)等。
在本申请的组合物中，还包含至少一种转染剂。
在本申请中，术语“转染”通常是指将核酸(例如，外源核酸)从细胞膜外转移至细胞膜内的过程或动作，也可以指将核酸(例如，外源核酸)引入细胞(例如，真核细胞，如哺乳动物细胞)中的过程。在本申请中，“转染”包括例如，“稳定转染”和“瞬时转染”。“稳定转染”通常是指将外源核酸(例如DNA)引入并整合到被转染细胞的基因组中。“瞬时转染”通常是指将外源核酸(例如DNA)引入细胞中，而该外源核酸未与被转染细胞的基因组整合。可以通过各种方式进行核酸转染，例如电穿孔，使用转染试剂等。
在本申请中，“转染试剂”可以指用于介导核酸进入细胞的物质(例如，化合物)。例如，转染试剂可包括但不限于阳离子脂质体，脂类，聚胺，磷酸钙，聚乙烯亚胺，聚赖氨酸和/或它们的组合。所述转染试剂可以具有与核酸负电荷结合的正电荷。例如，阳离子脂质体或聚赖氨酸复合物具有净的正电荷，使得它们能够与DNA或RNA结合。
例如，所述转染试剂可以包含聚合物，如DNA聚阳离子复合物。其中，可包含阳离子蛋白，如组蛋白、精蛋白或者合成的聚合物，如聚赖氨酸、聚精氨酸、DEAE葡聚糖、聚苯醚、聚乙烯亚胺等。在某些实施方式中，所述转染试剂包含阳离子脂质，例如阳离子脂质体，如Lipofectamine2000或Lipofectamine3000等。
其他可用作转染试剂的物质可包括，例如pH敏感性的脂质，两亲性化合物，其他脂质体等。在某些实施方式中，所述转染试剂可包括DOTMA(二油酰丙基氯化三甲铵)，DOPE(二油酰磷脂酰乙醇胺)，DOSPA(三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵)，DOGS(N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺)和/或其他可形成脂质体的物质。
在某些实施方式中，所述组合物还包含一种或多种细胞培养基。例如，其可包含MEM培养基(最低必需培养基，最小基本培养基或低限量Eagle培养基)等。所述培养基的含量可以是，例如，至少约20μl，至少约30μl，至少约40μl，至少约50μl，至少约60μl，至少约70μl，至少约80μl，至少约90μl，至少约100μl，至少约110μl，至少约120μl，至少约130μl， 至少约140μl，至少约150μl，至少约160μl，至少约170μl，至少约180μl，至少约190μl，至少约200μl，至少约300μl，至少约400μl，至少约500μl，至少约600μl或更多。
在某些实施方式中，所述组合物中所述单链DNA(例如，环状单链DNA，或者线性单链DNA)的含量为约10ng至约1500ng。例如，约10ng至约1400ng，约10ng至约1300ng，约10ng至约1200ng，约10ng至约1100ng，约10ng至约1000ng，约10ng至约950ng，约10ng至约900ng，约10ng至约850ng，约10ng至约800ng，约10ng至约750ng，约10ng至约700ng，约10ng至约650ng，约10ng至约600ng，约10ng至约550ng，约10ng至约500ng，约10ng至约450ng，约10ng至约400ng，约10ng至约350ng，约10ng至约300ng，约10ng至约250ng，约10ng至约200ng，约10ng至约150ng，约10ng至约100ng，约10ng至约50ng等。
在某些实施方式中，所述组合物中所述单链DNA(例如，环状单链DNA，或者线性单链DNA)的浓度为约0.1nM至约5nM。例如，约0.2nM至约4.5nM，约0.3nM至约4nM，约0.4nM至约3.5nM，约0.5nM至约3nM，约0.6nM至约2.5nM，约0.7nM至约2nM，约0.8nM至约1.9nM，约0.9nM至约1.8nM，约1nM至约1.6nM，约1.1nM至约1.5nM，约1.2nM至约1.4nM，约1.3nM至约1.7nM等。
试剂盒
另一方面，本申请提供了一种试剂盒，其可包含本申请所述的组合物(例如，转染组合物)。
另一方面，本申请提供了一种试剂盒。所述试剂盒可包含：单链DNA(例如，本申请所述的单链DNA)，至少一种转染试剂(例如，本申请所述的转染试剂)，以及使用说明(例如使用说明书)。所述使用说明可记载将所述单链DNA与所述转染试剂按照约5至约0.1的质量比比值施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。例如，所述单链DNA与所述转染试剂质量比的比值范围可以为约4.5至约0.5，约4至约1，约3.5至约1.5，约3至约0.5，约2.5至约0.1，约2至约0.1，约1.5至约0.1，约1至约0.1，约1至约0.2，约1.5至约0.2，约1.5至约0.25，约1.5至约0.3，约1至约0.25，约1.25至约0.2，约0.75至约0.25，约0.5至约0.3等。例如，所述单链DNA与所述转染试剂质量比的比值范围可以为约100至约0.1、约50至约0.1、约20至约0.1、约10至约0.1、约5至约0.1、约100至约0.5、约50至约0.5、约20至约0.5、约10至约0.5、约5至约0.5、约100至约1、约50至约1、约20至约1、约10至约1、或约5至约1等。
在某些实施方式中，所述使用说明记载了将所述单链DNA与所述转染试剂按照约5:1至 约1:10的质量比施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。例如，所述单链DNA与所述转染试剂的质量比可以为约4:1至约1:10，约3:1至约1:10，约2:1至约1:10，约1:1至约1:10，约1:1至约1:9，约1:1至约1:8，约1:1至约1:7，约1:1至约1:6，约1:1至约1:5，约1:1至约1:4，约1:1至约1:3，约1:1至约1:2，约2:1至约1:2，约2:1至约1:3，约2:1至约1:4，约2:1至约1:5等。例如，约100:1至约1:10，约100:1至约1:10，约50:1至约1:10，约20:1至约1:10，约10:1至约1:10，约100:1至约1:5，约100:1至约1:5，约50:1至约1:5，约20:1至约1:5，约10:1至约1:5，约100:1至约1:1，约100:1至约1:1，约50:1至约1:1，约20:1至约1:1，或约10:1至约1:1等。
在某些实施方式中，所述使用说明记载了将所述单链DNA与所述转染试剂按照所述质量比比值或者按照所述质量比混合配制为转染组合物。也即，可以将所述单链DNA与所述转染试剂先按照本申请中所述的比例混合物为转染组合物(任选地，所述转染组合物中还可以包含缓冲剂或其他溶剂，例如本申请中所描述的细胞培养基等)。而后可将所述转染组合物施用至细胞或组织。
在某些实施方式中，直接分别或同时将所述单链DNA及所述转染试剂按照本申请中所述的质量比比值或者所述质量比施加至细胞或组织，而可以不预先混合配制成转染组合物。
在某些实施方式中，所述单链DNA与所述至少一种转染试剂以彼此不互相混合的方式被包含于所述试剂盒中。例如，在所述试剂盒中，所述单链DNA与所述至少一种转染试剂可以各自独立地存在于单独包装的容器中。例如，所述试剂盒中包含2个或更多个彼此独立包装的容器，其各自分别包含本申请中的所述单链DNA及所述转染试剂，以及任选地其他试剂(例如，本申请中所述的细胞培养基等)。
在所述试剂盒中，所述单链DNA(例如，环状单链DNA，或者线性单链DNA)的含量可以为约10ng至约1500ng。例如，约10ng至约1400ng，约10ng至约1300ng，约10ng至约1200ng，约10ng至约1100ng，约10ng至约1000ng，约10ng至约950ng，约10ng至约900ng，约10ng至约850ng，约10ng至约800ng，约10ng至约750ng，约10ng至约700ng，约10ng至约650ng，约10ng至约600ng，约10ng至约550ng，约10ng至约500ng，约10ng至约450ng，约10ng至约400ng，约10ng至约350ng，约10ng至约300ng，约10ng至约250ng，约10ng至约200ng，约10ng至约150ng，约10ng至约100ng，约10ng至约50ng等。
在所述试剂盒中，所述单链DNA(例如，环状单链DNA，或者线性单链DNA)的浓度可以为约0.1nM至约5nM。例如，约0.2nM至约4.5nM，约0.3nM至约4nM，约0.4nM至约3.5nM，约0.5nM至约3nM，约0.6nM至约2.5nM，约0.7nM至约2nM，约0.8nM至约 1.9nM，约0.9nM至约1.8nM，约1nM至约1.6nM，约1.1nM至约1.5nM，约1.2nM至约1.4nM，约1.3nM至约1.7nM等。
在所述试剂盒中，所述单链DNA可包含编码一种或多种RNA和/或蛋白质的核酸序列。例如，所述单链DNA可以在细胞内作为模板被转录/反转录为RNA分子(例如，miRNA，mRNA或其他RNA分子)。在某些情形中，所述单链DNA可以被转化为双链DNA，其可以游离存在于细胞中，也可以整合到细胞的基因组中(例如，以随机插入的方式，或者整合到特定的位点处)。所述单链DNA可以经转录和翻译后表达其所编码的目标蛋白质(例如，治疗性蛋白，标记性蛋白如荧光蛋白或发光蛋白等)。
在所述试剂盒中，所述单链DNA可包含编码两种或更多种RNA和/或蛋白质的核酸序列。例如，所述单链DNA可包含第一编码区和第二编码区，所述第一编码区可以编码第一RNA或第一蛋白质，所述第二编码区可以独立地编码与所述第一RNA或第一蛋白质相同或不同的第二RNA或者第二蛋白质。所述第一编码区与所述第二编码区之间可以包含一个或多个非编码区。在某些情形中，所述第一编码区与所述第二编码区之间基本上不包含非编码区。在某些情形中，所述单链DNA可包含第三编码区或更多编码区。各个编码区的表达产物之间可以形成蛋白复合物或蛋白/核酸复合物。
例如，所述单链DNA(例如，单链环状DNA或者单链线性DNA)可包含编码一种或多种RNA和/或蛋白质的核酸序列(如编码两种或更多种RNA和/或蛋白质的核酸序列)，其可包含至少约40个核苷酸，例如至少约50个核苷酸，至少约100个核苷酸，至少约200个核苷酸，至少约300个核苷酸，至少约400个核苷酸，至少约450个核苷酸，至少约500个核苷酸，至少约550个核苷酸，至少约600个核苷酸，至少约650个核苷酸，至少约700个核苷酸，至少约750个核苷酸，至少约800个核苷酸，至少约850个核苷酸，至少约900个核苷酸，至少约950个核苷酸，至少约1000个核苷酸，至少约1050个核苷酸，至少约1100个核苷酸，至少约1150个核苷酸，至少约1200个核苷酸，至少约1250个核苷酸，至少约1300个核苷酸，至少约1350个核苷酸，至少约1400个核苷酸，至少约1450个核苷酸，至少约1500个核苷酸，至少约1550个核苷酸，至少约1600个核苷酸，至少约1650个核苷酸，至少约1700个核苷酸，至少约1750个核苷酸，至少约1800个核苷酸，至少约1850个核苷酸，至少约1900个核苷酸，至少约1950个核苷酸，至少约2000个核苷酸，至少约2100个核苷酸，至少约2200个核苷酸，至少约2300个核苷酸，至少约2400个核苷酸，至少约2500个核苷酸，至少约2600个核苷酸，至少约2700个核苷酸，至少约2800个核苷酸，至少约2900个核苷酸，至少约3000个核苷酸，至少约3100个核苷酸，至少约3200个核苷酸，至 少约3300个核苷酸，至少约3400个核苷酸，至少约3500个核苷酸，至少约3600个核苷酸，至少约3700个核苷酸，至少约3800个核苷酸，至少约3900个核苷酸，至少约4000个核苷酸，至少约4100个核苷酸，至少约4200个核苷酸，至少约4300个核苷酸，至少约4400个核苷酸，至少约4500个核苷酸，至少约4600个核苷酸，至少约4700个核苷酸，至少约4800个核苷酸，至少约4900个核苷酸，至少约5000个核苷酸，至少约5100个核苷酸，至少约5200个核苷酸，至少约5300个核苷酸，至少约5400个核苷酸，至少约5500个核苷酸，至少约5600个核苷酸，至少约5700个核苷酸，至少约5800个核苷酸，至少约5900个核苷酸，至少约6000个核苷酸或更多。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的正义链(也称为编码链，有义链，正链或+链)核酸序列。例如，所述单链DNA中包含的核酸序列与其所表达的目标蛋白质的编码序列(例如，编码目标蛋白的mRNA的序列)基本上一致。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。例如，所述反义链核酸序列与正义链序列(例如，目标蛋白质的编码核酸序列)基本上互补。在某些实施方式中，所述反义链核酸序列与正义链序列完全互补。
本申请所述的单链DNA还可以包含其他元件，例如启动子序列(如CMV启动子)，增强子序列和/或polyA序列(如SV40polyA)等。
所述试剂盒中可以包含一种或多种转染试剂。
例如，转染试剂可包括但不限于阳离子脂质体，脂类，聚胺，磷酸钙，聚乙烯亚胺，聚赖氨酸和/或它们的组合。所述转染试剂可以具有与核酸负电荷结合的正电荷。例如，阳离子脂质体或聚赖氨酸复合物具有净的正电荷，使得它们能够与DNA或RNA结合。
例如，所述转染试剂可以包含聚合物，如DNA聚阳离子复合物。其中，可包含阳离子蛋白，如组蛋白、精蛋白或者合成的聚合物，如聚赖氨酸、聚精氨酸、DEAE葡聚糖、聚苯醚、聚乙烯亚胺等。在某些实施方式中，所述转染试剂包含阳离子脂质，例如阳离子脂质体，如Lipofectamine2000或Lipofectamine3000等。
其他可用作转染试剂的物质可包括，例如pH敏感性的脂质，两亲性化合物，其他脂质体等。在某些实施方式中，所述转染试剂可包括DOTMA(二油酰丙基氯化三甲铵)，DOPE(二油酰磷脂酰乙醇胺)，DOSPA(三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵)，DOGS(N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺)和/或其他可形成脂质体的物质。
所述试剂盒中还可以包含一种或多种细胞培养基。例如，其可包含MEM培养基(最低 必需培养基，最小基本培养基或低限量Eagle培养基)等。所述培养基的含量可以是，例如，至少约20μl，至少约30μl，至少约40μl，至少约50μl，至少约60μl，至少约70μl，至少约80μl，至少约90μl，至少约100μl，至少约110μl，至少约120μl，至少约130μl，至少约140μl，至少约150μl，至少约160μl，至少约170μl，至少约180μl，至少约190μl，至少约200μl，至少约300μl，至少约400μl，至少约500μl，至少约600μl或更多。
细胞处理方法
另一方面，本申请提供了一种向细胞中递送核酸的方法。所述方法可包括：使所述细胞与包含所述核酸(例如，待递送的核酸)的本申请所述的单链DNA(例如，本申请的环状单链DNA或本申请的线性单链DNA)及本申请所述的转染试剂接触。所述单链DNA与所述转染试剂质量比的比值可以为约5至约0.1。例如，所述单链DNA与所述转染试剂质量比的比值范围可以为约4.5至约0.5，约4至约1，约3.5至约1.5，约3至约0.5，约2.5至约0.1，约2至约0.1，约1.5至约0.1，约1至约0.1，约1至约0.2，约1.5至约0.2，约1.5至约0.25，约1.5至约0.3，约1至约0.25，约1.25至约0.2，约0.75至约0.25，约0.5至约0.3等。
在该方法的某些实施方式中，所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。例如，所述单链DNA与所述转染试剂的质量比可以为约4:1至约1:10，约3:1至约1:10，约2:1至约1:10，约1:1至约1:10，约1:1至约1:9，约1:1至约1:8，约1:1至约1:7，约1:1至约1:6，约1:1至约1:5，约1:1至约1:4，约1:1至约1:3，约1:1至约1:2，约2:1至约1:2，约2:1至约1:3，约2:1至约1:4，约2:1至约1:5等。
另一方面，本申请提供了一种在细胞内表达外源RNA和/或外源蛋白质的方法。所述方法可包括：向所述细胞中引入编码所述外源RNA和/或所述外源蛋白质的单链DNA。所述单链DNA(例如，环状单链DNA或线性单链DNA)可以是本申请所述的单链DNA。
在该方法的某些实施方式中，所述方法还包括使所述细胞在允许所述外源RNA和/或所述外源蛋白质表达的条件下生长。
另一方面，本申请提供了一种降低与DNA转染有关的细胞毒性的方法。所述方法可包括：转染时，向所述细胞中引入本申请所述的单链DNA而非相应的双链DNA。
另一方面，本申请提供了一种降低与DNA转染有关的细胞免疫原性的方法。所述方法可包括：转染时，向所述细胞中引入本申请所述的单链DNA而非相应的双链DNA。
另一方面，本申请提供了一种提高细胞的DNA转染效率的方法。所述方法可包括：转染时，向所述细胞中引入本申请所述的单链DNA而非相应的双链DNA。
在本申请的所述方法中，“引入本申请所述的单链DNA而非相应的双链DNA”通常是指 基本上不(例如，以通常方法难以准确检测的量)向所述细胞中引入具有相同或相应(例如，互补或至少80％同源，至少85％同源，至少90％同源)核苷酸序列的双链DNA(例如，质粒DNA),而是仅通过向所述细胞中引入本申请所述的单链DNA来使得在所述细胞中表达目的RNA或目的蛋白质。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA包括：使所述细胞与本申请所述的组合物接触。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA(例如，环状单链DNA或线性单链DNA)包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂质量比的比值为约5至约0.1。例如，所述单链DNA与所述转染试剂质量比的比值范围可以为约4.5至约0.5，约4至约1，约3.5至约1.5，约3至约0.5，约2.5至约0.1，约2至约0.1，约1.5至约0.1，约1至约0.1，约1至约0.2，约1.5至约0.2，约1.5至约0.25，约1.5至约0.3，约1至约0.25，约1.25至约0.2，约0.75至约0.25，约0.5至约0.3等。
在本申请所述方法的某些实施方式中，向所述细胞中引入所述单链DNA(例如，环状单链DNA或线性单链DNA)包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。例如，所述单链DNA与所述转染试剂的质量比可以为约4:1至约1:10，约3:1至约1:10，约2:1至约1:10，约1:1至约1:10，约1:1至约1:9，约1:1至约1:8，约1:1至约1:7，约1:1至约1:6，约1:1至约1:5，约1:1至约1:4，约1:1至约1:3，约1:1至约1:2，约2:1至约1:2，约2:1至约1:3，约2:1至约1:4，约2:1至约1:5等。
在本申请所述方法的某些实施方式中，所述接触(例如，使所述细胞与所述单链DNA及转染试剂接触，或者使所述细胞与本申请所述的组合物接触)包括在约20℃至约45℃下进行孵育。例如，在约22℃至约44℃下，在约25℃至约40℃下，在约27℃至约38℃下，在约28℃至约37℃下，在约30℃至约36℃下，在约29℃至约35℃下，在约31℃至约33℃下，或在约32℃至约34℃下等进行所述孵育。
在本申请所述方法的某些实施方式中，所述接触包括进行所述孵育至少4小时，例如进行所述孵育至少6小时，至少8小时，至少10小时，至少12小时，至少14小时，至少15小时，至少16小时，至少17小时，至少18小时，至少19小时，至少20小时，至少21小时，至少22小时，至少23小时，至少24小时，至少25小时，至少26小时，至少27小时，至少28小时，至少29小时，至少30小时，至少32小时，至少34小时，至少36小时，至 少38小时，至少40小时，至少42小时，至少44小时，至少45小时，至少46小时，至少47小时，至少48小时，至少49小时，至少50小时，至少51小时，至少52小时，至少53小时，至少54小时或更长时间等。
所述单链DNA可包含编码一种或多种RNA和/或蛋白质的核酸序列。例如，所述单链DNA可以在细胞内作为模板被转录/反转录为RNA分子(例如，miRNA，mRNA或其他RNA分子)。在某些情形中，所述单链DNA可以被转化为双链DNA，其可以游离存在于细胞中，也可以整合到细胞的基因组中(例如，以随机插入的方式，或者整合到特定的位点处)。所述单链DNA可以经转录和翻译后表达其所编码的目标蛋白质(例如，治疗性蛋白，标记性蛋白如荧光蛋白或发光蛋白等)。
例如，所述单链DNA可包含编码两种或更多种RNA和/或蛋白质的核酸序列。例如，所述单链DNA可包含第一编码区和第二编码区，所述第一编码区可以编码第一RNA或第一蛋白质，所述第二编码区可以独立地编码与所述第一RNA或第一蛋白质相同或不同的第二RNA或者第二蛋白质。所述第一编码区与所述第二编码区之间可以包含一个或多个非编码区。在某些情形中，所述第一编码区与所述第二编码区之间基本上不包含非编码区。在某些情形中，所述单链DNA可包含第三编码区或更多编码区。各个编码区的表达产物之间可以形成蛋白复合物或蛋白/核酸复合物。
例如，所述单链DNA(例如，单链环状DNA或者单链线性DNA)可包含编码一种或多种RNA和/或蛋白质的核酸序列(如编码两种或更多种RNA和/或蛋白质的核酸序列)，其可包含至少约40个核苷酸，例如至少约50个核苷酸，至少约100个核苷酸，至少约200个核苷酸，至少约300个核苷酸，至少约400个核苷酸，至少约450个核苷酸，至少约500个核苷酸，至少约550个核苷酸，至少约600个核苷酸，至少约650个核苷酸，至少约700个核苷酸，至少约750个核苷酸，至少约800个核苷酸，至少约850个核苷酸，至少约900个核苷酸，至少约950个核苷酸，至少约1000个核苷酸，至少约1050个核苷酸，至少约1100个核苷酸，至少约1150个核苷酸，至少约1200个核苷酸，至少约1250个核苷酸，至少约1300个核苷酸，至少约1350个核苷酸，至少约1400个核苷酸，至少约1450个核苷酸，至少约1500个核苷酸，至少约1550个核苷酸，至少约1600个核苷酸，至少约1650个核苷酸，至少约1700个核苷酸，至少约1750个核苷酸，至少约1800个核苷酸，至少约1850个核苷酸，至少约1900个核苷酸，至少约1950个核苷酸，至少约2000个核苷酸，至少约2100个核苷酸，至少约2200个核苷酸，至少约2300个核苷酸，至少约2400个核苷酸，至少约2500个核苷酸，至少约2600个核苷酸，至少约2700个核苷酸，至少约2800个核苷酸，至少约 2900个核苷酸，至少约3000个核苷酸，至少约3100个核苷酸，至少约3200个核苷酸，至少约3300个核苷酸，至少约3400个核苷酸，至少约3500个核苷酸，至少约3600个核苷酸，至少约3700个核苷酸，至少约3800个核苷酸，至少约3900个核苷酸，至少约4000个核苷酸，至少约4100个核苷酸，至少约4200个核苷酸，至少约4300个核苷酸，至少约4400个核苷酸，至少约4500个核苷酸，至少约4600个核苷酸，至少约4700个核苷酸，至少约4800个核苷酸，至少约4900个核苷酸，至少约5000个核苷酸，至少约5100个核苷酸，至少约5200个核苷酸，至少约5300个核苷酸，至少约5400个核苷酸，至少约5500个核苷酸，至少约5600个核苷酸，至少约5700个核苷酸，至少约5800个核苷酸，至少约5900个核苷酸，至少约6000个核苷酸或更多。
例如，所述单链DNA可包含编码RNA和/或蛋白质的正义链(也称为编码链，有义链，正链或+链)核酸序列。例如，所述单链DNA中包含的核酸序列与其所表达的目标蛋白质的编码序列(例如，编码目标蛋白的mRNA的序列)基本上一致。
在某些实施方式中，所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。例如，所述反义链核酸序列与正义链序列(例如，目标蛋白质的编码核酸序列)基本上互补。在某些实施方式中，所述反义链核酸序列与正义链序列完全互补。例如，本申请的EGFP cssDNA反义链序列(2Ambystoma laterale x Ambystoma jeffersonianum)的核苷酸序列可以如SEQ ID NO：7所示。
本申请所述的单链DNA还可以包含其他元件，例如启动子序列(如CMV启动子)，增强子序列和/或polyA序列(如SV40polyA)等。
所述“转染试剂”可以指用于介导核酸进入细胞的物质(例如，化合物)。例如，转染试剂可包括但不限于阳离子脂质体，脂类，聚胺，磷酸钙，聚乙烯亚胺，聚赖氨酸和/或它们的组合。所述转染试剂可以具有与核酸负电荷结合的正电荷。例如，阳离子脂质体或聚赖氨酸复合物具有净的正电荷，使得它们能够与DNA或RNA结合。
例如，所述转染试剂可以包含聚合物，如DNA聚阳离子复合物。其中，可包含阳离子蛋白，如组蛋白、精蛋白或者合成的聚合物，如聚赖氨酸、聚精氨酸、DEAE葡聚糖、聚苯醚、聚乙烯亚胺等。在某些实施方式中，所述转染试剂包含阳离子脂质，例如阳离子脂质体，如Lipofectamine2000或Lipofectamine3000等。
其他可用作转染试剂的物质可包括，例如pH敏感性的脂质，两亲性化合物，其他脂质体等。在某些实施方式中，所述转染试剂可包括DOTMA(二油酰丙基氯化三甲铵)，DOPE(二油酰磷脂酰乙醇胺)，DOSPA(三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨 基)乙基铵)，DOGS(N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺)和/或其他可形成脂质体的物质。
所述方法还可包括施用一种或多种细胞培养基。例如，其可包含施用MEM培养基(最低必需培养基，最小基本培养基或低限量Eagle培养基)等。所述培养基的含量可以是，例如，至少约20μl，至少约30μl，至少约40μl，至少约50μl，至少约60μl，至少约70μl，至少约80μl，至少约90μl，至少约100μl，至少约110μl，至少约120μl，至少约130μl，至少约140μl，至少约150μl，至少约160μl，至少约170μl，至少约180μl，至少约190μl，至少约200μl，至少约300μl，至少约400μl，至少约500μl，至少约600μl或更多。在施用之前，可将所述培养基与本申请的所述单链DNA和/或本申请的所述转染试剂进行孵育(例如，孵育至少约5min,至少约10min,至少约12min,至少约13min,至少约14min,至少约15min,至少约20min，至少约25min,至少约30min,至少约35min或更长时间等)，例如在约20℃至约45℃下进行孵育。例如，在约22℃至约44℃下，在约25℃至约40℃下，在约27℃至约38℃下，在约28℃至约37℃下，在约30℃至约36℃下，在约29℃至约35℃下，在约31℃至约33℃下，或在约32℃至约34℃下等进行所述孵育。
在某些实施方式中，所使用的所述单链DNA(例如，环状单链DNA，或者线性单链DNA)的含量为约10ng至约1500ng。例如，约10ng至约1400ng，约10ng至约1300ng，约10ng至约1200ng，约10ng至约1100ng，约10ng至约1000ng，约10ng至约950ng，约10ng至约900ng，约10ng至约850ng，约10ng至约800ng，约10ng至约750ng，约10ng至约700ng，约10ng至约650ng，约10ng至约600ng，约10ng至约550ng，约10ng至约500ng，约10ng至约450ng，约10ng至约400ng，约10ng至约350ng，约10ng至约300ng，约10ng至约250ng，约10ng至约200ng，约10ng至约150ng，约10ng至约100ng，约10ng至约50ng等。
在某些实施方式中，所使用的所述单链DNA(例如，环状单链DNA，或者线性单链DNA)的浓度为约0.1nM至约5nM。例如，约0.2nM至约4.5nM，约0.3nM至约4nM，约0.4nM至约3.5nM，约0.5nM至约3nM，约0.6nM至约2.5nM，约0.7nM至约2nM，约0.8nM至约1.9nM，约0.9nM至约1.8nM，约1nM至约1.6nM，约1.1nM至约1.5nM，约1.2nM至约1.4nM，约1.3nM至约1.7nM等。
在某些实施方式中，所使用的所述转染试剂的量为约10ng至约4500ng。例如，约10ng至约4000ng，约10ng至约3800ng，约10ng至约3600ng，约10ng至约3500ng，约10ng至约3300ng，约10ng至约3000ng，约10ng至约2800ng，约10ng至约2600ng，约10ng至约2400ng，约10ng至约2200ng，约10ng至约2000ng，约10ng至约1900ng，约10ng至约1800ng， 约10ng至约1700ng，约10ng至约1600ng，约10ng至约1500ng，约10ng至约1400ng，约10ng至约1300ng，约10ng至约1200ng，约10ng至约1100ng，约10ng至约1000ng，约10ng至约950ng，约10ng至约900ng，约10ng至约850ng，约10ng至约800ng，约10ng至约750ng，约10ng至约700ng，约10ng至约650ng，约10ng至约600ng，约10ng至约550ng，约10ng至约500ng，约10ng至约450ng，约10ng至约400ng，约10ng至约350ng，约10ng至约300ng，约10ng至约250ng，约10ng至约200ng，约10ng至约150ng，约10ng至约100ng，约10ng至约50ng等。
在某些实施方式中，本申请所述的方法为体内方法、体外方法或离体方法。
在本申请所述方法的某些实施方式中，所述细胞为体细胞，肿瘤细胞和/免疫细胞。
例如，所述细胞可选自：肺癌细胞，肾细胞，黑色素瘤细胞，肝癌细胞，宫颈癌细胞，胶质瘤细胞，上皮细胞，结肠癌细胞，成纤维细胞，乳腺癌细胞，胃癌细胞和免疫效应细胞。
在某些情形中，所述细胞可选自：A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞,MDCK细胞，B16细胞，Hep3B细胞，L929细胞，4T1细胞，293T细胞，MGC803细胞和巨噬细胞。
细胞、生物体或其部分
另一方面，本申请提供了经本申请中任一个方面的方法处理而得到的细胞。
例如，在所述细胞中引入了本申请的所述单链DNA而非相应的双链DNA。例如，使所述细胞与本申请的组合物接触。例如，在所述细胞中引入了本申请所述的单链DNA，从而表达外源RNA和/或外源蛋白质。
另一方面，本申请提供了生物体(例如，非人生物体)或其组成部分，其可包含本申请所述的细胞(例如，经本申请的方法处理的细胞)。
在某些实施方式中，所述组成部分选自：组织(例如，肿瘤组织等)、器官及其组合。
用途
另一方面，本申请提供了单链DNA用于制备组合物(例如转染组合物，如细胞转染组合物，或者核酸递送组合物)的用途。所述组合物可用于下述的一项或多项：将本申请所述的单链DNA中包含的至少部分核酸递送至细胞内；在细胞内表达由所述单链DNA编码的外源RNA或外源蛋白；提高细胞的DNA转染效率；降低与DNA转染有关的细胞毒性；和/或降低与DNA转染有关的细胞免疫原性。所述组合物可以是本申请的任一方面所述的组合物(例如，转染组合物)。
在某些情况下，本申请的所述单链DNA或本申请所述的组合物可用于制备药物(例如，核酸药物)，例如用于作为基因疗法和/或核酸疫苗。
在某些实施方式中，所述细胞为体细胞，肿瘤细胞和/免疫细胞。
在某些实施方式中，所述细胞选自：肺癌细胞，肾细胞，黑色素瘤细胞，肝癌细胞，宫颈癌细胞，胶质瘤细胞，上皮细胞，结肠癌细胞，成纤维细胞，乳腺癌细胞，胃癌细胞和免疫效应细胞。
在某些实施方式中，所述细胞选自：A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞,MDCK细胞，B16细胞，Hep3B细胞，L929细胞，4T1细胞，293T细胞，MGC803细胞和巨噬细胞。
本申请还涉及以下内容。
本申请的发明提供了一种单链DNA在基因转染中的应用，具体包括基于传统噬菌体载体生成的环状单链DNA以及通过不对称PCR方法得到的线性单链DNA在基因转染中得以应用，与传统的环状双链质粒的转染不同，环状单链DNA的转染效率和转染试剂的浓度成正比，同等浓度更易被转染试剂递送进较难转染的细胞且表达量更高，引起的细胞毒性更低，环状单链DNA能够明显降低聚合物引起的细胞毒性，使得转染效率得到有效提高，且不明显引起细胞的毒性；另外，线性单链DNA也证明了用于基因转染的可行性，因此，使用单链DNA进行基因转染，可以看到相关基因的表达，故说明单链DNA可以作为基因转染的一种载体进行基因转染。
在某些方面，本申请提供了一种单链DNA在基因转染中的应用。
所述单链DNA可包括基于噬菌体载体产生的环状单链DNA和不对称PCR产生的线性单链DNA；所述单链DNA和转染试剂孵育后可在细胞培养皿中培养24-48小时。
例如，所述单链DNA的核酸数可以为1000-50000nt。
例如，所述转染试剂可选自阳离子聚合物。在某些实施方式中，所述转染试剂选自Lipofectamine 2000或聚醚酰亚胺中的一种以上。
所述环状单链DNA的制备方法可包括以下步骤：(1)、将基因连接插入载体中，得到基因载体；(2)、将所述基因载体和辅助噬菌体载体共转染到感受态细胞中；(3)、根据单链DNA长度的不同，培养不同的时间得到单一条带的环状单链DNA。其中，步骤(2)中，所述噬菌体载体可以为基于M13的质粒载体。
所述线性单链DNA的制备方法可包括以下步骤：(a)、基于基因设计上游引物和下游引物；(b)、通过控制上游引物和下游引物的比例进行PCR反应；(c)、将所获PCR产 物通过琼脂糖凝胶电泳分离、纯化。其中，步骤(a)中，所述上游引物序列可以如SEQ ID NO：3所示，所述下游引物序列可以如SEQ ID NO：4所示。在某些情形中，步骤(a)中，所述上游引物和上游引物的摩尔比可以为50:1。
例如，针对现有技术中的不足，本申请的发明提供了一种单链DNA(ssDNA)在基因转染中的应用，具体地，基于pScaf载体的环状单链DNA将目的基因插入到pScaf载体中，通过控制培养时间获得条带单一的环状单链DNA(cssDNA)。线性单链DNA(lssDNA)通过传统的不对称PCR的方法产生。产生的lssDNA和cssDNA包含CMV增强子和启动子、目的基因、SV40polyA等序列，减少了其他无关序列的引入，因此可以最大程度缩短DNA片段长度。
一方面，本申请提供了一种单链DNA在基因转染中的应用。
例如，所述单链DNA可包括基于噬菌体载体产生的环状单链DNA和不对称PCR产生的线性单链DNA，单链DNA和转染试剂孵育后放入细胞培养皿中培养24-48h，即可看到目的蛋白的明显表达。
又例如，所述单链DNA的核酸数可以合成的长度在1000-50000nt之间。例如，转染试剂可选自阳离子聚合物，如Lipofectamine 2000(lip 2000)或聚醚酰亚胺(PEI)。
例如，基于pScaf载体产生的cssDNA的制备方法可包括如下步骤：(1)、将目的基因通过酶切位点连接插入pScaf载体中，得到pScaf-目的基因载体；(2)、将10-20ng pScaf-目的基因载体和20-40ng基于M13的辅助噬菌体载体(辅助质粒pSB4423)共转染到100μL XL1-Blue感受态细胞中，培养温度为30℃；(3)、挑取单克隆在1mL培养基中活化1h，再转移至5mL液体培养基中，30℃扩大培养4-5h；(4)、将步骤(3)中获得的菌液在200mL 2×YT培养基中培养8-24h，根据目的基因长度，控制培养时间以获得条带单一、无杂带的目的产物；(5)、利用nanodrop测目的产物的浓度，根据分子量计算目的产物的摩尔浓度；(6)、使用步骤(5)中获得的cssDNA进行基因转染。
其中，步骤(1)中，目的基因可包括EGFP基因和EGFP-mCherry基因。其中，EGFP基因序列可如SEQ ID NO：1所示。EGFP-mCherry基因序列可如SEQ ID NO：2所示。
在所述方法的步骤(1)中，酶切位点可以为KpnⅠ(GGTACC)和BamHⅠ(GGATCC)。在所述方法的步骤(2)中，可用pScaf-目的基因载体将M13复制子与单链DNA的起始位点和终止位点整合在一起，从而减少副产物的生成。
在所述方法的步骤(4)中，可通过S1核酸酶验证，通过控制培养时间得到的DNA确定为环状单链DNA。
例如，基于传统的不对称PCR产生的lssDNA的制备方法可包括如下步骤：(a)、以pEGFP-N1质粒为模板，使用Takara公司的PrimerSTAR(R045A，TAKARA)酶，设计的上游引物和下游引物进行PCR反应；(b)、将所获PCR产物进行琼脂糖凝胶电泳；(c)、根据DNA marker位置切下目标条带；(d)、回收目标DNA；(e)、利用nanodrop检测lssDNA的浓度；(f)、使用获得的lssDNA进行基因转染。例如，在步骤(a)中，EGFP基因序列可以如SEQ ID NO：1所示。例如，步骤(a)中，上游引物序列可以如SEQ ID NO：3所示，下游引物序列可以如SEQ ID NO：4所示。例如，上游引物和上游引物的摩尔比可以为50:1。
本申请的发明的有益效果可以包括：本申请的发明将ssDNA在基因转染中应用，与传统的环状双链质粒的转染不同，cssDNA的转染效率和DNA浓度以及转染试剂的浓度成正比，同等浓度的cssDNA更易被转染试剂递送进较难转染的细胞且表达量更高，引起的细胞毒性更低，且在使用商业化阳离子聚合物转染试剂进行转染时，cssDNA能够明显降低聚合物引起的细胞毒性，使得转染效率得到有效提高，并且不明显引起细胞的毒性；另外，lssDNA也证明了用于基因转染的可行性，因此，证明ssDNA可以作为一种基因转染以及基因递送的潜在载体。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的方法和系统的工作方式，而不用于限制本申请发明的范围。
实施例
实施例1获得环状单链DNA分子
通过以下步骤构建表达载体并获得环状单链DNA:
1)通过KpnI和BamHI限制性内切酶对pScaf载体(Addgene plasmid#111401)进行限制性酶切，接着将PCR扩增得到的EGFP基因片段以及EGFP-mCherry基因片段分别连接到pScaf载体中，获得pScaf-EGFP载体和pScaf-mCherry-EGFP载体(如图1所示)；
2)将10ng pScaf-EGFP载体、10ng pScaf-EGFP-mCherry载体和15ng基于M13的pSB4423质粒(受赠于美国Ke yonggang实验室)共转化到XL1-Blue感受态细胞中，涂板并在30℃下培养16小时；
3)挑取单克隆，在1mL 2×YT(含5mM MgCl 2)培养基中，于30℃、220rpm的条件下培养1小时；
4)将步骤3)的培养物接种到5mL 2×YT(含5mM MgCl 2，100μg/mL氨苄青霉素钠，20μg/mL氯霉素)培养基中，在30℃、220rpm的条件下培养4小时；
5)将步骤4)的培养物接种到200mL 2×YT培养基中，在30℃、220rpm的条件下继续培养，其中pScaf-EGFP培养8小时，pScaf-EGFP-mCherry培养12小时；
6)将步骤5)的培养物进行离心处理，8000×rcf，4℃下离心10min，收集上清；
7)在步骤6)获得的上清中加入3.2g PEG-8000及2.4g NaCl，混匀，并置于冰水浴中冰浴30min；
8)将步骤7)的混合液在10,000×rcf，4℃下离心30min，得到白色沉淀；
9)用1.5mL 10mM Tris(pH＝8.5)重悬步骤8)获得的沉淀，在10,000g×rcf，4℃下离心15min；
10)重复步骤9)三次，以去除残留的细菌；
11)向步骤10)获得的溶液中加入3mL裂解液(0.2mol/L NaOH，1％SDS)，上下颠倒混匀，在室温下裂解5min；
12)向步骤11)获得的溶液中加入2.5mL终止液(3mol/L醋酸钾，pH＝5.5)，上下颠倒混匀，冰水浴15min；
13)将步骤12)获得的溶液在10,000×rcf，4℃下离心30min，去除沉淀，并收集上清液；
14)向步骤13)获得的上清液中加入2倍体积的无水乙醇，将混合液置于-20℃下沉淀过夜；
15)将步骤14)中获得的溶液在10,000×rcf，4℃下离心30min，得到EGFP环状单链DNA(EGFP cssDNA)(其核酸序列如SEQ ID NO：5所示)和EGFP-mCherry环状单链DNA(EGFP-mCherry cssDNA)(其核酸序列如SEQ ID NO：6所示)；
16)用3mL 75％乙醇清洗所述EGFP cssDNA和EGFP-mCherry cssDNA；
17)使用nanodrop对所述EGFP cssDNA和EGFP-mCherry cssDNA进行定量测定；
18)向得到的所述EGFP cssDNA中加入单链DNA酶降解(S1核酸酶)并进行琼脂糖凝胶电泳验证(结果如图11所示)。简要地，分别取2μg所述EGFP cssDNA、2μg P7560(Scaffold DNA,公司：IDT，货号：1081309)、和2μg EGFP质粒双链DNA(pEGFP-N1-4x，Addgene plasmid#172283)，在37℃下与S1核酸酶共同孵育30分钟。其中，S1核酸酶从米曲霉(aspergill suoryzae)中提取得到，能够特异性降解单链DNA。由图11可知，所述EGFP cssDNA及P7560阳性对照(噬菌体环状单链DNA)可以被S1核酸酶快速降解，而双链质粒plDNA无法被降解。据此，可以确定本实施例中制备的EGFP cssDNA为单链DNA。类似地，确认了所述EGFP-mCherry cssDNA也为单链DNA。如图11所示，其中第1泳道显示5kb DNA标记，第2泳道为EFGP CssDNA未经过S1核酸酶处理，第3泳道为EFGP CssDNA经过S1 核酸酶处理，第4泳道为P7560未经过S1核酸酶处理，第5泳道为P7560经过S1核酸酶处理，第6泳道为EGFP质粒双链DNA未经过S1核酸酶处理，第7泳道为EGFP质粒双链DNA经过S1核酸酶处理。
实施例2用电穿孔法将实施例1的单链DNA递送到细胞中并检测其表达
通过以下步骤将所述单链DNA递送到细胞中：
1)在Gene PμLser Xcell电穿孔系统上设置所需的脉冲条件。
2)在电穿孔杯中加入20μg实施例1中获得的EGFP cssDNA。
3)将细胞添加到电穿孔杯中，并轻拍电穿孔杯的一侧进行混合。具体地，向0.2cm的电穿孔杯中加入200μl 5 x 10 6个细胞。
4)将电穿孔杯放入电穿孔舱中，盖上盖子，脉冲一次。
5)脉冲后立即加入0.5ml的细胞培养基，再用巴氏吸管将细胞转移到培养皿中。
6)轻轻摇动培养皿，以确保细胞均匀分布在培养皿表面。在37℃的培养箱中培养所述细胞。
7)在进行电穿孔后24-48小时，检测所述单链DNA的表达情况。
在本实施例中，验证了所述EGFP cssDNA在多种细胞系(Hela细胞,A549细胞,L929细胞,MGC803细胞,MDCK细胞,CT26细胞,MCF7细胞,4T1细胞和Helf细胞)中的表达情况。在培养24小时后进行观察，发现EGFP cssDNA可以在这些细胞系中进行表达(结果如图2A-2B所示)。图2A中显示了EGFP cssDNA在各细胞系中的荧光表达图像。图2B显示了EGFP cssDNA在各细胞系中的电穿孔转染/表达效率。可见，本申请的环状单链DNA能够在多种细胞系中有效表达目的蛋白。
实施例3用转染试剂将实施例1的单链DNA递送到细胞中并检测其表达
通过以下步骤将所述单链DNA递送到细胞中：
1)将1.5μg阳离子聚合物(Lipofectamine2000，赛默飞，货号：11668500)与MEM培养基在室温下混匀放置5min(总体积50μL)。
2)将1.3nM(50μL，约460ng)实施例1中制备的EGFP cssDNA正义链与步骤1)中在50μL MEM培养基中混匀的1.5μg阳离子聚合物(Lipofectamine2000)进行混合，用枪头轻微吹打。得到混合均匀的100μL溶液，室温下放置15min。
3)将1.3nM(50μL，约460ng)实施例1中制备的EGFP cssDNA反义链与步骤1)中在50μL MEM培养基中混匀的1.5μg阳离子聚合物(Lipofectamine2000)进行混合，用枪头 轻微吹打。得到混合均匀的100μL溶液，室温下放置15min。
4)将1.3nM(50μL，约460ng)实施例1中制备的EGFP-mCherry cssDNA与步骤1)中在50μL MEM培养基中混匀的1.5μg阳离子聚合物(Lipofectamine2000)进行混合，用枪头轻微吹打。得到混合均匀的100μL溶液，室温下放置15min。
5)预先接种细胞(1x10 5个细胞)于24孔板内，在培养箱内培养12小时。
6)将上述各混合溶液滴加到事先铺好在24孔板的细胞中，培养24小时。
7)使用荧光显微镜观察细胞的转染效率。
在本实施例中，验证了所述EGFP cssDNA(正义链或反义链)和EGFP-mCherry cssDNA与转染试剂分别混合后，在多种细胞系(例如，A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞和MDCK细胞)中的转染效率和表达情况。在培养24小时后进行观察，发现EGFP cssDNA的正义链和反义链均可以在这些细胞系中进行表达(结果如图3所示)，且包含多个蛋白编码区的长链单链DNA(如本实施例中的EGFP-mCherry cssDNA)也能被有效地转染和表达(结果如图4A-4C所示)。图4A显示了EGFP-mCherry cssDNA经转染后，其中EGFP蛋白的表达情况。图4B显示了EGFP-mCherry cssDNA经转染后，其中mCherry蛋白的表达情况。图4C表明，EGFP蛋白表达与mCherry蛋白表达的叠加情况，显示了二者的共表达。
实施例4单链DNA与双链质粒DNA转染效率的比较
通过以下步骤进行核酸转染(测试了不同的DNA:转染试剂比例下，转染效率的差别)：
1)比例1：将0.5μg阳离子聚合物(Lipofectamine2000)加入50μL MEM培养基中，室温混匀放置5min。随后，将1.3nM EGFP cssDNA(于50μL MEM培养基中，约460ng)和1.3nM相应的双链质粒plDNA(于50μL MEM培养基中，约460ng)分别与已准备好的所述0.5μg阳离子聚合物(Lipofectamine2000，已在50μL MEM培养基中混匀)进行混合，得到混合均匀的100μL转染组合物，在室温下放置15min。
2)比例2：将1μg阳离子聚合物(Lipofectamine2000)加入50μL MEM培养基中，室温混匀放置5min。随后，将1.3nM EGFP cssDNA(于50μL MEM培养基中，约460ng)和1.3nM相应的双链质粒plDNA(于50μL MEM培养基中，约460ng)分别与已准备好的所述1μg阳离子聚合物(Lipofectamine2000，已在50μL MEM培养基中混匀)进行混合，得到混合均匀的100μL转染组合物，在室温下放置15min。
3)比例3：将2μg阳离子聚合物(Lipofectamine2000)加入50μL MEM培养基中，室温混匀放置5min。随后，将1.3nM EGFP cssDNA(于50μL MEM培养基中，约460ng)和1.3 nM相应的双链质粒plDNA(于50μL MEM培养基中，约460ng)分别与已准备好的所述2μg阳离子聚合物(Lipofectamine2000，已在50μL MEM培养基中混匀)进行混合，得到混合均匀的100μL转染组合物，在室温下放置15min。
4)预先接种细胞(1x10 5个细胞)于24孔板内，在培养箱内培养12小时。
5)将上述各混合溶液滴加到事先铺好在24孔板的细胞中，培养24小时。
6)使用荧光显微镜观察细胞的转染效率。
在本实施例中，验证了所述EGFP cssDNA在多种细胞系(例如，A549细胞、B16细胞和HepG2细胞，包括较难转染的细胞系)中的转染效率和表达情况。其中，当使用同等浓度的DNA时，单链DNA的转染效率显著高于双链质粒DNA。此外，通过使用更高比例的转染试剂，可以提高单链DNA的转染效率(结果如图5-图7所示)。图5显示的是在A549细胞中的转染效果，图6显示的是在B16细胞系中的转染效果，图7显示的是在HepG2细胞系中的转染效果。
实施例5单链DNA与双链质粒DNA转染后的细胞毒性比较
实验步骤简要描述如下：
1)制备细胞悬液，并进行细胞计数。
2)将MDCK细胞接种到96孔板中：以5x10 3个细胞/孔的密度进行铺板，每孔中加入约100μL细胞悬液，同样的样本重复5次。
3)在培养箱中于37℃下培养12-24小时。
4)加入单链DNA(EGFP cssDNA)和双链质粒plDNA分别与转染试剂Lipofectamine2000形成复合物(DNA:600ng；Lipofectamine2000:3ug)
5)在培养箱中37℃下分别培养6小时、12小时、24小时和48小时。
6)加入10μL CCK8试剂进行毒性分析。
7)在培养箱中37℃下孵育4小时。
8)在振动器上轻轻混合1分钟，以确保颜色均匀分布。使用酶标仪测定各孔在450nm处的吸光度，随后进行数据分析处理。
在本实施例中，验证了所述单链DNA与大量转染试剂的组合物，在转染细胞时的细胞毒性，并与相应的双链质粒DNA转染组合物进行了比较。
由本实施例的结果可知，在同等的DNA浓度下，在高浓度转染试剂(例如Lipofectamine2000)的作用下，经过0-48小时的转染，可以看到，plDNA组相对于单链DNA组，在24小时后出现明显的细胞毒性，而单链DNA组并没有引起明显的细胞毒性(结果如 图8所示)。可见，可以通过使用单链DNA来替换相应的双链质粒DNA，降低与DNA转染有关的细胞毒性。此外，令人惊讶地，当把单链DNA与较大量的转染试剂进行混合后，可以显示出协同效果，一方面较大剂量转染试剂的细胞毒性被降低了，另一方面，单链DNA的转染效率提高了。
实施例6线性单链DNA的转染效果
实验步骤简要描述如下：
6.1获得线性单链DNA(EGFP lssDNA)
1)按照如下配方配制PCR反应体系：
反向产物扩增：pEGFP-N1质粒(Addgene plasmid#54767)：2μL(10ng/μL)、上游引物：2μL(0.2μM)、下游引物：2μL(10μM)、PrimeSTAR Max Premix(25μL，R045A，Takara)；ddH2O：19μL。
正向产物扩增：pEGFP-N1质粒：2μL(10ng/μL)、上游引物：2μL(10μM)、下游引物：2μL(0.2μM)、PrimeSTAR Max Premix(25μL，R045A，Takara)；ddH2O：19μL。
其中，正向产物扩增和反向产物扩增中的上游引物序列如SEQ ID NO：3所示，下游引物序列如SEQ ID NO：4所示。
2)按照如下反应程序进行扩增：
95℃反应10min，95℃反应30s，60℃反应30s，72℃反应1min(其中，95℃反应30s，60℃反应30s，72℃反应1min分别进行35个循环)，72℃反应10min。
3)反应结束后，将上述反应产物在1％琼脂糖凝胶中进行电泳，100V，40min；
4)电泳结束后，切下目的条带(如图9所示，其中1kb显示1kb DNA标记，第1泳道为相应的双链DNA,第2泳道为反向产物扩增结果，第3泳道为正向产物扩增结果)，使用切胶回收试剂盒进行DNA的回收；
5)使用nanodrop对EGFP lssDNA进行定量分析。
6.2线性单链DNA的表达效果
1)将1.5μg阳离子聚合物(Lipofectamine2000)加入50μL MEM培养基中，室温混匀放置5min。随后，将0.5μg EGFP lssDNA(于50μL MEM培养基中)与已准备好的所述1.5μg阳离子聚合物(Lipofectamine2000，已在50μL MEM培养基中混匀)进行混合，得到混合均匀的100μL转染组合物，在室温下放置15min。
2)将上述混合溶液滴加到事先铺好在24孔板的293T细胞和MGC803细胞中，培养24小时。
3)使用荧光显微镜观察EGFP lssDNA的表达情况(结果如图10所示)。
由图10的结果可知，线性单链DNA也可以在转染后在细胞中表达目的蛋白产物。
实施例7单链DNA与双链质粒DNA转染后的体外免疫反应比较
实验步骤简要描述如下：
1)制备巨噬细胞悬液，并进行细胞计数。
2)将巨噬细胞接种到6孔板中：以5x10 5个细胞/孔的密度进行铺板，每孔约2mL细胞悬液。
3)在培养箱中于37℃培养12-24小时。
4)分别加入Lipofectamine2000(2ug)、EGFP cssDNA(0.6ug)与转染试剂Lipofectamine2000(2ug)的组合物，以及双链质粒plDNA(1.2ug)与转染试剂Lipofectamine2000(2ug)的组合物。
5)在培养箱中37℃下培养24小时。
6)收集细胞悬液。
7)用经FITC荧光标记的IFN-α抗体(赛默飞，货号PA5-115430)和IFN-β抗体(赛默飞，货号PA5-95722)与每组的细胞悬液共同孵育半小时。
8)在流式细胞仪检测各组荧光强度，随后进行数据分析处理，结果如图12A(IFN-α)和图12B(IFN-β)所示。
本实施例的结果显示，单链DNA相比于相应的双链质粒plDNA在细胞层面对于巨噬细胞有较低的免疫刺激性。
实施例8单链DNA与双链质粒DNA转染后的体内免疫反应比较
实验步骤简要描述如下：
1)将20只雌性小白鼠随机分为4组，每组5只，分别为对照组，Lipofectamine2000给药组，EGFP cssDNA和Lipofectamine2000的复合物给药组和双链质粒plDNA Lipofectamine2000的复合物给药组。
2)制备EGFP cssDNA(9μg)与转染试剂Lipofectamine2000(100μL)的组合物，以及双链质粒plDNA(18μg)与转染试剂Lipofectamine2000(100μL)的组合物。
3)通过尾静脉注射分别对不同组别给药，对照组给予100μL的生理盐水，脂质体给药组给予等质量的100μL Lipofectamine2000溶液，单链DNA给药组按照9μg EGFP cssDNA/只尾静脉注射，plDNA给药组按照18μg plDNA/只尾静脉注射。
4)一周后，由各组取外周血，按照Elisa试剂盒(abcam，货号ab252352和ab252363)说明书处理，测定血液中I型干扰素(IFN-α与IFN-β)的浓度，结果如图13A(IFN-α)和图13B(IFN-β)所示。
本实施例的结果显示，单链DNA相比于相应的双链质粒plDNA，在体内有较低的免疫刺激性，表明其有较低的副作用，在临床实践中具有更好的安全性，和更大的临床应用价值。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本文所列举的实施方式的多种变化对本领域普通技术人员来说是容易理解的，且保留在所附的权利要求和其等同方案的范围内。

Claims (80)

  1. 组合物，其包含
    单链DNA；以及
    至少一种转染试剂；
    所述组合物中所述单链DNA与所述转染试剂质量比的比值为约100至约0.1。
  2. 根据权利要求1所述的组合物，所述组合物中所述单链DNA与所述转染试剂的质量比为约100:1至约1:10。
  3. 根据权利要求1-2中任一项所述的组合物，所述组合物中所述单链DNA与所述转染试剂质量比的比值为约1至约0.2。
  4. 根据权利要求1-3中任一项所述的组合物，所述组合物中所述单链DNA与所述转染试剂的质量比为约2:1至约1:5。
  5. 根据权利要求1-4中任一项所述的组合物，其中所述单链DNA为环状单链DNA。
  6. 根据权利要求1-4中任一项所述的组合物，其中所述单链DNA为线性单链DNA。
  7. 根据权利要求1-6中任一项所述的组合物，其中所述单链DNA包含编码一种或多种RNA和/或蛋白质的核酸序列。
  8. 根据权利要求1-7中任一项所述的组合物，其中所述单链DNA包含编码两种或更多种RNA和/或蛋白质的核酸序列。
  9. 根据权利要求1-8中任一项所述的组合物，其中所述单链DNA包含编码RNA和/或蛋白质的正义链核酸序列。
  10. 根据权利要求1-9中任一项所述的组合物，其中所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。
  11. 根据权利要求1-10中任一项所述的组合物，其中所述单链DNA包含至少40个核苷酸。
  12. 根据权利要求1-11中任一项所述的组合物，其中所述单链DNA包含至少1500个核苷酸。
  13. 根据权利要求1-12中任一项所述的组合物，其中所述转染试剂包含阳离子脂质和/或阳离子聚合物。
  14. 根据权利要求1-13中任一项所述的组合物，其中所述转染试剂包含阳离子脂质体。
  15. 根据权利要求1-14中任一项所述的组合物，其中还包含一种或多种细胞培养基。
  16. 根据权利要求1-15中任一项所述的组合物，其中所述单链DNA的含量为约10ng至约1000ng。
  17. 根据权利要求1-16中任一项所述的组合物，其中所述单链DNA的浓度为约0.5nM至约3nM。
  18. 试剂盒，其包含权利要求1-17中任一项所述的组合物。
  19. 试剂盒，其包含：
    单链DNA；
    至少一种转染试剂；以及
    使用说明；
    其中所述使用说明记载了将所述单链DNA与所述转染试剂按照约100至约0.1的质量比比值施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
  20. 根据权利要求19所述的试剂盒，其中所述使用说明记载了将所述单链DNA与所述转染试剂按照约5:1至约1:10的质量比施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
  21. 根据权利要求19-20中任一项所述的试剂盒，其中所述使用说明记载了将所述单链DNA与所述转染试剂按照约10至约0.1质量比比值施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
  22. 根据权利要求19-21中任一项所述的试剂盒，其中所述使用说明记载了将所述单链DNA与所述转染试剂按照约2:1至约1:5的质量比施用于靶细胞从而将所述单链DNA中包含的至少部分核酸递送至所述靶细胞中。
  23. 根据权利要求19-22中任一项所述的试剂盒，其中所述使用说明记载了将所述单链DNA与所述转染试剂按照所述质量比比值或者按照所述质量比混合配制为转染组合物。
  24. 根据权利要求19-23中任一项所述的试剂盒，其中所述单链DNA与所述至少一种转染试剂以彼此不互相混合的方式被包含于所述试剂盒中。
  25. 根据权利要求19-24中任一项所述的试剂盒，其中所述单链DNA与所述至少一种转染试剂各自独立地存在于单独包装的容器中。
  26. 根据权利要求19-25中任一项所述的试剂盒，其中所述单链DNA的含量为约10ng至约1000ng。
  27. 根据权利要求19-26中任一项所述的试剂盒，其中所述单链DNA的浓度为约0.5nM至约3nM。
  28. 根据权利要求19-27中任一项所述的试剂盒，其中所述单链DNA为环状单链DNA。
  29. 根据权利要求19-27中任一项所述的试剂盒，其中所述单链DNA为线性单链DNA。
  30. 根据权利要求19-29中任一项所述的试剂盒，其中所述单链DNA包含编码一种或多种RNA和/或蛋白质的核酸序列。
  31. 根据权利要求19-30中任一项所述的试剂盒，其中所述单链DNA包含编码两种或更多种RNA和/或蛋白质的核酸序列。
  32. 根据权利要求19-31中任一项所述的试剂盒，其中所述单链DNA包含编码RNA和/或蛋白质的正义链核酸序列。
  33. 根据权利要求19-32中任一项所述的试剂盒，其中所述单链DNA包含编码RNA和/或蛋白质的反义链核酸序列。
  34. 根据权利要求19-33中任一项所述的试剂盒，其中所述单链DNA包含至少40个核苷酸。
  35. 根据权利要求19-34中任一项所述的试剂盒，其中所述单链DNA包含至少1500个核苷酸。
  36. 根据权利要求19-35中任一项所述的试剂盒，其中所述转染试剂包含阳离子脂质和/或阳离子聚合物。
  37. 根据权利要求19-36中任一项所述的试剂盒，其中所述转染试剂包含阳离子脂质体。
  38. 根据权利要求19-37中任一项所述的试剂盒，其中还包含一种或多种细胞培养基。
  39. 向细胞中递送核酸的方法，所述方法包括：
    使所述细胞与包含所述核酸的单链DNA及转染试剂接触，其中所述单链DNA与所述转染试剂质量比的比值为约5至约0.1。
  40. 根据权利要求39所述的方法，其中所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。
  41. 根据权利要求39-40中任一项所述的方法，其中所述单链DNA与所述转染试剂质量比的比值为约1至约0.2。
  42. 根据权利要求39-41中任一项所述的方法，其中所述单链DNA与所述转染试剂的质量比为约2:1至约1:5。
  43. 在细胞内表达外源RNA和/或外源蛋白质的方法，所述方法包括：
    向所述细胞中引入编码所述外源RNA和/或所述外源蛋白质的单链DNA。
  44. 根据权利要求43所述的方法，其还包括使所述细胞在允许所述外源RNA和/或所述外源蛋白质表达的条件下生长。
  45. 降低与DNA转染有关的细胞毒性的方法，所述方法包括：转染时，向所述细胞中引入单链DNA而非相应的双链DNA。
  46. 降低与DNA转染有关的细胞免疫应答的方法，所述方法包括：转染时，向所述细胞中引入单链DNA而非相应的双链DNA。
  47. 提高细胞的DNA转染效率的方法，所述方法包括：转染时，向所述细胞中引入单链DNA而非相应的双链DNA。
  48. 根据权利要求43-47中任一项所述的方法，其中向所述细胞中引入所述单链DNA包括：使所述细胞与权利要求1-17中任一项所述的组合物接触。
  49. 根据权利要求43-47中任一项所述的方法，其中向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂质量比的比值为约5至约0.1。
  50. 根据权利要求43-47及49中任一项所述的方法，其中向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂的质量比为约5:1至约1:10。
  51. 根据权利要求43-47及49-50中任一项所述的方法，其中向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂质量比的比值为约1至约0.2。
  52. 根据权利要求43-47及49-51中任一项所述的方法，其中向所述细胞中引入所述单链DNA包括：使所述细胞与所述单链DNA及转染试剂接触，且所述单链DNA与所述转染试剂的质量比为约2:1至约1:5。
  53. 根据权利要求39及48-52中任一项所述的方法，其中所述接触包括在约25℃至约40℃下孵育。
  54. 根据权利要求39及48-53中任一项所述的方法，其中所述接触包括在约37℃下孵育。
  55. 根据权利要求39及48-54中任一项所述的方法，其中所述接触包括孵育至少4小时。
  56. 根据权利要求39-55中任一项所述的方法，其中所述单链DNA为环状单链DNA。
  57. 根据权利要求39-56中任一项所述的方法，其中所述单链DNA为线性单链DNA。
  58. 根据权利要求39-57中任一项所述的方法，其中所述单链DNA包含编码一种或多种外源RNA和/或外源蛋白质的核酸序列。
  59. 根据权利要求39-58中任一项所述的方法，其中所述单链DNA包含编码两种或更多种外源RNA和/或外源蛋白质的核酸序列。
  60. 根据权利要求39-59中任一项所述的方法，其中所述单链DNA包含编码外源RNA和/或外源蛋白质的正义链核酸序列。
  61. 根据权利要求39-60中任一项所述的方法，其中所述单链DNA包含编码外源RNA和/或外源蛋白质的反义链核酸序列。
  62. 根据权利要求39-61中任一项所述的方法，其中所述单链DNA包含至少40个核苷酸。
  63. 根据权利要求39-62中任一项所述的方法，其中所述单链DNA包含至少1500个核苷酸。
  64. 根据权利要求39-63中任一项所述的方法，其中所述单链DNA的含量为约10ng至约1000ng。
  65. 根据权利要求39-64中任一项所述的方法，其中所述单链DNA的浓度为约0.5nM至约3nM。
  66. 根据权利要求39-42及49-65中任一项所述的方法，其中所述转染试剂包含阳离子脂质和/或阳离子聚合物。
  67. 根据权利要求39-42及49-66中任一项所述的方法，其中所述转染试剂包含阳离子脂质体。
  68. 根据权利要求39-67中任一项所述的方法，所述方法为体内方法、体外方法或离体方法。
  69. 根据权利要求39-68中任一项所述的方法，其中所述细胞为体细胞，肿瘤细胞和/免疫细胞。
  70. 根据权利要求39-69中任一项所述的方法，其中所述细胞选自：肺癌细胞，肾细胞，黑色素瘤细胞，肝癌细胞，宫颈癌细胞，胶质瘤细胞，上皮细胞，结肠癌细胞，成纤维细胞，乳腺癌细胞，胃癌细胞和免疫效应细胞。
  71. 根据权利要求39-70中任一项所述的方法，其中所述细胞选自：A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞,MDCK细胞，B16细胞，Hep3B细胞，L929细胞，4T1细胞，293T细胞，MGC803细胞和巨噬细胞。
  72. 经权利要求39-71中任一项所述的方法处理得到的细胞。
  73. 生物体或其组成部分，其包含权利要求72所述的细胞。
  74. 根据权利要求73所述的生物体或其组成部分，其中所述生物体为非人生物体。
  75. 根据权利要求73-74中任一项所述的生物体或其组成部分，其中所述组成部分选自：组织、器官及其组合。
  76. 单链DNA用于制备转染组合物的用途，所述转染组合物用于下述的一项或多项：
    将所述单链DNA中包含的至少部分核酸递送至细胞内；
    在细胞内表达由所述单链DNA编码的外源RNA或外源蛋白；
    提高细胞的DNA转染效率；
    降低与DNA转染有关的细胞毒性；和/或
    降低与DNA转染有关的细胞免疫应答。
  77. 根据权利要求76所述的用途，其中所述转染组合物为权利要求1-17中任一项所述的组合 物。
  78. 根据权利要求76-77中任一项所述的用途，其中所述细胞为体细胞，肿瘤细胞和/免疫细胞。
  79. 根据权利要求76-78中任一项所述的用途，其中所述细胞选自：肺癌细胞，肾细胞，黑色素瘤细胞，肝癌细胞，宫颈癌细胞，胶质瘤细胞，上皮细胞，结肠癌细胞，成纤维细胞，乳腺癌细胞，胃癌细胞和免疫效应细胞。
  80. 根据权利要求76-79中任一项所述的用途，其中所述细胞选自：A549细胞,C6细胞,MCF-7细胞,HepG2细胞,CT26细胞,Hela细胞,Helf细胞,MDCK细胞，B16细胞，Hep3B细胞，L929细胞，4T1细胞，293T细胞，MGC803细胞和巨噬细胞。
PCT/CN2021/121765 2020-09-30 2021-09-29 核酸递送方法及系统 WO2022068884A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP21874539.6A EP4223881A4 (en) 2020-09-30 2021-09-29 NUCLEIC ACID DELIVERY METHOD AND SYSTEM
CN202180066876.4A CN116583606A (zh) 2020-09-30 2021-09-29 核酸递送方法及系统
US18/192,674 US20230272416A1 (en) 2020-09-30 2023-03-30 Method and system for delivering nucleic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011058515.9 2020-09-30
CN202011058515.9A CN112195193A (zh) 2020-09-30 2020-09-30 单链dna在基因转染中的应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/192,674 Continuation US20230272416A1 (en) 2020-09-30 2023-03-30 Method and system for delivering nucleic acid

Publications (1)

Publication Number Publication Date
WO2022068884A1 true WO2022068884A1 (zh) 2022-04-07

Family

ID=74007283

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/121765 WO2022068884A1 (zh) 2020-09-30 2021-09-29 核酸递送方法及系统

Country Status (4)

Country Link
US (1) US20230272416A1 (zh)
EP (1) EP4223881A4 (zh)
CN (2) CN112195193A (zh)
WO (1) WO2022068884A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112195193A (zh) * 2020-09-30 2021-01-08 上海交通大学 单链dna在基因转染中的应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000074635A2 (en) * 1999-06-07 2000-12-14 Mirus Corporation Delivery of single stranded dna for expression
CN101124316A (zh) * 2004-12-17 2008-02-13 日东电工株式会社 用于转染真核细胞的固定的可降解阳离子聚合物
CN101952434A (zh) * 2007-12-13 2011-01-19 聚加转染公司 使用合成的聚合物递送对基因沉默有活性的核酸的工具
WO2011144358A1 (en) * 2010-05-21 2011-11-24 Curevac Gmbh Histidine-containing solution for transfection and/or injection of nucleic acids and uses thereof
CN107980059A (zh) * 2015-04-13 2018-05-01 美克斯细胞有限公司 用于修饰基因组dna的方法和组合物
WO2019041344A1 (en) * 2017-09-04 2019-03-07 Hebei University Of Science And Technology METHODS AND COMPOSITIONS FOR THE TRANSFECTION OF SINGLE STRANDED DNA
CN112195193A (zh) * 2020-09-30 2021-01-08 上海交通大学 单链dna在基因转染中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100468316B1 (ko) * 2002-01-29 2005-01-27 주식회사 웰진 Ｄｎａ의 세포 또는 조직 내 전달 효율을 높이는 펩타이드
JP2020127382A (ja) * 2019-02-08 2020-08-27 森永乳業株式会社 インターフェロン産生促進物質のスクリーニング法及びキット

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000074635A2 (en) * 1999-06-07 2000-12-14 Mirus Corporation Delivery of single stranded dna for expression
CN101124316A (zh) * 2004-12-17 2008-02-13 日东电工株式会社 用于转染真核细胞的固定的可降解阳离子聚合物
CN101952434A (zh) * 2007-12-13 2011-01-19 聚加转染公司 使用合成的聚合物递送对基因沉默有活性的核酸的工具
WO2011144358A1 (en) * 2010-05-21 2011-11-24 Curevac Gmbh Histidine-containing solution for transfection and/or injection of nucleic acids and uses thereof
CN107980059A (zh) * 2015-04-13 2018-05-01 美克斯细胞有限公司 用于修饰基因组dna的方法和组合物
WO2019041344A1 (en) * 2017-09-04 2019-03-07 Hebei University Of Science And Technology METHODS AND COMPOSITIONS FOR THE TRANSFECTION OF SINGLE STRANDED DNA
CN112195193A (zh) * 2020-09-30 2021-01-08 上海交通大学 单链dna在基因转染中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4223881A4 *

Also Published As

Publication number Publication date
EP4223881A1 (en) 2023-08-09
CN112195193A (zh) 2021-01-08
US20230272416A1 (en) 2023-08-31
CN116583606A (zh) 2023-08-11
EP4223881A4 (en) 2024-04-03

Similar Documents

Publication Publication Date Title
US11692205B2 (en) Systems and methods for one-shot guide RNA (ogRNA) targeting of endogenous and source DNA
EP2831239B1 (en) Artificial nucleic acid molecules
JP6745272B2 (ja) 核酸を細胞内へ導入するための組成物
JP2020535805A (ja) 細胞の遺伝子修飾のための非組込みｄｎａベクター
KR20180104692A (ko) Angpt2 및 pdgfb를 표적화하는 rna 복합체를 사용하는 혈관신생 관련 질환의 치료
WO2021113494A1 (en) Synthetic guide rna, compositions, methods, and uses thereof
WO2022068884A1 (zh) 核酸递送方法及系统
US20240209362A1 (en) Linear DNA with Enhanced Resistance Against Exonucleases
EP2739738B1 (en) Use of integrase for targeted gene expression
WO2023006978A1 (en) Linear dna with enhanced resistance against exonucleases and methods for the production thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21874539

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202180066876.4

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021874539

Country of ref document: EP

Effective date: 20230502