WO2019062887A1 - 一种体外产生环状核酸分子的方法 - Google Patents

一种体外产生环状核酸分子的方法 Download PDF

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WO2019062887A1
WO2019062887A1 PCT/CN2018/108557 CN2018108557W WO2019062887A1 WO 2019062887 A1 WO2019062887 A1 WO 2019062887A1 CN 2018108557 W CN2018108557 W CN 2018108557W WO 2019062887 A1 WO2019062887 A1 WO 2019062887A1
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nucleic acid
cells
acid molecule
sequence
linear nucleic
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French (fr)
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王皓毅
程晨
唐娜
李佳鑫
操时伟
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中国科学院动物研究所
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    • 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
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the invention belongs to the field of biomedicine and relates to a method for producing a circular nucleic acid molecule in vitro.
  • the present invention relates to an in vitro method for efficiently producing microcircle DNA independent of bacteria, and to microcircle DNA produced by the method and uses thereof.
  • Gene therapy is an attractive treatment for diseases. At present, it is mainly through genetic recombination technology to increase gene copies, modify genes, inactivate genes or correct mutant genes to achieve disease treatment. In recent years, chimeric antigen receptor (CAR) engineered T cells have been successfully used to treat certain cancers.
  • CAR chimeric antigen receptor
  • gene therapy vectors have been mainly viral vectors such as intensive lentivirus (LV) and gamma-retrovirus (RV).
  • viral vectors such as intensive lentivirus (LV) and gamma-retrovirus (RV).
  • LV and RV vectors prefers integration into certain highly expressed genes as well as genes involved in the development and progression of cancer, posing a significant safety hazard in clinical applications.
  • the high cost of viral vector production and the high demands associated with clinical applications make it difficult to be widely accepted.
  • non-viral vector The most common non-viral vector is a plasmid, which carries a gene of interest to the target cell and expresses the target product at a high level very quickly, but the gene is expressed for a short period of time and will rapidly fall back to a very low level within a few days.
  • bacterial backbone DNA linked to the gene of interest is an important cause of transcriptional silencing of the target gene.
  • the plasmid backbone may also induce a natural immune response in the cell, causing cell death. Therefore, Chen et al.
  • microcircle DNA can be expressed 10 to 1000 times longer in vivo and in vitro, making them widely used in preclinical gene therapy research.
  • the existing microcircle DNA preparation methods still have the following defects: 1) relying on special bacterial strains and plasmids; 2) time-consuming and laborious; 3) safety hazards, such as in bacteria Endotoxin carried during preparation. Therefore, there is still a need in the art for efficient methods for preparing safer microcircle DNA.
  • the present invention establishes a simple and rapid method for preparing microcircle DNA without the aid of bacteria, which is significantly superior to existing preparation techniques (see Figure 10).
  • the method of the invention is based on some simple molecular biology experiments and is capable of completing the preparation of microcircle DNA in a matter of hours.
  • the safety of microcircle DNA products is enhanced by eliminating the need for bacterial strains and avoiding possible endotoxin contamination.
  • the microcircle DNA prepared by the method of the present invention is excellent in cell lines and stem cells.
  • the microcircle DNA-engineered CAR-T cells obtained by the method of the invention can significantly inhibit tumor development and kill tumor cells in vivo and in vitro, and have potential clinical application value.
  • the microcircle DNA prepared according to the method of the present invention can improve the safety of CAR-T cell therapy and gene therapy, and reduce production cost and production time.
  • the invention provides a method of producing a circular nucleic acid molecule comprising a target nucleotide sequence, such as a microcircle DNA, the method comprising:
  • the linear nucleic acid molecule in the mixture is self-ligated by its complementary single-stranded overhanging end with a nucleic acid ligase such as T4 ligase, thereby self-cyclizing to form a circular nucleic acid molecule such as microcircle DNA.
  • a nucleic acid ligase such as T4 ligase
  • the single stranded overhang comprises 2, 3, 4, 5 or 6 or more nucleotides.
  • step a) A mixture of nucleic acid molecules.
  • each linear nucleic acid molecule in the mixture is from 0.01 to 20 ng/ ⁇ l, preferably from 0.01 to 10 ng/ ⁇ l, more preferably from 0.01 to 5 ng/ ⁇ l, more preferably from 0.01 to 2.5 ng/ ⁇ l, more It is preferably 0.01-1 ng/ ⁇ l.
  • the total concentration of linear nucleic acid molecules in the mixture is from 0.01 to 200 ng/[mu]l or greater.
  • the method further comprises:
  • step 2 mixing and optionally purifying the amplification product of step 1);
  • step 3 Optionally purifying the digestion product of step 3).
  • step 2) amplifying the linear nucleic acid molecules obtained in step 1) with a plurality of sets of primer pairs directed against the universal sequence tag, wherein the primers in each set of the primer pairs are cleaved and formed by a restriction enzyme at the 5' end. a sequence of unique complementary single-stranded overhangs;
  • step 2 mixing and optionally purifying the amplification product of step 2);
  • step 4) The digestion product of step 4) is optionally purified.
  • restriction endonuclease is BbsI
  • the two primers of the primer pair comprise 5'-GAAGACNNN 1 N 2 N 3 N 4 -3' and 5'-GAAGACNNN 5 N 6 N 7 N 8 -3' at the 5' end, respectively.
  • the circular nucleic acid molecule is a microcircle DNA.
  • the target nucleotide sequence comprises a coding sequence operably linked to a transcriptional regulatory element, such as a promoter and/or a terminator.
  • the coding sequence encodes a protein or RNA of interest.
  • the invention provides a kit for use in practicing the methods of the invention.
  • the invention provides a circular nucleic acid molecule produced by the method of the invention.
  • the circular nucleic acid molecule is a microcircle DNA.
  • FIG. 1 Schematic diagram of the preparation of minicircle DNA of the present invention.
  • Figure 2 Flow diagram of the preparation of minicircle DNA of the present invention.
  • FIG. 3 The eGFP expression cassette was amplified from the parental plasmid with 1 pair of primers and 96 pairs of primers (the sticky ends formed by the different primer pairs are mutually incompatible). The concentration of the GFP fragment after digestion with the restriction endonuclease BbsI affects the efficiency of cyclization.
  • B The 019-CAR expression sequence was amplified from the parental plasmid using a primer containing a pair of linkers and a primer containing 96 pairs of linkers, respectively. The concentration of the 019-CAR fragment product after restriction endonuclease BbsI digestion affects the efficiency of cyclization.
  • FIG. 4 Target fragments amplified with 96 pairs of primers (the sticky ends formed by different primer pairs are incompatible with each other) increase the yield of minicircle DNA.
  • A Agarose gel electrophoresis pattern of the minicircle-eGFP DNA finally produced by the pair of primer-amplified eGFP fragments at concentrations of 1, 5, 10, 20, 40 and 60 ng/ ⁇ l (top).
  • 96 pairs of primer-amplified eGFP fragments were finally subjected to agarose gel electrophoresis of the minicircle-eGFP DNA at concentrations of 1, 30, 60, 90 and 180 ng/ ⁇ l (bottom).
  • the bands shown by the arrows represent the target minicircle-eGFP DNA.
  • the cyclization efficiency is quantified and shown as a numerical value.
  • B Agarose gel electrophoresis of the 019-CAR fragment amplified by 1 pair of primers at a concentration of 1, 5, 10, 20, 40 and 60 ng/ ⁇ l to finally produce minicircle-019 DNA (top).
  • the 019-CAR fragment amplified by 96 pairs of primers finally produced minicircle-019 DNA agarose gel electrophoresis at concentrations of 1, 30, 60, 90 and 180 ng/ ⁇ l (bottom).
  • the band shown by the arrow represents the target minicircle-019 DNA.
  • the cyclization efficiency is quantified and shown as a numerical value.
  • C Quantitative statistical results of the circularization efficiency of minicircle-eGFP DNA shown in A.
  • D Quantitative graph of the circularization efficiency of minicircle-019 DNA shown in B.
  • FIG. 5 Transgene expression levels and duration of minicircle-eGFP in K562 cell lines.
  • A The linear PCR product of eGFP, minicircle-eGFP DNA, and eGFP-encoding plasmid having a bacterial skeleton were electroporated into K562 cells, and cell viability was measured on the 2nd and 4th day after electroporation, which is the detection result.
  • B The linear PCR product of eGFP, minicircle-eGFP DNA, and eGFP-encoding plasmid having a bacterial skeleton were electroporated into K562 cells, and the proportion of eGFP-positive cells decreased with time.
  • FIG. 6 Minicircle DNA electroporation does not affect the multi-lineage differentiation ability of CD34 + HSC.
  • CD34 + HSC separates cord blood from two different donors, AE is the result of donor 1, and FJ is the result of donor 2.
  • the clones of CD34 + HSC were numbered to indicate the ability of CD34 + HSC to form clones, and the figure is the result of the assay.
  • FIG. 7 Electrocirculation of minicircle DNA into human embryonic stem cells.
  • A The minicircle-eGFP DNA or plasmid encoding eGFP was electroporated into human embryonic stem cell H9, and a fluorescence microscope photograph was taken 48 hours after electroporation, which is the result.
  • B The minicircle-eGFP or plasmid encoding eGFP was electroporated into H9 cells, and the ratio of eGFP was analyzed by flow cytometry 48 hours after electroporation, which is the analysis result.
  • FIG. 8 019-CAR-T cells (from Donor 1) prepared with minicircle-019 DNA can kill tumor cells specifically and efficiently.
  • A The minicircle-019 DNA and plasmid-019 were electrotransferred to T cells, and T cells were photographed by fluorescence microscopy 48 hours after electroporation, which is a photograph taken.
  • B Cell viability assay on days 2, 4, and 6 of T cells transfected with minicircle-019 DNA and plasmid-019.
  • C Expression level of 019-CAR in T cells transfected with minicircle-019 DNA by FACS.
  • (G) Bioluminescence of NGP mice carrying Raji-luciferase tumor cells treated with T cells, lenti-CAR-T cells, mini-CAR-T cells or the same volume of PBS for 3, 16 and 30 days Imaging results (n 3).
  • FIG. 9 019-CAR-T cells (from Donor 2) prepared with minicircle can specifically kill tumor cells with high efficiency.
  • A Expression level of 019-CAR in T cells transfected with minicircle-019 DNA by FACS.
  • B The concentration of IFN- ⁇ and IL2 released by mini-CAR-T cells prepared by minicircle after incubation of 019-CAR T cells and tumor cells for 24 hours.
  • C The ability of mini-CAR-T cells or lenti-CAR-T to lyse tumor cells at different effector-target cell (E:T) ratios.
  • D Flow chart of in vivo functional testing of CAR-T cells engineered with minicircle and lentiviral vectors. ****P ⁇ 0.0001.
  • FIG. 10 Comparison of the minicircle DNA preparation method of the present invention and the prior art.
  • the invention provides a method of producing a circular nucleic acid molecule comprising a target nucleotide sequence, the method comprising:
  • the linear nucleic acid molecule in the mixture is self-ligated by its complementary single-stranded overhanging end with a nucleic acid ligase such as T4 ligase, thereby self-cyclizing to form a circular nucleic acid molecule.
  • a nucleic acid ligase such as T4 ligase
  • unique complementary single-stranded overhang means that a single-stranded overhang of one end of a linear nucleic acid molecule is complementary to a single-stranded overhang at the other end (also commonly referred to as a sticky end), but not in a mixture
  • the single-stranded overhangs of other types of linear nucleic acid molecules are complementary (ie, incompatible).
  • sequences of the plurality of linear nucleic acid molecules other than the unique complementary single-stranded overhang are identical.
  • the plurality of linear nucleic acid molecules comprise the same target nucleotide sequence.
  • the total number of possible sequences for the overhanging ends of N nucleotides is 4 N (4 N ), and since the protruding ends of one end of each linear nucleic acid molecule need to be complementary to the other end to achieve cyclization, a possible unique complementary end It is 4 N /2.
  • the single stranded overhang can comprise 2, 3, 4, 5 or 6 or more nucleotides.
  • 2-8, 22-3, 2-128, 2-512, or 2-2048 or more of the target nucleotides are provided in step a) A mixture of linear nucleic acid molecules of a sequence.
  • step a) provides no less than 8, no less than 16, no less than 24, no less than 32, no less than 40, no less than 48, and a plurality of A mixture of 56, not less than 64, not less than 72, not less than 80, and not less than 96 linear nucleic acid molecules comprising the target nucleotide sequence.
  • each linear nucleic acid molecule in the mixture may be from 0.01 to 20 ng/ ⁇ l, preferably from 0.01 to 10 ng/ ⁇ l, more preferably from 0.01 to 5 ng/ ⁇ l, more preferably from 0.01 to 2.5 ng/ ⁇ l, More preferably, it is 0.01-1 ng/ ⁇ l.
  • the total concentration of linear nucleic acid molecules in the mixture can be from 0.01 to 200 ng/ ⁇ l or higher, such as 10 ng/ ⁇ l, 20 ng/ ⁇ l, 40 ng/ ⁇ l, 60 ng/ ⁇ l, 80 ng/ ⁇ l, 100 ng / ⁇ l, 120 ng/ ⁇ l, 140 ng/ ⁇ l, 160 ng/ ⁇ l, 180 ng/ ⁇ l, 200 ng/ ⁇ l or higher.
  • the method of the invention further comprises the steps of:
  • the step of purifying the resulting circular nucleic acid molecule is also optionally included.
  • the purification step may be after step b) or may be after step c).
  • the unique complementary single-stranded overhanging ends of the linear nucleic acid molecule can be produced by various methods, for example, by directly adding a linker to both ends of the linear nucleic acid molecule, or by designing a suitable primer through the polymerase. Chain reaction (PCR) amplification and subsequent restriction enzyme digestion were introduced.
  • PCR Chain reaction
  • nucleic acid linker pairs to the linear nucleic acid molecule, the plurality of sets of nucleic acid linkers comprising a 5' linker and a 3' linker for each set and the 5' linker and the 3' linker comprising a unique complementary single strand Highlight the end;
  • amplification eg, PCR amplification
  • a plurality of sets of target-specific primer pairs each of which contains a primer at the 5' end that can be a restriction endonuclease cleaves and forms a unique sequence of complementary single-stranded overhangs
  • step 2 mixing and optionally purifying the amplification product of step 1);
  • step 3 Optionally purifying the digestion product of step 3).
  • primer pairs multiple sets of target-specific primer pairs need to be designed and synthesized separately for different target nucleotide sequences.
  • the number of primer pairs that can be used depends on the number of nucleotides of the single-stranded overhang formed by the restriction endonuclease used (as described above). For example, 2-8, 2-32, 2-128, 2-512, or 2-2048 or more primer pairs can be used in step 1). For example, it may be used in no less than 8 kinds, not less than 16, not less than 24, not less than 32, not less than 40, not less than 48, not less than 56, not less than 64 Species, not less than 72, not less than 80, not less than 96 primer pairs.
  • the restriction endonuclease is BbsI
  • the two primers of the primer pair comprise 5'-GAAGACNNN 1 N 2 N 3 N 4 -3' and 5, respectively, at the 5' end A sequence of '-GAAGACNNN 5 N 6 N 7 N 8 -3', wherein N represents any one of A, T, C and G, and the sequence N 1 N 2 N 3 N 4 and the sequence N 5 N 6 N 7 N 8 Reverse complementation.
  • a universal sequence tag at both ends of the linear nucleic acid molecule comprising the target nucleotide sequence by PCR, and then to use multiple sets of primer pairs for the universal sequence tag ( Amplification is carried out comprising a sequence which is cleaved by a restriction enzyme and forms a unique complementary single-stranded overhang.
  • Amplification is carried out comprising a sequence which is cleaved by a restriction enzyme and forms a unique complementary single-stranded overhang.
  • step 2) using a plurality of sets of primer pairs for the universal sequence tag, respectively, using the linear nucleic acid molecule obtained in step 1) as a template for amplification (for example, PCR amplification), and the primers in each set of the primer pair are included at the 5' end. a sequence that is cleaved by a restriction enzyme and forms a unique complementary single-stranded overhang;
  • step 2 mixing and optionally purifying the amplification product of step 2);
  • step 4) The digestion product of step 4) is optionally purified.
  • the number of primer pairs that can be used depends on the number of nucleotides of the single-stranded overhang formed by the restriction endonuclease used (as described above). For example, 2-8, 2-32, 2-128, 2-512, or 2-2048 or more primer pairs can be used in step 2). For example, it may be used in no less than 8 kinds, not less than 16, not less than 24, not less than 32, not less than 40, not less than 48, not less than 56, not less than 64 Species, not less than 72, not less than 80, not less than 96 primer pairs.
  • the restriction endonuclease is BbsI
  • the two primers of the primer pair comprise 5'-GAAGACNNN 1 N 2 N 3 N 4 -3' and 5, respectively, at the 5' end A sequence of '-GAAGACNNN 5 N 6 N 7 N 8 -3', wherein N represents any one of A, T, C and G, and the sequence N 1 N 2 N 3 N 4 and the sequence N 5 N 6 N 7 N 8 Reverse complementation.
  • FIG. Fig. 1 exemplarily shows the recognition site and the cutting manner of BbsI.
  • amplification and enzymatic digestion yielded four target linear nucleic acid molecules comprising unique complementary single-stranded overhangs that are not linked to each other.
  • high cyclization efficiency can be achieved because they prefer a self-joining of one molecule rather than a linear connection between two identical molecules.
  • FIG. 1 is merely illustrative of the method of the present invention and is not intended to limit the scope of the invention.
  • Suitable primer sequences can be readily designed by those skilled in the art in light of the teachings of the present invention.
  • Additional restriction enzymes useful in the methods of the invention include, but are not limited to, ApaI, BbvI, BsaI, BfuAI, BglI, BsmAI, BsmBI, BsmFI, BspMI, BslI, BstAPI, BstXI, BtgZI, DraIII, EarI, HgaI, and the like.
  • the methods of the invention are suitable for obtaining any circular double stranded nucleic acid molecule.
  • the circular nucleic acid molecule is minicircle DNA.
  • minicircle DNA is used interchangeably with "microcircle DNA”, which is a non-viral gene vector that contains only the expression cassette of the gene of interest, excluding the backbone sequence from the bacterial plasmid, compared to conventional plasmid vectors. . Minicircle DNA generally exists in the form of a circular supercoil.
  • the target nucleotide sequence comprises a coding sequence operably linked to a transcriptional regulatory element.
  • regulatory sequence and “regulatory element” are used interchangeably and refer to either upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) of a coding sequence, and affecting the relevant coding sequence. Transcription, RNA processing or stability or translation of nucleotide sequences.
  • An expression control element refers to a nucleotide sequence capable of controlling transcription, RNA processing or stability or translation of a nucleotide sequence of interest.
  • Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences.
  • Promoter refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment.
  • a promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell.
  • operably linked refers to a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) linked to a nucleic acid sequence (eg, a coding sequence or an open reading frame) such that the nucleotide Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements.
  • a regulatory element such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.
  • nucleic acid sequence eg, a coding sequence or an open reading frame
  • the coding sequence encodes a protein of interest or an RNA of interest.
  • the protein of interest is, for example, a T cell receptor (TCR), a chimeric antigen receptor (CAR), a Cas9 nuclease or other therapeutic protein.
  • the RNA of interest is, for example, sgRNA, antisense RNA, antagomir, siRNA or shRNA or other therapeutic RNA.
  • the CAR is an anti-CD19-CAR.
  • the invention provides a circular nucleic acid molecule, in particular a minicircle DNA, produced according to the method of the invention.
  • the invention provides the use of a circular nucleic acid molecule, in particular minicircle DNA, produced according to the methods of the invention in the preparation of modified T cells.
  • the modified T cell is, for example, a CAR-T cell.
  • the CAR-T cell is an anti-CD19-CAR-T cell.
  • the invention provides the use of a circular nucleic acid molecule, in particular a minicircle DNA, produced according to the methods of the invention in gene therapy.
  • the invention provides the use of a circular nucleic acid molecule, in particular a minicircle DNA, produced by the method of the invention, for introducing a target nucleotide sequence into a cell.
  • a circular nucleic acid molecule in particular a minicircle DNA
  • Such cells include, but are not limited to, cell lines, primary cells, stem cells such as embryonic stem cells or hematopoietic stem cells, or T cells.
  • the circular nucleic acid molecules produced by the methods of the invention can be introduced into cells by methods known in the art, such as: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, and the like.
  • the invention provides a kit for producing a circular nucleic acid molecule, such as minicircle DNA, by the methods of the invention.
  • the kit can include a plurality of sets of nucleic acid linkers, each set comprising a 5' linker and a 3' linker and the 5' linker and 3' linker comprise unique complementary single stranded overhangs.
  • the kit can include sets of primer pairs for universal sequence tags, each set of primers comprising a primer at the 5' end that is cleaved by a restriction enzyme and forms a unique complementary single-stranded overhang. sequence.
  • the kit of the present invention may further comprise various reagents for use in the method of the present invention, such as a polymerase for amplification, a ligase for ligation such as T ligase, and an exonuclease for removing single-stranded nucleic acid.
  • various reagents for use in the method of the present invention such as a polymerase for amplification, a ligase for ligation such as T ligase, and an exonuclease for removing single-stranded nucleic acid.
  • T5 exonuclease such as BbsI
  • reagents and devices for purifying nucleic acid molecules such as BbsI
  • various buffers such as BbsI
  • the invention also provides the use of minicircle DNA in the preparation of modified T cells.
  • the modified T cell is, for example, a CAR-T cell.
  • the CAR-T cell is an anti-CD19-CAR-T cell.
  • each primer contained a restriction endonuclease BbsI recognition site followed by a 6 bp unique sequence such that the BbsI-digested PCR product would have a 4 bp single-stranded overhang at both ends.
  • the total number of possible combinations of these 4 bp overhangs is 256 (44), and since the overhanging ends of one end of each PCR product need to be complementary to the other end to achieve cyclization, the number of possible unique end pairs is 128.
  • 96 design primer pairs were randomly selected from these 128 combinations, and the primers used in the experiments are shown in Table 1.
  • Amplification of these 96 pairs of primers from AAVS1-2KB-EF1a-019-2A-eGFP/AAVS1-1KB-CMV-eGFP (TAKARA, PrimeSTAR@HS DNA Polymerase, Cat: #R010B) target fragment (EF1a-019-2A) -eGFP/cmv-eGFP).
  • the amplification procedure was: 98 ° C for 2 min; 35 x (98 ° C for 10 s, 60 ° C for 30 s, 72 ° C for 2 / 4 min; 72 ° C for 3 min; 4 ° C retention.
  • the PCR products of 96 PCR reactions were all mixed and purified using Qiagen, QIAquick PCR Purififation Kit (Cat No./ID: 28106).
  • the PCR product (New ENGLAND, Cat: #R0539L) was digested with restriction endonuclease Bbs1 and the digested product was purified again (QIAquick PCR Purififation Kit).
  • the obtained target fragment was ligated with T4 ligase (New ENGLAND, Cat: #M0202L) for 2 hours at 16 ° C, followed by treatment with T5 exonuclease ENGLAND, Cat: #M0363L) at 37 ° C for 2 hours.
  • the purified product was collected to obtain a minicircle-DNA of non-bacterial origin.
  • the BbsI recognition site is shown in italics and the unique end sequence of 4 bp is underlined.
  • K562 erythroleukemia cell line
  • Raji Burkitt's lymphoma cell line
  • ATCC American Type Culture Collection
  • Raji-fluc cells for bioluminescence imaging and K562-CD19 cells expressing tumor antigen CD19 were constructed as previously described (Chen et al., Mol Ther 2003; 8:495-500). All of the above cells were cultured in RPMI1640 medium (ThermoFisher Scientific).
  • the lentivirus producing cell line 293T was cultured in DMEM (ThermoFisher Scientific).
  • All media were supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. All cell lines were grown at 37 ° C, 5% CO 2 .
  • Fresh cord blood was obtained from the Beijing Cord Blood Bank (Beijing, China) with informed consent from healthy volunteer donors.
  • Mononuclear cells were collected by Hispanaque-1077 (Sigma-Aldrich) gradient separation, and T cells were isolated using an EasySep Human T Cell Enrichment Kit (Stemcell Technologies).
  • T cells were co-cultured with T-cells in a ratio of 1:1 with anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) and cultured in X-vivo15 medium (Lonza) supplemented with 5% (v/v) heat.
  • Fresh cord blood was obtained from the Beijing Cord Blood Bank (Beijing, China) with informed consent from healthy volunteer donors.
  • Mononuclear cells were collected by Histopaque-1077 (Sigma-Aldrich) gradient separation and CD34 + HSCs were isolated using the EasySep Human Cord Blood CD34 Positive Selection Kit (Stemcell Technologies). After separation, CD34 + cells were cultured in a stem span H3000 (Stemcell Technologies, #09800) supplemented with a small factor supplement that maintained dryness (Stemcell Technologies, #02691).
  • CD34 cells were electroporated with 1 ⁇ g of Mini-eGFP/Pasmid-eGFP by 4D-Nucleofector System N (Lonza) using P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to the instructions.
  • Use Program EO-100 5 ⁇ 10 4 CD34 cells were electroporated with 1 ⁇ g of Mini-eGFP/Pasmid-eGFP by 4D-Nucleofector System N (Lonza) using P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to the instructions.
  • Use Program EO-100 Use Program EO-100.
  • the cells were resuspended in 37 ° C prewarmed cell culture medium and transferred to a Petri dish, and cultured at 37 ° C in 5% CO 2 . Cell viability and transfection efficiency were assessed by cell counting and FACS analysis 2 days after electroporation, respectively.
  • Freshly purified primary T cells were activated for 1 day and then infested with a lentiviral vector carrying the second generation CD19 CAR.
  • the structure of CAR is as previously described (Chen et al., Mol Ther 2003; 8: 495-500). Infection efficiency was assessed by FACS analysis two days after transduction.
  • T cells were activated for 3 days according to the above method, and then 1 ⁇ 10 6 cells were electroporated with 3 ⁇ g of Mini-019 DNA by 4D-Nucleofector System N (Lonza) using P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza) according to the instructions. perforation. Use Program EO-115.
  • the cells were resuspended in 1 ml of pre-warmed cell culture medium and transferred to a 12-well cell plate and cultured in 5% CO 2 at 37 °C. Cell viability and transfection efficiency were assessed by cell counting and FACS analysis 2 days after electroporation, respectively.
  • CAR-T cells The cytotoxicity of CAR-T cells was assessed by a luciferase-based CTL assay as previously described (Chen et al., Gene Ther 2004; 11: 856-864).
  • K562-1uc and K562-CD19-1uc cell lines were generated by infecting K562 and K562-CD19 cells with lentivirus expressing luciferase.
  • K562-1uc and K562-CD19-1uc cells were resuspended in 100 ⁇ l of RPMI1640 medium at 1 ⁇ 10 5 cells/ml and mixed with effector cells in different ratios (for example, 25:1, 15:1, etc.) in a total volume of 200 ⁇ l. After incubation for 16 hours at 37 ° C in 5% CO 2 , 10 ⁇ l of substrate was added and fluorescence was measured after 5 minutes.
  • the efficiency of CAR-T cell lysis on target cells is:
  • Kill % 100-((RLU of effector cell and target cell co-culture) / (RLU of target cell)) x100
  • Effector cells T, Mini-019-CART
  • target tumor cells K562-CD19, K562
  • T mice
  • K562-CD19, K562 target tumor cells
  • Example 1 Rapid preparation of microcircle DNA containing a target sequence by a simple and safe method.
  • a minicircle DNA vector expressing eGFP or anti-CD19 CAR (019-CAR) was prepared.
  • eGFP and CAR-019 were amplified with a pair of primers or 96 pairs of primers, respectively, and the primer design was as described above.
  • the product amplified with a pair of primers was purified and directly digested with Bbs1 enzyme, and 96 pairs of different primers were separately amplified, and all 96 PCR products were first mixed, and then purified and then digested with Bbs1 enzyme.
  • the self-cyclization efficiency rapidly decreases as the concentration of the substrate is increased.
  • the self-cyclization efficiency was maintained at a high level even if the concentration of the substrate was as high as 180 ng/ ⁇ l.
  • the concentration of the substrate was the same, the self-cyclization efficiency of the 96 pairs of primer sets was significantly higher than that of the pair of primer sets.
  • the minicircle DNA vector can be efficiently produced by the method of the present invention.
  • minicircle DNA prepared by the method of the present invention.
  • eGFP PCR product minicircle-eGFP DNA and eGFP plasmid with bacterial backbone sequence into K562 cells by electroporation.
  • the minicircle-eGFP DNA group On days 2 and 4 after electroporation, it was found that the K562 cells in the minicircle-eGFP DNA group were significantly more viable than the plasmid group (Fig. 5A). At the same time, eGFP-positive K562 cells were observed for 12 days (Fig. 5B). In the PCR product group and the plasmid group, the eGFP-positive cells of K562 cells quickly decreased to a low ratio, while the expression level of the transgene in the minicircle-eGFP DNA group remained high for the first 8 days, and then gradually decreased in the last 4 days (Fig. 5B). ). As shown in Figure 5C, the same trend of change was also observed for the mean fluorescence intensity (MFI). As can be seen from the above data, the minicircle-eGFP DNA group has a significant advantage in maintaining cell viability and transgene expression stability compared to the other groups.
  • MFI mean fluorescence intensity
  • HSC hematopoietic stem cells
  • the transcircle expression level of the minicircle DNA group after 48 hours of electroporation was significantly higher than that of the plasmid group, although the proportion of eGFP positive cells in the two groups was similar (Fig. 6A, B, F and G).
  • the cell viability of the minicircle DNA group was significantly higher than that of the plasmid group (Fig. 6C, H).
  • CFU colony forming unit
  • Example 4 Human embryonic stem cells transfected with minicircle DNA have higher transgene expression levels and better cell viability.
  • minicircle-eGFP DNA and plasmid-eGFP were electroporated into H9 cells.
  • H9 cells died significantly after 48 hours in the plasmid-eGFP transfection group, and only some tiny clones were formed.
  • the H9 cells of the minicircle-eGFP DNA transfection group performed much better.
  • the proportion of eGFP-positive cells in the minicircle-eGFP electroporated H9 cells was much higher compared to the plasmid-eGFP electroporated H9 cells (Fig. 7B).
  • Cell viability decreased after electrocirculation of minicircle DNA and plasmid Fig.
  • Example 5 Reliable antitumor activity of CD3 positive T cells transfected with minicircle-019 DNA in vitro and in vivo
  • CAR-T cells prepared by LV and RV have demonstrated encouraging anti-tumor effects in experimental studies and clinical trials.
  • This example further demonstrates that non-integrated transgenic CAR-T cells can be prepared using minicircle-019 DNA encoding anti-CD19CAR, and such CAR-T cells are capable of specifically killing tumor cells.
  • CAR-019-T cells from two different donors prepared with minicircle-019 DNA was analyzed (Fig. 8D-G, Fig. 9B-C).
  • the CAR T cells secrete high levels of IL2 and IFN- ⁇ after incubation with target tumor cells (Fig. 8D and Fig. 9B).
  • CAR-T cells were able to specifically and efficiently kill target tumor cells, even though both donor cells were used at low E:T ratios (Fig. 8E and Fig. 9C).
  • NPG mice were inoculated intraperitoneally with Raji-luci cells. After 3 days, these mice were divided into 4 groups based on fluorescence values. The T cell group, the minicircle-019 DNA engineered CAR-T cell group, and the lentiviral vector engineered CAR-T cell group were treated on days 3 and 9 (intraperitoneal cells). Control mice were injected with the same volume of PBS. Tumor burden was monitored weekly by luciferase in vivo imaging.
  • tumor burden was significantly reduced in the mini-CAR-T group and the lenti-CAR-T group compared to the T cell group, particularly the PBS group. From the above data, it was demonstrated that CAR-T cells engineered with minicircle-019 DNA have reliable ability to alleviate tumors in vivo and in vitro.

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Abstract

提供一种体外产生环状核酸分子的方法。具体而言，提供一种不依赖于细菌而高效产生minicircle DNA的体外方法，还涉及通过该方法产生的minicircle DNA及其用途。

Description

一种体外产生环状核酸分子的方法 技术领域
本发明属于生物医药领域，涉及一种体外产生环状核酸分子的方法。具体而言，本发明涉及一种不依赖于细菌而高效产生微环DNA的体外方法，还涉及通过该方法产生的微环DNA及其用途。
发明背景
基因治疗是极具吸引力的疾病治疗方法，目前主要是通过基因重组技术增加基因拷贝、修饰基因、失活基因或校正突变基因等来实现疾病治疗。近年来，嵌合抗原受体(CAR)工程化的T细胞已经被成功的用于治疗某些癌症。
目前为止，基因治疗载体主要是整合性慢病毒(LV)和γ-逆转录病毒(RV)等病毒载体。然而，使用LV和RV载体递送的DNA偏好于整合进某些高表达的基因以及与癌症发生、发展相关的基因，在临床应用中存在很大的安全隐患。同时，病毒载体生产的高成本以及与临床应用相关的高要求使得其难以被广泛接受。
数十年来，人们一直在致力于寻找高效的非病毒载体。最常见的非病毒载体是质粒，其携带的目的基因被递送到靶细胞后会很快高水平地表达目的产物，但是基因的表达时间较短，会在几天内迅速回落至很低的水平。研究表明与目的基因连接的细菌骨架DNA是导目的基因转录沉默的重要原因。此外，质粒骨架还可能诱发细胞天然免疫反应，造成细胞死亡。因此，Chen等开发了一种产生不含细菌相关骨架序列的微环(minicircle)DNA载体技术(Chen et al.，Mol Ther 2003；8：495-500)。与具有细菌骨架序列的质粒相比，微环DNA在体内和体外的表达时间可延长10至1000倍，这使得它们在临床前基因治疗研究中被广泛使用。然而，对于真正临床上的基因治疗，已有的微环DNA制备方法仍存在以下缺陷：1)依赖于特殊的细菌株和质粒；2)耗时费力；3)存在安全隐患，如在细菌中制备过程中携带的内毒素。因此，本领域仍然需要高效的方法制备更为安全的微环DNA。
发明简述
本发明建立了一种简单快速的、无需借助细菌的方法制备微环DNA，该方法显著优于现有的制备技术(见图10)。本发明的方法基于一些简单的分子生物学实验，并能够在几个小时内完成微环DNA的制备。由于不需要使用细菌菌株，避免了可能的内毒素污染，从而提高了微环DNA产品的安全性。通过本发明的方法制备的微环DNA在细胞系和干细胞中表现优异。用本发明的方法获得的微环DNA改造的CAR-T细胞能够在体内和体外显著抑制肿瘤发展并杀死肿瘤细胞，具有潜在的临床应用价值。根据本发明的方法所制备的微环DNA能够提高CAR-T细胞治疗和基因治疗的安全性，并且降低生产成本和生产时间。
在第一方面，本发明提供一种产生包含靶核苷酸序列的环状核酸分子例如微环DNA的方法，所述方法包括：
a)提供多种包含所述靶核苷酸序列的线性核酸分子的混合物，所述多种线性核酸分子每种在两端包含独特的互补单链突出末端；
b)用核酸连接酶如T4连接酶使所述混合物中的线性核酸分子通过其互补单链突出末端而自身连接，由此自身环化形成环状核酸分子例如微环DNA。
在一些实施方案中，其中所述单链突出末端包含2个、3个、4个、5个或6个或更多个核苷酸。
在一些实施方案中，其中步骤a)中提供2-8种、2-32种、2-128种、2-512种或2-2048种或更多种包含所述靶核苷酸序列的线性核酸分子的混合物。
在一些实施方案中，其中所述混合物中每种线性核酸分子的浓度为0.01-20ng/μl，优选0.01-10ng/μl，更优选0.01-5ng/μl，更优选0.01-2.5ng/μl，更优选0.01-1ng/μl。
在一些实施方案中，，其中所述混合物中线性核酸分子的总浓度为0.01-200ng/μl或更高。
在一些实施方案中，所述方法进一步包括：
c)使用核酸外切酶例如T5核酸外切酶去除混合物中的线性核酸分子。
在一些实施方案中，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
1)提供包含所述靶核苷酸序列的线性核酸分子；
2)将多组接头对分别连接至所述线性核酸分子，所述多组接头对每组包含5’接头和3’接头且所述5’接头和3’接头包含独特的互补单链突出末端；
3)混合并任选地纯化步骤2)中的产物。
在一些实施方案中，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
1)用多组靶特异性引物对分别扩增获得包含所述靶核苷酸序列的线性核酸分子，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列；
2)混合并任选地纯化步骤1)的扩增产物；
3)用所述限制性内切酶消化步骤2)获得的混合物；和
4)任选地纯化步骤3)的消化产物。
在一些实施方案中，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
1)用包含通用序列标签的靶特异性引物对扩增获得包含所述靶核苷酸序列的线性核酸分子，
2)用多组针对所述通用序列标签的引物对分别扩增步骤1)获得的线性核酸分子，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列；
3)混合并任选地纯化步骤2)的扩增产物；
4)用所述限制性内切酶消化步骤3)获得的混合物；和
5)任选地纯化步骤4)的消化产物。
在一些实施方案中，其中所述限制性内切酶是BbsI。
在一些实施方案中，所述引物对中的两种引物分别在5’端包含5’-GAAGACNNN 1N 2N 3N 4-3’和5’-GAAGACNNN 5N 6N 7N 8-3’的序列，其中N代表A、T、C和G中任一个，且序列N 1N 2N 3N 4与序列N 5N 6N 7N 8反向互补。
在一些实施方案中，其中所述环状核酸分子是微环DNA。
在一些实施方案中，其中所述靶核苷酸序列包含与转录调控元件例如启动子和/或终止子可操作连接的编码序列。
在一些实施方案中，其中所述编码序列编码感兴趣的蛋白质或RNA。
在第二方面，本发明提供一种试剂盒，其用于实施本发明的方法。
在第三方面，本发明提供环状核酸分子，其通过本发明的方法产生。 在一些实施方案中，所述环状核酸分子是微环DNA。
附图说明
图1.本发明制备minicircle DNA的原理示意图。
图2.本发明制备minicircle DNA的流程图。
图3.(A)：分别用1对引物和96对引物(不同引物对形成的粘末端相互不兼容)从亲本质粒上扩增eGFP表达盒。限制性核酸内切酶BbsI消化后的GFP片段浓度影响环化效率。(B)：分别用含有1对接头的引物和含有96对接头的引物从亲本质粒上扩增019-CAR表达序列。限制性核酸内切酶BbsI消化后的019-CAR片段产物的浓度影响环化效率。
图4.用96对引物(不同引物对形成的粘末端相互不兼容)所扩增的靶片段提高了minicircle DNA的产量。(A)1对引物所扩增的eGFP片段在浓度为1、5、10、20、40和60ng/μl时最终产生minicircle-eGFP DNA的琼脂糖凝胶电泳图(上)。96对引物所扩增的eGFP片段在浓度为1、30、60、90和180ng/μl时最终产生minicircle-eGFP DNA的琼脂糖凝胶电泳(下)。箭头示出的条带代表靶minicircle-eGFP DNA。定量环化效率并以数值示出。(B)1对引物所扩增的019-CAR片段在浓度为1、5、10、20、40和60ng/μl时最终产生minicircle-019 DNA的琼脂糖凝胶电泳(上)。96对引物所扩增的019-CAR片段在浓度为1、30、60、90和180ng/μl时最终产生minicircle-019DNA的琼脂糖凝胶电泳(下)。箭头示出的条带代表靶minicircle-019 DNA。定量环化效率并以数值示出。(C)A中显示的minicircle-eGFP DNA的环化效率定量的统计结果。(D)B中显示的minicircle-019 DNA的环化效率定量的统计图。
图5.minicircle-eGFP在K562细胞系中的转基因表达水平和持续时间。(A)将eGFP的线性PCR产物、minicircle-eGFP DNA、和具有细菌骨架的eGFP编码质粒电转至K562细胞，在电穿孔后第2天和第4天检测细胞活力，该图为检测结果。(B)将eGFP的线性PCR产物、minicircle-eGFP DNA、和具有细菌骨架的eGFP编码质粒电转至K562细胞中，eGFP阳性细胞的比例随时间降低。(C)将eGFP的线性PCR产物、minicircle-eGFP DNA、和具有细菌骨架的eGFP编码质粒电转至K562细胞中，eGFP阳性细胞的平均荧光强度随时间降低。**P＜0.01；***P＜0.001；****P＜0.0001。
图6.Minicircle DNA电穿孔并不影响CD34 +HSC的多谱系分化能力。CD34 +HSC分离来自两个不同供体的脐带血，A-E为供体1的结果，F-J为供体2的结果。(A)和(F)：将编码eGFP的minicircle-eGFP DNA或质粒电转至CD34 +HSC中，电穿孔后48小时检测CD34和eGFP的表达水平，该图为检测结果。(B)和(G)：将编码eGFP的minicircle-eGFP DNA或质粒转染至CD34 +HSC中，电穿孔后分别在第2、4、6天检测的eGFP阳性细胞的平均荧光强度，该图为检测的统计结果。(C)和(H)：将编码eGFP的minicircle-eGFP DNA或质粒转染至CD34 +HSC中，电穿孔后48小时检测细胞活力，该图为检测结果。(D)和(I)：将编码eGFP的minicircle-eGFP DNA或质粒转染至CD34 +HSC中，随后将300个电转的细胞与特殊培养基混匀种于6孔板中，两周后检测CD34 +HSC的克隆形成个数以表示CD34 +HSC的克隆形成能力，该图为检测结果。(E)和(J)：该图分别是对(D)图(I)的统计图。。*P＜0.05；**P＜0.01；***P＜0.001。
图7.向人胚胎干细胞中电转minicircle DNA。(A)将编码eGFP的minicircle-eGFP DNA或质粒电转至人类胚胎干细胞H9中，电穿孔后48小时进行荧光显微镜拍照，该图为所得结果。(B)将编码eGFP的minicircle-eGFP或质粒电转至H9细胞中，在电穿孔后48小时用流式细胞仪分析eGFP的比例，该图为分析结果。(C)将编码eGFP的minicircle-eGFP或质粒电转至H9细胞中，在电穿孔后第2天和第6天检测细胞活力，该图为检测结果。(D)H9中在电穿孔后第2天、第4天和第6天的GFP阳性细胞的比例的统计结果。*P＜0.05。
图8.用minicircle-019 DNA制备的019-CAR-T细胞(来自供体1)可以特异高效地杀死肿瘤细胞。(A)将minicircle-019 DNA和质粒-019电转至T细胞，在电穿孔后48小时，用荧光显微镜对T细胞进行拍照，该图为所拍荧光照片。(B)电转了minicircle-019 DNA和质粒-019的T细胞在第2、4、6天的细胞活力检测。(C)通过FACS分析用minicircle-019 DNA转染的T细胞中019-CAR的表达水平。(D)019-CAR T细胞和肿瘤细胞孵育24小时后，minicircle-019 DNA制备的CAR-T(mini-CAR-T)细胞所释放的IFN-γ和IL2的浓度。(E)mini-CAR-T细胞或lenti-CAR-T在不同效应细胞-靶细胞(E：T)比例下对肿瘤细胞的裂解能力。(G)用T细胞、lenti-CAR-T细胞、mini-CAR-T细胞或相同体积的PBS处理3天、16天和30天的携带Raji- 荧光素酶肿瘤细胞的NPG小鼠的生物荧光成像结果(n＝3)。(F)不同时间点的荧光定量。**P＜0.01；***P＜0.001；****P＜0.0001。
图9.用minicircle制备的019-CAR-T细胞(来自供体2)可以特异性高效地杀死肿瘤细胞。(A)通过FACS分析用minicircle-019 DNA转染的T细胞中019-CAR的表达水平。(B)019-CAR T细胞和肿瘤细胞孵育24小时后，minicircle制备的CAR-T(mini-CAR-T)细胞释所放的IFN-γ和IL2的浓度。(C)mini-CAR-T细胞或lenti-CAR-T在不同效应细胞-靶细胞(E∶T)比例下对肿瘤细胞的裂解能力。(D)用minicircle和慢病毒载体工程化的CAR-T细胞体内功能测试的流程图。****P＜0.0001。
图10.本发明的minicircle DNA制备方法和现有技术的比较。
发明详述
在第一方面，本发明提供了一种产生包含靶核苷酸序列的环状核酸分子的方法，所述方法包括：
a)提供多种包含所述靶核苷酸序列的线性核酸分子的混合物，所述多种线性核酸分子每种在两端包含独特的互补单链突出末端；
b)用核酸连接酶如T4连接酶使所述混合物中的线性核酸分子通过其互补单链突出末端而自身连接，由此自身环化形成环状核酸分子。
如本文所用，“独特的互补单链突出末端”意指一种线性核酸分子的一端的单链突出末端与其另一端的单链突出末端互补(通常也称为粘末端)，而不与混合物中其它种类的线性核酸分子的单链突出末端互补(即不相容)。优选地，所述多种线性核酸分子除了所述独特的互补单链突出末端外的序列是相同的。例如，所述多种线性核酸分子包含相同的靶核苷酸序列。
当将仅仅一种两端具有互补单链突出末端(粘末端)的线性核酸分子以高浓度放在一起时，它们将倾向于相互线性地连接，因此很少环状产物。而当这些线性核酸分子的浓度降低时，由于分子之间相互接触的可能性降低，其更倾向于自身连接，因此环化效率会增加，然而由于使用低浓度，无法大量产生环状分子。申请人令人惊奇地发现，如果将多种具有不相容的互补粘末端的线性核酸分子各自以低浓度放在一起，由于不同种分子之间的粘末端不相容，无法相互之间连接，环化效率将大大提高。并且尽管每种线性分子都是低浓度，核酸分子的总浓度却可以相对很高，从而提高 了环状分子的得率。一般而言，具有不相容的互补粘末端的线性核酸分子的种数越多，在各自确保高环化效率的特定低浓度下，可以实现越高的总核酸分子浓度，获得更多的环状核酸分子。
N个核苷酸的突出末端的全部可能的序列数目是4 N种(4 N)，而由于每种线性核酸分子一端的突出末端需要与另一端互补以实现环化，因此可能的独特互补末端对是4 N/2种。在一些实施方案中，其中所述单链突出末端可以包含2个、3个、4个、5个或6个或更多个核苷酸。因此，在一些实施方案中，其中步骤a)中提供2-8种、2-32种、2-128种、2-512种、或2-2048种或更多种包含所述靶核苷酸序列的线性核酸分子的混合物。
在一些实施方案中，其中步骤a)中提供不少于8种、不少于16种、不少于24种、不少于32种、不少于40种、不少于48种、不少于56种、不少于64种、不少于72种、不少于80种、不少于96种包含所述靶核苷酸序列的线性核酸分子的混合物。
在一些实施方案中，其中所述混合物中每种线性核酸分子的浓度可以为0.01-20ng/μl，优选0.01-10ng/μl，更优选0.01-5ng/μl，更优选0.01-2.5ng/μl，更优选0.01-1ng/μl。
在一些实施方案中，其中所述混合物中线性核酸分子的总浓度可以为0.01-200ng/μl或更高，例如10ng/μl、20ng/μl、40ng/μl、60ng/μl、80ng/μl、100ng/μl、120ng/μl、140ng/μl、160ng/μl、180ng/μl、200ng/μl或更高。
在一些实施方案中，本发明的方法其进一步包括以下步骤：
c)使用核酸外切酶例如T5核酸酶去除b)的产物中的线性核酸分子。
在本发明的方法中，还任选地包括纯化所得的环状核酸分子的步骤。所述纯化步骤可以在步骤b)之后或可以在步骤c)之后。
本发明的方法中，所述线性核酸分子两端的独特的互补单链突出末端可以通过多种方法产生，例如，可以通过直接向线性核酸分子两端添加接头，或者通过设计合适的引物通过聚合酶链反应(PCR)扩增和随后的限制性酶消化导入。
例如，在本发明方法的一些实施方案中，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
1)提供包含所述靶核苷酸序列的线性核酸分子；
2)将多组核酸接头对分别连接至所述线性核酸分子，所述多组核酸接 头对每组包含5’接头和3’接头且所述5’接头和3’接头包含独特的互补单链突出末端；
3)混合并任选地纯化步骤2)中的产物。
例如，在本发明方法的一些优选实施方案中，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
1)用多组靶特异性引物对分别通过扩增(如PCR扩增)获得包含所述靶核苷酸序列的线性核酸分子，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列；
2)混合并任选地纯化步骤1)的扩增产物；
3)用所述限制性内切酶消化步骤2)获得的混合物；和
4)任选地纯化步骤3)的消化产物。
根据该实施方案，对于不同的靶核苷酸序列，需要各自设计和合成多组靶特异性引物对。可以使用的引物对数目取决于使用的限制性内切酶形成的单链突出末端的核苷酸数目(如上所述)。例如，步骤1)中可以使用2-8种、2-32种、2-128种、2-512种、或2-2048种或更多种引物对。例如，可以使用不少于8种、不少于16种、不少于24种、不少于32种、不少于40种、不少于48种、不少于56种、不少于64种、不少于72种、不少于80种、不少于96种引物对。
在一些具体实施方案中，其中所述限制性内切酶是BbsI，且所述引物对中的两种引物分别在5’端包含5’-GAAGACNNN 1N 2N 3N 4-3’和5’-GAAGACNNN 5N 6N 7N 8-3’的序列，其中N代表A、T、C和G中任一个，且序列N 1N 2N 3N 4与序列N 5N 6N 7N 8反向互补。
而在一些更为便利的实施方案中，可以考虑通过PCR在所述包含靶核苷酸序列的线性核酸分子两端引入通用序列标签，然后再用针对所述通用序列标签的多组引物对(包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列)扩增。这样，针对一种靶核苷酸序列，仅需设计一对靶特异性引物(两种引物在5’端包含不同的通用序列标签)。而针对所述通用序列标签的多组引物对则可以用于产生包含不同靶核苷酸序列的环状核酸分子。
因此，在本发明方法的一些更优选实施方案中，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
1)用包含通用序列标签的靶特异性引物对扩增获得包含所述靶核苷酸序列的线性核酸分子，
2)用多组针对所述通用序列标签的引物对分别以步骤1)获得的线性核酸分子为模板进行扩增(例如PCR扩增)，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列；
3)混合并任选地纯化步骤2)的扩增产物；
4)用所述限制性内切酶消化步骤3)获得的混合物；和
5)任选地纯化步骤4)的消化产物。
可以使用的引物对数目取决于使用的限制性内切酶形成的单链突出末端的核苷酸数目(如上所述)。例如，步骤2)中可以使用2-8种、2-32种、2-128种、2-512种、或2-2048种或更多种引物对。例如，可以使用不少于8种、不少于16种、不少于24种、不少于32种、不少于40种、不少于48种、不少于56种、不少于64种、不少于72种、不少于80种、不少于96种引物对。
在一些具体实施方案中，其中所述限制性内切酶是BbsI，且所述引物对中的两种引物分别在5’端包含5’-GAAGACNNN 1N 2N 3N 4-3’和5’-GAAGACNNN 5N 6N 7N 8-3’的序列，其中N代表A、T、C和G中任一个，且序列N 1N 2N 3N 4与序列N 5N 6N 7N 8反向互补。
BbsI在本发明的方法中的应用原理如图1所示。图1示例性示出了BbsI的识别位点和切割方式。通过设计4组包含BbsI位点的引物对，扩增并酶消化产生四种包含独特的互补单链突出末端的靶线性核酸分子，这四种靶线性核酸分子相互之间不会连接。而如果各自以低浓度存在，由于其更倾向于一个分子的自身连接而不是两个相同分子之间的线性连接，可以实现高的环化效率。应理解的是，图1仅是对本发明方法的示例性说明，并不旨在限制本发明的范围。
原则上，识别、切割特定序列并产生若干(两个或更多个)连续N(N＝A、T、G或C)组成的粘末端的限制性内切酶都可用于本发明。本领域技术人员根据本发明的教导可以容易地设计出合适的引物序列。可用于本发明的方法的另外限制性内切酶包括但不限于ApaI、BbvI、BsaI、BfuAI、BglI、BsmAI、BsmBI、BsmFI、BspMI、BslI、BstAPI、BstXI、BtgZI、DraIII、EarI、HgaI等。
本发明的方法适于获得任何环状双链核酸分子。在本发明方法的一些实施方案中，其中所述环状核酸分子是minicircle DNA。
术语“minicircle DNA”与“微环DNA”可互换使用，其是一种非病毒基因载体，与传统的质粒载体相比，其仅含有目的基因的表达盒，不包括来自细菌质粒的骨架序列。Minicircle DNA一般以环状超螺旋的形式存在。
在一些实施方案中，所述靶核苷酸序列包含与转录调控元件可操作连接的编码序列。
如本发明所用，“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5′非编码序列)、中间或下游(3′非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。表达调控元件指的是能够控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。
调控序列可包括但不限于启动子、翻译前导序列、内含子、增强子和多腺苷酸化识别序列。
“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方式中，启动子是能够控制细胞中基因转录的启动子，无论其是否来源于所述细胞。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
在一些实施方案中，其中所述编码序列编码感兴趣的蛋白质或感兴趣的RNA。所述感兴趣的蛋白质例如T细胞受体(TCR)、嵌合抗原受体(CAR)、Cas9核酸酶或其他治疗性蛋白。所述感兴趣的RNA例如是sgRNA、反义RNA、antagomir、siRNA或shRNA或其他治疗性RNA。在一具体实施方案中，所述CAR是抗CD19-CAR。
在第二方面，本发明提供一种根据本发明的方法产生的环状核酸分子特别是minicircle DNA。
在第三方面，本发明提供根据本发明的方法产生的环状核酸分子特别是minicircle DNA在制备修饰的T细胞中的用途。所述修饰的T细胞例如是CAR-T细胞。在一优选实施方案中，所述CAR-T细胞是抗CD19-CAR-T 细胞。
在第四方面，本发明提供根据本发明的方法产生的环状核酸分子特别是minicircle DNA在基因治疗中的用途。
在第五方面，本发明提供根据本发明的方法产生的环状核酸分子特别是minicircle DNA在将靶核苷酸序列导入细胞中的用途。所述细胞包括但不限于细胞系、原代细胞、干细胞如胚胎干细胞或造血干细胞、或T细胞。
根据本发明的方法产生的环状核酸分子特别是minicircle DNA可以通过本领域已知的方法导入细胞，例如：磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射等。
在第六方面，本发明提供一种用于通过本发明的方法产生环状核酸分子例如minicircle DNA的试剂盒。例如，所述试剂盒可以包括多组核酸接头对，每组包含5’接头和3’接头且所述5’接头和3’接头包含独特的互补单链突出末端。例如，所述试剂盒可以包括多组针对通用序列标签的引物对，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列。本发明的试剂盒还可以包括用于本发明的方法的各种试剂，例如用于扩增的聚合酶、用于连接的连接酶如T连接酶、用于去除单链核酸的核酸外切酶如T5核酸外切酶、适于本发明的限制性内切酶如BbsI、用于纯化核酸分子的试剂和装置、各种缓冲液和/或描述如何实施本发明的方法的说明书。
在第七方面，本发明还提供minicircle DNA在制备修饰的T细胞中的用途。所述修饰的T细胞例如是CAR-T细胞。在一优选实施方案中，所述CAR-T细胞是抗CD19-CAR-T细胞。本领域中没有关于用minicircle DNA制备CAR-T细胞的报道。本发明人首次证明利用minicircle DNA可以制备CAR-T细胞，所制备的CAR-T细胞具有良好活力和肿瘤杀伤效果，优于根据现有技术用慢病毒载体制备的CAR-T细胞。
实施例
通过参考在此给出的一些具体实施例可获得对本发明的进一步的理解，这些实施例仅用于说明本发明，其无意于对本发明的范围做出任何限制。显然，可以对本发明作出多种改动和变化而不脱离本发明的实质，因此，这些改动和变化同样在本申请要求保护的范围内。
一般材料和方法
产生非细菌来源的minicircle-DNA
设计了96对引物来扩增靶转基因。每一引物的5’末端含有限制性核酸内切酶BbsI识别位点和紧接着的6bp独特序列，使得BbsI消化后的PCR产物在两端都会具有4bp的单链突出末端。这些4bp突出末端的全部可能的组合数目是256种(44)，而由于每个PCR产物一端的突出末端需要与另一端互补以实现环化，因此可能的独特末端对是128种。从这些128种组合中随机选择96种设计引物对，实验中所用的这些引物示于于表1中。
用这96对引物分别从AAVS1-2KB-EF1a-019-2A-eGFP/AAVS1-1KB-CMV-eGFP扩增(TAKARA，PrimeSTAR@HS DNA Polymerase，Cat：#R010B)目标片段(EF1a-019-2A-eGFP/cmv-eGFP)。扩增程序为：98℃2min；35x(98℃10s，60℃30s，72℃2/4min；72℃3min；4℃保持。
将96个PCR反应的PCR产物全部混合并使用Qiagen，QIAquick PCR Purififcation Kit(Cat No./ID：28106)纯化。使用限制性内切酶Bbs1消化PCR产物(New ENGLAND，Cat：#R0539L)，消化产物再次纯化(QIAquick PCR Purififcation Kit)。获得的靶片段用T4连接酶(New ENGLAND，Cat：#M0202L)在16℃连接2小时，随后用T5核酸外切酶ENGLAND，Cat：#M0363L)在37℃处理2小时。收集纯化后的产物，获得非细菌来源的minicircle-DNA。
表1.用于本实验的96种引物对的列表
Figure PCTCN2018108557-appb-000001
Figure PCTCN2018108557-appb-000002
Figure PCTCN2018108557-appb-000003
Figure PCTCN2018108557-appb-000004
Figure PCTCN2018108557-appb-000005
Figure PCTCN2018108557-appb-000006
Figure PCTCN2018108557-appb-000007
斜体示出BbsI识别位点，下划线示出4bp的独特末端序列。
细胞系
K562(红白血病细胞系)和Raji(Burkitt’s淋巴瘤细胞系)购自美国典型菌种保藏中心(ATCC)。如之前所描述(Chen et al.，Mol Ther 2003；8：495-500)构建用于生物荧光成像的Raji-fluc细胞和表达肿瘤抗原CD19的K562-CD19细胞。全部上述细胞用RPMI1640培养基(ThermoFisher Scientific)培养。慢病毒生产细胞系293T(ATCC-CRL3216)用DMEM(ThermoFisher Scientific)培养。全部培养基补充有10％(v/v)胎牛血清、100U/mL青霉素和链霉素、2mM L-谷氨酰胺、和1mM丙酮酸钠。全部细胞系在37℃、5％CO 2条件下生长。
原代人UCB-衍生的T细胞
新鲜脐带血(UCB)获得自北京脐带血库(中国北京)，具有健康志愿供体的知情同意书。用Histopaque-1077(Sigma-Aldrich)梯度分离收集单个核细胞，并用EasySep人T细胞富集试剂盒(Stemcell Technologies)分离获得T细胞。用抗-CD3/CD28 Dynabeads(Thermo Fisher Scientific)以1∶1的比例与T细胞共培养激活T细胞，并用X-vivo15培养基(Lonza)培养，培养基补充有5％(v/v)热灭活的胎牛血清、2mM L-谷氨酰胺和1mM丙酮酸钠，以及含有300IU/mL重组人IL-2(全部来自Thermo Fisher Scientific)。
原代人UCB-衍生的CD34 +HSCs
新鲜脐带血(UCB)获得自北京脐带血库(中国北京)，具有健康志愿供体的知情同意书。用Histopaque-1077(Sigma-Aldrich)梯度分离收集单个核细胞，并用EasySep人脐带血CD34阳性选择试剂盒(Stemcell Technologies)分离CD34 +HSCs。分离后，在添加有维持干性的小因子补充剂(Stemcell Technologies，#02691)的stem span H3000(Stemcell Technologies，#09800)中 培养CD34 +细胞。
K562、CD34 +HSCs和H9细胞的电穿孔
2×10 5K562细胞用3μg PCR-eGFP/Mini-eGFP/Pasmid-eGFP通过4D-Nucleofector System N(Lonza)使用Amaxa TM SF Cell line 4D-NucleofectorTM X Kit S(V4XC-2032)根据说明书进行电穿孔。使用Program FF-120。
5×10 4CD34细胞用1μg Mini-eGFP/Pasmid-eGFP通过4D-Nucleofector System N(Lonza)使用P3Primary Cell 4D-Nucleofector X Kit(V4XP-3024，Lonza)根据说明书进行电穿孔。使用Program EO-100。
4×10 5ES H9细胞用1μg Mini-eGFP/Pasmid-eGFP通过4D-Nucleofector System N(Lonza)使用P3Primary Cell 4D-Nucleofector X Kit(V4XP-3024，Lonza)根据说明书进行电穿孔。使用Program CB-150。
电穿孔后，细胞用37摄氏度预热的细胞培养基重悬并转移至培养皿中，置于5％CO 2中37℃培养。电穿孔2天后分别通过细胞计数和FACS分析评估细胞活力和转染效率。
产生Lenti-CAR-T细胞
新鲜纯化的原代T细胞激活1天，然后用携带第二代CD19 CAR的慢病毒载体侵染。CAR的结构如先前描述(Chen et al.，Mol Ther 2003；8：495-500)。侵染效率在转导两天后通过FACS分析评估。
产生Mini-019-CAR-T细胞
T细胞根据上述方法激活3天，然后1×10 6个细胞用3μg Mini-019 DNA通过4D-Nucleofector System N(Lonza)使用P3Primary Cell 4D-Nucleofector X Kit(V4XP-3024，Lonza)根据说明书进行电穿孔。使用Program EO-115。
电穿孔后，细胞用1ml预热的细胞培养基重悬并转移至12孔细胞板中置于5％CO 2中37℃培养。电穿孔2天后分别通过细胞计数和FACS分析评估细胞活力和转染效率。
基于荧光素酶的CTL测定
通过先前描述的(Chen et al.，Gene Ther 2004；11：856-864)基于荧光素酶的CTL测定评估CAR-T细胞的细胞毒性。
通过用表达荧光素酶的慢病毒侵染K562和K562-CD19细胞产生K562-1uc和K562-CD19-1uc细胞系。
K562-1uc和K562-CD19-1uc细胞在100μl RPMI1640培养基中以1x10 5细胞/ml重悬，并以不同比例和效应细胞混合(例如，25∶1、15∶1等)，总体积200μl。5％CO 2中37℃孵育16小时后，添加10μl底物，并在5分钟后测量荧光。CAR-T细胞对靶细胞裂解效率为：
杀死％＝100-((效应细胞和靶细胞共培养的RLU)/(靶细胞的RLU))x100
细胞因子的酶联免疫吸附测定(ELISA)
将效应细胞(T、Mini-019-CART)和靶肿瘤细胞(K562-CD19、K562)以1∶1比例(各10 4细胞)在每孔终体积200μl的完全RPMI1640培养基中共培养。24小时后，使用ELISA试剂盒(Biolegend)测定上清液中IL-2和IFN-γ的产生。
鼠异种移植研究
为了建立Raji-fluc肿瘤模型，6-12周龄的NOD-Prkdcscid Il2rgnull(NPG)小鼠(VITALSTAR，Beijing，China)在第0天腹膜内注射2×10 5Raji-fluc细胞。注射3天后，使用NightOWL LB983体内成像系统(Berthold Technologies)通过系列生物光子成像评估肿瘤移植入。将具有可比的肿瘤负荷的小鼠分进不同的处理组并接受不同处理。分组为：PBS组、T细胞组、lenti-019 CAR T细胞组和Mini-019 CAR-T细胞组。以5×10 6细胞/小鼠的剂量经腹膜内注射施用细胞。第一次处理后6天评估肿瘤负荷，并用5×10 6细胞/小鼠处理第二次。然后每7天评估肿瘤负荷。
实施例1、用简易安全的方法快速制备含靶序列的微环DNA。
在本实施例中，制备了表达eGFP或抗-CD19 CAR(019-CAR)的minicircle DNA载体。
首先，用一对引物或96对引物分别扩增eGFP和CAR-019，引物设计 如上文所述。用一对引物扩增的产物纯化后直接用Bbs1酶进行消化，而用96对不同引物分别扩增则先将所有96种PCR产物进行混合，然后在纯化后用Bbs1酶进行消化。由图3和图4可知，在一对引物组中，自身环化效率随着连接底物浓度的增加而迅速降低。然而，在96对接头引物组中，自身环化效率保持在较高水平，即使连接底物的浓度高达180ng/μl。此外，连接底物浓度相同时，96对引物组的自身环化效率显著高于一对引物组。由上述数据可以看出，通过本发明的方法可以高效地制备minicircle DNA载体。
实施例2、minicircle DNA在细胞系中具有高水平和长时间的基因表达
为了验证本发明方法制备的minicircleDNA的基因表达水平、持续时间以及对细胞活力的影响，我们将eGFP的PCR产物、minicircle-eGFP DNA和具有细菌骨架序列的eGFP质粒通过电穿孔转染至K562细胞。
在电穿孔后第2天和第4天检测发现，minicircle-eGFP DNA组的K562细胞活力显著好于质粒组(图5A)。同时，持续12天都观察到eGFP阳性K562细胞(图5B)。PCR产物组和质粒组中，K562细胞的eGFP阳性细胞很快下降至低比例，而minicircle-eGFP DNA组中转基因表达水平在最初8天都保持高水平，然后在最后4天逐渐下降(图5B)。如图5C所示，针对平均荧光强度(MFI)也观察到相同的变化趋势。从上述数据可看出，与其他组相比，minicircle-eGFP DNA组在保持细胞活力和转基因表达稳定性方面具有显著优势。
实施例3、minicircleDNA转染后的CD34阳性HSC具有更高转基因表达和更优细胞活力
进一步研究了minicircle DNA在来自脐带血的CD34阳性造血干细胞(HSC)中的转染效率，以及minicircle DNA对HSC的活力和功能的影响。
与质粒组相比，minicircle DNA组在电穿孔48小时后的转基因表达水平显著高于质粒组，尽管两组的eGFP阳性细胞比例相似(图6A、B、F和G)。同时，minicircle DNA组的细胞活力要显著高于质粒组(图6C、H)。
为了表征HSC的分化能力，进行了集落形成单位(CFU)分析。经电转的HSC细胞成功形成集落(图6D、I)，并且各组中均存在红细胞集落、粒 细胞集落、巨噬细胞集落和巨核细胞集落(图6E、J)，表明HSC的分化能力没有受到影响。
实施例4、minicircle DNA转染后的人类胚胎干细胞具有更高的转基因表达水平和更好的细胞活力
为确认本发明的minicircle DNA可应用于胚胎干细胞，将minicircle-eGFP DNA和质粒-eGFP电穿孔至H9细胞。如图7A所示，质粒-eGFP转染组48小时后H9细胞死亡显著，仅仅形成一些微小的克隆，然而，minicircle-eGFP DNA转染组的H9细胞表现要好很多。同时，与质粒-eGFP电穿孔的H9细胞相比，minicircle-eGFP电穿孔的H9细胞中的eGFP阳性细胞的比例要高很多(图7B)。在minicircle DNA和质粒电穿孔后细胞活力有所下降(图7C)，存活的H9细胞均匀地表达OCT4和NANOG(数据未显示)，表明这些细胞仍然具有多能性。从上述数据，证明minicircle DNA在胚胎干细胞中在转基因表达水平和保持细胞活力方面都优于质粒。
实施例5、用minicircle-019 DNA转染的CD3阳性T细胞在体外和体内的可靠抗肿瘤活性
通过LV和RV制备的CAR-T细胞已经在实验研究和临床实验中证明具有令人鼓舞的抗肿瘤效果。本实施例进一步说明可以用编码抗-CD19CAR的minicircle-019 DNA制备无整合的转基因的CAR-T细胞，且这样的CAR-T细胞能够特异性杀死肿瘤细胞。
首先比较了通过minicircle-019 DNA和质粒递送的CAR-019在CD3阳性T细胞中的转基因表达水平。如图8A所示，在两组中的细胞在电穿孔后48小时都变成CD19-CAR阳性。然而，质粒组的T细胞死亡显著，其电穿孔后48小时活细胞少于5％。minicircle组的细胞活力显著好于质粒组。有超过20％的活T细胞，其能够迅速增殖以满足功能分析所需的细胞数目(图8B)。
然后，分析了用minicircle-019 DNA制备的来自两个不同供体的CAR-019-T细胞的抗肿瘤能力(图8D-G，图9B-C)。所述CAR T细胞在和靶肿瘤细胞孵育后分泌高水平的IL2和IFN-γ(图8D和图9B)。同时，CAR-T细胞能够特异性高效的杀死靶肿瘤细胞，即使两种供体细胞都是以低E∶T 比例使用(图8E和图9C)。
为了比较用minicircle DNA载体和慢病毒载体改造的CAR-T细胞在体内缓解肿瘤的能力，设计了图9D中的实验。NPG小鼠用Raji-luci细胞腹膜内接种。3天后，将这些小鼠基于荧光值分为4组。T细胞组、minicircle-019DNA改造的CAR-T细胞组以及慢病毒载体改造的CAR-T细胞组在第3天和第9天进行治疗(腹腔注射细胞)。对照组小鼠注射相同体积的PBS。通过荧光素酶体内成像每周监测肿瘤负荷。如图8F、G所示，与T细胞组特别是PBS组小鼠相比，mini-CAR-T组和lenti-CAR-T组小鼠中的肿瘤负荷显著降低。从上述数据证明，用minicircle-019 DNA改造的CAR-T细胞具有可靠的体内和体外缓解肿瘤的能力。

Claims (17)

  1. 一种产生包含靶核苷酸序列的环状核酸分子的方法，所述方法包括：
    a)提供多种包含所述靶核苷酸序列的线性核酸分子的混合物，所述多种线性核酸分子每种在两端包含独特的互补单链突出末端；
    b)用核酸连接酶如T4连接酶使所述混合物中的线性核酸分子通过其互补单链突出末端而自身连接，由此自身环化形成环状核酸分子。
  2. 权利要求1的方法，其中所述单链突出末端包含2个、3个、4个、5个或6个或更多个核苷酸。
  3. 权利要求1或2的方法，其中步骤a)中提供2-8种、2-32种、2-128种、2-512种、或2-2048种或更多种包含所述靶核苷酸序列的线性核酸分子的混合物。
  4. 权利要求1-3中任一项的方法，其中所述混合物中每种线性核酸分子的浓度为0.01-20ng/μl，优选0.01-10ng/μl，更优选0.01-5ng/μl，更优选0.01-2.5ng/μl，更优选0.01-1ng/μl。
  5. 权利要求1-4中任一项的方法，其中所述混合物中线性核酸分子的总浓度为0.01-200ng/μl或更高。
  6. 权利要求1-5中任一项的方法，其进一步包括：
    c)使用核酸外切酶例如T5核酸外切酶去除混合物中的线性核酸分子。
  7. 权利要求1-6中任一项的方法，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
    1)提供包含所述靶核苷酸序列的线性核酸分子；
    2)将多组接头对分别连接至所述线性核酸分子，所述多组接头对每组包含5’接头和3’接头且所述5’接头和3’接头包含独特的互补单链突出末端；
    3)混合并任选地纯化步骤2)中的产物。
  8. 权利要求1-6中任一项的方法，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
    1)用多组靶特异性引物对分别扩增获得包含所述靶核苷酸序列的线性核酸分子，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列；
    2)混合并任选地纯化步骤1)的扩增产物；
    3)用所述限制性内切酶消化步骤2)获得的混合物；和
    4)任选地纯化步骤3)的消化产物。
  9. 权利要求1-6中任一项的方法，其中a)中通过以下步骤提供所述多种线性核酸分子的混合物：
    1)用包含通用序列标签的靶特异性引物对扩增获得包含所述靶核苷酸序列的线性核酸分子，
    2)用多组针对所述通用序列标签的引物对分别扩增步骤1)获得的线性核酸分子，每组所述引物对中的引物在5’端包含能被限制性内切酶切割并形成独特的互补单链突出末端的序列；
    3)混合并任选地纯化步骤2)的扩增产物；
    4)用所述限制性内切酶消化步骤3)获得的混合物；和
    5)任选地纯化步骤4)的消化产物。
  10. 权利要求8或9的方法，其中所述限制性内切酶是BbsI。
  11. 权利要求10的方法，所述引物对中的两种引物分别在5’端包含5’-GAAGACNNN 1N 2N 3N 4-3’和5’-GAAGACNNN 5N 6N 7N 8-3’的序列，其中N代表A、T、C和G中任一个，且序列N 1N 2N 3N 4与序列N 5N 6N 7N 8反向互补。
  12. 权利要求1-11中任一项的方法，其中所述环状核酸分子是minicircle DNA。
  13. 权利要求1-12中任一项的方法，其中所述靶核苷酸序列包含与转录调控元件例如启动子和/或终止子可操作连接的编码序列。
  14. 权利要求13的方法，其中所述编码序列编码感兴趣的蛋白质或RNA。
  15. 一种试剂盒，其用于通过权利要求1-14中任一项的方法产生包含靶核苷酸序列的环状核酸分子。
  16. 环状核酸分子，其通过权利要求1-14的方法产生。
  17. 权利要求16的环状核酸分子，其是minicircle DNA。
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