Novel hybrid DNA vector and application thereof

Abstract

The invention relates to a novel DNA carrier and application thereof, and belongs to the technical field of biology. The DNA vector is of a hybrid DNA structure formed by linear or annular single-stranded DNA and one or more oligonucleotides with complementary sequences. The oligonucleotide can carry a functional sequence outside a complementary region for imparting a specific biological function to the hybrid DNA. The invention also provides an efficient preparation method of the hybrid DNA. The hybrid DNA can be used as a donor template to be combined with a CRISPR (clustered regularly interspaced short palindromic repeats) system based on homologous recombination, is used for realizing accurate knock-in of large-fragment genes, is also suitable for gene editing in CAR-T cell therapy, and can be used as an expression vector to realize efficient exogenous gene expression in vivo and in vitro.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel hybrid DNA vector and application thereof. Background Art

[0002] In genome editing technologies, homology-directed repair (HDR) is a mechanism that achieves precise insertion through homologous recombination with a donor DNA template and is widely used in gene function research and therapeutic gene editing. The efficiency of HDR is highly dependent on the type, structural stability, and homology match of the exogenous donor DNA with the target sequence. Traditional donors, such as linear double-stranded DNA (ldsDNA), are easy to construct, but their application is often limited by nonspecific insertion and cytotoxicity. In recent years, single-stranded DNA donors, such as lssDNA, have attracted widespread attention due to their low off-target rates and high editing precision. However, lssDNA can typically only carry inserts smaller than 2 kb and exhibits poor stability and susceptibility to degradation, particularly in primary cells and in vivo. Circular single-stranded DNA (cssDNA), a novel donor template, offers improved structural stability and greater carrying capacity, but its immunogenicity and transcriptional regulation mechanisms in cells are not fully understood, limiting its application. Therefore, developing an HDR donor template with large capacity, low toxicity, high efficiency and high specificity has become the key to improving gene knock-in efficiency and editing accuracy.

[0003] At the same time, gene expression vectors, as important tools in molecular biology and genetic engineering, are the core platforms for driving the expression of exogenous genes in cells. Commonly used vectors such as plasmids and minicircles are widely used in eukaryotic and prokaryotic systems. Although these vectors have met the needs of research and industrialization to a certain extent, they still have problems such as low transfection efficiency, unstable expression, fixed module functions, and lack of tissue-specific regulation, making it difficult to adapt to the ever-expanding precise expression and multi-gene regulation scenarios. Some studies have attempted to use cssDNA directly as an expression vector, but due to its lack of regulatory elements or stable expression structures, there are still many challenges in practical applications. For example, patent CN113271984A discloses a scheme for circular single-stranded DNA for gene knock-in, but the knock-in efficiency and expression stability are still unsatisfactory. For this reason, it is urgent to develop a DNA construction system with novel structure, flexible function, and both efficient expression and editing capabilities. The hybrid DNA vector proposed in this invention combines the stability of cssDNA with the functionality of oligonucleotides, and has the dual characteristics of HDR donor and expression vector. It aims to solve the core bottlenecks of existing technologies in precise insertion and efficient expression, and expand its application prospects in fields such as cell engineering, gene therapy and vaccine development. Summary of the Invention

[0004] The present invention provides a design of a hybrid DNA expression vector and its application scheme. The vector adopts a circular structure, and a double-stranded circular DNA structure is formed by annealing a long single-stranded DNA with a complementary oligonucleotide. This construction reduces the size of the vector, avoids unnecessary sequences on traditional plasmids, and can enter cells more efficiently and drive gene expression. The hybrid DNA vector can be used as a homologous recombination donor template in gene editing, and can also be used as an efficient gene expression vector for gene delivery and expression in and outside cells or animals. The vector of the present invention is small in size and stable, and can be introduced into cells or animals through delivery methods such as electroporation, and achieve higher transfection and expression efficiency. The hybrid DNA expression vector of the present invention is suitable for the expression of a variety of genes, is not limited to specific diseases or cell types, and can be widely used in gene therapy, vaccine development, biological research and other fields. To achieve the above purpose, the present invention provides the following scheme: The present invention provides a hybrid DNA vector comprising: a) a long single-stranded DNA of ≥200 nucleotides in length, comprising a target sequence; b) at least one oligonucleotide of ≤200 nucleotides in length, said oligonucleotide comprising a reverse complementary sequence to said long single-stranded DNA; The long single-stranded DNA and the oligonucleotide form hybrid DNA by annealing, and the long single-stranded DNA and the oligonucleotide form a hybrid structure by complementary base pairing.

[0005] The method for preparing hybrid DNA comprises the following steps: first, constructing a circular single-stranded DNA containing a target sequence; then, mixing the circular single-stranded DNA with oligonucleotides of different lengths and quantities at a molar ratio of 1:2 as shown in the example; adding 10× annealing buffer to stabilize the single-stranded structure and promote base complementary pairing to obtain solution b; diluting the solution to the target concentration to form system c; performing annealing PCR to generate circular hybrid DNA; and purifying the product; similarly, constructing a linear single-stranded DNA containing the same functional element; mixing the solution with oligonucleotides of different parameters at a molar ratio of 1:2 to form solution d; adding 10× annealing buffer to stabilize the single-stranded structure and promote base complementary pairing to obtain solution e; diluting the solution to the target concentration to form system f; performing annealing PCR to generate linear hybrid DNA, and purifying the product. Finally, hybrid DNA is obtained.

[0006] In some specific embodiments, the long single-stranded DNA is circular single-stranded DNA. Currently, there are multiple methods for producing circular single-stranded DNA, such as methods for producing long circular single-stranded DNA based on M13 bacteriophage, methods for producing circular single-stranded DNA by specific cleavage and DNase digestion, methods for producing circular single-stranded DNA by electrophoretic capture of heat-denatured dsDNA plasmids, and methods for synthesizing circular single-stranded DNA by chemical ligation or enzyme-mediated methods. In the present invention, a method for producing circular single-stranded DNA based on M13 bacteriophage is used.

[0007] In some specific embodiments, the long single-stranded DNA is linear single-stranded DNA. Currently, there are many methods for producing linear single-stranded DNA, such as chemical synthesis, asymmetric PCR, isolation of avidin-labeled single-stranded DNA using streptavidin particles, digestion of phosphorylated single-stranded DNA with lambda exonuclease, rolling circle PCR amplification, and plasmid cleavage with restriction endonucleases followed by denaturation. In the present invention, the method of digesting phosphorylated single-stranded DNA with lambda exonuclease is employed.

[0008] In some specific embodiments, the long single-stranded DNA in the hybrid DNA is not less than 200 nucleotides in length. Preferably, the length of the long single-stranded DNA is 0.2 kb to 200 kb, and can be extended to about 0.5 kb, 2 kb, 2.5 kb, 5 kb, 10 kb, 20 kb, 40 kb, 80 kb, 100 kb, 150 kb, or even 200 kb according to application requirements.

[0009] In certain embodiments, the oligonucleotide in the hybridizing DNA is less than about 200 nucleotides in length, including at least one nucleotide, and can be about 10 nt, 50 nt, 100 nt, 150 nt, or 200 nt in length, preferably about 50 nt.

[0010] In other embodiments, the linear or circular hybrid DNA vector can be complementary annealed with one or more oligonucleotides, and the number of oligonucleotides can be about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, preferably about 35.

[0011] For example, as shown in Examples 1 and 2 below, using RAB11A-cssDNA (SEQ ID NO: 2) as an example, RAB11A-cssDNA (SEQ ID NO: 2) is an exemplary sequence of the long single-stranded DNA, and optionally one or more of RAB11A-oligo-1 (SEQ ID NO: 3) to RAB11A-oligo-35 (SEQ ID NO: 37) are the one or more oligonucleotides. Any of RAB11A-oligo-1 (SEQ ID NO: 3) to RAB11A-oligo-35 (SEQ ID NO: 37) can reverse-complement the long single-stranded RAB11A-cssDNA (SEQ ID NO: 2) to form a complementary region, for a total of 35 complementary regions. A complementary region is a continuous region formed by continuous complementarity between an oligonucleotide and a long single-stranded DNA.

[0012] In some embodiments, the entire length of the oligonucleotide in the hybridizing DNA is reverse complementary to the long single-stranded DNA. For example, the entire length of the nucleotides in any one of RAB11A-oligo-1 (SEQ ID NO: 3) to RAB11A-oligo-35 (SEQ ID NO: 37) is reverse complementary to the long single-stranded DNA.

[0013] In some specific embodiments, there is more than one oligonucleotide, and the long single-stranded DNA forms more than one hybridization structure with the oligonucleotide through complementary base pairing, and the hybridization structures are spaced 0-100 nucleotides apart on the long single-stranded DNA. For example, the long single strand of the hybrid structure has 0 nucleotides, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides 0 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, 80 nucleotides. For example, when the oligonucleotide includes RAB11A-oligo-1 and RAB11A-oligo-2, the complementary region formed by RAB11A-oligo-1 (SEQ ID NO: 3) and the long single-stranded RAB11A-cssDNA (SEQ ID NO: 2) and the complementary region formed by RAB11A-oligo-2 (SEQ ID NO: 4) and the long single-stranded RAB11A-cssDNA (SEQ ID NO: 2) are separated by 0 nucleotides.For another example, when the oligonucleotide includes RAB11A-oligo-1 and RAB11A-oligo-3, the complementary region formed by RAB11A-oligo-1 (SEQ ID NO: 3) and the long single-stranded RAB11A-cssDNA (SEQ ID NO: 2) and the complementary region formed by RAB11A-oligo-3 (SEQ ID NO: 5) and the long single-stranded RAB11A-cssDNA (SEQ ID NO: 2) are separated by 50 nucleotides.

[0014] In some specific embodiments, there is more than one oligonucleotide, and the long single-stranded DNA forms more than one hybridization structure with the oligonucleotide through complementary base pairing, and the total length of the more than one hybridization structure accounts for 1% to 100% of the length of the long single-stranded DNA, for example, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100% or 100%.

[0015] In some embodiments, there is more than one oligonucleotide, and the long single-stranded DNA forms more than one hybrid structure with the oligonucleotide through complementary base pairing, the hybrid structures are separated by 0 nucleotides, and there is a gap structure between the hybrid structures. The gap structure is a structure in which no phosphodiester bond is formed between the two nucleotides. For example, RAB11A-oligo-1 and RAB11A-oligo-2 do not form a phosphodiester bond in the hybrid DNA.

[0016] In some specific embodiments, the annealing molar ratio of the long single-stranded DNA to the oligonucleotide is 1:0.1 to 1:10, preferably 1:2.

[0017] In the embodiment, the annealing step includes: mixing the long single-stranded DNA and the oligonucleotide according to a set molar ratio, heating at 85°C for 3-5 minutes to eliminate the secondary structure; then lowering the temperature to 80°C and maintaining it for 2 minutes to provide initial annealing conditions; then, gradually cooling to 20°C at a rate of 1°C per cycle for a total of 60 cycles to achieve gradual annealing of the oligonucleotide and the long single-stranded DNA and form a stable hybrid structure.

[0018] In some specific embodiments, the annealing buffer used comprises 10 mM Tris-HCl (pH 8.0) and 10 mM MgCl 2 .

[0019] In some embodiments, the oligonucleotide used for hybridization is selected from DNA, RNA and analogs thereof, such as non-natural nucleic acids LNA, 2'-O-methyl RNA, PNA, etc.

[0020] In some embodiments, the oligonucleotide is linked to a functional ligand that is used to confer a specific biological function on the hybrid DNA. The functional ligand includes, but is not limited to, one or a combination of a short peptide, a protein binding domain, a small molecule ligand, a nucleic acid aptamer, or a nanoparticle.

[0021] The ligand can be introduced through the 5' or 3' end of the oligonucleotide, and the connection methods include thiol modification and metal surface coupling, amide bond connection, click chemistry reaction or double-stranded hybridization complementary connection, etc. Furthermore, the oligonucleotide contains a functional ligand at the 5' end to hybridize the gene knock-in efficiency of the DNA donor template.

[0022] The ligand can be used to enhance the uptake efficiency of hybrid DNA in target cells, improve intracellular stability, impart targeted recognition ability, or promote its entry into the cell nucleus, etc., and includes, for example, cell-penetrating peptides (such as TAT), Cas9 recognition sequences (CTS, the specific sequence is shown in SEQ ID NO: 224 or SEQ ID NO: 225), albumin-binding peptides, folic acid, small molecule lipids, HER2 nucleic acid aptamers, or structural units covalently linked to gold nanoparticles, liposomes, or polymer nanoparticles.

[0023] In some embodiments, to enhance the stability and delivery efficiency of oligonucleotides in cells, gold nanoparticles are used as carriers, and oligonucleotides are covalently coupled to gold nanoparticles to construct hybrid DNA nanocomplexes, and their application effects in gene editing are evaluated.

[0024] In some embodiments, the hybrid DNA is used as a donor template for gene knock-in. The DNA insert sequence of the long single-stranded DNA is selected from sequences encoding proteins, RNAs, DNA regulatory elements, or variants thereof.

[0025] In some embodiments, when hybrid DNA is used as a donor template for gene knock-in, the length of the DNA insert is varied but comprises at least one nucleotide. In other experiments, the length is extended to approximately 0.5 kilobase pairs (kb), 2 kb, 2.5 kb, 5 kb, 10 kb, 20 kb, 40 kb, 80 kb, 100 kb, or even 150 kb.

[0026] In some embodiments, when hybrid DNA is used as a donor template for gene knock-in, the DNA insert may comprise a nucleotide sequence encoding a transcription unit, each of which can produce a cellular product. In some embodiments, the DNA insert may comprise a sequence encoding a protein such as a chimeric antigen receptor (CAR), a T cell receptor, an immunomodulatory protein, a growth factor, an antibody, or other protein.

[0027] In some embodiments, when the hybrid DNA is used as a donor template for gene knock-in, it comprises a DNA insert sequence located between the 5' end and the 3' end, wherein the 5' end and the 3' end respectively comprise nucleotide sequences having homology to the genomic sequence of the insertion site; In some specific embodiments, when hybrid DNA is used as a donor template for gene knock-in, the 5' homology arm and the 3' homology arm may be of different lengths, and the lengths of the nucleotide sequences with homology at the 5' and 3' ends of the long single-stranded DNA are 50 to 3000 nucleotides, respectively. For example, the lengths of the homology arms are approximately 50 to 550 nucleotides, 550 to 1050 nucleotides, 1050 to 1550 nucleotides, 1550 to 2050 nucleotides, 2050 to 2550 nucleotides, and 2550 to 3050 nucleotides, respectively. If the homology arms are too long, the melting point of the primers may be too high, thereby reducing the amplification efficiency; on the contrary, if the homology arms are too short, their specific binding ability to the target sequence may be weakened, resulting in non-specific or non-targeted integration.

[0028] In some embodiments, the hybridization region of the oligonucleotide covers at least one of a nucleotide sequence having homology to the long single-stranded DNA, a DNA insert sequence, a target sequence, or other auxiliary sequences.

[0029] In another aspect, the present invention provides a method for constructing a donor template using the hybrid DNA or achieving genome targeted editing using the hybrid DNA prepared above, comprising the following steps: (1) introducing the hybrid DNA donor template into the target cell, i.e., transferring the hybrid DNA having the DNA insert, 5' homology arm and 3' homology arm into the cell, wherein the 5' homology arm and the 3' homology arm complementarily bind to the polynucleotide sequence in the target region of the genome in the cell; (2) introducing a nuclease and its accessories into the target cell, wherein the nuclease induces nucleotide cleavage in the target region of the genome; (3) The 5' homology arm and 3' homology arm of the hybrid DNA donor template are complementary to the genomic break region through homologous recombination to achieve targeted integration of the DNA insert.

[0030] In step (1), the DNA insert is placed between two homology arms: one is the 5' homology arm and the other is the 3' homology arm, which are complementary to the corresponding polynucleotide sequences in the target region of the genomic DNA in the cell.

[0031] In some embodiments, the method for introducing the hybrid DNA donor template and the nuclease is selected from electroporation, lentiviral transduction, or liposome transfection.

[0032] In some embodiments, the break is a DNA double-strand break (DSB).

[0033] In some embodiments, the break is a DNA single-strand break or nick.

[0034] In the present invention, the gene editing method is selected from the group consisting of a meganuclease system, a zinc finger nuclease system, a transcription activator-like effector nuclease (TALEN) system, an Argonaute nuclease system, and a CRISPR / Cas system. This precise gene editing technology creates a break near the target region to be edited, inducing homologous recombination or nonhomologous end joining, thereby knocking in the gene.

[0035] In the above applications, it is only necessary to compare the knock-in efficiency with that of the normal donor DNA in the control group to analyze whether the hybrid DNA can improve the gene knock-in efficiency.

[0036] In the present invention, targeted integration aims to “knock-in” (KI) DNA into a predetermined genomic location.

[0037] The term “approximately” used in the present invention is intended to cover a range of ±10% of the numerical value to reflect the actual operational flexibility in the experiment and the tolerance range of the data.

[0038] In this context, homology arms are defined as a series of nucleotides that exactly match the corresponding nucleotide sequence in the endogenous DNA sequence within the target region. These two homology arms, located at either end of the DNA insert, allow the DNA insert to be precisely positioned and integrated into the target region. The target region, in turn, is the region of nucleic acid sequence where a specific insertion or modification is desired.

[0039] For purposes of this invention, the terms "complementarity" and "hybridization" specifically refer to the process by which two complementary regions within a polynucleotide molecule bind to each other through hydrogen bonds, thereby forming a double-stranded structure. Specifically, the two complementary regions may reside within the same polynucleotide chain (i.e., intramolecular hybridization) or may originate from different polynucleotide chains (i.e., intermolecular hybridization).

[0040] In some embodiments, the target cells are mammalian cells, including but not limited to in vitro cultured cell lines, primary cells, or in vivo cells. Specific target cells can be selected from one or a combination of the following types: (1) Primary hematopoietic cells, such as CD34 derived from peripheral blood, umbilical cord blood, or bone marrow +Hematopoietic stem / progenitor cells, T cells, B cells, etc.; (2) Immune effector cells, such as T cells, NK cells, NKT cells, γ / δ T cells, macrophages, dendritic cells, etc.; (3) Cancer cells, including human solid tumor cells or hematological tumor cells, such as K562, Jurkat, HeLa, HepG2, etc.; (4) Commonly used mammalian cell lines, such as HEK293T, CHO, NIH-3T3, etc.

[0041] In a preferred embodiment, the target cells are human T cells or hematopoietic stem cells, which are suitable for in situ gene editing or gene-modified cell therapy scenarios.

[0042] In some embodiments, the inserted fragment of the hybrid DNA can encode a chimeric antigen receptor (CAR) and be used to construct a genetically modified immune cell. In a specific implementation, the genetically modified cell can be selected from: (1) CAR-modified T cells; (2) CAR-modified natural killer (NK) cells; (3) CAR-modified macrophages; (4) CAR-modified hematopoietic stem / progenitor cells; The cells are preferably human cells, but may also be derived from other mammals.

[0043] The method of targeted gene editing using hybrid DNA described herein can be widely applied to the genetic modification of T cells. In recent years, CAR-T and TCR-T cells have become important approaches to tumor treatment as highly specific and low-toxic immunotherapies in T cell adoptive transfer therapy. In some embodiments, the T cells carry exogenous T cell receptors (TCRs) or chimeric antigen receptors (CARs) to confer the ability to specifically recognize tumor antigens.

[0044] In the present invention, "T cells" refer to lymphocytes expressing TCR, including but not limited to naive T cells, stimulatory T cells, helper T cells (such as Th1, Th2, Th3, Th9, Th17, TFH subtypes), cytotoxic T cells, memory T cells, regulatory T cells (FOXP3 + or FOXP3 - ), natural killer T cells (NKT cells), α / β T cells, γ / δ T cells, and combinations or subsets thereof.

[0045] The T cells may be genetically modified recombinant T cells. In some embodiments, recombinant T cells include the following types: (1) recombinant TCR-T cells, in which the TCR is mutated in the CDR region to change the antigen recognition repertoire, or the signal strength is modulated by intracellular domain modification; (2) CAR-T cells, in which the TCR is replaced by a heterologous structure to construct a chimeric antigen receptor (CAR). The CAR structure generally includes: a targeting domain, such as a single-chain antibody (scFv); a transmembrane domain, such as CD28 or a CD8 hinge region; and an intracellular signaling domain, including a costimulatory domain (such as CD28, 4-1BB) and an activation domain (such as CD3ζ).

[0046] In certain preferred embodiments, the CAR comprises an antigen recognition structure for mesothelin, such as that derived from a monoclonal antibody, a single domain antibody, a humanized antibody, or an antigen-binding fragment thereof (such as scFv, VH, VL, scFab, etc.).

[0047] In some embodiments, gene editing can be achieved by using methods such as antisense RNA, antagomir (miRNA antagonist), siRNA, shRNA, large-range nucleases, zinc finger nucleases, transcription activator-like effector nucleases, or CRISPR systems.

[0048] In a further embodiment of the present invention, the transfection of the hybrid DNA can be carried out in primary hematopoietic cells or primary hematopoietic stem cells. The transfection dose is preferably 1×10 5 to 2×10 6 The primary hematopoietic cells are preferably T cell subsets, including regulatory T cells (Treg), effector T cells (Teff) or naive T cells. In addition, these T cells can be further characterized as CD8 + Single positive T cells or CD4 + CD8 + Double-positive T cells are suitable for different gene editing application requirements.

[0049] The present invention also provides for the use of the gene-edited cells in the preparation of tumor immunotherapy drugs. In some specific embodiments, CAR-T cells that meet quality standards are sterilized and prepared; according to the treatment plan, the CAR-T cells are administered to patients via intravenous infusion or other methods to implement tumor immunotherapy.

[0050] The present invention also provides a method for using hybrid DNA as a gene expression vector to achieve gene expression in vivo and in vitro. The hybrid DNA can be used not only as a donor template in gene editing systems such as CRISPR, but also as an independent expression vector to efficiently drive the expression of exogenous genes in mammalian cells and animals in vitro and in vivo.

[0051] In the above, whether it is gene editing or the application of exogenous gene expression, the application includes in vivo, in vitro or ex vivo.

[0052] In some embodiments, the hybrid DNA is formed by annealing a long single-stranded DNA obtained by synthesis or amplification with at least one complementary short oligonucleotide to form a hybrid structure. The hybrid DNA molecule is a closed circular or stable structure, and has good molecular stability and transfection compatibility.

[0053] In a preferred embodiment, the hybrid DNA contains a complete gene expression cassette, which includes at least the following elements: At least one enhancer to increase promoter activity and enhance transcription efficiency; A promoter, which serves as the main promoter driving the expression of the exogenous gene; A heterologous coding sequence, which can be an open reading frame of any target protein, such as green fluorescent protein (EGFP), luciferase, reporter protein, functional enzyme, or therapeutic protein; A polyadenylation signal that stabilizes mRNA and increases translation efficiency.

[0054] In addition, to facilitate the amplification and preparation of hybrid DNA in a prokaryotic system, in some embodiments, the vector further comprises an M13 origin replication site to support ssDNA copy amplification in Escherichia coli.

[0055] In other optional embodiments, the enhancer and promoter in the above expression cassette can be replaced with other commonly used regulatory elements, such as the SV40 enhancer, EF1α promoter, Ubiquitin promoter (UbC), and hSyn promoter, to adapt to different tissues, cell types, or expression intensity requirements. Auxiliary regulatory elements, such as IRES sequences, 2A peptide sequences, and transcription termination signals, can also be introduced to achieve multi-gene co-expression, auto-cleavage, or regulated expression.

[0056] That is, when used, the hybrid DNA is introduced into cells or living organisms, and at least one heterologous coding sequence is expressed under the drive of the regulatory elements of the expression vector.

[0057] In some embodiments, the hybrid DNA can be used for gene editing or gene expression experiments in mammals. Applicable animal models include, but are not limited to, mice, rats, rabbits, dogs, pigs, and non-human primates (e.g., macaques or marmosets). These animal models are widely used in gene therapy, vaccine evaluation, immune response research, and other fields, and have a strong experimental foundation and translational potential.

[0058] In in vivo applications, hybrid DNA can be introduced into animals via tail vein injection, intramuscular injection, subcutaneous injection, adeno-associated virus (AAV), or liposome delivery to achieve exogenous gene expression or gene knock-in in target tissues. In mouse models, intravenous delivery of the hybrid DNA can achieve stable expression in the liver or spleen.

[0059] In certain embodiments, the hybrid DNA described herein can be used for gene editing in human cells, and is particularly suitable for the construction of therapeutic cells, including but not limited to the development of therapeutic products such as chimeric antigen receptor T cells (CAR-T), T cell receptor engineered T cells (TCR-T), and CAR-NK cells. Such gene editing can be used to insert exogenous receptor genes, knock out endogenous immunosuppressive factors, or integrate reporter genes / tracking tags to enhance function, reduce toxicity, or improve targeting.

[0060] In clinical applications, the above therapies have been widely studied and used to treat a variety of human diseases, especially showing significant research potential in the following indications: (1) hematological malignancies: such as acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM); (2) solid tumors: such as pancreatic cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, mesothelioma, etc.; (3) autoimmune diseases: such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes (T1D); (4) inherited immunodeficiency or metabolic diseases: such as severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), etc.

[0061] The hybrid DNA structure described in the present invention not only has excellent stability and designability, but also shows good versatility and scalability in constructing expression systems. It is suitable for basic research, cell function verification, disease model construction, gene therapy and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the preparation scheme of the hybrid DNA donor template of the present invention; Figure 2 The hybrid DNA donor template is formed by complementary pairing of circular single-stranded DNA and oligonucleotides of different lengths and numbers. RAB11A Comparison of knock-in efficiency of sites; Figure 3 Different forms of DNA templates in K562 cells RAB11A Gene editing efficiency at the site; Figure 4 CRISPR / Cas9 gene editing efficiency of hybrid DNA donors with functional CTS sequences; Figure 5 Functionalized CTS hybridized DNA donor coupled to gold nanoparticles affects CRISPR / Cas9 gene editing efficiency; Figure 6 Different forms of DNA templates in human primary T cells TRAC Gene editing efficiency at the site; Figure 7 Flow cytometry of T cells expressing CAR and TCR; Figure 8 cssDNA and hybrid DNA in NK92MI cell line CLTA Gene editing efficiency at the site; Figure 9 The expression of EGFP in cells by hybrid DNA expression vector; Figure 10 This is a bioluminescent imaging image of luciferase expression induced by the hybrid DNA expression vector in mice; The scattered points in the above figures represent parallel samples. DETAILED DESCRIPTION

[0063] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0064] The present invention first synthesizes a hybrid DNA donor and uses this product in combination with CRISPR technology to achieve efficient gene knock-in. At the same time, a hybrid DNA vector is constructed and combined with electroporation technology to achieve in vivo and in vitro gene expression.

[0065] To achieve the above objectives, the present invention provides a method for hybridizing DNA, wherein circular or linear single-stranded DNA is annealed with oligonucleotides of different lengths and numbers by PCR to obtain hybrid DNA, and finally the hybrid DNA is purified.

[0066] On the other hand, the present invention provides a solution for achieving targeted and efficient gene knock-in using CRISPR / Cas9 technology with or without functional hybrid DNA. After Cas9 protein cuts genomic DNA, a break is generated. The homologous arms of the hybrid DNA are homologous to the break sequence. Under the action of the HDR repair pathway in the cell, the hybrid DNA is copied as a template to the break to achieve the purpose of gene knock-in. We choose RAB11AThe target gene is knocked into the gene locus. If the donor DNA sequence is successfully inserted, the cells will express green fluorescent protein, and the results can be observed by flow cytometry. Next, we also provide a method for T cell gene editing using a hybrid DNA donor template, which is suitable for gene editing needs in CAR-T cell therapy.

[0067] Finally, the present invention also provides a hybrid DNA vector with or without function, which is introduced into cells or animals using electroporation technology to express the heterologous coding sequence under the drive of the vector.

[0068] The following is a detailed description of the method for synthesizing hybrid DNA and the scheme for achieving efficient gene knock-in and efficient gene expression using the product.

[0069] Example 1: Synthetic hybrid DNA First, we synthesized linear and circular single-stranded DNA. We used Lambda exonuclease to digest phosphorylated single-stranded DNA to produce long linear single-stranded DNA. We also used the M13 phage method to produce circular single-stranded DNA.

[0070] Long linear single-stranded DNA (lssDNA) was synthesized using conventional primers (primer sequences shown in SEQ ID NOs: 226-227) for the target strand and 5' phosphorylated primers (phosphorylated at the 5' end of the reverse primer sequence, sequence shown in SEQ ID NO: 228) for the digested strand.

[0071] The first step is to design phosphate-modified primers based on the insertion target site. RAB11A For example, a donor with 300-nt homology arms was used. The donor sequence and its complementary oligonucleotide sequences are shown in SEQ ID NOs: 2-37, where the donor sequence is RAB11A-lssDNA. dsDNA (RAB11A-plasmid sequence is shown in SEQ ID NO: 1) was amplified by PCR. The dsDNA was then recovered using SPRIselect Bead-Based Reagent (1.0×). The non-target strand was digested with Lambda Exonuclease and Exonuclease III at 37°C for 5 minutes, followed by incubation at 80°C for 5 minutes to inactivate the enzymes. Linear single-stranded DNA (RAB11A-lssDNA, sequence shown in SEQ ID NO: 2) was recovered again using SPRIselect Bead-Based Reagent (1.0×) and verified by agarose gel electrophoresis.

[0072] The synthesis of circular single-stranded DNA is produced by the M13 phage method. In simple terms, the first step is to transform the RAB11AThe site-recombination plasmid (sequence shown in SEQ ID NO: 1) and the helper plasmid pSB4423 were co-transformed into XL1-Blue competent cells and plated onto inverted solid culture plates containing the corresponding resistance. Single colonies were selected and cultured in 2xYT medium for 18-20 hours. Escherichia coli were removed by centrifugation at 15,000 × g for 15 minutes at 4°C. The supernatant was collected and filtered, and 0.25 volumes of 5x PEG buffer were added to the filtrate. The mixture was thoroughly shaken and centrifuged at 17,000 × g for 35 minutes at 4°C. The supernatant was discarded to obtain a white precipitate of adherent phage. Circular single-stranded DNA (RAB11A-cssDNA, sequence shown in SEQ ID NO: 2) was extracted using the QIAGEN EndoFree Plasmid Maxi Kit and verified by agarose gel electrophoresis.

[0073] After obtaining linear or circular single-stranded DNA, specific oligonucleotides of different lengths and quantities synthesized by the company (sequences of RAB11A-oligo-1 to RAB11A-oligo-35 are shown in SEQ ID NO. 3 to 37, respectively) were combined with the linear DNA single-stranded template (molar ratio of ssDNA:oligo = 1:2). 10× Tris MgCl2 was added to the system. 2+ , stabilizing the single-stranded structure and promoting complementary base pairing. PCR was then performed with the following annealing procedure to form hybrid DNA: long single-stranded DNA and oligonucleotide were mixed at a molar ratio of 1:2 and heated at 85°C for 3-5 minutes to fully denature and eliminate possible secondary structures; the temperature was then lowered to 80°C and maintained for 2 minutes to create an initial mild environment for the annealing reaction; then, the temperature was lowered by 1°C per cycle for 60 cycles until it dropped to 20°C, allowing the long single-stranded DNA and oligonucleotide to gradually anneal and form a stable hybrid structure ( Figure 1 ).

[0074] In the same way, we also constructed CLTA The cssDNA and hybrid DNA at the locus were synthesized. The long single-stranded DNA was CLTA-cssDNA (sequence shown in SEQ ID NO: 100), the oligonucleotides were CLTA-oligo-1 to CLTA-oligo-36 (sequence shown in SEQ ID NOs: 101-106, 9-23, 107-113, and 30-37, respectively), and the plasmid sequence used was shown in SEQ ID NO: 99.

[0075] Example 2: CRISPR / Cas9 gene knock-in experiment Our goal was to demonstrate that hybrid DNA is a better and more stable DNA donor template. RAB11AFor example, to compare the gene editing efficiency of the hybrid DNA donor template, we also synthesized a DNA template with the same sequence as the circular single-stranded DNA. RAB11A Circular double-stranded DNA at the locus was synthesized as follows: RAB11A-oligo-3 (SEQ ID NO: 5) was selected as a primer and mixed with the synthesized circular single-stranded DNA. 2× Phanta Max Master Mix was added to the desired solution system and then extended at 95°C for 2 minutes, 55°C for 1 minute, 72°C for 20 minutes, and 12°C for ∞. Circular double-stranded DNA was recovered using SPRIselect Bead-Based Reagent (1.0×).

[0076] To induce DNA double-strand breaks, we used RAB11A The sgRNA targeting the target site (sequence shown in Table 1, SEQ ID NO: 221) and the Cas9 protein (Novoprotein, Catalog No. E365-02B) with NLS at both ends were used. K562 cells were used as a model organism to test insertion efficiency and cell viability. K562 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum at 37°C and 5% carbon dioxide.

[0077] Table 1

[0078] To compare the efficiency and stability of hybrid DNA knock-in in K562 cells, we used the Lonza 4D nucleofection system to deliver Cas9 RNP and homologous recombinant DNA template. Before electroporation, 25 pmol of recombinant Cas9 protein (Novoprotein, E365-02B) and 75 pmol of chemically synthesized sgRNA (GENEWIZ) were incubated at room temperature for 15 minutes per well to form the Cas9 RNP complex. 2 × 10 5 10,000 K562 cells were added. RNPs were mixed with different forms of DNA donor templates and transferred to an electroporation cuvette along with the K562 cell suspension. The EO138 transfection protocol was selected. After electroporation, 85 μl of prewarmed 1640 medium was added to the cuvette. The electroporated cells were seeded in a 48-well plate containing 400 ml of prewarmed 1640 medium supplemented with 10% fetal bovine serum. After 24 hours, the K562 cells were transferred from the 48-well plate to a 12-well plate for expansion and culture for 5-7 days. Flow cytometric analysis was performed using a Beckman CytoFLEX LX to assess gene editing efficiency.

[0079] The gene editing efficiency of the DNA donor template was calculated by measuring the percentage of EGFP-positive cells, as only cells that have correctly integrated the donor template sequence can produce the EGFP-RAB11A fusion protein. The geometric mean of EGFP intensity was measured from the EGFP-positive cells in each sample. The results showed that the proportion of EGFP-positive cells reached the highest when the oligonucleotide length was 50 nt and 35 oligonucleotides, and the proportion of EGFP-positive cells gradually increased with increasing DNA template dosage ( Figure 2 ).in Figure 2 The horizontal axis represents the hybridization of long single-stranded DNA with RAB11A-oligo-1 to RAB11A-oligo-4 (4 oligos), RAB11A-oligo-1 to RAB11A-oligo-8 (8 oligos), RAB11A-oligo-1 to RAB11A-oligo-12 (12 oligos), RAB11A-oligo-1 to RAB11A-oligo-16 (16 oligos), and RAB11A-oligo-1 to RAB11A-oligo-1. AB11A-oligo-20 hybridization (20 oligos), RAB11A-oligo-1 to RAB11A-oligo-24 hybridization (24 oligos), RAB11A-oligo-1 to RAB11A-oligo-28 hybridization (28 oligos), RAB11A-oligo-1 to RAB11A-oligo-32 hybridization (32 oligos), RAB11A-oligo-1 to RAB11A-oligo-35 hybridization (35 oligos).

[0080] Among them, the proportion of EGFP-positive cells in the hybrid DNA+Cas9 RNP group was significantly higher than that in the long circular single-stranded DNA+Cas9RNP and long linear single-stranded DNA+Cas9 RNP groups ( Figure 3 ).in Figure 3 The horizontal axes represent the experimental results of linear single-stranded DNA (lssDNA), linear double-stranded DNA (ldsDNA), hybrid linear single-stranded DNA (lssDNA-oligos) described in the present application, plasmid, circular single-stranded DNA (cssDNA), circular double-stranded DNA (cdsDNA), and hybrid circular single-stranded DNA (cssDNA-oligos, 100% length hybridization, i.e., 35 oligos) described in the present application.

[0081] Example 3: CRISPR / Cas9 gene knock-in experiment with functional hybrid DNA donor We conjugated the Cas9 recognition sequence (CTS) to oligonucleotides, enabling them to bind to the Cas9 RNP and thereby increase DNA nucleofection efficiency. The CTS sequence (shown in SEQ ID NOs: 224 and 225) was attached to the oligonucleotides (odd-numbered oligonucleotides from RAB11A-oligo-1 to RAB11A-oligo-35) as an overhang to form functional hybrid DNA with long single-stranded DNA. We tested the effects of introducing the double-stranded CTS sequence into the oligonucleotides, introducing the CTS functional sequence at the 5' or 3' end of the oligonucleotide, or introducing the CTS functional sequence using two oligonucleotides, and hybridizing with circular single-stranded DNA to form css-CTS oligo templates, and evaluated their effects on gene editing efficiency. The remaining procedures were the same as in Examples 1 and 2. The geometric mean of the proportion of EGFP-positive cells was measured. The results showed that the proportion of EGFP-positive cells with 5'CTS hybrid DNA + Cas9 + gRNA was significantly higher than that with long circular single-stranded DNA + Cas9 + gRNA and hybrid DNA + Cas9 + gRNA groups, and was stronger than the other two CTS design methods ( Figure 4 ).

[0082] We also verified the gene editing efficiency of hybridized DNA using oligonucleotide-conjugated gold nanoparticles (NanoSeeds, Cat. No. NS-C-13-20). A C6 HS-SH modification (containing a disulfide bond) was introduced at the 5' end of the oligonucleotide and dissolved in ultrapure water to prepare a 100 μM working solution. Tricarboxyphosphine (TCEP) was then added to this oligonucleotide solution to a final concentration of 30 mM and allowed to react at room temperature for 1 hour to reduce and cleave the disulfide bonds. Following the reaction, the solution was ultrafiltered three times through a 3 kDa ultrafiltration centrifuge tube and washed with ultrapure water to remove residual TCEP. 100 μL of 40 nM gold nanoparticles (13 nm in diameter) was mixed with 30 μL of 35 μM activated oligonucleotide and allowed to react at room temperature for 1 hour. The following components were added to the reaction system in sequence to stabilize the coupling system and prevent aggregation: sodium dodecyl sulfate (SDS) was added to a final concentration of 0.01% and allowed to react at room temperature for 30 minutes, with sonication for 1 minute before and after addition; 0.1 M PBS was added to a final concentration of 10 mM and allowed to react at room temperature for 30 minutes, with sonication for 1 minute before and after addition; and 5 M PBS was added to a final concentration of 300 mM and allowed to react at room temperature overnight (approximately 12-16 hours), with sonication for 1 minute before and after addition. After the reaction, 0.01% SDS was added to a total volume of 1 mL, and the gold nanoparticles were collected by centrifugation at 6000 × g (room temperature, 20 minutes). The supernatant was removed and the mixture was washed three times in the same manner to remove uncoupled oligonucleotide. The coupling between the oligonucleotide and the gold nanoparticles was confirmed by agarose gel electrophoresis.

[0083] Next, the CTS-anti oligonucleotide conjugated with gold nanoparticles was annealed with the circular single-stranded DNA template to form a hybrid DNA complex of gold nanoparticles and CTS. This complex was used to evaluate as a gene editing donor. The experimental results showed that compared with the CTS hybrid DNA template without gold nanoparticles, the gold nanoparticle modification significantly improved the gene editing efficiency ( Figure 5 ).

[0084] Example 4: Application of hybrid DNA in gene editing of primary human T cells to prepare CAR-T cells Frozen PMBCs were thawed and cultured overnight in X-VIVO 15 complete medium without cytokines. TM Primary human T cells were isolated by magnetic sorting using CD3 Nanobeads (Genscript). The desired number of cells was transferred to a tube and resuspended in separation buffer. The appropriate amount of magnetic beads was added based on the number of cells. After gently tapping the bottom of the tube to mix the beads and cells, the cells were incubated at 2-8°C for 15 minutes. 7 Wash the cells once with 1-2 ml isolation buffer for each mononuclear cell (MNC), centrifuge at 300 × g for 10 minutes, and discard the supernatant. Finally, resuspend the cells in 500 μL isolation buffer for 10 min. 8 cells. Using CytoSinct TM T cells were sorted using a manual magnetic separation system. A separation column was placed on the magnetic stand, and the cell suspension was passed through the column. The cells were then washed twice with separation buffer. The separation column was removed from the magnet and placed in a new centrifuge tube of appropriate size. The magnetic bead-labeled T cells were eluted with separation buffer and counted. Depending on the number of isolated primary human T cells, anti-CD3 / CD28 Dynabeads (ThermoFisher) were added at a 1:1 ratio to activate the T cells for 48 hours before use in subsequent experiments.

[0085] Unless otherwise stated, human primary T cells were cultured in X-VIVO 15 medium (Lonza) containing 10% fetal bovine serum (Gibco), 1% NEAA (Gibco), 55 μM 2-mercaptoethanol (Absin), and 1 mM sodium pyruvate (Gibco). 10 ng / mL IL-7 (Novoprotein) and 10 ng / mL IL-15 (Novoprotein) were added to the medium to maintain T cell activity and expansion. Cells were cultured at 37°C and 5% carbon dioxide. When the cell density exceeded 2.5 × 10 6 When the culture medium reaches 0.5 μg / ml or turns yellow, the cells are transferred to a larger culture dish and fresh IL-7 and IL-15 are added.

[0086] Targeting the human primary T cell genome TRAC A highly specific sgRNA (sequence shown in SEQ ID NO: 222 in Table 1) was designed to ensure precise cutting at the safe site, facilitating the CAR Gene knock-in. Mix 25 pmol of Cas9 protein (Novoprotein, E365-02B) with 75 pmol of sgRNA per well and incubate at room temperature for 15 min to assemble into RNPs. Construct a donor DNA template for inserting EGFP or CAR gene, which is flanked by TRAC The left and right homology arms corresponding to the site are used to achieve precise knock-in using the HDR mechanism ( Figure 6 ).in Figure 6 The horizontal axis represents the experimental results of linear single-stranded DNA (lssDNA), linear double-stranded DNA (ldsDNA), hybrid linear single-stranded DNA (lssDNA-oligos) described in this application, plasmid (TRAC-EGFP-plasmid), circular single-stranded DNA (CssDNA), circular double-stranded DNA (CdsDNA), and hybrid circular single-stranded DNA (CssDNA-oligos) described in this application, respectively. The donor template adopts the hybrid DNA form described above, and the donor sequence and its complementary oligonucleotide are as follows: TRAC-EGFP-cssDNA (SEQ ID NO: 39) is the EGFP donor DNA template (long single-stranded DNA) for insertion, TRAC-EGFP-CAR-19bbz-cssDNA (SEQ ID NO: 40) is the CAR donor DNA template for insertion, and TRAC-EGFP-BBZ-1 to TRAC-EGFP-BBZ-35 (represented in SEQ ID NOs: 41-62 and 30-37, respectively) are hybrid oligonucleotides commonly used for insertion into the EGFP or CAR gene. TRAC-EGFP-1 to TRAC-EGFP-16 (shown in SEQ ID NOs: 63, 10-22, and 64-65, respectively) are hybrid oligonucleotides inserted into EGFP. TRAC-19bbz-1 to TRAC-19bbz-33 (shown in SEQ ID NOs: 66-98, respectively) are hybrid oligonucleotides inserted into CAR.

[0087] Activated T cells were washed and resuspended in B1mix buffer. The assembled Cas9 RNPs were mixed with the donor DNA template and electroporated with the T cell suspension (Lonza Nucleofector) using appropriate voltage and pulse parameters. Following electroporation, the cells were quickly transferred to prewarmed culture medium containing IL-7 and IL-15 and incubated at 37°C with 5% CO2 to promote cell recovery and HDR repair.

[0088] Flow cytometry was used to detect gene editing efficiency, cell viability 72 hours after transfection, live cell count 120 hours after transfection, and EGFP positive cell count 120 hours after transfection. The results are shown in Table 2. The experimental results show that circular hybrid DNA TRAC The site achieved the highest gene editing efficiency and obtained the largest number of EGFP-positive cells. After sorting the positive cells on the 7th day, PacBio amplicon sequencing was used to evaluate the gene editing accuracy of different forms of HDR templates in T cells. Knock-Knock analysis was used to distinguish accurate HDR from unexpected events. The results showed that long linear single-stranded DNA had the lowest accuracy, accompanied by a large number of incomplete insertions, which may be due to its poor stability and easy degradation; long linear double-stranded DNA and linear hybrid DNA had blunt-end insertions, which may be related to NHEJ-mediated misconnection; and the HDR template based on circular single-stranded DNA was the most accurate insertion, which significantly improved the editing accuracy and provided support for efficient homologous recombination (Table 2). For T cells with knocked-in CAR genes, flow cytometry was used to detect the CAR expression level and CD3 knockout level to test the gene knock-in efficiency of CAR ( Figure 7 The results showed that hybrid DNA donor templates exhibited higher gene editing efficiency than circular single-stranded DNA.

[0089] Table 2 Gene editing accuracy of human primary T cells transfected with different forms of DNA templates detected by third-generation amplicon sequencing

[0090] Example 5: Application of hybrid DNA as a donor for gene knock-in in NK92MI cell line The same operation as in Example 2 was used to induce DNA double-strand breaks. CLTA The sgRNA targeting the target site (sequence shown in Table 1 as SEQ ID NO: 223) and Cas9 mRNA (Novoprotein, catalog number: MR109) were injected. NK92MI cells were used to test insertion efficiency and cell viability. NK92MI cells were cultured in NK92MI cell-specific culture medium (Pneusai) supplemented with 200 U / ml IL-2 at 37°C and 5% CO2.

[0091] To compare the efficiency and stability of hybrid DNA knock-in in NK92MI cells, we used the Lonza 4D Nucleofection System to deliver Cas9 mRNA, sgRNA, and homologous recombination DNA template. Before electroporation, 0.3 μg of Cas9 mRNA was mixed with 2.4 μg of chemically synthesized sgRNA (GENEWIZ) and 0.6 μg of different forms of DNA donor template per well. 5 × 10 5 10,000 NK92MI cells were plated. The mixed DNA donor template, Cas9 mRNA, sgRNA, and NK92MI cell suspension were transferred to an electroporation cuvette and transfected using the CA137 protocol. After electroporation, 85 μl of prewarmed NK92MI cell-specific culture medium was added to the cuvette. The electroporated cells were seeded into a 48-well plate containing 400 ml of prewarmed NK92MI cell-specific culture medium. After 24 hours, the NK92MI cells were transferred from the 48-well plate to a 12-well plate for expansion and culture for 5-7 days. Flow cytometric analysis was performed using a Beckman CytoFLEX LX instrument to assess gene editing efficiency.

[0092] The editing efficiency of the DNA donor template was calculated by measuring the percentage of EGFP-positive cells, as only cells that have correctly integrated the donor template sequence can produce the EGFP-CLTA fusion protein. The geometric mean of EGFP intensity was measured from the EGFP-positive cells in each sample. The results showed that in the NK92MI cell line, the proportion of EGFP-positive cells in the hybrid DNA group was significantly higher than that in the long circular single-stranded DNA group ( Figure 8 ).

[0093] Example 6: Application of hybrid DNA as a vector for gene expression in cells or animals Taking K562 as an example, the constructed hybrid DNA vector containing the EGFP gene (wherein CMV-EGFP-cssDNA is a long single-stranded DNA sequence, the sequence is shown in SEQ ID NO: 114, and CMV-EGFP-oligo-1 to CMV-EGFP-oligo-40 are oligonucleotide sequences shown in SEQ ID NO: 115-154, respectively) is mixed with the cell suspension in the electrotransfection buffer, and the oligonucleotide with the sequence shown in SEQ ID NO: 229 is used as the oligonucleotide control. Transfection is performed using an electroporator (Lonza Nucleofector). Electroporation program: EO138. After transfection, the cells are revived and cultured for 24-48 hours to allow the expression of EGFP protein to reach a detectable level. Expression verification can be completed by flow cytometry to analyze the EGFP positivity and fluorescence intensity in the transfected cells. Generally, the transfection group using the vector of the present invention will show a green fluorescence positive signal that is significantly higher than that of the negative control group ( Figure 9 ).

[0094] In animal experiments, adult BALB / C mice weighing approximately 20 grams were injected and electroporated. A luciferase-encoding circular hybrid DNA vector (pscaf-Luc2-cssDNA is a long single-stranded DNA sequence (labeled C0-Css Luc in the figure), 40 (C40-Css Luc), or 65 (C65-Css Luc) 50-nt oligonucleotides was diluted in saline (e.g., 20 μg DNA in 20 μL saline) and injected into the tibialis anterior or quadriceps femoris muscles of the mice via syringe. Immediately after injection, place an electrode (needle or plate) at the injection site and perform electroporation. If using a BTX ECM830 electric pulse generator, set the parameters to 50 V pulse voltage, 20 ms pulse width, 5 pulses, and 100 ms pulse interval. After electroporation, return the mouse to its normal cage. Luciferase signal imaging is performed multiple times after transfection. The imaging steps are as follows: Prepare D-luciferin (luciferase substrate) injection solution, typically at 15 mg / mL (in phosphate buffered saline). Following the IVIS imaging system protocol, inject the mouse intraperitoneally with 150 mg / kg D-luciferin (approximately 150-200 μL per 20 g mouse). When the signal peaks 10-15 minutes after injection, anesthetize the mouse with isoflurane. Place the anesthetized mouse in a live imaging system (such as the IVIS Lumina) and set the light field of view and exposure time (30 to 60 seconds, binning 4-8, F-stop 1). Collect bioluminescent signal images recorded by a gamma counter or CCD camera. The positive expression group will show obvious bioluminescent signals in the abdomen or muscle area, while the control group will show no or weak signals, thus verifying the expression function of the hybrid DNA vector in vivo. The experimental results show that the expression efficiency of circular hybrid DNA is significantly higher than that of circular single-stranded DNA. Among them, the hybrid DNA vector with 65 complementary oligonucleotides (i.e., 100% hybridization) has the highest expression efficiency ( Figure 10 ).

[0095] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A hybrid DNA vector, characterized in that include: a) a long single-stranded DNA comprising a target sequence; b) at least one oligonucleotide, comprising a nucleotide sequence complementary to the long single-stranded DNA, wherein the length of the nucleotide sequence complementary to the long single-stranded DNA is shorter than that of the long single-stranded DNA; The long single-stranded DNA and the oligonucleotide form a hybrid structure through complementary base pairing.

2. The hybrid DNA vector according to claim 1, characterized in that The long single-stranded DNA is circular single-stranded DNA or linear single-stranded DNA.

3. The hybrid DNA vector according to claim 1, characterized in that There are more than one oligonucleotide, and the long single-stranded DNA forms more than one hybridization structure with the oligonucleotide through complementary base pairing, and the hybridization structure is spaced 0-80 nucleotides apart on the long single-stranded DNA.

4. The hybrid DNA vector according to claim 1, characterized in that There are more than one oligonucleotide, and the long single-stranded DNA and the oligonucleotide form more than one hybridization structure through base complementary pairing, and the total length of the more than one hybridization structure accounts for 1% to 100% of the length of the long single-stranded DNA.

5. The hybrid DNA vector according to claim 1, characterized in that There are more than one oligonucleotide, and the long single-stranded DNA forms more than one hybridization structure with the oligonucleotide through complementary base pairing. There are 0 nucleotides between the hybridization structures, and there are gap structures between the hybridization structures.

6. The hybrid DNA vector according to claim 1, wherein The length of the long single-stranded DNA is 0.2 kb to 200 kb, the length of the oligonucleotide is 1 to 200 nucleotides; and the number of the oligonucleotides is 10 to 100.

7. The hybrid DNA vector according to claim 1, characterized in that The oligonucleotide is linked to a functional ligand, and the functional ligand is selected from at least one of a short peptide, a protein binding domain, a small molecule ligand, a nucleic acid aptamer and a nanoparticle.

8. The hybrid DNA vector according to claim 7, characterized in that The functional ligand is a Cas9 recognition sequence, which is connected to the 5' end of the oligonucleotide; And / or the oligonucleotide is covalently coupled to gold nanoparticles.

9. The hybrid DNA vector according to claim 7, characterized in that The hybrid DNA vector further comprises a 5' homology arm and a 3' homology arm.

10. Use of the hybrid DNA vector according to any one of claims 1 to 9 in gene editing.

11. The use according to claim 10, characterized in that The hybrid DNA vector is used as a donor template and is integrated into the target gene site.

12. The use according to claim 11, characterized in that The donor template comprises a 5' homology arm, a 3' homology arm and a DNA insert located therebetween; The DNA insert encodes a protein, RNA, DNA regulatory element, or a sequence encoding a variant of a protein, RNA, or DNA regulatory element; The protein is a chimeric antigen receptor or a T cell receptor.

13. The use according to claim 12, characterized in that The hybrid DNA vector is used to construct immune cells carrying chimeric antigen receptors.

14. The use according to claim 13, characterized in that The constructed immune cells carrying chimeric antigen receptors are used to prepare drugs for cell immunotherapy; The immune cells are CAR-T or CAR-NK.

15. Use of the hybrid DNA vector according to any one of claims 1 to 9 in heterologous gene expression.

16. The use according to claim 15, characterized in that During application, the hybrid DNA vector is introduced into cells or living organisms, and at least one heterologous coding sequence is expressed under the drive of the regulatory elements of the expression vector.

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