Polynucleotide constructs encoding DNA polymerases and pores

Abstract

The present invention relates to techniques for replicating and transferring polynucleotides within eukaryotic cells, including a polynucleotide sequence encoding a DNA secretion pore, and a gene encoding a protein required for DNA replication. The polynucleotide may also include a polynucleotide sequence that provides a desired function, such as a therapeutic effect, or a polynucleotide sequence that can supplement or directly replace a mutant gene in a eukaryotic cell. Methods of treatment comprising administering the polynucleotides are also disclosed.

Description

Technical Field

[0001] The present invention relates to techniques for replicating polynucleotides within eukaryotic cells and transferring the polynucleotides between eukaryotic cells. The polynucleotides can be used to deliver polynucleotide sequences that provide desired functions such as therapeutic effects, or polynucleotide sequences that are capable of complementing or directly replacing mutant genes in eukaryotic cells. Therapeutic methods including administering the polynucleotides are also disclosed. Background Art

[0002] Many therapeutic methods require the transfer of nucleic acid molecules into humans or other animals for subsequent expression of the genes encoded by the nucleic acid molecules. These therapeutic methods include gene therapy, cancer therapy, and DNA vaccination. In gene therapy, a functional copy of a gene mutated in the host chromosome is delivered by chromosomal integration or gene editing to correct a defective phenotype temporarily or permanently (Anguela & High, 2019). Cancer is typically caused by mutations in genes responsible for DNA repair or regulating cell division, and these genes can be introduced to prophylactically correct mutations representative of cancer predisposition or to kill tumors (Anguela & High, 2019). DNA vaccination relies on the expression of genes encoding immunogenic proteins from pathogens in the host's antigen-presenting cells (Hobernik & Bros, 2018).

[0003] Nucleic acids can be formulated in solutions, encapsulated in liposomes, adhered to microbeads or other carriers, or packaged into viruses (Ates et al., 2020). For gene and cancer therapy, the transfected DNA ideally needs to reach most cells in the target organ to produce a beneficial effect. However, the proportion of transfected cells using existing techniques is very low, which greatly limits the development of gene therapy and cancer gene therapy. The fundamental problem is that transfected DNA is restricted to the cells in which it was initially transfected. Attempting to transfect more cells requires high doses of DNA or virus, which makes production expensive and there are tolerance problems in the host.

[0004] Immunotherapy most commonly involves ex vivo modification of an individual's T cells to target tumor antigens (CAR-T therapy). T cells are extracted, genetically modified to express T cell receptors, and then reintroduced into the patient (Miliotou & Papadopoulou, 2018). This patient-specific approach is costly and time-consuming. T cells are produced from progenitor cells in the bone marrow and mature in the thymus. Modifying T cells at the source would be simpler and more economical, but existing techniques are unable to modify a sufficient number of progenitor cells.

[0005] Direct treatment of pathogens involves delivering antimicrobial agents, including antibiotics, antifungal, and antiviral compounds. Gene editing technologies such as CRISPR-Cas9 can also be used to target viruses and eliminate viral genomes from infected individuals (Doudna & Charpentier, 2014). However, there is currently no technology that enables DNA-encoded antimicrobial agents to spread through infected organs and target invading pathogens.

[0006] Recombinant protein expression involves inserting DNA containing a target gene regulated by a promoter and a polyadenylation signal sequence into cell cultures from in vitro multicellular eukaryotes. The generation and selection of high-expression clones is a long process, so transient transfection can be used to achieve transient gene expression in a shorter time (Bandaranayake & Almo, 2014). Ensuring that as many cells as possible take up the DNA maximizes the production of recombinant proteins, but currently high concentrations of DNA are used to achieve this.

[0007] All of the above areas are limited because recombinant DNA molecules cannot reach most cells in target tissues, organs, or cell cultures. Therefore, a technology capable of achieving intercellular DNA transfer needs to be developed. Summary of the Invention

[0008] The inventors have developed a technology for intercellular transfer of polynucleotides such as DNA. The inventors unexpectedly found that the combination of a gene expressing a DNA secretion pore and a gene providing replication function enables polynucleotides to diffuse between eukaryotic cells, thus making it possible to proliferate polynucleotides such as DNA to most target cells in a tissue or organ. Applications of the present invention include gene therapy, cancer therapy, DNA vaccination, immunotherapy, antibacterial therapy, and in vitro recombinant protein production.

[0009] Accordingly, in a first aspect of the present invention, there is provided:

[0010] - A polynucleotide comprising:

[0011] a) A polynucleotide sequence encoding a DNA-dependent DNA polymerase; and

[0012] b) A polynucleotide sequence encoding a pore that enables eukaryotic cells to secrete DNA.

[0013] In another aspect of the present invention, there is provided:

[0014] - A polynucleotide comprising:

[0015] a) An origin of replication;

[0016] b) A polynucleotide sequence encoding a DNA-dependent DNA polymerase;

[0017] c) Encoding the following polynucleotide sequences:

[0018] i) Protelomerase; or

[0019] ii) Terminal proteins and DNA-binding proteins required for plasmid replication in eukaryotic cells; and

[0020] d) A polynucleotide sequence encoding a pore that enables eukaryotic cells to secrete DNA.

[0021] In another aspect of the present invention, there is provided:

[0022] - Circular or linear plasmids containing the polynucleotides defined herein.

[0023] In another aspect of the present invention, there is provided:

[0024] - A pharmaceutical composition comprising the polynucleotides defined herein, or the plasmids defined herein, and a pharmaceutically acceptable excipient.

[0025] In another aspect of the present invention, there is provided:

[0026] - A method of treatment comprising administering to an individual in need thereof the polynucleotides defined herein, the plasmids defined herein, or the pharmaceutical composition defined herein.

[0027] Brief Description of the Sequence Listing

[0028] SEQ ID NO: 1 - N15 repA amino acid sequence

[0029] SEQ ID NO: 2 - N15 repA cistron

[0030] SEQ ID NO: 3 - N15 telN amino acid sequence

[0031] SEQ ID NO: 4 - N15 telN cistron

[0032] SEQ ID NO: 5 - N15 telRL

[0033] SEQ ID NO: 6 - N15 sopA amino acid sequence

[0034] SEQ ID NO: 7 - N15 sopA cistron

[0035] SEQ ID NO: 8 - N15 sopB amino acid sequence

[0036] SEQ ID NO: 9 - N15 sopB cistron

[0037] SEQ ID NO: 10 - Amino Acid Sequence of Phi29 DNA-Dependent DNA Polymerase (Gene 2)

[0038] SEQ ID NO: 11 - Phi29 DNA-Dependent DNA Polymerase Cistron (Gene 2)

[0039] SEQ ID NO: 12 - Amino Acid Sequence of Phi29 Terminal Protein (Gene 3)

[0040] SEQ ID NO: 13 - Phi29 Terminal Protein Cistron (Gene 3)

[0041] SEQ ID NO: 14 - Amino Acid Sequence of Phi29 Single-Stranded DNA-Binding Protein (Gene 5)

[0042] SEQ ID NO: 15 - Phi29 Single-Stranded DNA-Binding Protein Cistron (Gene 5)

[0043] SEQ ID NO: 16 - Amino Acid Sequence of Phi29 Double-Stranded DNA-Binding Protein (Gene 6)

[0044] SEQ ID NO: 17 - Phi29 Double-Stranded DNA-Binding Protein Cistron (Gene 6)

[0045] SEQ ID NO: 18 - Amino Acid Sequence of Adenovirus 5 DNA-Dependent DNA Polymerase

[0046] SEQ ID NO: 19 - Adenovirus 5 DNA-Dependent DNA Polymerase Cistron

[0047] SEQ ID NO: 20 - Amino Acid Sequence of Adenovirus 5 Precursor Terminal Protein (pTP)

[0048] SEQ ID NO: 21 - Adenovirus 5 Precursor Terminal Protein (pTP) Cistron

[0049] SEQ ID NO: 22 - Amino Acid Sequence of Adenovirus 5 DNA-Binding Protein (DBP)

[0050] SEQ ID NO: 23 - Adenovirus 5 DNA-Binding Protein (DBP) Cistron

[0051] SEQ ID NO: 24 - Amino Acid Sequence of TraB of Streptomyces venezuelae pSVH1

[0052] SEQ ID NO: 25 - Streptomyces venezuelae pSVH1 traB Cistron

[0053] SEQ ID NO: 26 - Streptomyces venezuelae pSVH1 clt Locus

[0054] SEQ ID NO: 27 - Streptomyces venezuelae pSVH1 clt Repeat

[0055] SEQ ID NO: 28 - Amino Acid Sequence of TdtA from Thermus thermophilus

[0056] SEQ ID NO: 29 - Thermus thermophilus tdtA Cistron

[0057] SEQ ID NO: 30 - 2A “Ribosome Skipping” Peptide Consensus Sequence

[0058] SEQ ID NO: 31 - E2A “Ribosome Skipping” Peptide Sequence

[0059] SEQ ID NO: 32 - P2A “Ribosome Skipping” Peptide Sequence

[0060] SEQ ID NO: 33 - T2A “Ribosome Skipping” Peptide Sequence

[0061] SEQ ID NO: 34 - pBITREP Nucleotide Sequence

[0062] SEQ ID NO: 35 – pBITREPA2 Nucleotide Sequence

[0063] SEQ ID NO: 36 – pBITREPB2 Nucleotide Sequence

[0064] SEQ ID NO: 37 - Nucleotide Sequence

[0065] SEQ ID NO: 38 - Nucleotide Sequence

[0066] SEQ ID NO: 39 - Nucleotide Sequence

[0067] SEQ ID NO: 40 - Nucleotide Sequence

[0068] SEQ ID NO: 41 - Nucleotide Sequence

[0069] SEQ ID NO: 42 - Nucleotide Sequence

[0070] SEQ ID NO: 43 - Nucleotide Sequence

[0071] SEQ ID NO: 44 - Nucleotide Sequence

[0072] SEQ ID NO: 45 - pLUCK Nucleotide Sequence

[0073] SEQ ID NO: 46 - Nucleotide sequence of pLUCKREP

[0074] SEQ ID NO: 47 - Nucleotide sequence of pLUCKB

[0075] SEQ ID NO: 48 - Nucleotide sequence of pLUCKCB

[0076] SEQ ID NO: 49 - Nucleotide sequence of pLUCKOB

[0077] SEQ ID NO: 50 - Nucleotide sequence of pLUCKTB

[0078] SEQ ID NO: 51 - Nucleotide sequence of pLUCKRB

[0079] SEQ ID NO: 52 – NoTelNRepA primer

[0080] SEQ ID NO: 53 - NoTelNR primer BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 - A) and B) show the inter - cellular DNA transport mechanism of the present invention, including the phage N15 DNA replication function and the DNA secretion pore.

[0082] Figure 2 - shows A) the processing of the telRL site on circular DNA by the N15 front - telomerase TelN to produce covalently - closed hairpin ends (thus resulting in linear DNA), and B) the mechanism of DNA replication by phage N15 repA and TelN (adapted from Ravin, 2014).

[0083] Figure 3 - shows the inter - cellular DNA transport mechanism of the present invention, including the Phi29 or AdV DNA replication function and the DNA secretion pore (TP: terminal protein; DBP: DNA - binding protein).

[0084] Figure 4 - shows the mechanism of linear DNA replication using the Phi29 - based terminal protein. The Phi29 polymerase uses the TP (covalently linked to each 5' end) to initiate the synthesis of each linear DNA strand. When the DNA polymerases meet, the strands separate, and replication continues to produce two linear DNA molecules (adapted from Choi et al., 2016).

[0085] Figure 5- Shows the plasmids used in the experiments to study membrane binding and DNA secretion through the TdtA and TraB pores: A) pBITTdtA and B) pBITTraB2. Experiments were performed to show that the plasmids transfected into HEK 293 cells expressed FLAG-tagged porins. Cells were stained with wheat germ agglutinin Alexa Fluor 647 conjugate (membrane), DAPI (DNA), and anti-FLAG antibody to detect the porins, i.e., C) TdtA expressed from pBITTdtA, and D) TraB expressed from pBITTraB2.

[0086] Figure 6 – Shows the experimental results where HEK 293 cells were transfected with two plasmids expressing mCherry: the negative control pMCPK and pBITTdtA expressing TdtA, and subsequently transfected with pdClover2-N1 expressing Clover2 (a green fluorescent protein). Recorded were: A) the total percentage of cells expressing Clover2 and also expressing mCherry, and; B) the total percentage of cells expressing Clover2 and adjacent to cells expressing both fluorescent reporter proteins.

[0087] Figure 7 – Shows the experimental results of transfecting HEK 293 cells with the TraB pore-expressing plasmid pBITTraB2, and subsequently transfecting HEK 293 cells with pdClover2-N1 and the plasmid pCMV-Clover2-CLT containing the clt locus. Recorded were: A) the total percentage of cells expressing Clover2 and also expressing mCherry, and; B) the total percentage of cells expressing Clover2 and adjacent to cells expressing both fluorescent reporter proteins.

[0088] Figure 8 - Shows the plasmids expressing the Gentrafix system components: A) pBITREPA2 expressing telN, repA, and tdtA; B) pBITREPB2 expressing telN, repA, and traB.

[0089] Figure 9 - Shows the Western blots demonstrating the expression of the Gentrafix component proteins TelN, RepA, TdtA, and TraB in the human cell line HEK293.

[0090] Figure 10 - Shows the other plasmids used in the experiments to provide evidence of intercellular DNA secretion: A) pBITREP, and B) pMCPK.

[0091] Figure 11-Shows the plasmids used in the experiments to study membrane binding and DNA secretion through the TdtA and TraB pores: A) pdClover2-N1, and B) pCMV-Clover2-CLT.

[0092] Figure 12 -Shows the experimental results of transfecting HEK 293 cells with plasmids pMCPK, pBITREP or pBITREPA2. Positive (plasmid-containing) cells are red (because the two plasmids also express mCherry), and clusters are defined as groups of three or more adjacent red cells: A) The number of cells expressing mCherry in each image, B) The number of clusters of cells expressing mCherry in each image, C) The number of cells expressing mCherry in the clusters in each image, D) The number of cells expressing mCherry that form clusters, and E) Representative images of the red cells indicated by the arrows.

[0093] Figure 13 -Shows the experimental results of transfecting HEK 293 cells with pBITREP, pBITREPA2 and pBITREPB2, and then adding cells of the second cell line, HEK 293GFP, to the culture. Red cells are indicated by white arrows, and cells that are both red and green are indicated by dashed arrows.

[0094] Figure 14 -Shows the experimental results of transfecting MDCK-GFP cells with pMCPK and pBITREPB2, and then adding cells of the second cell line, MDCK, to the culture.

[0095] Figure 15 -Shows the plasmids containing the firefly luciferase gene.

[0096] Figure 16 -Shows the experimental results of transfecting HEK 293 cells with the plasmids containing the firefly luciferase gene. Detailed Description

[0097] General Definitions

[0098] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0099] It should be understood that the different applications of the disclosed invention can be adjusted according to the specific needs of the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the invention only and are not intended to be limiting.

[0100] When referring to a value, the term "about" or "approximately" means that value, but within reasonable scientific error. Alternatively, if the value is within 10%, 5%, or 1% of x, then the value is "about x" or "approximately x".

[0101] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, "a pore" includes "a plurality of pores" and the like.

[0102] In general, the term "comprising" is intended to mean including but not limited to. For example, the phrase "a polynucleotide comprising a polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA, an origin of replication, a polynucleotide sequence encoding a DNA-dependent DNA polymerase, and a polynucleotide sequence encoding a telomerase, or a terminal protein and a DNA-binding protein required for plasmid replication in a eukaryotic cell" should be interpreted to mean that the polynucleotide includes at least one polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA, an origin of replication, a polynucleotide sequence encoding a DNA-dependent DNA polymerase, and a polynucleotide sequence encoding a telomerase, or a terminal protein and a DNA-binding protein required for plasmid replication in a eukaryotic cell, but the polynucleotide may also contain other polynucleotide sequences.

[0103] In some embodiments of the present invention, the word "comprising" is replaced by the phrase "consisting of". The term "consisting of" is intended to be limiting. For example, the phrase "a polynucleotide consisting of: a polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA, an origin of replication, a polynucleotide sequence encoding a DNA-dependent DNA polymerase, and a polynucleotide sequence encoding a telomerase, or a terminal protein and a DNA-binding protein required for plasmid replication in a eukaryotic cell" should be interpreted to mean that the polynucleotide contains at least one polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA, an origin of replication, a polynucleotide sequence encoding a DNA-dependent DNA polymerase, and a polynucleotide sequence encoding a telomerase, or a terminal protein and a DNA-binding protein required for plasmid replication in a eukaryotic cell, and does not contain other polynucleotide sequences.

[0104] In some embodiments of the present invention, the term "comprising" is replaced by the phrase "consisting essentially of". The term "consisting essentially of" means that certain other components may be present, namely those that do not materially affect the basic characteristics of the subject matter. For example, the phrase "a polynucleotide consisting essentially of: a polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA, an origin of replication, a polynucleotide sequence encoding a DNA-dependent DNA polymerase, and a polynucleotide sequence encoding a telomerase front end, or a terminal protein and a DNA-binding protein required for plasmid replication in a eukaryotic cell" may also include polynucleotide sequences such as linker sequences between the claimed polynucleotide sequences.

[0105] The terms "nucleic acid molecule", "nucleic acid sequence", "polynucleotide", and "nucleotide sequence" are used interchangeably herein and refer to a polymeric chain of nucleotides of any length, such as deoxyribonucleotides, ribonucleotides, or analogs thereof. For example, a polynucleotide may include DNA (deoxyribonucleotides) or RNA (ribonucleotides). A polynucleotide may consist of DNA. A polynucleotide may be mRNA. Since polynucleotides may contain RNA or DNA, all references to T (thymine) nucleotides may be replaced by U (uracil).

[0106] It is standard in the art for nucleotide sequences to be written from 5' to 3', i.e., the first nucleotide in any given sequence may be considered to be at the 5' end, and the last nucleotide may be considered to be at the 3' end of any given nucleotide. Thus, in a nucleotide sequence, a sequence element located 5' of a second sequence element is located before the second sequence element. In a nucleotide sequence, a first sequence element located 5' of a second sequence element may be immediately before the second sequence element. Alternatively, in a nucleotide sequence, a first sequence element located 5' of a second sequence element may not be immediately before the second sequence element, i.e., the nucleotide sequence may include an insertion sequence between the first sequence element and the second sequence element. Similarly, if the length of the insertion sequence is less than 10 nucleotides, the first sequence element is less than 10 nucleotides 5' from the second sequence element.

[0107] In a nucleotide sequence, a first sequence element located 3' of a second sequence element is located after the second sequence element. In a nucleotide sequence, a first sequence element located 3' of a second sequence element may be immediately after the second sequence element, i.e., there are no inserted nucleotides between the two sequence elements. Alternatively, in a nucleotide sequence, a first sequence element located 3' of a second sequence element may not be immediately after the second sequence element, i.e., the nucleotide sequence may include an insertion sequence between the first sequence element and the second sequence element. Similarly, if the length of the insertion sequence is less than 10 nucleotides, the first sequence element is less than 10 nucleotides 3' from the second sequence element.

[0108] For the purposes of the present invention, to determine the percent identity between two sequences (e.g., two polynucleotides or two polynucleotide sequences), the sequences are aligned for optimal alignment purposes (e.g., gaps can be introduced in the first sequence for optimal alignment with the second sequence). The nucleotides at each position are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the nucleotides at that position are identical. The percent identity between the two sequences is a function of the number of positions having identical nucleotides (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0109] Typically, sequence comparisons are performed over the entire length of the reference sequence. For example, if a user wishes to determine whether a given ("test") sequence is 95% identical to SEQ ID NO:1, then SEQ ID NO:1 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO:1 (an example of a reference sequence), one of ordinary skill in the art would align the sequences based on the length of SEQ ID NO:1 and determine how many positions in the test sequence are identical to positions in SEQ ID NO:1. If at least 95% of the positions are identical, then the test sequence is at least 95% identical to SEQ ID NO:1. If the test sequence is shorter than SEQ ID NO:1, gaps or missing positions should be considered non-identical positions.

[0110] Those skilled in the art know of various computer programs that can be used to align two sequences. For example, mathematical algorithms can be used to perform the alignment between two sequences. In one embodiment, the Needleman and Wunsch (1970) algorithm from the National Center for Biotechnology Information, U.S.A., or the BLAST 2 (Basic Local Alignment Search Tool) algorithm is used to align two nucleic acid sequences.

[0111] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0112] Polynucleotide Replication of Bacteriophage N15 and Equivalent Components

[0113] The inventors have developed a technique for transferring polynucleotides such as DNA between cells. In a preferred embodiment of the present invention, the polynucleotide is DNA. In a preferred embodiment of the present invention, the polynucleotide is in the form of a plasmid. In a preferred embodiment of the present invention, the polynucleotide is in the form of a circular plasmid or a linear plasmid.

[0114] The inventors unexpectedly discovered that the combination of a polynucleotide sequence encoding a DNA secretion pore and a polynucleotide sequence providing plasmid replication function enables the diffusion of polynucleotides between eukaryotic cells, thus achieving a technique for the polynucleotides to reach most target cells of a tissue or organ (Figure 1). The present invention aims to exert a prophylactic or therapeutic function in target cells by expressing the protein and RNA encoded by the polynucleotide.

[0115] The polynucleotide of the present invention comprises a plurality of polynucleotide sequences. These polynucleotide sequences can be designated as sequence elements or components. These components at least include:

[0116] - A polynucleotide sequence encoding a DNA-dependent DNA polymerase; and

[0117] - A polynucleotide sequence encoding a pore that enables eukaryotic cells to secrete DNA.

[0118] In a preferred embodiment, these components at least include:

[0119] - An origin of replication;

[0120] - A polynucleotide sequence encoding a DNA-dependent DNA polymerase;

[0121] - A polynucleotide sequence encoding:

[0122] i) Telomerase front; or

[0123] ii) The terminal protein and DNA binding protein required for plasmid replication in eukaryotic cells; and

[0124] - A polynucleotide sequence encoding a pore that enables eukaryotic cells to secrete DNA.

[0125] Each component can be positioned in any order along the polynucleotide of the present invention in the 5' to 3' direction. Other components may also be present at the 5' of the above-listed components, at the 3' of the above-listed components, or at the 5' of some of the above-listed components but at the 3' of other of the above-listed components.

[0126] The components can be positioned in the following 5' to 3' direction: DNA-dependent DNA polymerase; a pore enabling a eukaryotic cell to secrete DNA. The components can be positioned in the following 5' to 3' direction: a pore enabling a eukaryotic cell to secrete DNA; DNA-dependent DNA polymerase. The components can be positioned in the following 5' to 3' direction: telomerase or terminal protein and DNA-binding protein; DNA-dependent DNA polymerase; a pore enabling a eukaryotic cell to secrete DNA. The components can be positioned in the following 5' to 3' direction: DNA-dependent DNA polymerase; telomerase or terminal protein and DNA-binding protein; a pore enabling a eukaryotic cell to secrete DNA. The components can be positioned in the following 5' to 3' direction: telomerase or terminal protein and DNA-binding protein; a pore enabling a eukaryotic cell to secrete DNA; DNA-dependent DNA polymerase. The components can be positioned in the following 5' to 3' direction: DNA-dependent DNA polymerase; a pore enabling a eukaryotic cell to secrete DNA; telomerase or terminal protein and DNA-binding protein. The components can be positioned in the following 5' to 3' direction: a pore enabling a eukaryotic cell to secrete DNA; telomerase or terminal protein and DNA-binding protein; DNA-dependent DNA polymerase. The components can be positioned in the following 5' to 3' direction: a pore enabling a eukaryotic cell to secrete DNA; DNA-dependent DNA polymerase; telomerase or terminal protein and DNA-binding protein.

[0127] The components can be positioned in the following 5' to 3' direction: origin of replication; telomerase or terminal protein and DNA-binding protein; DNA-dependent DNA polymerase; a pore enabling a eukaryotic cell to secrete DNA. The components can be positioned in the following 5' to 3' direction: telomerase or terminal protein and DNA-binding protein; origin of replication; DNA-dependent DNA polymerase; a pore enabling a eukaryotic cell to secrete DNA. The components can be positioned in the following 5' to 3' direction: telomerase or terminal protein and DNA-binding protein; DNA-dependent DNA polymerase; origin of replication; a pore enabling a eukaryotic cell to secrete DNA.

[0128] The components can be positioned in the following 5' to 3' direction: RepA; TdtA.

[0129] The components can be positioned in the following 5' to 3' direction: RepA; TraB.

[0130] The components can be positioned in the following 5' to 3' direction: TelN; RepA; TdtA. The components can be positioned in the following 5' to 3' direction: TelN; RepA; TraB.

[0131] The polynucleotide of the present invention can be referred to herein as the Gentrafix system, Gentrafix cassette or the like.

[0132] The first aspect of the present invention relates to the mechanism for replicating polynucleotides within eukaryotic cells.

[0133] The polynucleotides of the present invention present in eukaryotic cells can be in the form of linear plasmids or circular plasmids. Replication of the polynucleotides of the present invention is achieved by a DNA-dependent DNA polymerase that binds, when required, to one or more other proteins necessary for replication. These components can have a phage, bacterial, archaeal, viral, or eukaryotic origin.

[0134] The enzyme for plasmid replication is preferably a DNA polymerase RepA from a phage, including PY54 of Yersinia enterocolitica, φKO2 of Klebsiella oxytoca, and Escherichia coli phage N15.

[0135] The repA gene sequence of Escherichia coli phage N15 contains an origin of replication (ori) where replication is initiated. N15RepA alone is sufficient to replicate circular DNA in a bidirectional theta mode as it has primase, helicase, and origin-binding activities (Ravin, 2014). Thus, plasmids containing only RepA will replicate as covalently closed circular double-stranded DNA molecules.

[0136] Thus, in the polynucleotides of the present invention, the polynucleotide contains a polynucleotide sequence encoding a DNA-dependent DNA polymerase. In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase is encoded by the repA gene.

[0137] In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase expressed by the polynucleotide sequence comprises:

[0138] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a continuous fragment of at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 amino acids of SEQ ID NO: 1; or

[0139] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1; or

[0140] (iii) SEQ ID NO: 1.

[0141] In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase polynucleotide sequence comprises:

[0142] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 2000, at least 2500, at least 3000, at least 3500, at least 3700, at least 3800 or at least 3900 nucleotides of SEQ ID NO: 2; or

[0143] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2; or

[0144] (iii) SEQ ID NO: 2.

[0145] The said sequence variant retains the ability to act as a DNA-dependent DNA polymerase upon expression. Suitable assays for determining DNA-dependent DNA polymerase activity are known to those skilled in the art, including quantitative PCR assays using primers and probes that bind to the DNA sequence replicated by the DNA polymerase.

[0146] In one embodiment of the present invention, the polynucleotide of the present invention is a linear double-stranded DNA molecule with covalently closed ends that form a so-called "hairpin loop" and prevent the DNA ends from being exposed to exonucleases, thereby increasing its stability. In this embodiment, the polynucleotide will further include a polynucleotide sequence containing telN from a phage (including phage N15), which encodes a single copy of the front telomerase and its target site telRL, and telRL is a 56bp inverted repeat sequence.

[0147] In a preferred embodiment of the present invention, the polynucleotide of the present invention includes a polynucleotide sequence encoding the front telomerase TelN from phage N15.

[0148] In a preferred embodiment of the present invention, the front telomerase expressed by this polynucleotide sequence includes:

[0149] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 300, at least 400, at least 450, at least 500, at least 550 or at least 600 amino acids of SEQ ID NO: 3; or

[0150] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 3; or

[0151] (iii) SEQ ID NO: 3.

[0152] In a preferred embodiment of the present invention, the TelN polynucleotide sequence includes:

[0153] (i) A polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 1000, at least 1500, at least 1600, at least 1700 or at least 1800 nucleotides of SEQ ID NO: 4; or

[0154] (ii) A polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 4; or

[0155] (iii) SEQ ID NO: 4.

[0156] The sequence variant retains the ability to act as a telomerase when expressed. Suitable assays for determining telomerase activity are known to those skilled in the art and include agarose gel electrophoresis of plasmid DNA containing the TelRL sequence, since treatment with TelN generates a linear form that migrates at its correct size compared to circular plasmid DNA, which can be determined using a "size marker" containing DNA fragments of known length. In addition, restriction endonuclease digestion using an enzyme that cuts once on the plasmid will convert the circular plasmid into a single linear form, while the linear plasmid will be cut into two fragments.

[0157] In a preferred embodiment of the present invention, the polynucleotide of the present invention contains a telRL locus. In one embodiment, the telRL locus polynucleotide sequence contains SEQ ID NO: 5, or a variant of SEQ ID NO: 5 that differs by 1, 2, 3, 4 or 5 nucleotides.

[0158] The sequence variant retains the ability to act as a telRL locus. Suitable assays for determining the function of the telRL locus are known to those skilled in the art and include agarose gel electrophoresis of plasmid DNA containing the TelRL sequence, since treatment with TelN generates a linear form that migrates at its correct size compared to circular plasmid DNA, which can be determined using a "size marker" containing DNA fragments of known length. In addition, restriction endonuclease digestion using an enzyme that cuts once on the plasmid will convert the circular plasmid into a single linear form, while the linear plasmid will be cut into two fragments.

[0159] The replication mechanism of linear plasmids is as follows. The front telomerase TelN cuts a single telRL site on the plasmid in a staggered cleavage manner, generating a 6bp extension that folds back and is ligated to the complementary DNA strand via a phosphodiester bond to form a terminal hairpin loop on the linear double-stranded DNA molecule. These loops are called telL and telR. This linear plasmid is replicated by RepA. When telL is converted from single-stranded to double-stranded in the replication bubble, the resulting site telLL is cut by TelN and self-annealed to generate a Y-shaped molecule or a circular plasmid dimer replication intermediate. When telR is converted from single-stranded to double-stranded in the replication bubble, the resulting site telRR is also cut and self-annealed by TelN to generate a linear plasmid with opposite telL and telR hairpin ends (Ravin, 2014). Then, the newly generated linear plasmid is replicated by RepA and processed by TelN to create further copies. The plasmid replication mechanism of phage N15 repA and TelN is as Figure 2 shown.

[0160] As is well known in the art, therapeutic plasmid DNA can be produced and purified in prokaryotic cells (such as Escherichia coli) and then introduced into eukaryotic (such as Homo sapiens) target cells. In one embodiment of the present invention, the repA and telN polynucleotide sequences are expressed by a standard promoter, a hybrid promoter, or a dual promoter capable of transcribing in both prokaryotic and eukaryotic cells.

[0161] In another embodiment of the present invention, the repA and telN polynucleotide sequences are expressed by a promoter that functions only in eukaryotic cells, and additional repA and telN polynucleotide sequences are trans-expressed by a promoter that functions in prokaryotic cells. Additionally, the genes sopA and sopB from phage N15 can be present in trans to stabilize the linear form of the polynucleotides of the present invention in prokaryotic cells. When the polynucleotide sequences containing one or more of repA, telN, sopA, and sopB are present in trans, they can be located on a second plasmid or integrated into the chromosome, and their expression is regulated by a prokaryotic promoter, or a promoter that can be constitutive or inducible.

[0162] In a preferred embodiment of the present invention, a host cell containing the polynucleotide of the present invention is provided. In a preferred embodiment, the host cell is an Escherichia coli cell. In a preferred embodiment, the Escherichia coli cell expresses the genes telN and repA from phage N15. In a preferred embodiment, the Escherichia coli cell expresses the genes sopA and sopB from phage N15. In a preferred embodiment, the Escherichia coli cell includes a polynucleotide containing a polynucleotide sequence encoding sopA and sopB.

[0163] In a preferred embodiment of the present invention, the SopA expressed by the polynucleotide sequence comprises:

[0164] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 200, at least 250, at least 275, at least 300, at least 325, at least 350 or at least 375 amino acids of SEQ ID NO: 6; or

[0165] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6; or

[0166] (iii) SEQ ID NO: 6.

[0167] In a preferred embodiment of the present invention, the sopA polynucleotide sequence comprises:

[0168] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 or at least 1100 nucleotides of SEQ ID NO: 7; or

[0169] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7; or

[0170] (iii) SEQ ID NO: 7.

[0171] The sequence variants retain the ability to act as SopA protein when expressed. Suitable assays for determining SopA protein activity are known to those skilled in the art and include culturing Escherichia coli strains that replicate plasmids for multiple generations by inoculating at low density into nutrient broth flasks, growing to high density and repeating for several days. By comparing plasmid preparations from the strains daily by agarose gel electrophoresis or quantitative PCR, any plasmid loss can be detected.

[0172] In a preferred embodiment of the present invention, the SopB expressed by the polynucleotide sequence comprises:

[0173] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 200, at least 250, at least 275, at least 300, at least 325 or at least 340 amino acids of SEQ ID NO: 8; or

[0174] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8; or

[0175] (iii) SEQ ID NO: 8.

[0176] In a preferred embodiment of the present invention, the sopB polynucleotide sequence comprises;

[0177] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 nucleotides of SEQ ID NO: 9; or

[0178] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9; or

[0179] (iii) SEQ ID NO: 9.

[0180] The sequence variants retain the ability to act as SopB protein when expressed. Suitable assays for determining SopB protein activity are known to those skilled in the art and include culturing Escherichia coli strains that replicate plasmids for multiple generations by inoculating at low density into nutrient broth flasks, growing to high density and repeating for several days. By comparing plasmid preparations from the strains daily by agarose gel electrophoresis or quantitative PCR, any plasmid loss can be detected.

[0181] Alternative methods for producing the polynucleotides of the present invention include cell-free systems, whereby linear DNA with TelR and TelL hairpin ends is replicated in vitro using TelN and Phi29 polymerase.

[0182] Linear DNA replication involving terminal proteins

[0183] In one embodiment of the present invention, the polynucleotide of the present invention is a linear double-stranded DNA molecule, each end of which is protected by covalent linkage to a terminal protein (TP) at the 5'-phosphate, and the inverted repeat sequence acts as an origin of replication at the DNA ends. These systems also require a DNA polymerase for DNA replication initiated by TP, and one or more DNA-binding proteins necessary for DNA replication. In bacteria, plasmids are circular and replication begins at the bacterial origin of replication, which may be N15 repA, pMB1, ColEI, p15A or pSC101 in Escherichia coli. The circular plasmid is linearized using a restriction endonuclease to generate a linear plasmid with terminal inverted repeat sequences for transfection into target eukaryotic cells. The linear plasmid can also be ligated to TP in vitro before transfection.

[0184] In other embodiments of the present invention, the linear replication system included as part of the polynucleotide of the present invention is from Phi29 of the Bacillus subtilis phage group, including PZA, BS32, B103, Nf, M2Y and GA-1 (Meijer et al., 2001). The replication mechanism includes a DNA-dependent DNA polymerase (gene 2), TP (gene 3), a single-stranded DNA-binding protein p5 (gene 5) and a double-stranded DNA-binding protein p6 (gene 6); the DNA-binding protein is essential for DNA amplification (Salas et al., 2016).

[0185] In a preferred embodiment of the present invention, the polynucleotide of the present invention includes a polynucleotide sequence encoding a DNA-dependent DNA polymerase from phage Phi29. In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase is encoded by gene 2.

[0186] In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase expressed by the polynucleotide sequence includes:

[0187] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 300, at least 350, at least 400, at least 450, at least 500 or at least 550 amino acids of SEQ ID NO: 10; or

[0188] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 10; or

[0189] (iii) SEQ ID NO: 10.

[0190] In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase polynucleotide sequence includes:

[0191] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600 or at least 1700 nucleotides of SEQ ID NO: 11; or

[0192] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11; or

[0193] (iii) SEQ ID NO: 11.

[0194] The sequence variant retains the ability to act as a DNA-dependent DNA polymerase upon expression. Suitable assays for determining DNA-dependent DNA polymerase activity are known to those skilled in the art and include expressing the DNA-dependent DNA polymerase in an Escherichia coli strain and a plasmid containing its corresponding origin of replication. An increase in the total DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis, or quantitative PCR indicates that the functional DNA-dependent DNA polymerase is replicating the plasmid.

[0195] In a preferred embodiment of the present invention, the polynucleotide of the present invention comprises a polynucleotide sequence encoding a terminal protein TP and DNA-binding proteins p5 and p6 from a Bacillus subtilis phage of the Phi29 group.

[0196] In a preferred embodiment of the present invention, the terminal protein expressed by the polynucleotide sequence comprises:

[0197] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 125, at least 150, at least 175, at least 200, at least 225, at least 250 or at least 260 amino acids of SEQ ID NO: 12; or

[0198] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12; or

[0199] (iii) SEQ ID NO: 12.

[0200] In a preferred embodiment of the present invention, the terminal protein polynucleotide sequence comprises;

[0201] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 400, at least 500, at least 600, at least 700, at least 750 or at least 800 nucleotides of SEQ ID NO: 13; or

[0202] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 13; or

[0203] (iii) SEQ ID NO: 13.

[0204] When expressed, the said sequence variant retains the ability to act as a terminal protein. Suitable assays for determining terminal protein activity are known to those skilled in the art, including the increase in the total linear DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis, or quantitative PCR, which indicates that the functional terminal protein contributes to plasmid replication. In addition, electrophoretic mobility shift assays can be used to detect the terminal protein that binds to DNA.

[0205] In a preferred embodiment of the present invention, the DNA-binding protein p5 expressed by the polynucleotide sequence comprises:

[0206] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 or at least 120 amino acids of SEQ ID NO: 14; or

[0207] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14; or

[0208] (iii) SEQ ID NO: 14.

[0209] In a preferred embodiment of the present invention, the polynucleotide sequence of the DNA-binding protein p5 comprises;

[0210] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 250, at least 300, at least 350, at least 360 or at least 370 nucleotides of SEQ ID NO: 15; or

[0211] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 15; or

[0212] (iii) SEQ ID NO: 15.

[0213] When expressed, the said sequence variant retains the ability to act as the DNA-binding protein p5. Suitable assays for determining the activity of the DNA-binding protein p5 are known to those skilled in the art, including the increase in the total linear DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis, or quantitative PCR, which indicates that the functional p5 contributes to plasmid replication. In addition, electrophoretic mobility shift assays can be used to detect the terminal protein that binds to DNA.

[0214] In a preferred embodiment of the present invention, the DNA-binding protein p6 expressed by the polynucleotide sequence comprises:

[0215] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 50, at least 60, at least 70, at least 80, at least 90, at least 95 or at least 100 amino acids of SEQ ID NO: 16; or

[0216] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 16; or

[0217] (iii) SEQ ID NO: 16.

[0218] In a preferred embodiment of the present invention, the polynucleotide sequence of the DNA-binding protein p6 comprises;

[0219] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 250, at least 260, at least 270, at least 280, at least 290, at least 300 or at least 310 nucleotides of SEQ ID NO: 17; or

[0220] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 17; or

[0221] (iii) SEQ ID NO: 17.

[0222] The said sequence variants retain the ability to act as the DNA-binding protein p6 upon expression. Suitable assays for determining the activity of the DNA-binding protein p6 are known to those skilled in the art and include an increase in the total linear DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis or quantitative PCR, which indicates that functional p6 contributes to plasmid replication. In addition, electrophoretic mobility shift assays can be used to detect the terminal protein that binds to DNA.

[0223] The first stage of DNA replication involves the formation of a heterodimer between TP and DNA polymerase, which recognizes and binds to the replication origin sequence located at both ends of the linear plasmid. DNA is unwound by the binding of p6 to the entire plasmid, and DNA replication is initiated by DNA polymerase, forming a phosphodiester bond between the hydroxyl group of TP Ser232 and dAMP. Initiation occurs at the second nucleotide of the template, which starts with a T repeat (TTT), so when the first dAMP is added to the new DNA strand, the TP-A complex slides back to the starting position to ensure no information is lost. DNA polymerase dissociates from TP after inserting the tenth nucleotide and continues DNA elongation through the single-stranded region of the replication bubble bound to p5. When the replication forks from both ends meet, the linear plasmid is divided into two, and replication of each plasmid terminates when DNA polymerase reaches the end of the template and dissociates (Salas et al., 2016). This results in two linear plasmids, each with TP attached to both ends, which can initiate subsequent rounds of replication.

[0224] In other embodiments of the present invention, the linear replication system included as part of the polynucleotides of the present invention is from an adenovirus (AdV) that infects vertebrate cells. Adenoviruses use a precursor terminal protein (pTP) to initiate DNA replication through its DNA polymerase AdV Pol (both are expressed by the same gene E2B); the plasmid also requires the AdV DNA binding protein (DBP) encoded by E2A.

[0225] In a preferred embodiment of the present invention, the polynucleotides of the present invention include a polynucleotide sequence encoding a DNA-dependent DNA polymerase from an adenovirus. In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase is encoded by the gene E2B.

[0226] In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase expressed by the polynucleotide sequence includes:

[0227] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 700, at least 800, at least 900, at least 950, at least 1000, at least 1050, at least 1100 or at least 1150 amino acids of SEQ ID NO: 18; or

[0228] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 18; or

[0229] (iii) SEQ ID NO: 18.

[0230] In a preferred embodiment of the present invention, the DNA-dependent DNA polymerase polynucleotide sequence comprises;

[0231] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 2600, at least 2700, at least 2800, at least 2900, at least 3000, at least 3100, at least 3200, at least 3300, at least 3400 or at least 3500 nucleotides of SEQ ID NO: 19; or

[0232] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 19; or

[0233] (iii) SEQ ID NO: 19.

[0234] The sequence variants retain the ability to act as a DNA-dependent DNA polymerase upon expression. Suitable assays for determining DNA-dependent DNA polymerase activity are known to those skilled in the art and include expressing the DNA-dependent DNA polymerase in an Escherichia coli strain and a plasmid containing its corresponding origin of replication. An increase in the total DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis or quantitative PCR indicates that the functional DNA-dependent DNA polymerase is replicating the plasmid.

[0235] In a preferred embodiment of the present invention, the polynucleotide of the present invention comprises a polynucleotide sequence encoding the terminal protein pTP and the DNA-binding protein E2A from an adenovirus.

[0236] In a preferred embodiment of the present invention, the terminal protein expressed by the polynucleotide sequence comprises:

[0237] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 350, at least 400, at least 450, at least 500, at least 550, at least 600 or at least 650 amino acids of SEQ ID NO: 20; or

[0238] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 20; or

[0239] (iii) SEQ ID NO: 20.

[0240] In a preferred embodiment of the present invention, the terminal protein polynucleotide sequence comprises;

[0241] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 1500, at least 1600, at least 1700, at least 1800, at least 1900 or at least 2000 nucleotides of SEQ ID NO: 21; or

[0242] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 21; or

[0243] (iii) SEQ ID NO: 21.

[0244] The said sequence variant retains the ability to act as a terminal protein upon expression. Suitable assays for determining terminal protein activity are known to those skilled in the art, including the increase in total linear DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis or quantitative PCR, which indicates that the functional terminal protein contributes to plasmid replication. In addition, electrophoretic mobility shift assays can be used to detect the terminal protein that binds to DNA.

[0245] In a preferred embodiment of the present invention, the DNA-binding protein expressed by the polynucleotide sequence comprises:

[0246] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 250, at least 300, at least 350, at least 400, at least 450, at least 500 or at least 520 amino acids of SEQ ID NO: 22; or

[0247] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 22; or

[0248] (iii) SEQ ID NO: 22.

[0249] In a preferred embodiment of the present invention, the DNA-binding protein polynucleotide sequence comprises;

[0250] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 1000, at least 1100, at least 1200, at least 1300, at least 1400 or at least 1500 nucleotides of SEQ ID NO: 23; or

[0251] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 23; or

[0252] (iii) SEQ ID NO: 23.

[0253] The said sequence variant retains the ability to act as a DNA-binding protein upon expression. Suitable assays for determining DNA-binding protein activity are known to those skilled in the art, including an increase in the total linear DNA production measured by ultraviolet spectrophotometry, agarose gel electrophoresis, or quantitative PCR, which indicates that functional E2A contributes to plasmid replication. Additionally, electrophoretic mobility shift assays can be used to detect the terminal protein binding to DNA.

[0254] Two host transcription factors (NFI and Oct-1) are involved in enhancing the initiation of DNA replication (Hoeben & Uil, 2013), but are not essential for the plasmid. It includes inverted terminal repeats containing the origin of replication. The DNA replication mechanism is similar to that of the bacteriophage Phi29 group. DBP binds to dsDNA and unwinds it through polymerization. pTP is covalently linked to the 5'-phosphate, and AdV Pol adds dCMP to the hydroxyl group of pTP Ser580 (except for avian adenovirus-A, which incorporates dGMP) to initiate DNA replication. Initiation most frequently occurs at the 4th nucleotide of the template starting with a 3-nt repeat (GTAGTA), so once the third nucleotide is added to the new DNA strand, the pTP-CAT complex jumps back to the origin to ensure no information is lost. Then, AdV Pol and DBP start replicating the sequence, thus creating two linear plasmids, each with pTP linked to both ends, which can initiate subsequent rounds of replication (Hoeben & Uil, 2013).

[0255] In other embodiments of the present invention, the linear replication system included as part of the polynucleotides of the present invention is from other organisms having linear plasmids, genomes, or other replicons, including Escherichia coli bacteriophage PRD1, Streptococcus pneumoniae bacteriophage Cp-1, Streptomyces spp., viruses, archaea, linear plasmids of bacteria, fungi, and plants, transposable elements, and mitochondrial DNA (Salas et al., 2016). Figure 3 Shows the mechanism of plasmid replication using a linear DNA replication system characterized by a terminal protein.

[0256] DNA secretion pore

[0257] The second aspect of the present invention relates to a pore that can secrete DNA from one eukaryotic cell to an adjacent eukaryotic cell.

[0258] "DNA secretion pore" refers to a structure composed of one or more protein subunits that spans the membrane between two regions of a cell or between two cells and can transfer DNA molecules across the transmembrane junction.

[0259] Preferably, the DNA secretion pore polynucleotide sequence included as part of the polynucleotide of the present invention is TraB from Streptomyces species (including Streptomyces lividans and Streptomyces venezuelae plasmid pSVH1).

[0260] In a preferred embodiment of the present invention, the pore expressed by the polynucleotide sequence includes;

[0261] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700 or at least 750 amino acids of SEQ ID NO: 24; or

[0262] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 24; or

[0263] (iii) SEQ ID NO: 24.

[0264] In a preferred embodiment of the present invention, the pore polynucleotide sequence includes;

[0265] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200 or at least 2300 nucleotides of SEQ ID NO: 25; or

[0266] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 25; or

[0267] (iii) SEQ ID NO: 25.

[0268] The sequence variants retain the ability to act as DNA secretion pores upon expression. Suitable assays for determining DNA secretion pore activity are known to those skilled in the art and include transferring a plasmid encoding a fluorescently labeled gene from a donor to a recipient cell line.

[0269] TraB consists of a single subunit that assembles into a hexameric pore-forming ATPase, similar to the chromosome segregation protein FtsK, and translocates DNA by recognizing a specific 8-bp clt repeat sequence (GACCCGGA - SEQ ID NO: 27) present in the plasmid clt locus (Thoma & Muth, 2012).

[0270] In a preferred embodiment of the present invention, the polynucleotide of the present invention comprises the clt locus polynucleotide sequence. In a preferred embodiment of the present invention, the clt locus polynucleotide sequence comprises SEQ ID NO: 26, or a variant of SEQ ID NO: 26 that differs by 1, 2, 3, 4, or 5 nucleotides. The sequence variant retains the ability to act as the clt locus. Suitable assays for determining the functionality of the clt locus are known to those skilled in the art, including electrophoretic mobility shift assays (EMSA) using DNA sequences with and without the clt locus (only the former is inhibited by TraB) (Amado et al. 2019).

[0271] TraB transfers plasmid DNA through binding between the hyphal tips of Streptomyces hyphae, and proteins encoded by the spd family genes are involved in subsequent diffusion through the septal cross-walls, but only TraB is required for the primary transfer from donor to recipient (Thoma & Muth, 2015).

[0272] In one embodiment of the present invention, the DNA secretion pore is the single-protein DNA translocase TdtA from Thermus spp. (including Thermus thermophilus), which actively extrudes DNA without a specific sequence from donor cells (Blesa et al. 2017). TdtA does not require a specific DNA sequence for secretion, such as the clt repeat sequence.

[0273] In one embodiment of the present invention, the DNA secretion pore is a single-protein DNA translocase of bacterial or archaeal origin belonging to the FtsK-HerA superfamily, including FtsK and SpoIIIE that recognize the 8-bp motifs KOPS and SRS, respectively (Amado et al. 2019).

[0274] In a preferred embodiment of the present invention, the pore expressed by the polynucleotide sequence comprises;

[0275] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a continuous fragment of at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, or at least 560 amino acids of SEQ ID NO: 28; or

[0276] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 28; or

[0277] (iii) SEQ ID NO: 28.

[0278] In a preferred embodiment of the present invention, the pore polynucleotide sequence comprises;

[0279] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 1300, at least 1400, at least 1450, at least 1500, at least 1550, at least 1600, at least 1650 or at least 1700 nucleotides of SEQ ID NO: 29; or

[0280] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 29; or

[0281] (iii) SEQ ID NO: 29.

[0282] In one embodiment, the DNA secretion pore is from a type VI secretion system (T4SS) of bacterial genera including Agrobacterium, Bartonella, Brucella, Escherichia, Legionella, Helicobacter, Rickettsia, Salmonella and Shigella, the DNA release system of Neisseria spp., the Helicobacter pylori ComB system or the Bordetella pertussis pertussis toxin export (Ptl) system (Christie et al., 2014). These multi-component protein pores do not require a specific DNA sequence for secretion.

[0283] In vivo gene expression regulation

[0284] The third aspect of the present invention relates to promoters and other sequences involved in expressing the polynucleotide sequences contained in the polynucleotides of the present invention in eukaryotic cells. A promoter is a binding site for RNA polymerase and transcription factors and is essential for initiating mRNA synthesis. It should be understood in the art that a promoter can use its complete wild-type sequence or a truncated derivative.

[0285] In a preferred embodiment of the present invention, the polynucleotide of the present invention comprises a promoter sequence that functions in most cells of the target organism: for mammals, these promoters include those from human cytomegalovirus (major immediate early promoter hCMV-MIE), Rous sarcoma virus (RSV), simian virus 40 (SV40), Moloney murine leukemia virus long terminal repeat, elongation factor 1α (EF-1α), cytokeratins 18 and 19 (K18 and K19), amylase (AMY), and rat aquaporin-5 (rAQP5) (Zheng & Baum, 2005).

[0286] Preferably, the promoter sequence contained in the polynucleotide of the present invention restricts gene expression to a specific organ or tissue to limit the spread of the polynucleotide of the present invention to areas where its activity has a therapeutic benefit. Genes regulated by these tissue-specific promoters include human muscle creatine kinase (MCK) (Wang et al., 2008), mammary gland-specific murine whey acidic protein (WAP) (Oztürk-Winder et al., 2002) or small breast epithelial mucin gene promoter (SBEM) (Hub et al., 2006), lung ciliated cell-specific gene FOXJ1 (hepatocyte nuclear factor-3 / forkhead homolog 4) (Ostrowski et al., 2003), and the proximal promoter of WASp (Wiskott-Aldrich syndrome) in hematopoietic cells (Martín et al., 2005).

[0287] Internal ribosome entry site (IRES) sequences enable two or more cistrons to be regulated by the same promoter by initiating translation within a single mRNA transcript, thereby generating shorter expression cassettes. In one embodiment of the present invention, IRES sequences (including IRES sequences of encephalomyocarditis virus (EMCV) (Al-Allaf et al., 2019) and poliovirus (PV) (Malnou et al., 2002)) can be incorporated into the polynucleotide of the present invention.

[0288] An alternative method of expressing two or more cistrons from the same promoter is to use 2A "ribosome skipping" peptides, including F2A (foot-and-mouth disease virus), E2A (equine rhinitis A virus), P2A (porcine teschovirus-12A), and T2A (Dendrolimus punctatuswenshanensis virus 2A) (Liu et al., 2017). These require the addition of the highly conserved sequence GDVEXNPGP (SEQ ID NO: 30) to the C-terminus of the protein encoded by the upstream gene. After translation, this sequence remains at the C-terminus of the "upstream" protein, with the last proline removed, which becomes the first amino acid of the "downstream" protein. The 2A peptide can be separated from the upstream protein by a short linker sequence (such as GSG) to increase the skipping frequency (Szymczak-Workman et al., 2012). In one embodiment of the present invention, the polynucleotide of the present invention comprises one or more polynucleotides encoding a 2A 'ribosome skipping' peptide.

[0289] It is known in the art that incorporation of a polyadenylation signal (poly(A)) sequence containing the central sequence motif AAUAAA increases the steady-state level of the mRNA of a gene expressed in eukaryotic cells (Proudfoot, 2011), thereby increasing the expression level of the recombinant protein. The poly(A) sequence is located downstream of the cistron and can include those from late simian virus 40 (SV40), human or bovine growth hormone genes (hGH or bGH), and human or rabbit β-globin genes. In an embodiment of the present invention, the polynucleotide of the present invention comprises one or more polyadenylation signal (poly(A)) sequences.

[0290] Selectable marker gene

[0291] A fourth aspect of the present invention relates to the presence of a selectable marker gene in the polynucleotide of the present invention and a mechanism for subsequent removal of the selectable marker gene if desired.

[0292] It is well established in the art that plasmids require selectable marker genes for initial selection in bacterial (usually Escherichia coli) host cells and to ensure that cells that have lost the plasmid do not proliferate in the culture and thus compromise the cells containing the plasmid. The most common of these genes are antibiotic resistance genes, including those conferring resistance to β-lactam antibiotics (β-lactamase: bla); aminoglycoside antibiotics such as kanamycin or neomycin (kanamycin phosphotransferase: kan, neomycin phosphotransferase: neo); chloramphenicol (chloramphenicol acetyltransferase: cat); and tetracycline (tetracycline efflux pump: tetA). Preferably, the antibiotic resistance gene on the plasmid is kan.

[0293] An alternative to antibiotic resistance genes is an antibiotic-free plasmid selection system. These include complementing auxotrophy of the bacterial host by the presence of functional genes (e.g., dapD) or inhibitor tRNAs, toxin-antitoxin systems (e.g., hok / sok and ccdB / ccdA), operon-repressor titration (ORT), and various RNA-based selection systems (Vandermeulen et al., 2011). In an embodiment of the present invention, the polynucleotide of the present invention comprises one or more selection marker genes as described above.

[0294] Preferably, an antibiotic resistance gene is used during plasmid construction and removed after transformation into the final E. coli production strain. This can be achieved by using the native XerCD site-specific recombination system by ligating the antibiotic resistance gene to the recognition sequences of XerC and XerD (cer, psi or dif); thereby enabling the XerCD recombinase to excise the inserted gene by site-specific recombination. The "X-mark" technique is employed on circular plasmids, whereby the antibiotic resistance gene is flanked by cer or psi sites and adjacent binding sites for the accessory proteins PepA and ArgR / ArcA - the antibiotic resistance gene is retained when cultured in pepA mutant E. coli, but is excised when transformed into any E. coli strain with functional pepA (Cranenburgh & Leckenby, 2010). On circular plasmids containing telRL (which will be converted to linear plasmids by the action of TelN), the "Xer-cise" technique is used, where the antibiotic resistance gene is flanked by dif sites and the Xer recombination event excising the antibiotic resistance gene only occurs when the plasmid reaches a linear conformation (Bloor & Cranenburgh, 2006). In an embodiment of the present invention, the polynucleotide of the present invention comprises one or more antibiotic resistance genes as described above.

[0295] Controlling the duration of protein function and intercellular DNA transfer

[0296] The fifth aspect of the present invention relates to mechanisms for restricting the duration of replication and gene expression to enhance the biosafety of the polynucleotide of the present invention, and potentially removing the TP if it impedes DNA secretion through the intermembrane pore.

[0297] To limit the time that the protein expressed by the polynucleotides of the present invention exists in a cell, the half-life of components such as RepA, TelN, TdtA, or TraB can be reduced, such that a limited number of replication cycles are allowed to achieve a therapeutic effect and transfer the polynucleotides of the present invention to adjacent cells. To limit the half-life of the component protein, it can be fused with a peptide sequence targeted for degradation. These include degrons and destabilizing domains (DDs). Degrons directly bind ubiquitin ligases or proteasomes, targeting the fusion protein to the ubiquitin-proteasome system (UPS) (Wu et al., 2020). DDs can be N-terminal or C-terminal fusions and, when expressed in mammalian cells, rapidly degrade the fusion protein through an undefined quality control pathway unless a ligand that blocks degradation is provided. Examples of DD sequences are from the rapamycin-binding protein (FKBP12), and its ligands include Shld1 (Wu et al., 2020). In an embodiment of the present invention, the polynucleotides of the present invention comprise one or more polynucleotide sequences that, when expressed, serve to limit the half-life of a protein (such as RepA, TelN, TdtA, or TraB).

[0298] In applications of the CRISPR-Cas9 technology for gene editing (see below), the polynucleotides of the present invention are self-limiting because the Cas9 RNA-guided nuclease will cleave and thus inactivate the polynucleotides of the present invention in the cell nucleus; for applications that do not involve gene editing, the polynucleotide sequence encoding plasmid-targeted Cas9 can be incorporated into the polynucleotides of the present invention as an additional safety feature.

[0299] The polynucleotides of the present invention can comprise a transgene sequence, also referred to as a payload sequence. The payload sequence can be a therapeutic gene, a polynucleotide sequence having a therapeutic effect on eukaryotic cells, or a sequence encoding a protein having a therapeutic effect on eukaryotic cells. The following aspects of the present invention illustrate examples of payload sequences. For example, the payload sequence can be a therapeutic gene, a CRISPR RNA-guided nuclease (optionally including CRISPR donor DNA), a zinc finger nuclease, or a TALEN, an antigen gene, or a gene encoding an immunogenic protein or a protein from a pathogen or tumor, or an antibiotic, antifungal, or antiviral compound, or an antibody, or a chimeric antigen or T cell receptor, or a B cell receptor.

[0300] Gene editing

[0301] The sixth aspect of the present invention relates to gene editing to replace a mutant copy of a chromosomal gene that causes a genetic disease, represents an increased cancer risk, or causes cancer with a copy of the gene having the correct function; alternatively, when a chromosomal gene is inactivated, to mutate or excise it, thereby producing the desired effect.

[0302] Gene editing involves cutting specific gene sequences in the genome to inactivate genes, or allowing the insertion of foreign genes, and restoring the break through homology-directed repair (HDR) or non-homologous end joining (NHEJ). Synthetic nucleases can be used for gene editing in the polynucleotides of the present invention, including restriction enzymes, for example, FokI fused with a modular DNA recognition protein subunit (such as a zinc finger) to produce zinc finger nucleases (ZFNs), or transcription activator-like effector proteins (TALEs) from Xanthomonas spp. to create TAL nucleases (TALENs) (Adli, 2018), or members of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family such as TnpB (Nety et al., 2023), or eukaryotic transposon-encoded Fanzor (Fz) proteins (Saito et al., 2023), or artificial peptide genome editing tools (ApGet) (GB2114453.0). Preferably, components of the CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune system are used for gene editing, whereby a specific double-strand break is introduced into the target host genome using an RNA-guided nuclease (such as Cas9 from Streptococcus pyogenes (SpCas9)). In an embodiment of the present invention, the polynucleotide of the present invention comprises a polynucleotide sequence encoding one or more gene editing proteins (such as an RNA-guided nuclease, such as Cas9).

[0303] The CRISPR-associated RNA-guided nuclease Cas9, or a Cas9 functional equivalent (such as Cas13 or CPf1), or a variant thereof, can be from prokaryotes, including the bacterial genera Acidaminococcus spp., Campylobacter spp., Francisella spp., Lachnospiraceae spp., Neisseria spp., Staphylococcus spp., Streptococcus spp. (Adli, 2018). In an embodiment of the present invention, the nuclease used is the MAD7 nuclease, which is a type V CRISPR nuclease isolated from Eubacterium rectale. For gene editing applications, wild-type CRISPR nucleases (such as Cas9) cause unwanted mutations at off-target sites that are homologous to the desired target sequence.

[0304] Preferably, the present invention will use Cas9 with an altered amino acid sequence to reduce or eliminate off-target effects, such as SpCas9-HF1 (Kleinstiver et al., 2016) or eSpCas9 (Slaymaker et al., 2016). Introducing an N-terminal or C-terminal nuclear localization signal (NLS) such as the SV40 large T antigen NLS may improve the efficiency of gene editing (Hu et al., 2018).

[0305] Cas9 requires CRISPR RNA (crRNA) to recognize the complementary DNA sequence adjacent to the protospacer adjacent motif (PAM, consensus sequence: NGG), and trans-activating CRISPR RNA (tracrRNA) to bind to Cas9 - they are combined into a single guide RNA (sgRNA) for gene editing applications (Jinek et al., 2013). The sgRNA sequence can be expressed by mammalian promoters that initiate RNA polymerase III transcription, including the U6, H1, and 7SK promoters (Yin et al., 2020). In an embodiment of the present invention, the polynucleotide comprises a polynucleotide sequence encoding an RNA-guided nuclease such as SpCas9-HF1 or eSpCas9. In an embodiment of the present invention, the polynucleotide of the present invention contains one or more polynucleotide sequences encoding sgRNA.

[0306] The polynucleotides of the present invention can be used to replace mutant genes in gene therapy and cancer therapy applications in humans and other animals (including embryos) through gene editing. The polynucleotides of the present invention contain the wild-type or cDNA cistron of the defective gene, flanked by approximately 1 kb of target-site homologous sequences on each side, and on each side of the homologous sequences there is a pair of complementary sites for sgRNA, and the sgRNA is also located on both sides of the chromosomal target site. The target site may be a defective gene (especially when its removal may be beneficial), or it may be an intergenic region. When the polynucleotides of the present invention enter the cell, the first polynucleotide to cross the nuclear membrane into the nucleus undergoes gene transcription, and the resulting mRNA crosses the nuclear membrane and is exported back to the cytoplasm for translation. Then, the Cas9 protein enters the nucleus, binds to the sgRNA and cleaves the polynucleotide and the chromosome, releasing the donor DNA and the defective gene respectively. The donor DNA is integrated into the chromosomal break by HDR, and possibly also by NHEJ.

[0307] In vivo gene expression

[0308] The seventh aspect of the present invention relates to the expression of therapeutic genes without chromosomal gene editing. In this application, the therapeutic gene is expressed in target cells to continuously produce therapeutic proteins. This can be achieved by using the single polynucleotide or plasmid of the present invention, or by placing a functional gene (encoding a DNA replication protein and a pore) on the first polynucleotide of the present invention, which is degraded after a limited time, for example, by Cas9 or another endonuclease, so that the second polynucleotide expresses the therapeutic gene, but cannot be transferred to other cells without the first plasmid. Therefore, the polynucleotides of the present invention can be used to treat genetic diseases and cancers. In an embodiment of the present invention, the polynucleotides of the present invention can be used to treat genetic diseases and cancers caused by coding mutation inactivation, or as a DNA vaccine.

[0309] Gene mutations leading to genetic diseases

[0310] Potential genetic disease targets (and mutant genes) include: achromatopsia (genes encoding cone phototransduction cascade components: CNGA3, CNGB3, GNAT2, PDE6C, PDE6H; activating transcription factor 6: ATF6); alpha-1-antitrypsin deficiency (serine protease inhibitor A1: SERPINA1); Angelman syndrome (ubiquitin ligase: UBE3A); aromatic L-amino acid decarboxylase (AADC) deficiency (dopa decarboxylase: DDC); Batten disease (neuronal ceroid lipofuscinosis) (genes include: PPT1, TPP1, CLN3, DNAJC5, CLN5, CLN6, MFSD8, CLN8, CTSD, GRN, ATP13A2, CTSF, KCTD7); beta-thalassemia (beta-globin: HBB); Charcot-Marie-Tooth disease type 1A (peripheral myelin protein 22: PMP22); choroideremia (Rab escort protein 1: CHM); Crigler–Najjar syndrome (UDP-glucuronosyltransferase: UGT1A1); cystic fibrosis (cystic fibrosis transmembrane conductance regulator: CFTR); diabetes (insulin: INS); Duchenne muscular dystrophy (dystrophin: DMD); giant axonal neuropathy (giant axonal protein: GAN); dysferlinopathy (dysferlin: DYSF); glycogen storage disease type 1a (glucose-6-phosphatase: G6PC); hemophilia A (coagulation factor 8: FVIII); hemophilia B (coagulation factor 9: FIX); Huntington's disease (huntingtin: HTT); hypercholesterolemia (low density lipoprotein receptor: LDLR, or apolipoprotein B: APOB); hypophosphatemic rickets (X-linked phosphate-regulating endopeptidase homolog: PHEX); Leber congenital amaurosis (retinol isomerohydrolase: RPE65); Leber hereditary optic neuropathy (NADH dehydrogenase 4: MT-ND4); long-chain fatty acid oxidation disorders (medium-chain acyl-CoA dehydrogenase: ACADM; very long-chain acyl-CoA dehydrogenase: ACADVL; long-chain 3-hydroxyacyl-CoA dehydrogenase: HADHA; carnitine palmitoyltransferase 1: CPT1A; carnitine-acylcarnitine translocase: SLC25A20; carnitine palmitoyltransferase 2: CPT2; carnitine transporter: SLC22A5; short-chain acyl-CoA dehydrogenase: ACADS; multiple acyl-CoA dehydrogenase deficiency: ETF A, ETF B, ETFDH; 3-hydroxyacyl-CoA dehydrogenase: HADH); limb-girdle muscular dystrophy type 2E (sarcoglycan genes: SGCB, SGCC, SGCD); Marfan syndrome (fibrillin: FBN1); mucopolysaccharidosis (alpha-L-iduronidase: IDUA; iduronate-2-sulfatase: IDS; N-sulfo-glucosamine sulfohydrolase: SGSH; alpha-N-acetylglucosaminidase: NAGLU;Heparin-α-glucosaminide N-acetyltransferase: HGSNAT; N-acetylglucosamine-6-sulfatase: GNS; Galactose-6-sulfate sulfatase: GALNS; β-galactosidase: GLB1; N-acetylgalactosamine-4-sulfatase: ARSB; β-glucuronidase: GUSB; HYAL1 (hyaluronidase); myotonia congenita (chloride channel 1: CLCN1); myotonic dystrophy type 1 (myotonic dystrophy protein kinase: DMPK); neurofibromatosis type 1 (neurofibromin 1: NF1); phenylketonuria (phenylalanine hydroxylase: PAH); ornithine transcarbamylase (OCT) deficiency; polycystic kidney disease 1 and 2 (polycystin 1, transient receptor potential channel interacting: PKD1; polycystin 2, transient receptor potential cation channel: PKD2); Pompe disease (α-glucosidase: GAA); retinitis pigmentosa (cellular retinaldehyde-binding protein: RLBP1); Rett syndrome (methyl-CpG-binding protein 2: MECP2); sickle cell disease (β-globin: HBB); spinal muscular atrophy (survival motor neuron 1: SMN1); Tay–Sachs disease (hexosaminidase A: HEXA); Wiskott–Aldrich syndrome (WASp); X-linked myotubular myopathy (myotubularin 1: MTM1); X-linked retinitis pigmentosa (X-linked retinitis pigmentosa GTPase regulator: RPGR); X-linked retinoschisis (retinoschisin: RS1); X-linked severe combined immunodeficiency (common γ-chain-encoding gene: IL2RG).;

[0311] Gene duplication leading to genetic diseases

[0312] Genetic diseases treatable with the polynucleotides of the present invention can include genetic diseases caused by gene duplication or amplification, where treatment represents deleting one or more irrelevant copies or replacing one or more duplicated copies with a single copy. An example is the most common type 1A Charcot-Marie-Tooth disease (peripheral myelin protein 22: PMP22).

[0313] Genetic modification to eliminate susceptibility to infectious diseases

[0314] In some cases, the removal of an antigen from a host cell may reduce or eliminate susceptibility to an infectious disease. The CCR5 receptor is a co-receptor for CD4, and when human immunodeficiency viruses (HIV 1 and 2) bind to it, they enter T helper cells, leading to acquired immunodeficiency syndrome (AIDS). The human CCR5 gene mutation has no known harmful effects, so it can be mutated or deleted by gene editing of hematopoietic stem cells (in the bone marrow where they originate, in the thymus where they mature), or modified ex vivo and re-implanted; this will prevent HIV infection and may lead to the clearance of HIV in infected individuals (Epah& 2021). In an embodiment of the present invention, the polynucleotides of the present invention are used for the treatment of HIV infection.

[0315] Loss-of-function diseases - exogenous gene expression is therapeutic

[0316] Some diseases are not caused by gene mutations, but rather by gene function loss due to cell death, senescence, or abnormal immune responses. These diseases can be treated by introducing functional genes, which can exist in a free form or be inserted into chromosomes by the polynucleotides of the present invention. These diseases include: Parkinson's disease (artemin: ARTN; dopa decarboxylase: DDC; glial cell line-derived neurotrophic factor: GDNF; neurotrophin: NRTN; persephin: PSPN); wet age-related macular degeneration (anti-vascular endothelial growth factor proteins and antibodies).

[0317] Gene mutations cause and actively trigger cancer

[0318] Gene targets that may lead to cancer after mutation include genes encoding DNA repair enzymes BRCA1, BRCA2 (breast and ovarian cancers), and TP53 (multiple cancers).

[0319] Other potential targets include genes that regulate cell growth or division and thus become oncogenes upon mutation, such as ACRV2A (activin A receptor type 2A); APC (adenomatous polyposis coli); ATRX (X-linked alpha-thalassemia / mental retardation syndrome); CDKN2A (cyclin-dependent kinase inhibitor 2A); CTNNB1 (beta-catenin 1); DAXX (death domain-associated protein); EGFR (epidermal growth factor receptor); FBXW7 (F-box with 7 tandem WD40s); MEN1 (multiple endocrine neoplasia type 1); PCBP1 (poly C-binding protein 1); PIK3CA (phosphatidylinositol 3-kinase); PTEN (phosphatase and tensin homolog); RAS gene family (HRAS, NRAS, and KRAS); RB1 (RB transcriptional corepressor 1); SMAD2, SMAD3, and SMAD4; SOX (sex-determining region Y-box) gene family, including SOX2 and SOX9; TCF7L2 (transcription factor 7-like 2); ZFP36L2 (ZFP36 ring finger protein-like 2) (Gerstung et al., 2020).

[0320] Other potential targets include genes that can enable tumor proliferation through immune evasion upon mutation, including B2M (beta-2-microglobulin).

[0321] Other potential targets include genes that can enable tumor proliferation when present in extra copies, such as through gene rearrangement or amplification, such as HER2 (human epidermal growth factor receptor 2) and TERT (telomerase reverse transcriptase). In one embodiment of the present invention, the polynucleotide of the present invention comprises the genes described above. In one embodiment of the present invention, the polynucleotide of the present invention is used for treating cancer.

[0322] Immunotherapy

[0323] The eighth aspect of the present invention is applicable to immunotherapy by genetically modifying progenitor cells of T cells and B cells such that the resulting T cells express receptors capable of targeting antigens on pathogens or cancer cells, and the B cells produce antibodies targeting antigens on pathogens or cancer cells. The present invention achieves this method by the ability to modify most of the cells within the target tissue. The progenitor cells can be modified in the bone marrow where they originate, or for T cells, also in the thymus where they mature; or modified ex vivo and re-implanted. This hematopoietic stem and progenitor cell (HSPC) gene therapy method can be achieved through gene editing or gene expression (Epah& In principle, the engineered T cells and B cells generated by engineering HSPCs with the polynucleotides of the present invention can be used to target all tumor-associated antigens and pathogen-specific antigens listed below (under "DNA vaccines").

[0324] DNA vaccines

[0325] In a ninth aspect of the present invention, one or more immune proteins from a pathogen or cancer cell are expressed within antigen-presenting cells of a host to trigger the immune system to target the pathogen or cancer cell, thereby serving as a DNA vaccine. Compared with current DNA vaccine delivery methods, the present invention will allow significantly more antigen-presenting cells to express the antigen and will use less DNA, which will improve tolerance at the injection site. The tumor-associated antigen sequences contained in the polynucleotides of the present invention can be selected from cancer cells having any of the above mutations and include: AFP: alpha-fetoprotein; AIM-2: interferon-inducible protein 2 lacking in melanoma; ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; 707-AP: 707-alanine proline; APL: acute promyelocytic leukemia; ART-4: adenocarcinoma antigen 4 recognized by T cells; BAGE: B antigen; bcr-abl: breakpoint cluster region-Abelson; CAMEL: CTL-recognized antigen on melanoma; CAP-1: carcinoembryonic antigen peptide-1; CASP-8: caspase 8; CDC27: cell division cycle 27; CDK4: cyclin-dependent kinase 4; CEA: carcinoembryonic antigen; CLCA2: calcium-activated chloride channel 2; CML: chronic myelogenous leukemia; CT: testicular cancer (antigen); CTL: cytotoxic T lymphocyte; Cyp-B: cyclophilin B; DAM: differentiation antigen melanoma (the epitopes of DAM-6 and DAM-10 are identical, but the gene sequences are different. DAM-6 is also known as MAGE-B2, and DAM-10 is also known as MAGE-B1); ELF2: elongation factor 2; Ep-CAM: epithelial cell adhesion molecule; EphA2, 3: Ephrin A-type receptor 2, 3; Ets: E-26 transformation-specific (transcription factor family); ETV6-AML1: Ets variant gene 6 / acute myeloid leukemia 1 gene ETS; FGF-5: fibroblast growth factor 5; FN: fibronectin; G250: glycoprotein 250; GAGE: G antigen; GnT-V: N-acetylglucosaminyltransferase V; Gp100: glycoprotein 100 kDa; HAGE: helicase antigen; HER-2 / neu: human epidermal receptor 2 / nervous system; HLA-A*0201-R170I: arginine (R) is exchanged with isoleucine (I) at residue 170 of the alpha helix in the alpha2 domain of the HLA-A2 gene; H / N: head and neck; HSP70-2 M: heat shock protein 70-2 mutation; HST-2: human signet ring cell carcinoma 2; hTERT: human telomerase reverse transcriptase; iCE: intestinal carboxylesterase; IL-13Rα2: interleukin 13 receptor alpha 2 chain; KIAA0205; LAGE: L antigen; LDLR / FUT: low density lipid receptor / GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase; MAGE: melanoma antigen;MART-1 / Melan-A: Melanoma antigen recognized by T cells-1 / Melanoma antigen A; MART-2: Melanoma antigen recognized by T cells-2; MC1R: Melanocortin 1 receptor; M-CSF: Macrophage colony-stimulating factor gene; MHC: Major histocompatibility complex; MSI: Microsatellite instability; MUC1, 2: Mucin 1, 2; MUM-1, -2, -3: Melanoma ubiquitous mutation 1, 2, 3; NA88-A: NA cDNA clone of patient M88; Neo-PAP: Neo-poly(A) polymerase; NPM / ALK: Nucleophosmin / anaplastic lymphoma kinase fusion protein; NSCLC: Non-small cell lung cancer; NY-ESO-1: New York esophageal cancer 1; OA1: Ocular albinism type 1 protein; OGT: O-linked N-acetylglucosamine transferase gene; OS-9; P15: Protein 15; p190 minor bcr-abl: 190kD abcr-abl protein; Pml / RARα: Promyelocytic leukemia / retinoic acid receptor α; PRAME: Melanoma preferentially expressed antigen; PSA: Prostate-specific antigen; PSMA: Prostate-specific membrane antigen; PTPRK: Receptor-type protein tyrosine phosphatase κ; RAGE: Renal antigen; RCC: Renal cell carcinoma; RU1, 2: Renal ubiquitous 1, 2; SAGE: Sarcoma antigen; SART-1, -2, -3: Squamous antigen-rejecting tumor 1, 2, 3; SCC: Squamous cell carcinoma; SSX-2: Synovial sarcoma, X breakpoint 2; Survivin-2B: Survivin with intron 2 retention; SYT / SSX: Synaptotagmin I / Synovial sarcoma, X fusion protein; TAA: Tumor-associated antigen; TEL / AML1: Translocated Ets family leukemia / Acute myeloid leukemia 1; TGFβRII: Transforming growth factor β receptor 2; TPI: Triosephosphate isomerase; TRAG-3: Paclitaxel resistance-related protein 3; TRG: Testin-related gene; TRP-1: Tyrosinase-related protein 1 or gp75; TRP-2: Tyrosinase-related protein 2; TRP-2 / INT2: TRP-2 / Intron 2; TRP-2 / 6b: TRP-2 / New exon 6b; TSTA: Tumor-specific transplantation antigen; WT1: Wilms tumor gene (Novellino et al., 2004).;

[0326] The antigen can be from a virus, bacterium, fungus, or eukaryotic parasite, including Acinetobacter baumannii; Actinomyces israelii, Actinomyces gerencseriae, and Propionibacterium propionicus; Trypanosoma brucei; HIV (human immunodeficiency virus); Entamoeba histolytica; Anaplasma spp.; Angiostrongylus; Anisakis; Bacillus anthracis; Arcanobacterium haemolyticum; Junin virus; Ascaris lumbricoides; Aspergillus spp.; Astroviridae spp.; Babesia spp.; Bacillus cereus; Bacteroides spp.; Balantidium coli; Bartonella; Baylisascaris spp.; BK virus; Piedraia hortae; Blastocystis spp.; Blastomyces dermatitidis; Machupo virus; Clostridium botulinum (botulinum toxin); Sabia virus; Brucella spp.; Yersinia pestis; Burkholderia cepacia, Burkholderia spp.; Mycobacterium ulcerans; Caliciviridae spp.; Campylobacter spp.) ; Candida albicans and other Candida species; intestinal diseases caused by Capillaria philippinensis, liver diseases caused by Capillaria hepatica, and lung diseases caused by Capillaria aerophila; Bartonella bacilliformis; Bartonella henselae; group A streptococci and staphylococci; Trypanosoma cruzi; Haemophilus ducreyi; varicella-zoster virus (VZV); alphavirus genus; Chlamydia trachomatis; Chlamydophila pneumoniae; Vibrio cholerae; Fonsecaea pedrosoi; Batrachochytrium dendrobatidis; Clonorchis sinensis; Clostridium difficile; Coccidioides immitis and Coccidioides posadasii; Colorado tick fever virus (CTFV); rhinoviruses and coronaviruses; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); Crimean-Congo hemorrhagic fever virus; Cryptococcus neoformans; Cryptosporidium spp.; Ancylostoma braziliense; Cyclospora cayetanensis; Taenia solium; cytomegalovirus; dengue viruses (DEN-1, DEN-2, DEN-3, and DEN-4) - flaviviruses; Desmodesmus armatus; Dientamoeba fragilis; Corynebacterium diphtheriae; Diphyllobothrium; Dracunculus medinensis; Ebola virus (EBOV); Echinococcus spp.; Ehrlichia spp.); Enterobius vermicularis; Enterococcus spp.; Enterovirus spp.; Rickettsia prowazekii; Parvovirus B19; Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7); Fasciola hepatica and Fasciola gigantica; Fasciolopsis buski; Filarioidea superfamily; Clostridium perfringens; Fusobacterium spp.; Clostridium perfringens; Clostridium spp.; Geotrichum candidum; Giardia lamblia; Burkholderia mallei; Gnathostoma spinigerum and Gnathostoma hispidum; Neisseria gonorrhoeae; Klebsiella granulomatis; Streptococcus pyogenes; Streptococcus agalactiae; Haemophilus influenzae; Enterovirus Coxsackievirus A and Enterovirus 71 (EV71); Sin Nombre virus; Heartland virus; Helicobacter pylori; Escherichia coli ETEC, O157:H7, O111, and O104:H4; Bunyaviridae spp.);Hendra virus; Hepatitis A virus; Hepatitis B virus; Hepatitis C virus; Hepatitis D virus; Hepatitis E virus; Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2); Histoplasma capsulatum; Ancylostoma duodenale and Necator americanus; Human bocavirus (HBoV); Ehrlichia ewingii; Anaplasma phagocytophilum; Human metapneumovirus (hMPV); Ehrlichia chaffeensis; Human papillomavirus; Human parainfluenza virus (HPIV); Hymenolepis nana and Hymenolepis diminuta; Epstein–Barr virus (EBV); Orthomyxoviridae spp.; Isospora belli; Kingella kingae; Lassa virus; Legionella pneumophila; Legionella pneumophila; Leishmania spp.; Mycobacterium leprae and Mycobacterium lepromatosis; Leptospira spp.; Listeria monocytogenes; Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii; Wuchereria bancrofti and Brugia malayi; Lymphocytic choriomeningitis virus (LCMV); Plasmodium spp.);Marburg virus; Measles virus; Middle East Respiratory Syndrome (MERS) coronavirus; Burkholderia pseudomallei; Neisseria meningitidis; Metagonimus yokagawai; Microsporidia phylum; Molluscum contagiosum virus (MCV); Monkeypox virus; Mumps virus; Rickettsia typhi; Mycoplasma pneumoniae; Mycoplasma genitalium; Chlamydia trachomatis; Neisseria gonorrhoeae; Nipah virus; Norovirus; Nocardia asteroides and Nocardia spp.; Onchocerca volvulus; Opisthorchis viverrini and Opisthorchis felineus; Paracoccidioides brasiliensis; Paragonimus spp. (Paragonimus westermani and other Paragonimus species); Pasteurella spp.; Pediculus humanus capitis; Pediculus humanus corporis; Pthirus pubis; Bordetella pertussis; Yersinia pestis; Streptococcus pneumoniae; Pneumocystis jirovecii; Poliovirus; Prevotella spp.; Naegleria fowleri; JC virus; Chlamydophila psittaci; Coxiella burnetii; Rabies virus; Borrelia hermsii, Borrelia recurrentis, Borrelia spp.); Respiratory syncytial virus (RSV); Rhinosporidium seeberi; Rhinovirus; Rickettsia spp.; Rickettsia akari; Rift Valley fever virus; Rickettsia rickettsii; Rotavirus; Rubella virus; Salmonella spp.; SARS coronavirus; Sarcoptes scabiei; Group A Streptococcus spp.; Schistosoma spp.; Shigella spp.; Varicella zoster virus (VZV); Variola major or Variola minor; Sporothrix schenckii; Staphylococcus spp.; Strongyloides stercoralis; Measles virus; Treponema pallidum; Taenia spp.; Clostridium tetani; Trichophyton spp.; Trichophyton tonsurans; Trichophyton spp.; Epidermophyton floccosum, Trichophyton rubrum and Trichophyton mentagrophytes; Trichophyton rubrum; Hortaea werneckii; Malassezia spp.);Streptococcus pyogenes; Toxocara canis, Toxocara cati; Toxoplasma gondii; Trichinella spiralis; Trichomonas vaginalis; Trichuris trichiura; Mycobacterium tuberculosis; Francisella tularensis; Salmonella enterica serotypes Typhi, Paratyphi, and Typhimurium; Ureaplasma urealyticum; Coccidioides immitis, Coccidioides posadasii; Venezuelan equine encephalitis virus; Guanarito virus; Vibrio vulnificus; Vibrio parahaemolyticus; West Nile virus; Trichosporon beigelii; Yersinia pseudotuberculosis; Yersinia enterocolitica; Yellow fever virus; Zeaspora fungus; Zika virus.

[0327] The antigen or antigen gene on the polynucleotide of the DNA vaccine of the present invention can be co-expressed or fused with an immunogenic protein as an adjuvant to increase the level of the immune response. Examples of such proteins include the subunit B of the lethal toxin (LT-B) from pathogenic strains of Escherichia coli, the subunit B of the cholera toxin (CT-B) from Vibrio cholerae, and the tetanus toxin (tetanospasmin) from Clostridium tetani; these may contain mutations to reduce toxicity. Optionally, the DNA vaccine plasmid can be co-administered with one of these adjuvants or with adjuvants including aluminum-based mineral salts (aluminum phosphate, aluminum hydroxide), calcium phosphate, MF59 (submicron oil-in-water emulsion), monophosphoryl lipid A (MPL: AS03, AS04).

[0328] RNA therapy

[0329] In other aspects, the present invention aims to express RNAs that achieve a therapeutic effect upon translation into proteins and serve as CRISPR guide RNAs. In a tenth aspect of the present invention, the expressed RNAs themselves are therapeutic products. The encoded RNAs can be single-stranded antisense RNAs, including antisense oligonucleotides (ASOs) or double-stranded small interfering RNAs (siRNAs), which are designed to alter the expression of host chromosomal genes to achieve the desired therapeutic effect (Zhu et al., 2022). Antisense RNAs bind to the target mRNA through Watson-Crick base pairing or downregulate expression through steric hindrance to reduce or prevent translation, or induce exon skipping: converting out-of-frame mutations into in-frame mutations for therapeutic applications, such as in a few cases of Duchenne muscular dystrophy (Aartsma-Rus et al., 2007).

[0330] Host mRNA can be degraded through RNA interference (RNAi) (using siRNAs or hairpin microRNAs (miRNAs)) to achieve a therapeutic effect. miRNAs are first sequentially processed by the RNase III enzymes DICER1 and DROSHA to generate double-stranded RNAs similar to siRNAs. Their mechanism of action is achieved through the Argonaute 2 protein (AGO2, a part of the RNA-induced silencing complex RISC). The double-stranded siRNA binds to AGO2, one strand (the passenger strand) is removed, and the remaining antisense guide strand directs the RISC complex to the corresponding mRNA target, which is then cleaved by AG02 (Roberts et al., 2020).

[0331] ASOs and siRNAs are usually chemically modified, typically by introducing phosphorothioate (PS) bonds in place of phosphodiester bonds to reduce their degradation by ribonucleases (Roberts et al., 2020), but the nuclear location of the Gentrafix plasmid and its ability to continuously express RNA will enable unmodified RNAs to produce a therapeutic effect.

[0332] Recombinant protein production

[0333] It is known in the art that recombinant proteins are produced in vitro using cell lines from multicellular eukaryotes by transfection with plasmids carrying genes encoding proteins - which represents the eleventh aspect of the present invention. The classes of recombinant proteins include antibodies, antibody fragments, antigens, enzymes, and hormones. Mammalian cell lines commonly used for recombinant protein production include cell lines from rodents: CHO (Chinese hamster ovary) and NS0 (mouse myeloma), and cell lines from humans: HEK (human embryonic kidney), PER.C6 (human retinal cells), and CAP-T (primary human amniotic fluid cells) (Bandaranayake and Almo, 2014). Transfection of adherent cell lines with DNA usually requires a high DNA-to-cell ratio and generally does not result in transgene expression in most of the cells in the culture medium. The present invention will modify most cells, resulting in higher yields of recombinant proteins in the transfected cell bank (transient gene expression), and more cells expressing at a high level for subsequent clonal selection.

[0334] Germline modification and gene drive

[0335] In a further embodiment of the present invention, the polynucleotides of the present invention are designed to modify animal germline cells by modifying spermatogenic cells (spermatogonia) or oocytes by expressing components of the polynucleotides of the present invention using constitutive or tissue-specific promoters. This can be used for co-expression of one or more genes encoding therapeutic compounds. Optionally, gene editing functions (such as CRISPR) can be expressed, enabling such modification to be passed on to the offspring of the gene-edited animal. This can be used to correct gene mutations to prevent their transmission to offspring or to introduce beneficial traits.

[0336] Another aspect of germline modification is the introduction of a "gene drive" cassette, which will then spread to most or even all offspring (Wedell et al., 2019). Gene drives may be based on natural selfish genetic elements, such as transmission distorters overexpressed in eggs or sperm targeting gametogenesis. Optionally, synthetic meiotic drives based on CRISPR-mediated gene editing can be used to copy the gene drive onto homologous chromosomes so that it spreads rapidly in the resulting population. These can be used to control disease vectors, such as mosquitoes, or invasive mammals that have an adverse impact on the local population, for example, by skewing the sex ratio to produce only males. This can be achieved in rodent populations by moving the Sry sex-determining gene from the X chromosome to an autosome.

[0337] Pharmaceutical compositions

[0338] In another aspect of the present invention, there is provided a pharmaceutical composition comprising the polynucleotide of the present invention, or a plasmid embodiment of the polynucleotide of the present invention, and a pharmaceutically acceptable excipient.

[0339] The pharmaceutically acceptable excipient may include a carrier, a diluent, and / or other drugs, medicaments, or adjuvants, etc. Optionally, the pharmaceutically acceptable excipient includes a salt solution. Optionally, the pharmaceutically acceptable excipient includes human serum albumin.

[0340] Typical "pharmaceutically acceptable excipients" include any carrier that does not itself cause a harmful reaction in the individual receiving the composition. The pharmaceutically acceptable excipient may also contain diluents such as water, saline, glycerol, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffering substances, etc. may also be present. Typical pharmaceutical excipients may include one or more of Tris buffer, histidine, sodium chloride, and sodium phosphate.

[0341] Method of treatment

[0342] The present invention further provides the polynucleotide of the present invention, or a plasmid embodiment of the polynucleotide of the present invention, or the pharmaceutical composition of the present invention for use in a method of treatment / method of treating a disease. Optionally, the method of treatment includes administering to a patient an effective amount of the polynucleotide of the present invention, or a plasmid embodiment of the polynucleotide of the present invention, or the pharmaceutical composition of the present invention.

[0343] The present invention also provides a method of treatment comprising administering to a patient an effective amount of the polynucleotide of the present invention, or a plasmid embodiment of the polynucleotide of the present invention, or the pharmaceutical composition of the present invention.

[0344] The present invention further provides the use of the polynucleotide of the present invention, or a plasmid embodiment of the polynucleotide of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a medicament for use in a method of treatment / method of treating a disease. For the avoidance of doubt, the terms "method of treatment" and "method of treating a disease" are used interchangeably herein. Optionally, the method of treatment / method of treating a disease includes administering to a patient an effective amount of the composition or polynucleotide of the present invention, or a plasmid embodiment of the polynucleotide of the present invention.

[0345] The term "treatment" includes therapeutic treatment and prophylactic or preventive treatment, the purpose of which is to prevent or mitigate infection. For example, treatment can include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of symptoms associated with, for example, infection, delaying the onset of symptoms associated with, for example, infection, reducing the symptoms associated with, for example, infection, or a combination thereof. "Prevention" can refer to delaying the onset of symptoms, preventing disease recurrence, etc. "Treatment" can also include "suppressing" or "inhibiting" an infection or disease, such as reducing the severity, quantity, incidence or latency of symptoms, improving symptoms, reducing secondary symptoms, reducing secondary infections, prolonging the survival period of a patient, or a combination thereof.

[0346] "Therapeutically effective amount" means an amount that achieves the desired therapeutic result, such as increasing the transgenic level of an object (thus resulting in a production level of a functional transgenic sufficient to improve the symptoms of a disease or disorder), within the necessary dosage and time period.

[0347] In a preferred embodiment of the present invention, the diseases and disorders to be treated are those discussed herein.

[0348] Method of administration

[0349] In a preferred embodiment of the present invention, the polynucleotide of the present invention, or the plasmid embodiment of the polynucleotide of the present invention, or the pharmaceutical composition of the present invention is administered to a patient in need thereof by injection, microinjection, inhalation, jet injection, ingestion, liposome, lipid nanoparticle, virus, virus-like particle or microcarrier-mediated delivery.

[0350] Method of manufacture

[0351] In another aspect of the present invention, there is provided a host cell comprising the polynucleotide of the present invention or the plasmid embodiment of the polynucleotide of the present invention. Suitable host cells are described herein, such as Escherichia coli. In a preferred embodiment of the present invention, the Escherichia coli cells express the genes repA, telN, sopA and sopB from bacteriophage N15.

[0352] In a preferred embodiment of the present invention, the SopA expressed by the polynucleotide sequence comprises:

[0353] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 200, at least 250, at least 275, at least 300, at least 325, at least 350 or at least 375 amino acids of SEQ ID NO: 6; or

[0354] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6; or

[0355] (iii) SEQ ID NO: 6.

[0356] In a preferred embodiment of the present invention, the sopA polynucleotide sequence comprises:

[0357] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 or at least 1100 nucleotides of SEQ ID NO: 7; or

[0358] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7; or

[0359] (iii) SEQ ID NO: 7.

[0360] The sequence variant retains the ability to act as a SopA protein when expressed. Suitable assays for determining SopA protein activity are known to those skilled in the art and include culturing an Escherichia coli strain that replicates a plasmid for multiple generations by inoculating at low density into a nutrient broth flask, growing to high density and repeating for several days. Any plasmid loss can be detected by comparing plasmid preparations from the strain daily by agarose gel electrophoresis or quantitative PCR.

[0361] In a preferred embodiment of the present invention, the SopB expressed by the polynucleotide sequence comprises:

[0362] (i) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 200, at least 250, at least 275, at least 300, at least 325 or at least 340 amino acids of SEQ ID NO: 8; or

[0363] (ii) an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 8; or

[0364] (iii) SEQ ID NO: 8.

[0365] In a preferred embodiment of the present invention, the sopB polynucleotide sequence comprises;

[0366] (i) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a continuous fragment of at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 nucleotides of SEQ ID NO: 9; or

[0367] (ii) a polynucleotide sequence that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9; or

[0368] (iii) SEQ ID NO: 9.

[0369] The said sequence variant retains the ability to act as the SopB protein upon expression. Suitable assays for determining SopB protein activity are known to those skilled in the art, including culturing in vitro strains that replicate plasmids for multiple generations by inoculating at low density into a nutrient broth flask, growing to high density and repeating for several days. By comparing plasmid preparations from the strains daily by agarose gel electrophoresis or quantitative PCR, any plasmid loss can be detected.

[0370] In another aspect of the present invention, a method for producing the plasmid embodiments of the present invention is provided, including culturing a host cell as defined herein, lysing the cell and purifying the plasmid from the cell lysate.

[0371] In another aspect of the present invention, eukaryotic cells containing the polynucleotide of the present invention or the plasmid embodiments of the present invention are provided.

[0372] Embodiment

[0373] Example 1

[0374] Construct plasmids pBITTdtA( Figure 5 A) and pBITTraB2( Figure 5 B) to express pore TdtA and TraB respectively to determine whether they are membrane-associated in human cells.

[0375] To generate pBITTdtA, the pBITREPB plasmid (see Example 4) was cut with SpeI and XbaI and ligated with the tdtA cistron flanked by SpeI and XbaI restriction sites. To generate pBITTraB, the traB cistron was inserted into the SpeI and XbaI sites of the pBITREPB plasmid. Then, the PCR-generated FLAG tag was added to the 3'-end of the traB cistron by ligating BamHI-XbaI to pBITTraB to create pBITTraB2.

[0376] Human embryonic kidney cells (HEK 293) were cultured on glass coverslips in standard six-well plates. When the cell confluence reached 70 - 80%, they were transfected with 1 μg of pBITTraB2 or pBITTdtA using TurboFect reagent (Life Technologies, UK) according to the manufacturer's instructions. As a negative control, we used the pMCPK plasmid that expresses mCherry but lacks the pore cassette. After 72 hours, the cells were incubated with 200 μg / ml of wheat germ agglutinin (WGA) conjugated with Alexa Fluor 647 (Invitrogen) dissolved in DMEM medium containing 10% FBS at 37 °C and 5% CO2 for 30 minutes, and then washed three times with PBS. Next, the cells were fixed with a phosphate-buffered saline (PBS) solution of 4% paraformaldehyde (PFA; Merck, UK) for 10 minutes at room temperature and washed three times with PBS. The cells were permeabilized by incubation in PBS containing 0.05% Triton X-100 (PBST) for 15 minutes and then blocked in PBST containing 10% FBS for 1 hour. The primary antibody: mouse anti-FLAG (Merck, UK) was applied at a dilution of 1:1000 in PBST-10% FBS and incubated for 1 hour at ambient temperature. The unbound antibody was removed by washing three times with PBST, and then the secondary antibody (goat anti-mouse, Alexa Fluor 488; Abcam, UK) was applied at a dilution of 1:1000 for 1 hour at ambient temperature. The unbound antibody was removed by washing three times with PBST, the first wash containing 0.1 μg / ml DAPI (Life Technologies, UK), and then the coverslips were mounted with FluorSave reagent (EMD Millipore, USA), left to dry in the dark, and photographed at 63-fold magnification.

[0377] Figure 5 The micrographs in Figure 5 C-TdtA, Figure 5 D-TraB) co-localization with WGA cell membrane staining (red).

[0378] Example 2

[0379] An experiment was conducted to determine whether a plasmid with the membrane pore TdtA could transfer plasmid DNA to adjacent cells compared to the control plasmid pMCPK ( Figure 10 B) lacking the pore.

[0380] HEK 293 cells were cultured in six-well plates on glass coverslips with Dulbecco's Modified Eagle Medium (DMEM - GlutaMAX; Life Technologies, UK) and 10% fetal bovine serum (FBS; Merck, UK) until reaching a density of 70 - 80%. Cells were transfected with 1 μg of each plasmid DNA expressing mCherry (pMCPK (negative control) or pBITTdtA (TdtA-expressing wells)). After 24 hours, cells were transfected with 0.5 μg of pdClover2 - N1()( Figure 11 A) (Addgene, USA) that expresses green fluorescent protein Clover2. After another 48 hours, the cells were rinsed with PBS buffer and fixed with 4% PFA for 10 minutes, then washed three times with PBS for 5 minutes each to remove PFA. Then the cells were permeabilized with PBS containing 0.05% TritonX - 100 for 15 minutes and blocked with PBS, 0.05% TritonX - 100, and 10% FBS for one hour. The cells were incubated with a 1:1000 rabbit anti - mCherry antibody (Abcam, UK) in PBS containing 0.05% TritonX - 100 and 10% FBS at room temperature for 1 hour, then washed three times with PBS containing 0.05% TritonX - 100 for 10 minutes each. The cells were incubated with a 1:1000 goat anti - rabbit antibody Alexa Fluor 594 (Abcam, UK) in PBS containing 0.05% TritonX - 100 and 10% FBS for 1 hour (protected from light). The coverslips were mounted with FluorSave reagent (Merck, UK) and left to dry in the dark.

[0381] The total number of cells expressing both mCherry and Clover2 was recorded. For the two mCherry plasmids, these cell numbers were equal, approximately 25% of the total cells, indicating equal transfection efficiency( Figure 6 A). Then the number of Clover2 cells (green) adjacent to the double - fluorescent cells was recorded. These cell numbers were significantly (T - test, p = 0.049) in excess for pBITTdtA compared to pMCPK. This indicates that cells containing pBITTdtA secrete pdClover2 - N1.

[0382] Example 3

[0383] An experiment was conducted using plasmids with and without the clt locus required for TraB to secrete DNA to test the specificity of output through TraB pores.

[0384] Plasmid pCMV - Clover2 - CLT( Figure 11B) was constructed by synthesizing the clt locus (SED ID NO: 26; ThermoFisher, Germany) and cloning it into a single AseI site of pdClover2-N1.

[0385] HEK 293 cells were cultured on glass coverslips in six-well plates with Dulbecco's Modified Eagle Medium (DMEM-GlutaMAX; Life Technologies, UK) and 10% fetal bovine serum (FBS; Merck, UK) until they reached a density of 70-80%. The cells were first transfected with 1 μg of pBITTraB2, which expresses mCherry and TraB pores. After 24 hours, the cells were transfected with 0.5 μg of a plasmid expressing green fluorescent protein Clover2: the negative control pdClover2-N1 or pCMV-Clover2-CLT, which additionally contains the clt locus. After another 48 hours, the cells were rinsed with PBS buffer and fixed with 4% PFA for 10 minutes, then washed three times with PBS for 5 minutes each to remove the PFA. The cells were then permeabilized with PBS containing 0.05% TritonX-100 for 15 minutes and blocked with PBS, 0.05% TritonX-100, and 10% FBS for one hour. The cells were incubated with a 1:1000 rabbit anti-mCherry antibody (Abcam, UK) in PBS containing 0.05% TritonX-100 and 10% FBS at room temperature for 1 hour, then washed three times with PBS containing 0.05% TritonX-100 for 10 minutes each. The cells were incubated with a 1:1000 goat anti-rabbit antibody Alexa Fluor 594 (Abcam, UK) in PBS containing 0.05% TritonX-100 and 10% FBS for 1 hour (protected from light). The coverslips were mounted with FluorSave reagent (Milipore EMD, USA) and allowed to dry.

[0386] The total number of cells containing both pBITTraB2 and Clover2 plasmids was recorded, and it was found that the proportion of pCMV-Clover2-CLT in double-fluorescent cells was higher than that of pdClover2-N1 (T-test, p = 0.029) - one explanation for this is the import of the plasmid containing clt from adjacent cells ( Figure 7 A). Then the number of Clover2 cells (green) adjacent to double-fluorescent cells was recorded, and the number of Clover2 cells of pCMV-Clover2-CLT was significantly in excess compared to pdClover2-N1 (p = 0.0046), which may indicate that the former is preferentially exported from cells containing TraB pores ( Figure 7 B).

[0387] Example 4

[0388] Western blotting was performed to detect the expression of two components of the N15 replication system, RepA and TelN, and the pore proteins TraB and TdtA in human cell cultures.

[0389] Two synthetic gene cassettes were generated, one in plasmid pET5R containing the EF-1a promoter upstream of telN and the 5' end of the repA gene separated by a P2A peptide, and the other in p3RTmP containing the 3' end of repA followed by an IRES (Internal Ribosome Entry Site) element and traB. They were digested with HindIII and NdeI (all restriction enzymes were from NEB, UK) and ligated to create pBITREPB. pBITREPA was generated using pBITREPB by replacing the traB cistron with a synthetic tdtA with a C-terminal FLAG tag as an NheI-XbaI fragment. Since the IRES could not express either pore cistron, pBITREPA and pBITREPB were further modified by replacing the IRES with an E2A peptide, which was done by synthesizing a region encoding the C-terminus of repA, E2A, and the N-terminal region of the pore cistron (the HindIII-Bsu36I fragment for tdtA and the HindIII-PpuMI fragment for traB); they were ligated into pBITREPA and pBITREPB cut with the same enzymes to create plasmids pBITREPA2( Figure 8 A) and pBITREPB2( Figure 8 B).

[0390] The purified plasmid was transfected into low passage HEK 293 cells, and the transfection efficiency was visually verified by detecting mCherry fluorescence under a microscope 72 hours after transfection. The cells were then rinsed with PBS, lysed in radioimmunoprecipitation assay (RIPA) buffer on ice, then heat-denatured (100 °C for 5 minutes) and reduced with dithiothreitol (DTT), and then size-separated in a sodium dodecyl sulfate (SDS)-acrylamide gel. After electrophoresis, the proteins were electrotransferred onto a nitrocellulose membrane, blocked for 1 hour in Tris-buffered solution containing 3% non-fat milk (containing 0.1% Tween20 detergent (TBST)), and then immunoblotted with the following antibodies at a dilution of 1:1000: mouse anti-mCherry (Abcam, UK); anti-2A peptide (Merck, UK), anti-V5 tag (Abcam, UK) and anti-FLAG (Abcam, UK), blocked for 1 hour at ambient temperature. Next, the membrane was washed three times with TBST for 10 minutes each, and a secondary antibody was applied for 1 hour: goat anti-mouse antibody conjugated with alkaline phosphatase (Abcam, UK) was diluted at a ratio of 1:1000 in a TBST solution of 3% milk. The signal was developed by applying WesternBlue stabilized alkaline phosphatase substrate (Promega, UK). The developed membrane was photographed and the protein size was verified by reference to a prestained protein ladder broad molecular weight (10 - 245 kDa) (Abcam, UK).

[0391] Figure 9 The results showed that RepA, TelN, TraB and TdtA were expressed in the human cell line.

[0392] Example 5

[0393] Experiments were conducted to determine whether a plasmid with N15 replication functional components and a DNA secretion pore gene could be transferred to adjacent cells compared to a control plasmid lacking pores.

[0394] HEK 293 cells were cultured on glass coverslips in six-well plates with Dulbecco's Modified Eagle Medium (DMEM-GlutaMAX; Life Technologies, UK) and 10% fetal bovine serum (FBS; Merck, UK) until they reached a density of 70 - 80%. The cells were transfected with 1 μg of Gentrafix plasmids pBITREPA2 (TelN, RepA, and TdtA wells) and pBITREPB2 (TelN, RepA, and TraB wells), plus the negative control plasmid pMCPK (without Gentrafix components); all plasmids also expressed the red fluorescent reporter gene mCherry. After 72 hours, the cells were rinsed with PBS buffer and fixed with 4% PFA for 10 minutes, then washed three times with PBS for 5 minutes each to remove the PFA. The cells were then permeabilized with PBS containing 0.05% TritonX-100 for 15 minutes and blocked with PBS, 0.05% TritonX-100, and 10% FBS for one hour. The cells were incubated with a 1:1000 rabbit anti-mCherry antibody (Abcam, UK) in PBS containing 0.05% TritonX-100 and 10% FBS at room temperature for 1 hour, then washed three times with PBS containing 0.05% TritonX-100 for 10 minutes each. The cells were incubated with a 1:1000 goat anti-rabbit antibody Alexa Fluor 594 (Abcam, UK) in PBS containing 0.05% TritonX-100 and 10% FBS for 1 hour (protected from light). The coverslips were mounted with FluorSave reagent (Milipore EMD, USA) and allowed to dry.

[0395] Micrographs representing 30 different fields of view were taken randomly, and the total number of red blood cells (expressing mCherry) and the number of clusters per image were analyzed ( Figure 12 A - D). Figure 12 E shows a representative image with a cluster indicated by an arrow.

[0396] Compared with the negative control plasmid, a greater total number of red cells were seen in the Gentrafix plasmids ( Figure 12 A; t-test for pBITREPA2, p = 3.44x10 -8 ; t-test for pBITREPB2, p = 2.64x10 -14 ), and a greater total number of clusters were formed ( Figure 12 B; t-test for pBITREPA2, p = 3.51x10 -10 ; t-test for pBITREPB2, p = 3.34x10 -19)。Compared to the negative control, the number ( Figure 12 C; T-test for pBITREPA2, p = 5.06x10 -14 ; T-test for pBITREPB2, p = 3.54x10 -16 ) and proportion ( Figure 12 D; T-test for pBITREPA2, p = 1.30x10 -13 ; T-test for pBITREPB2, p = 5.21x10 -21 ) of Gentrafix mCherry total cells present in clusters were significantly greater. Among the two plasmids, pBITREPB2 expressing the TraB pore produced more transfected cells and cell clusters than pBITREPA2 expressing the TdtA pore.

[0397] The higher incidence of clusters with DNA secretion pores supports the evidence of intercellular DNA transfer via the Gentrafix platform. It was expected that the smaller control plasmid would represent a higher proportion of transfected cells as it has a higher copy number per unit mass and smaller plasmids are more efficiently taken up by cells, yet the Gentrafix plasmid was more abundant.

[0398] Example 6

[0399] Experiments were conducted to detect the direct transfer of plasmids from one cell line to another.

[0400] Low-density HEK 293 cells were transfected with pBITREP( Figure 10 A), pBITREPA2( Figure 8 A) and pBITREPB2( Figure 8 B). After 24 hours, the plasmid-containing medium was removed, the cells were washed twice, and then fresh medium containing DNaseI was applied. After another 24 hours, the cell line HEK 293GFP (amsbio, UK) containing the chromosomally integrated green fluorescent protein (GFP) gene was added to each culture and incubated for 72 hours.

[0401] The mixed culture was fixed with PBS solution containing 4% PFA, blocked with 10% FBS, and incubated with primary antibodies (mouse anti-GFP and rabbit anti-mCherry, 1:500) at 4°C for 24 hours, followed by application of secondary antibodies (goat anti-mouse AF488 and goat anti-rabbit AF594). Images were obtained using a Kern & Sohn OCM 167 fluorescence microscope. White arrows indicate cells expressing only mCherry, while dashed arrows show cells co-expressing GFP and mCherry - the latter being the result of intercellular gene transfer, visible only in pBITREPA2 and pBITREP2 but not in pBITREP lacking the pore gene( Figure 13 ).

[0402] Example 7

[0403] Experiments were conducted to detect the direct transfer of plasmids from one cell line to another through connections between two cell lines.

[0404] Confluent adherent MDCK-GFP cells (Innoprot, Spain) were trypsinized, washed with fresh DMEM / FBS medium containing trypsin, and resuspended in 1 ml of fresh DMEM / FBS medium. MDCK-GFP cells are Madin-Darby canine kidney cells that constitutively express GFP by chromosomally integrated genes. 20 μl of the cell suspension was applied to each well of a 6-well plate, and the cells were then incubated overnight (37°C, 5% CO2) until they reattached. Next, MDCK-GFP cells were transfected with 1 μg of pMCPK and pBITREPB2 plasmids. The next day, the medium was replaced with fresh DMEM / FBS containing 5 units / ml of DNaseI for two hours. Then the transfected MDCK-GFP cells were overlaid with non-fluorescent MDCK cells (UKHSA, UK). After the MDCK cells reattached, 3 ml of fresh DMEM / FBS was added, and the cells were incubated for another 48 hours. Then the cells were fixed with 4% PFA, permeabilized, and treated with anti-mCherry antibody. These images were taken using a Kern & Sohn OCM167 fluorescence microscope.

[0405] The negative control pMCPK plasmid remained in the initially transfected MDCK-GFP cells (all red cells also showed green), while in Figure 14 the Gentrafix plasmid pBITREPB2 diffused into adjacent MDCK cells (cells visible in the merged micrograph but not in the GFP micrograph).

[0406] Example 8

[0407] Experiments were conducted to determine which components of the TraB-based Gentrafix system (repA, telN, traB, and clt loci) are essential by constructing luciferase expression plasmids containing these components, as well as additional plasmids omitting one or more of each component.

[0408] The firefly luciferase gene was cloned into the negative control plasmids pMCPK and pBITREP, as well as the Gentrafix plasmid pBITREP2. This was achieved by digesting with the BstBI and AvrII restriction enzymes to remove the mCherry-puromycin resistance gene cassette, and then ligating a de novo synthesized firefly luciferase cistron flanked by BstBI and AvrII restriction sites on both sides. The new plasmids were pLUCK (without Gentrafix genes), pLUCKREP (telN and repA), and pLUCKB (telN, repA, traB, clt).

[0409] Next, plasmids lacking Gentrafix components were generated using pLUCKB. To generate a plasmid lacking the clt locus, pLUCKB was cut with AvrII and BstBI, blunt-ended using the NEB Quick Blunting Kit (NEB, UK), and then self-ligated to produce pLUCKCB (telN, repA, traB).

[0410] To create a plasmid lacking the N15 replication components repA and telN (i.e., containing traB and clt), pLUCKB was cut with SpeI and MreI to remove the 5' part of traB as well as the repA and telN cistrons, and the plasmid pBITTraBclt containing traB was cut with the same enzymes to release the N-terminus of traB, which was then ligated to restore traB, generating the plasmid pLUCKOB (traB, clt).

[0411] A plasmid lacking repA was constructed as follows: pLUCKB was cut with MreI, which removed the 3' end of telN, all of repA, and the 5' end of traB - then the synthetic MreI-cut fragment "NoRepA" was ligated into it to restore the telN and traB cistrons and insert the P2A peptide sequence, generating the plasmid pLUCKTB (telN, traB, clt).

[0412] A telN - deficient plasmid was created by cutting pLUCKB with SpeI and SbfI (removing the 5'-ends of telN and repA). Subsequently, using pLUCKB as a template, PCR products were generated with primers NoTelNRepA (ATAGGACTAGTGCCGCCACCATGACCTTACAAGAATTCTACGCGG) and NoTelNR (GCGCCCCCTGCAGGTCGCCA). The PCR products (containing the 5'-end of repA) were digested with SpeI and SbfI and ligated into pLUCKB. The luciferase plasmids are as Figure 15 shown.

[0413] For luciferase assays, HEK 293 cells were seeded in white, clear-bottom 96-well plates, four plates as a set, and cultured in a tissue culture incubator at 37 °C and 5% CO2 until 50% confluence was reached. Subsequently, cells were transfected with equal copy numbers of each plasmid (equivalent to 200 ng pLUCKB). Over the next four days, the cell culture medium in one plate was replaced daily with fresh medium supplemented with 150 μg / ml luciferin in each well, and then luminescence was measured using a GloMax microplate reader. The signal integration time was empirically set to 10 seconds on day 0 (six hours after transfection). Data obtained on subsequent days were first normalized to the luminescence of untransfected control cells and then to the luminescence of each plasmid observed on day 0. Each plasmid was analyzed by five biological replicates ( Figure 16 ).

[0414] Plasmids containing both repA and traB as well as clt, namely pLUCKB (telN, repA, traB, clt) and pLUCKRB (repA, traB, clt), showed progressively more pronounced luminescence signals throughout the experiment, which were associated with replication and intercellular transfer, demonstrating the importance of the combination of DNA replication components and DNA secretion pores. In contrast, four plasmids lacking repA and traB showed minimal increases in fluorescence during the experiment. The relative effect of the clt locus was seen by comparing pLUCKB (telN, repA, traB, clt) with pLUCKCB (repA, traB, clt): the latter plasmid lacking the clt locus produced a significantly lower signal than pLUCKB but higher than plasmids lacking repA and traB, indicating that the clt locus enhanced intercellular transfer but was not essential for its achievement. A decrease in cell viability in all cultures led to a reduction in all fluorescence signals on day 5.

[0415] Sequence Listing

[0416] SEQ ID NO:1 - N15 RepA amino acid sequence

[0417] MTLQEFYAERFGSDPFSLLDAARDELTELAKMAGINWPACADKIQLNPRGGVERYTTYNNSSPEALQKSL

[0418] KGRVEIYSRLEQSKDGISYPFVNFVEKAHDAGSWSGFSFLFSEYRREQQRNGATVVAQPEEERARMERQA

[0419] EARRLRAEQQRVNDLKNNQMEQERLLGWLAFHRAWEHSPAEDGSWPYAVKKGIRDVFGACDIRRVTSH

[0420] DSAKWSRGPTTYMAIPLSHLDGRKDGRIVGWQRIDLNGGKFQTSAITNGDFVGACFVIGDLQGAQKIATA

[0421] EGFATGASIWLATRNDPKKRFDAVVIAVSANNMIHVVEQLVNMYPAAQITCALDNDRKSSAEGKGNTGL

[0422] RTGFDIMEKFSGVKCVYPTFEDDPELECSDFNDLHSLRGLKEVARQLTRNHLSRATDLLSITLNKLRTLPRL

[0423] NRRTFAKELLRAVDIGMLTCPVPNSPKELMRLFSSTLRDMGIAEIYNGTVKDHITRRLNRKCRAAQTSRSF

[0424] SERITNPNLRPSHITYKRFETSRMTDEVMTYAAQLQGIVIVRAGMGSGKSTGLLRPLMLQSTRGVSVAHR

[0425] VSLIGGLHEMMTEGKGAKADILHYQDPGYQEMAPYANKLTICINSILKGCWQPLMRQHDFFGFDEATQG

[0426] LRAILAGRAMENPVGVFNTLIDALARTEEHAIMVDADANDLLVDLAELAMKRREELGLPAWLQIHVIELP

[0427] VDVRNRETNKPIRVFYTEKNRIMTEVIAAVQRGERIMLATDSSTFAEDVTMQLRLQFPDKKFLCVNQKNK

[0428] QEKEVDDFTNQPKVMVKKYDGLIYSPSISSGVSIEEKHFHRHFGMFCGEVVPSDAIQMLRRDRTAQEYIIG

[0429] FDKLRGKRETDPEKIKRAYAQALLETAGHSGLLTDVVFDGDRISLGVANSSFMQLKIKAAALEASARNDY

[0430] ASNMICIMHDDGYQVAPMATDALANSIGKDLRKEARELVFEQLMERHLSVDTPDQAEHDELIKKRTLSL

[0431] DEQAQLVRWDIEKELQLDVDEVALKFYFDGGLKKVRLFETMQLDEITARRLDREEALIHFTYAYRVAGR

[0432] WQQFVTTAMTREQADAEFQAKFPAITDYRVKSTPAVEIGMRGFYTLKSATLQQYFRDCGIDPKTLEGEAD

[0433] MDALKRARDNLLTPERRDLLNNVLRIGGFNTEKGKKKAPADLCIGILESMGLSSKTRRARDGDARPTMR

[0434] SIDPDSVEFLMNIVEKRREAGLSIHARKVEKTTIEVDRDLDLNIDIHGNPRSKTEHVPDAPQSVIIQALEAIP

[0435] VAVPEAWAENALPATEMEAVRLWPVASIARTFASLYMTEFMDLLSVREIRLLKAFLSQRQAVAI

[0436] SEQ ID NO:2 - N15 repA cistron

[0437]

[0438] SEQ ID NO:3 - N15 TelN amino acid sequence

[0439] MSKVKIGELINTLVNEVEAIDASDRPQGDKTKRIKAAAARYKNALFNDKRKFRGKGLQKRITANTFNAYMSRARKRFDDKLHHSFDKNINKLSEKYPLYSEELSSWLSMPTANIRQHMSSLQSKLKEIMPLAEELSNVRIGSKGSDAKIARLIKKYPDWSFALSDLNSDDWKERRDYLYKLFQQGSALLEELHQLKVNHEVLYHLQLSPAERTSIQQRWADVLREKKRNVVVIDYPTYMQSIYDILNNPATLFSLNTRSGMAPLAFALAAVSGRRMIEIMFQGEFAVSGKYTVNFSGQAKKRSEDKSVTRTIYTLCEAKLFVELLTELRSCSAASDFDEVVKGYGKDDTRSENGRINAILAKAFNPWVKSFFGDDRRVYKDSRAIYARIAYEMFFRVDPRWKNVDEDVFFMEILGHDDENTQLHYKQFKLANFSRTWRPEVGDENTRLVALQKLDDEMPGFARGDAGVRLHETVKQLVEQDPSAKITNSTLRAFKFSPTMISRYLEFAADALGQFVGENGQWQLKIETPAIVLPDEESVETIDEPDDESQDDELDEDEIELDEGGGDEPTEEEGPEEHQPTALKPVFKPAKNNGDGTYKIEFEYDGKHYAWSGPADSPMAAMRSAWETYYS

[0440] SEQ ID NO:4 - N15 telN cistron

[0441]

[0442] SEQ ID NO:5 - N15 telRL

[0443] TATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA

[0444] SEQ ID NO:6 - N15 SopA amino acid sequence

[0445] MSLINLLKDCINRGQEMTRAIAIAQFGDDSPEARRITRRWGITEVADLIGVTPQAIRDAEKAGRLPAPDFEMRGRVERRAGYTIDQISHMRSVFGNPNQRPDDKNPVVLSVMSHKGGVYKTSSAVHQAQWLALQGHRVLLVEGNDPQGTASMYHGYVPDLHIHADDTLLPFYLGKRDNAEYAIKPTCWPGLDIIPSCLALHRIETDLMQYHSEGKLPHPPHLMLRAAIESVWDNYDIIVIDSAPNLGTGTINVVCAADVIVVATPAELFDYASVLQFFTMLLDLLETVDLGGFEPVVRLLLTKYSLTNGNQSRWMEEQIRNTWGAMVLRQVVRVTDEVGKGQIKMRTVFEQAANQRSTLNAWRNAVEIWEPVCKEIFEDLIKPRWED

[0446] SEQ ID NO:7 - N15 sopA cistron

[0447]

[0448] SEQ ID NO:8 - N15 SopB amino acid sequence

[0449] MKNRSILKNAPNIETFMSNNHNAPQKAPSVSPMVGDLQSKLSSLSGNSITLPVCGRNVTFKLETIPADKVEKATMVWLGNERDQELLNESALADLIPSFLTSGQQNPAFARRTSGIIEIADGSRRRKTAIITGSDYRVLVGELDDEQMQQLSQLGNDYRPTSAYERGKRYLRRLKEFDGNVKALAEAEGIDRNIVNRCMNTAGLPREILSIFKHPGELSARAGDALSKVYKGNEQTMLDGAKQLLRMKQAGEDFEPARIIQALQDFILVDKEEMPKTEKKYGEGVVAKYKGSFVTLKVDSRKIPSNLIKKIEALLEAELGAAEQVNRDLDKLENIIKNKEKK

[0450] SEQ ID NO:9 - N15 sopB cistron

[0451]

[0452] SEQ ID NO:10 - Amino acid sequence of Phi29 DNA - dependent DNA polymerase (gene 2)

[0453] MPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK

[0454] SEQ ID NO:11 - Phi29 DNA - dependent DNA polymerase cistron (gene 2)

[0455]

[0456] SEQ ID NO: 12 - Amino acid sequence of the Phi29 terminal protein (gene 3)

[0457] MARSPRIRIKDNDKAEYARLVKNTKAKIARTKKKYGVDLTAEIDIPDLDSFETRAQFNKWKEQASSFTNRANMRYQFEKNAYGVVASKAKIAEIERNTKEVQRLVDEKIKAMKDKEYYAGGKPQGTIEQRIAMTSPAHVTGINRPHDFDFSKVRSYSRLRTLEESMEMRTDPQYYEKKMIQLQLNFIKSVEGSFNSFDAADELIEELKKIPPDDFYELFLRISEISFEEFDSEGNTVENVEGNVYKILSYLEQYRRGDFDLSLKGF

[0458] SEQ ID NO: 13 - Phi29 terminal protein cistron (gene 3)

[0459] ATGGCGAGAAGTCCACGTATACGCATTAAGGATAATGACAAAGCCGAATACGCTCGATTGGTCAAGAATACAAAAGCCAAGATTGCGAGAACGAAGAAAAAGTATGGTGTAGACCTTACCGCTGAAATTGATATACCTGACCTTGATTCATTTGAAACACGGGCGCAGTTCAATAAGTGGAAGGAACAAGCGTCCTCTTTCACTAACCGTGCTAATATGCGTTATCAGTTCGAAAAGAATGCATACGGTGTGGTGGCTAGTAAAGCTAAGATAGCTGAGATTGAACGTAACACAAAAGAGGTTCAGCGGTTAGTAGATGAGAAAATCAAGGCTATGAAAGACAAAGAATACTATGCAGGCGGTAAGCCGCAAGGGACAATTGAACAACGGATAGCTATGACAAGTCCTGCACACGTTACAGGAATTAATAGACCCCATGATTTTGACTTTAGCAAGGTGCGAAGCTATAGCCGTTTGCGAACCCTAGAAGAAAGCATGGAGATGAGAACAGACCCTCAGTATTATGAAAAGAAAATGATACAGTTACAGTTAAACTTTATTAAGAGCGTTGAGGGTAGTTTCAATTCATTTGATGCGGCAGATGAACTGATCGAAGAATTAAAAAAGATACCTCCTGATGACTTCTATGAATTGTTTCTCAGAATATCAGAAATATCCTTTGAGGAATTTGATAGTGAGGGAAACACAGTGGAGAACGTAGAAGGTAATGTATATAAAATACTGTCATACTTGGAACAGTATCGAAGGGGTGACTTTGATCTAAGCTTAAAGGGGTTCTAG

[0460] SEQ ID NO:14 - Amino acid sequence of Phi29 single - stranded DNA - binding protein (gene 5)

[0461] MENTNIVKATFDTETLEGQIKIFNAQTGGGQSFKNLPDGTIIEANAIAQYKQVSDTYGDAKEETVTTIFAADGSLYSAISKTVAEAASDLIDLVTRHKLETFKVKVVQGTSSKGNVFFSLQLSL

[0462] SEQ ID NO:15 - Phi29 single - stranded DNA - binding protein cistron (gene 5) ATGGAAAACACAAACATCGTAAAGGCTACTTTTGACACAGAAACTCTTGAAGGACAAATCAAAATCTTTAATGCTCAGACAGGCGGCGGACAATCTTTTAAAAACCTTCCAGATGGAACAATTATAGAAGCCAACGCCATTGCTCAATATAAGCAAGTGTCCGATACATACGGGGACGCTAAGGAAGAAACAGTTACTACTATTTTTGCGGCTGACGGGTCGTTATATTCCGCTATCTCTAAGACTGTAGCAGAAGCCGCATCTGACTTAATTGACCTTGTGACTCGTCATAAGCTTGAAACGTTTAAGGTTAAAGTGGTTCAAGGAACATCTAGTAAAGGTAACGTATTCTTTAGCTTACAACTATCCCTATAA

[0463] SEQ ID NO:16 - Amino acid sequence of Phi29 double - stranded DNA - binding protein (gene 6)

[0464] MAKMMQREITKTTVNVAKMVMVDGEVQVEQLPSETFVGNLTMEQAQWRMKRKYKGEPVQVVSVEPNTEVYELPVEKFLEVATVRVEKDEDQEEQTEAPEEQVAE

[0465] SEQ ID NO:17 - Phi29 double - stranded DNA - binding protein cistron (gene 6)

[0466] ATGGCAAAAATGATGCAGAGAGAAATCACAAAGACAACCGTCAACGTTGCCAAAATGGTGATGGTGGACGGAGAGGTTCAGGTAGAGCAACTACCATCTGAAACATTTGTGGGTAATCTGACAATGGAACAGGCTCAATGGAGAATGAAGCGCAAATATAAAGGCGAACCTGTTCAAGTGGTAAGCGTTGAACCTAACACAGAGGTTTATGAGCTACCTGTAGAAAAATTCCTTGAAGTTGCTACCGTTCGGGTAGAGAAAGACGAAGATCAAGAGGAACAAACAGAAGCTCCAGAAGAACAGGTTGCTGAATGA

[0467] SEQ ID NO: 18 - Amino acid sequence of adenovirus 5 DNA - dependent DNA polymerase

[0468]

[0469] SEQ ID NO: 19 - Adenovirus 5 DNA - dependent DNA polymerase cistron

[0470]

[0471] SEQ ID NO: 20 - Amino Acid Sequence of Adenovirus 5 Precursor Terminal Protein (pTP)

[0472] MALSVNDCARLTGQSVPTMEHFLPLRNIWNRVRDFPRASTTAAGITWMSRYIYGYHRLMLEDLAPGAPATLRWPLYRQPPPHFLVGYQYLVRTCNDYVFDSRAYSRLRYTELSQPGHQTVNWSVMANCTYTINTGAYHRFVDMDDFQSTLTQVQQAILAERVVADLALLQPMRGFGVTRMGGRGRHLRPNSAAAAAIDARDAGQEEGEEEVPVERLMQDYYKDLRRCQNEAWGMADRLRIQQAGPKDMVLLSTIRRLKTAYFNYIISSTSARNNPDRRPLPPATVLSLPCDCDWLDAFLERFSDPVDADSLRSLGGGVPTQQLLRCIVSAVSLPHGSPPPTHNRDMTGGVFQLRPRENGRAVTETMRRRRGEMIERFVDRLPVRRRRRRVPPPPPPPEEEEGEALMEEEIEEEEEAPVAFEREVRDTVAELIRLLEEELTVSARNSQFFNFAVDFYEAMERLEALGDINESTLRRWVMYFFVAEHTATTLNYLFQRLRNYAVFARHVELNLAQVVMRARDAEGGVVYSRVWNEGGLNAFSQLMARISNDLAATVERAGRGDLQEEEIEQFMAEIAYQDNSGDVQEILRQAAVNDTEIDSVELSFRLKLTGPVVFTQRRQIQEINRRVVAFASNLRAQHQLLPARGADVPLPPLPAGPEPPLPPGARPRHRF

[0473] SEQ ID NO: 21 - Adenovirus 5 Precursor Terminal Protein (pTP) Cistron

[0474]

[0475] SEQ ID NO: 22 - Amino acid sequence of Adenovirus 5 DNA binding protein (DBP)

[0476] MASREEEQRETTPERGRGAARRPPTMEDVSSPSPSPPPPRAPPKKRMRRRIESEDEEDSSQDALVPRTPSPRPSTSAADLAIAPKKKKKRPSPKPERPPSPEVIVDSEEEREDVALQMVGFSNPPVLIKHGKGGKRTVRRLNEDDPVARGMRTQEEEEEPSEAESEITVMNPLSVPIVSAWEKGMEAARALMDKYHVDNDLKANFKLLPDQVEALAAVCKTWLNEEHRGLQLTFTSNKTFVTMMGRFLQAYLQSFAEVTYKHHEPTGCALWLHRCAEIEGELKCLHGSIMINKEHVIEMDVTSENGQRALKEQSSKAKIVKNRWGRNVVQISNTDARCCVHDAACPANQFSGKSCGMFFSEGAKAQVAFKQIKAFMQALYPNAQTGHGHLLMPLRCECNSKPGHAPFLGRQLPKLTPFALSNAEDLDADLISDKSVLASVHHPALIVFQCCNPVYRNSRAQGGGPNCDFKISAPDLLNALVMVRSLWSENFTELPRMVVPEFKWSTKHQYRNVSLPVAHSDARQNPFDF

[0477] SEQ ID NO: 23 - Cistron of Adenovirus 5 DNA binding protein (DBP)

[0478]

[0479] SEQ ID NO: 24 - Amino Acid Sequence of Streptomyces venezuelae pSVH1 TraB

[0480] MTEHLTEKNPAPADFDSIATVTFLRPTLPAPASVPEDNTWSDMPDDTDPAVVIPPMPTVAPAIGSDERTVVLETARVILDKTPAVEPVVRPGRFAGTVDLDDFDDAPLIPAWMYSAEGWAAWAGVFYRARRRDFRRWVRRQPTQHGHVRQFGRGTRRAHEWVVGFEGVRVQSAAHTAHVLTREARLAARHARFTPRILGSKKELAMKAADKATKAAEEAVLLHKKAKKDRNRVRNLRAGVVYGPPLTAIGTGYVMGGGLGLAAGLLSTFAGGAFVGRKPYDEEADWTSDWRSLGDGDRMTAPMLDASFRAAKVIGAEETLGVVQMPMLDTRGAWTAVLDLPPGVPAKKAIRATDELAAAFGVEEAQVSVAKRGRAGRIELYVARELPFTDKAAPGPLLALESAADFWGRISIGPDVRGIHQSISVVERSGLIGGEPGAGKSASGNTILLAAALDPRVILWLADGKGGGDLEPFEHLCERYEGDADPEAFNAMLDELLDVMKARYALLKKLGKRKVTEELANKYPELRQLLLWVDELMLYTTAEEFGKKITRKLRNLVSRGRAAGIITFCATQKPGSDVVDTSLRDLLSIRWALRCTTPEASDTILGKGAAASGYSAKTIQSEMRGAGLLWAEGTNPTMVRADYYDDDQVTALIERATEWRRKAGTLPNGPVPLVDQLRARGDDDALLLAAVLEVFAAQATEDDPVDWLPGQLLVDELKAAGHSVTAEKLGALVVRTDEEKAKRPWGEKKTRVTGYPLARVEAAATDRFGLTA

[0481] SEQ ID NO: 25 - Streptomyces venezuelae pSVH1 traB Cistron

[0482]

[0483] SEQ ID NO:26 - Streptomyces venezuelae pSVH1 clt locus

[0484] TTCCGACCCGGCCGCGACCCGGACAGGCGCCCGGATTGACCCGGAATGCCCCCGGGCGGACCGGGCCCCGACCCGGACGCCGGAGTGCCCGACCCGGACGCCCGCGTCCGGGTCGGGCCCGGA

[0485] SEQ ID NO:27 - Streptomyces venezuelae pSVH1 clt repeat sequence

[0486] GACCCGGA

[0487] SEQ ID NO:28 - Thermus thermophilus TdtA amino acid sequence

[0488] MTDQELLQKRVIGRASATERDPNSADRFAFWIRPGERVNPFDIVAAEHFDGSWTYGLVTNIRHVTDAASHLANFISNDFGESVDEPNTPRQGANVAEVTVLDNDKDIFMPVQSEARVYFADEYGVHVALGIDTMKEKEERTGRKIRVPAGIIRMSNGTEVVAYLDVEYVLGPESAHVNVSGISGLATKTSYIMFLIQSILQTVGASSIATILLNVKYDDLLHIHEPGTLSEEERRMYERMGLRPEPFPEDRVHYLLPWGKHTQVTGRPNVFIPEGEPIPPYKVYAYDLRSTVDKLDLLFSHVPDPWDTLGSLIGEIANGIQNDEPKWRDILTWDDLLSQEPLVKQGIPQKVGNVAASSVGRFLRILRRVVKTRQSGIFVPHLSTRMTTIGRELSRIRGGHVYVVDIARLADEEQTLVFGDILRTIYGLYSGELLLEDEEVELPEKVIIFVDELNKYAPARGEASKSPILEQVLDISERGRSFGIVLFSAQQFLSAVHPRVTGNAATKVLGRTDSVELSDSVYRFLDPDIKMHLTRLEKGELILSHPIYRQPVKVNFPKPPFRQGRVRT

[0489] SEQ ID NO:29 - Thermus thermophilus tdtA cistron

[0490]

[0491] SEQ ID NO: 30 - 2A "Ribosomal skipping" peptide consensus sequence

[0492] GDVEXNPGP

[0493] SEQ ID NO: 31 - E2A "Ribosomal skipping" peptide sequence

[0494] QCTNYALLKLAGDVESNPGP

[0495] SEQ ID NO: 32 - P2A "Ribosomal skipping" peptide sequence

[0496] ATNFSLLKQAGDVEENPGP

[0497] SEQ ID NO: 33 - T2A "Ribosomal skipping" peptide sequence

[0498] EGRGSLLTCGDVEENPGP

[0499] SEQ ID NO: 34 - pBITREP nucleotide sequence

[0500]

[0501] SEQ ID NO: 35 - Nucleotide sequence of pBITREPA2

[0502]

[0503] SEQ ID NO: 36 - Nucleotide sequence of pBITREPB2

[0504]

[0505] SEQ ID NO:37 - TATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA

[0506] SEQ ID NO:38 - TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATA

[0507] SEQ ID NO:39 - TATCAGCACACAATTGCCCATTATA

[0508] SEQ ID NO:40 - CGCGCGTATAATGGACTATTGTGTGCTGATA

[0509] SEQ ID NO:41 - TATCAGCACACAATAGTCCATTATA

[0510] SEQ ID NO:42 - CGCGCGTATAATGGGCAATTGTGTGCTGATA

[0511] SEQ ID NO:43 - TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA

[0512] SEQ ID NO:44 - TATCAGCACACAATTGCCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATA

[0513]

[0514] SEQ ID NO: 46 - Nucleotide sequence of pLUCKREP

[0515]

[0516] SEQ ID NO: 47 - Nucleotide sequence of pLUCKB

[0517]

[0518] SEQ ID NO: 48 - Nucleotide sequence of pLUCKCB

[0519]

[0520] SEQ ID NO: 49 - Nucleotide sequence of pLUCKOB

[0521]

[0522] SEQ ID NO:50 - pLUCKTB nucleotide sequence

[0523]

[0524] SEQ ID NO: 51 - Nucleotide sequence of pLUCKRB

[0525]

[0526] SEQ ID NO: 52 - NoTelNRepA primer

[0527] ATAGGACTAGTGCCGCCACCATGACCTTACAAGAATTCTACGCGG

[0528] SEQ ID NO: 53 - NoTelNR primer

[0529] GCGCCCCCTGCAGGTCGCCA

[0530] Other Embodiments of the Present Invention

[0531] 1. A polynucleotide, comprising:

[0532] a) A polynucleotide sequence encoding a DNA - dependent DNA polymerase; and

[0533] b) A polynucleotide sequence encoding a pore that enables eukaryotic cells to secrete DNA.

[0534] 2. The polynucleotide of embodiment 1, comprising:

[0535] a) An origin of replication;

[0536] b) A polynucleotide sequence encoding a DNA - dependent DNA polymerase;

[0537] c) A polynucleotide sequence encoding:

[0538] i) Telomerase front; or

[0539] ii) Terminal proteins and DNA - binding proteins required for plasmid replication in eukaryotic cells; and

[0540] d) A polynucleotide sequence encoding a pore that enables eukaryotic cells to secrete DNA.

[0541] 3. The polynucleotide of embodiment 2, wherein the origin of replication is from phage N15.

[0542] 4. The polynucleotide of any one of the foregoing embodiments, wherein the DNA - dependent DNA polymerase is from phage N15.

[0543] 5. The polynucleotide of any one of the foregoing embodiments, wherein the DNA - dependent DNA polymerase is encoded by the repA gene.

[0544] 6. A polynucleotide of any one of the foregoing embodiments, wherein the DNA-dependent DNA polymerase expressed by the polynucleotide sequence comprises SEQ ID NO: 1, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and retains the ability to act as a DNA-dependent DNA polymerase.

[0545] 7. A polynucleotide of any one of the foregoing embodiments, wherein the DNA-dependent DNA polymerase polynucleotide sequence comprises SEQ ID NO: 2, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 2 and retains the ability to act as a DNA-dependent DNA polymerase upon expression.

[0546] 8. A polynucleotide of any one of the foregoing embodiments, wherein the telomere primase is TelN from bacteriophage N15.

[0547] 9. A polynucleotide of any one of the foregoing embodiments, wherein the telomere primase expressed by the polynucleotide sequence comprises SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and retains the ability to act as a telomere primase.

[0548] 10. A polynucleotide of any one of the foregoing embodiments, wherein the telomere primase polynucleotide sequence comprises SEQ ID NO: 4, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 4 and retains the ability to act as a telomere primase upon expression.

[0549] 11. A polynucleotide of any one of the foregoing embodiments, further comprising a telRL site, optionally wherein the telRL site polynucleotide sequence comprises SEQ ID NO: 5.

[0550] 12. The polynucleotide of embodiment 1 or embodiment 2, wherein the DNA-dependent DNA polymerase is from bacteriophage Phi29.

[0551] 13. The polynucleotide of embodiment 12, wherein the DNA-dependent DNA polymerase is encoded by gene 2.

[0552] 14. The polynucleotide of embodiment 12 or embodiment 13, wherein the DNA-dependent DNA polymerase expressed by the polynucleotide sequence comprises SEQ ID NO: 10, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 and retains the ability to act as a DNA-dependent DNA polymerase.

[0553] 15. The polynucleotide of embodiment 14, wherein the DNA-dependent DNA polymerase polynucleotide sequence comprises SEQ ID NO: 11, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 11 and retains the ability to act as a DNA-dependent DNA polymerase upon expression.

[0554] 16. The polynucleotide of any one of embodiments 12 to 15, wherein the terminal protein and the DNA-binding protein are the terminal protein TP and the DNA-binding proteins p5 and p6 of a phage of the Phi29 group from Bacillus subtilis.

[0555] 17. The polynucleotide of any one of embodiments 12 to 16, wherein the terminal protein expressed by the polynucleotide sequence comprises SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 and retains the ability to act as a terminal protein.

[0556] 18. The polynucleotide of embodiment 17, wherein the terminal protein polynucleotide sequence comprises SEQ ID NO: 13, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 13 and retains the ability to act as a terminal protein upon expression.

[0557] 19. The polynucleotide of any one of embodiments 16 to 18, wherein the DNA-binding proteins p5 and p6 expressed by the polynucleotide sequence comprise SEQ ID NO: 14 and SEQ ID NO: 16, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NO: 14 and SEQ ID NO: 16 and retain the ability to act as DNA-binding proteins.

[0558] 20. The polynucleotide of any one of embodiments 16 to 19, wherein the DNA-binding protein p5 and p6 polynucleotide sequences comprise SEQ ID NO: 15 and 17, respectively, or polynucleotide sequences that are at least 90% identical to SEQ ID NO: 15 and 17, respectively, and retain the ability to act as DNA-binding proteins upon expression.

[0559] 21. The polynucleotide of embodiment 1 or embodiment 2, wherein the DNA-dependent DNA polymerase is from an adenovirus.

[0560] 22. The polynucleotide of embodiment 21, wherein the DNA-dependent DNA polymerase from the adenovirus is encoded by gene E2B.

[0561] 23. The polynucleotide of Embodiment 21 or Embodiment 22, wherein the DNA-dependent DNA polymerase expressed by the polynucleotide sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 18 and retains the ability to act as a DNA-dependent DNA polymerase.

[0562] 24. The polynucleotide of any one of Embodiments 21 to 23, wherein the DNA-dependent DNA polymerase polynucleotide sequence comprises SEQ ID NO: 19, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 19 and retains the ability to act as a DNA-dependent DNA polymerase upon expression.

[0563] 25. The polynucleotide of any one of Embodiments 21 to 24, wherein the terminal protein and the DNA-binding protein are the terminal protein pTP and the DNA-binding protein E2A from adenovirus.

[0564] 26. The polynucleotide of any one of Embodiments 21 to 25, wherein the terminal protein expressed by the polynucleotide sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 20 and retains the ability to act as a terminal protein.

[0565] 27. The polynucleotide of any one of Embodiments 21 to 26, wherein the terminal protein polynucleotide sequence comprises SEQ ID NO: 21, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 21 and retains the ability to act as a terminal protein.

[0566] 28. The polynucleotide of any one of Embodiments 21 to 27, wherein the DNA-binding protein expressed by the polynucleotide sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 22 and retains the ability to act as a DNA-binding protein upon expression.

[0567] 29. The polynucleotide of any one of Embodiments 21 to 28, wherein the DNA-binding protein polynucleotide sequence comprises SEQ ID NO: 23, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 23 and retains the ability to act as a DNA-binding protein upon expression.

[0568] 30. The polynucleotide of any one of the foregoing embodiments, wherein the pore is TraB from a Streptomyces species.

[0569] 31. The polynucleotide of embodiment 30, wherein the pore expressed by the polynucleotide sequence comprises SEQ ID NO: 24, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 and retains the ability to act as a pore.

[0570] 32. The polynucleotide of embodiment 30 or embodiment 31, wherein the pore polynucleotide sequence comprises SEQ ID NO: 25, or a polynucleotide sequence that has at least 90% sequence identity to SEQ ID NO: 25 and retains the ability to act as a pore upon expression.

[0571] 33. The polynucleotide of any one of embodiments 30 to 32, further comprising a clt locus.

[0572] 34. The polynucleotide according to embodiment 33, wherein the clt locus comprises SEQ ID NO: 26, or a variant of SEQ ID NO: 26 that differs by 1, 2, 3, 4, or 5 nucleotides, and wherein the variant retains the ability to act as a clt locus.

[0573] 35. The polynucleotide of any one of embodiments 1 to 29, wherein the pore is TdtA from a Thermus species.

[0574] 36. The polynucleotide of embodiment 35, wherein the pore expressed by the polynucleotide sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 28 and retains the ability to act as a pore.

[0575] 37. The polynucleotide of embodiment 35 or embodiment 36, wherein the pore polynucleotide sequence comprises SEQ ID NO: 29, or a polynucleotide sequence that is at least 90% sequence identical to SEQ ID NO: 29 and retains the ability to act as a pore upon expression.

[0576] 38. The polynucleotide of any one of the foregoing embodiments, further comprising a promoter.

[0577] 39. The polynucleotide of embodiment 38, wherein the promoter is tissue-specific.

[0578] 40. The polynucleotide of any one of the foregoing embodiments, further comprising a payload sequence.

[0579] 41. The polynucleotide of embodiment 40, wherein the payload sequence is a therapeutic gene, a CRISPR RNA-guided nuclease, optionally including a CRISPR donor DNA, a zinc finger nuclease or a TALEN, an antigen gene or a gene encoding an immunogenic protein or a protein from a pathogen or a tumor, or an antibiotic, an antifungal or an antiviral compound, or an antibody, or a chimeric antigen or a T cell receptor, or a B cell receptor.

[0580] 42. A circular or linear plasmid comprising the polynucleotide defined in any one of the foregoing embodiments.

[0581] 43. A linear plasmid comprising the polynucleotide defined in any one of embodiments 1 to 10, further comprising hairpin ends having the sequences telR and telL.

[0582] 44. A host cell comprising the polynucleotide defined in any one of embodiments 1 to 41, or the plasmid defined in embodiment 42 or embodiment 43.

[0583] 45. The host cell of embodiment 44, wherein the host cell is an Escherichia coli cell.

[0584] 46. The host cell of embodiment 45, wherein the Escherichia coli cell expresses the genes telN, repA, sopA and sopB from bacteriophage N15.

[0585] 47. The host cell of embodiment 46, wherein the SopA expressed from the polynucleotide sequence comprises SEQ ID NO: 6, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 6 and retains the ability to act as a SopA protein, and the SopB expressed from the polynucleotide sequence comprises SEQ ID NO: 8, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 and retains the ability to act as a SopB protein.

[0586] 48. The host cell of embodiment 46 or embodiment 47, wherein the sopA polynucleotide sequence comprises SEQ ID NO: 7, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 7 and retains the ability to act as a SopA protein upon expression, and the sopB polynucleotide sequence comprises SEQ ID NO: 9, or a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 9 and retains the ability to act as a SopB protein upon expression.

[0587] 49. A pharmaceutical composition comprising the polynucleotide defined in any one of embodiments 1 to 41, or the plasmid defined in embodiment 42 or embodiment 43, and a pharmaceutically acceptable excipient.

[0588] 50. A method of treatment, comprising administering to an individual in need thereof a polynucleotide as defined in any one of embodiments 1 to 41, or a plasmid as defined in embodiment 42 or embodiment 43, or a pharmaceutical composition as defined in embodiment 49.

[0589] 51. The method of embodiment 50, wherein the polynucleotide, plasmid or pharmaceutical composition is administered by injection, microinjection, inhalation, jet injection, ingestion, liposome or microcarrier-mediated delivery.

[0590] 52. A method of producing the plasmid of embodiment 42 or embodiment 43, comprising culturing a host cell as defined in any one of embodiments 44 to 48, lysing the cells and purifying the plasmid from the cell lysate.

[0591] 53. A eukaryotic cell, comprising a polynucleotide as defined in any one of embodiments 1 to 41, or a plasmid as defined in embodiment 42 or embodiment 43.

[0592] 54. The polynucleotide of embodiment 1 or embodiment 2, wherein the pore is from a type VI secretion system of bacteria, such as bacteria of the genus Agrobacterium, Bartonella, Bordetella, Brucella, Escherichia, Legionella, Helicobacter, Neisseria, Rickettsia, Salmonella and Shigella.

[0593] 55. The polynucleotide of embodiment 1 or embodiment 2, wherein the origin of replication is from a bacterial plasmid such as pMB1, ColE1, p15A or pSC101.

[0594] 56. The polynucleotide of embodiment 1 or embodiment 2, wherein the terminal protein, DNA polymerase and DNA binding protein are from organisms such as bacteriophage PRD1 of Escherichia coli, bacteriophage Cp-1 of Streptococcus pneumoniae, Streptomyces species, viruses and archaea, or from linear plasmids of bacteria, fungi and plants, or from transposable elements, or from mitochondrial DNA.

[0595] 57. The polynucleotide of embodiment 1 or embodiment 2, which further comprises:

[0596] a) recognition sites of restriction endonucleases such as homing endonucleases that are not present in the target host chromosome; and

[0597] b) a polynucleotide sequence encoding a homologous restriction endonuclease.

[0598] 58. A composition, comprising:

[0599] a) a first plasmid, which comprises the polynucleotide of embodiment 1 or embodiment 2; and

[0600] b) A second plasmid, which contains a telRL locus, or telR and telL loci, and an origin of replication, or inverted terminal repeats and a clt sequence, wherein the telRL locus, or telR and telL loci, and the origin of replication, or inverted terminal repeats and the clt sequence are the same as those of the first plasmid.

[0601] 59. The polynucleotide of Embodiment 1 or Embodiment 2 further comprises a 2A 'ribosome skipping' peptide sequence.

[0602] 60. The polynucleotide of Embodiment 59, wherein the 'ribosome skipping' peptide comprises SEQ ID NO: 30, 31, 32 or 33.

[0603] Reference

[0604] Patent documents cited in the specification

[0605] Cranenburgh RM and Leckenby MW, 2010. Self - deleting plasmid, GB201011046A.

[0606] Ibrahim MRM, Kanwar N, Oleg R, 2021. Synthetic genome editing system, GB2114453.0.

[0607] Non - patent literature cited in the specification

[0608] Aartsma - Rus A, Janson AA, van Ommen GJ, van Deutekom JC. Antisense - induced exon skipping for duplications in Duchenne muscular dystrophy. BMC Med Genet. 2007 Jul 5; 8:43. doi:10.1186 / 1471 - 2350 - 8 - 43. PMID:17612397; PMCID:PMC1931584.

[0609] Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun. 2018 May 15; 9(1):1911. doi:10.1038 / s41467 - 018 - 04252 - 2. PMID:29765029; PMCID:PMC5953931.

[0610] Al-Allaf FA, Abduljaleel Z, Athar M, Taher MM, Khan W, Mehmet H, Colakogullari M, Apostolidou S, Bigger B, Waddington S, Coutelle C, Themis M, Al-Ahdal MN, Al-Mohanna FA, Al-Hassnan ZN, Bouazzaoui A. Modifying inter-cistronic sequence significantly enhances IRES dependent second gene expression in bicistronic vector: Construction of optimised cassette for gene therapy of familial hypercholesterolemia. Noncoding RNA Res. 2018 Nov 22;4(1):1-14. doi:10.1016 / j.ncrna.2018.11.005. PMID:30891532; PMCID:PMC6404380.

[0611] Amado E, Muth G, Arechaga I, Cabezón E. The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies. J Biol Chem. 2019 Mar 29;294(13):5050-5059. doi:10.1074 / jbc.RA119.007459. Epub 2019 Feb 5. PMID:30723158; PMCID:PMC6442058.

[0612] Anguela XM, High KA. Entering the Modern Era of Gene Therapy. Annu Rev Med. 2019 Jan 27;70:273-288. doi:10.1146 / annurev-med-012017-043332. Epub 2018 Nov 26. PMID:30477394.

[0613] Ates I, Rathbone T, Stuart C, Bridges PH, Cottle RN. Delivery Approaches for Therapeutic Genome Editing and Challenges. Genes (Basel). 2020 Sep 23;11(10):1113. doi:10.3390 / genes11101113. PMID:32977396; PMCID:PMC7597956.

[0614] Bandaranayake AD, Almo SC. Recent advances in mammalian protein production. FEBS Lett. 2014 Jan 21;588(2):253 - 60. doi:10.1016 / j.febslet.2013.11.035. Epub 2013 Dec 6. PMID:24316512; PMCID:PMC3924552.

[0615] Blesa A, Baquedano I, Quintáns NG, Mata CP, Castón JR, Berenguer J. The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation. PLoS Genet. 2017 Mar 10;13(3):e1006669. doi:10.1371 / journal.pgen.1006669. PMID:28282376; PMCID:PMC5365140.

[0616] Bloor AE, Cranenburgh RM. An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol. 2006 Apr;72(4):2520 - 5. doi:10.1128 / AEM.72.4.2520 - 2525.2006. PMID:16597952; PMCID:PMC1449051.

[0617] Choi KH. Viral polymerases. Adv Exp Med Biol. 2012;726:267 - 304. doi:10.1007 / 978 - 1 - 4614 - 0980 - 9_12. PMID:22297518; PMCID:PMC4711277.

[0618] Christie PJ, Whitaker N, González - Rivera C. Mechanism and structure of the bacterial type IV secretion systems. Biochim Biophys Acta. 2014 Aug;1843(8):1578 - 91. doi:10.1016 / j.bbamcr.2013.12.019. Epub 2014 Jan 2. PMID:24389247; PMCID:PMC4061277.

[0619] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR - Cas9. Science. 2014 Nov 28;346(6213):1258096. doi:10.1126 / science.1258096. PMID:25430774.

[0620] Epah J, R. Implications of hematopoietic stem cells heterogeneity for gene therapies. Gene Ther. 2021 Feb 15. doi:10.1038 / s41434 - 021 - 00229 - x. Epub ahead of print. Erratum in: Gene Ther. 2021 Mar 4;: PMID:33589780.

[0621] Gerstung M, Jolly C, Leshchiner I, Dentro SC, Gonzalez S, Rosebrock D, Mitchell TJ, Rubanova Y, Anur P, Yu K, Tarabichi M, Deshwar A, Wintersinger J, Kleinheinz K, Vázquez-García I, Haase K, Jerman L, Sengupta S, Macintyre G, Malikic S, Donmez N, Livitz DG, Cmero M, Demeulemeester J, Schumacher S, Fan Y, Yao X, Lee J, Schlesner M, Boutros PC, Bowtell DD, Zhu H, Getz G, Imielinski M, Beroukhim R, Sahinalp SC, Ji Y, Peifer M, Markowetz F, Mustonen V, Yuan K, Wang W, Morris QD; PCAWG Evolution&Heterogeneity Working Group, Spellman PT, Wedge DC, Van Loo P; PCAWG Consortium. The evolutionary history of 2,658 cancers. Nature. 2020 Feb; 578(7793): 122-128. doi: 10.1038 / s41586-019-1907-7. Epub 2020 Feb 6. PMID: 32025013; PMCID: PMC7054212.

[0622] Hafez M, Hausner G. Homing endonucleases: DNA scissors on a mission. Genome. 2012 Aug; 55(8): 553-69. doi: 10.1139 / g2012-049. Epub 2012 Aug 14. PMID: 22891613.

[0623] Hobernik D, Bros M. DNA Vaccines - How Far From Clinical Use? Int J Mol Sci. 2018 Nov 15; 19(11): 3605. doi: 10.3390 / ijms19113605. PMID: 30445702; PMCID: PMC6274812.

[0624] Hoeben RC, Uil TG. Adenovirus DNA replication. Cold Spring Harb Perspect Biol. 2013 Mar 1; 5(3): a013003. doi: 10.1101 / cshperspect.a013003. PMID: 23388625; PMCID: PMC3578361.

[0625] Hu P, Zhao X, Zhang Q, Li W, Zu Y. Comparison of Various Nuclear Localization Signal - Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish. G3 (Bethesda). 2018 Mar 2; 8(3): 823 - 831. doi: 10.1534 / g3.117.300359. PMID: 29295818; PMCID: PMC5844304.

[0626] Hubé F, Chooniedass - Kothari S, Hamedani MK, Miksicek RJ, Leygue E, Myal Y. Identification of an octamer - binding site controlling the activity of the small breast epithelial mucin gene promoter. Front Biosci. 2006 Sep 1; 11: 2483 - 95. doi: 10.2741 / 1984. PMID: 16720387.

[0627] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21. doi:10.1126 / science.1225829. Epub 2012 Jun 28. PMID:22745249; PMCID:PMC6286148.

[0628] Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016 Jan 28;529(7587):490-5. doi:10.1038 / nature16526. Epub 2016 Jan 6. PMID:26735016; PMCID:PMC4851738.

[0629] Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Vaseghi HR, Qian L, Liu J. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep. 2017 May 19;7(1):2193. doi:10.1038 / s41598-017-02460-2. PMID:28526819; PMCID:PMC5438344.

[0630] Malnou CE, TA, Jackson RJ, Kean KM. Poliovirus internal ribosome entry segment structure alterations that specifically affect function in neuronal cells: molecular genetic analysis. J Virol. 2002 Nov;76(21):10617 - 26. doi:10.1128 / jvi.76.21.10617 - 10626.2002. PMID:12368304; PMCID:PMC136602.

[0631] Martín F, Toscano MG, Blundell M, Frecha C, Srivastava GK, Santamaría M, Thrasher AJ, Molina IJ. Lentiviral vectors transcriptionally targeted to hematopoietic cells by WASP gene proximal promoter sequences. Gene Ther. 2005 Apr;12(8):715 - 23. doi:10.1038 / sj.gt.3302457. PMID:15750617.

[0632] Meijer WJ, Horcajadas JA, Salas M. Phi29 family of phages. Microbiol Mol Biol Rev. 2001 Jun;65(2):261 - 87; second page, table of contents. doi:10.1128 / MMBR.65.2.261 - 287.2001. PMID:11381102; PMCID:PMC99027.

[0633] Miliotou AN, Papadopoulou LC. CAR T - cell Therapy: A New Era in Cancer Immunotherapy. Curr Pharm Biotechnol. 2018;19(1):5 - 18. doi:10.2174 / 1389201019666180418095526. PMID:29667553.

[0634] Nety SP, Altae-Tran H, Kannan S, Demircioglu FE, Faure G, Hirano S, Mears K, Zhang Y, Macrae RK, Zhang F. The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity. CRISPR J. 2023 Jun;6(3):232-242. doi:10.1089 / crispr.2023.0015. PMID:37272862; PMCID:PMC10278001.

[0635] Novellino L, Castelli C, Parmiani G. A listing of human tumour antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother. 2005 Mar;54(3):187-207. doi:10.1007 / s00262-004-0560-6. Epub 2004 Aug 7. PMID:15309328.

[0636] Oztürk-Winder F, Renner M, Klein D, Müller M, Salmons B, Günzburg WH. The murine whey acidic protein promoter directs expression to human mammary tumours after retroviral transduction. Cancer Gene Ther. 2002 May;9(5):421-31. doi:10.1038 / sj.cgt.7700456. PMID:11961665.

[0637] Ostrowski LE, Hutchins JR, Zakel K, O'Neal WK. Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter. Mol Ther. 2003 Oct;8(4):637-45. doi:10.1016 / s1525-0016(03)00221-1. PMID:14529837.

[0638] Proudfoot NJ. Ending the message: poly(A) signals then and now. Genes Dev. 2011;25(17):1770-1782. doi:10.1101 / gad.17268411.

[0639] Ravin NV. Replication and Maintenance of Linear Phage-Plasmid N15. Microbiol Spectr. 2015 Feb;3(1):PLAS-0032-2014. doi:10.1128 / microbiolspec.PLAS-0032-2014. PMID:26104561.

[0640] Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020 Oct;19(10):673-694. doi:10.1038 / s41573-020-0075-7. Epub 2020 Aug 11. PMID:32782413; PMCID:PMC7419031.

[0641] Saito M, Xu P, Faure G, Maguire S, Kannan S, Altae-Tran H, Vo S, Desimone A, Macrae RK, Zhang F. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature. 2023 Jun 28. doi:10.1038 / s41586-023-06356-2. Epub ahead of print. PMID: 37380027.

[0642] Salas M, Holguera I, Redrejo-Rodríguez M, de Vega M. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication. Front Mol Biosci. 2016 Aug 5; 3:37. doi:10.3389 / fmolb.2016.00037. PMID: 27547754; PMCID: PMC4974454.

[0643] Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016 Jan 1; 351(6268):84-8. doi:10.1126 / science.aad5227. Epub 2015 Dec 1. PMID: 26628643; PMCID: PMC4714946.

[0644] Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc. 2012 Feb 1; 2012(2):199-204. doi:10.1101 / pdb.ip067876. PMID: 22301656.

[0645] Thoma L, Muth G. Conjugative DNA transfer in Streptomyces by TraB: is one protein enough? FEMS Microbiol Lett. 2012 Dec; 337(2): 81 - 8. doi: 10.1111 / 1574 - 6968.12031. PMID: 23082971.

[0646] Thoma L, Muth G. The conjugative DNA - transfer apparatus of Streptomyces. Int J Med Microbiol. 2015 Feb; 305(2): 224 - 9. doi: 10.1016 / j.ijmm.2014.12.020. Epub 2014 Dec 24. PMID: 25592263.

[0647] Vandermeulen G, Marie C, Scherman D, Préat V. New generation of plasmid backbones devoid of antibiotic resistance marker for gene therapy trials. Mol Ther. 2011 Nov; 19(11): 1942 - 9. doi: 10.1038 / mt.2011.182. Epub 2011 Aug 30. PMID: 21878901; PMCID: PMC3222533.

[0648] Wang B, Li J, Fu FH, Chen C, Zhu X, Zhou L, Jiang X, Xiao X. Construction and analysis of compact muscle - specific promoters for AAV vectors. Gene Ther. 2008 Nov; 15(22): 1489 - 99. doi: 10.1038 / gt.2008.104. Epub 2008 Jun 19. PMID: 18563184.

[0649] Wedell N, Price TAR, Lindholm AK. Gene drive: progress and prospects. Proc Biol Sci. 2019 Dec 18; 286(1917): 20192709. doi:10.1098 / rspb.2019.2709. Epub 2019 Dec 18. PMID:31847764; PMCID:PMC6939923.

[0650] Wu T, Yoon H, Xiong Y, Dixon-Clarke SE, Nowak RP, Fischer ES. Targeted protein degradation as a powerful research tool in basic biology and drug target discovery. Nat Struct Mol Biol. 2020 Jul; 27(7): 605-614. doi:10.1038 / s41594-020-0438-0. Epub 2020 Jun 15. PMID:32541897; PMCID:PMC7923177.

[0651] Yin HC, Chen XY, Wang W, Meng QW. Identification and comparison of the porcine H1, U6, and 7SK RNA polymerase III promoters for short hairpin RNA expression. Mamm Genome. 2020 Apr; 31(3-4): 110-116. doi:10.1007 / s00335-020-09838-0. Epub 2020 Apr 21. PMID:32318815.

[0652] Zheng C, Baum BJ. Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands. Mol Ther. 2005 Sep; 12(3): 528-36. doi:10.1016 / j.ymthe.2005.03.008. PMID:16099414.

[0653] Zhu Y, Zhu L, Wang X, Jin H. RNA-based therapeutics: an overview and prospectus. Cell Death Dis. 2022 Jul 23;13(7):644. doi: 10.1038 / s41419-022-05075-2. PMID: 35871216; PMCID: PMC9308039.

Claims

1. A polynucleotide, comprising: a) a polynucleotide sequence encoding a DNA-dependent DNA polymerase; and b) a polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA.

2. The polynucleotide according to claim 1, comprising: a) an origin of replication; b) a polynucleotide sequence encoding a DNA-dependent DNA polymerase; c) a polynucleotide sequence encoding: i) a telomerase; or ii) a terminal protein and a DNA-binding protein required for plasmid replication in a eukaryotic cell; and d) a polynucleotide sequence encoding a pore that enables a eukaryotic cell to secrete DNA.

3. The polynucleotide according to claim 2, wherein the origin of replication is from bacteriophage N15.

4. The polynucleotide according to any one of the preceding claims, wherein the DNA-dependent DNA polymerase is from bacteriophage N15.

5. The polynucleotide according to any one of the preceding claims, wherein the DNA-dependent DNA polymerase is encoded by the repA gene.

6. The polynucleotide according to any one of the preceding claims, wherein the DNA-dependent DNA polymerase expressed by the polynucleotide sequence comprises SEQ ID NO: 1, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and retains the ability to act as a DNA-dependent DNA polymerase.

7. The polynucleotide according to any one of the preceding claims, wherein the telomerase is TelN from bacteriophage N15.

8. The polynucleotide according to any one of the preceding claims, wherein the telomerase expressed by the polynucleotide sequence comprises SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 and retains the ability to act as a telomerase.

9. The polynucleotide according to any one of the preceding claims, further comprising a telRL locus, optionally wherein the polynucleotide sequence of the telRL locus comprises SEQ ID NO:

5.

10. The polynucleotide according to claim 1 or claim 2, wherein the DNA-dependent DNA polymerase is from bacteriophage Phi29.

11. The polynucleotide according to claim 10, wherein the DNA-dependent DNA polymerase is encoded by gene 2.

12. The polynucleotide according to claim 10 or claim 11, wherein the DNA-dependent DNA polymerase expressed by the polynucleotide sequence comprises SEQ ID NO: 10, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 and retains the ability to act as a DNA-dependent DNA polymerase.

13. The polynucleotide according to any one of claims 10 to 12, wherein the terminal protein and the DNA-binding protein are the terminal protein TP and the DNA-binding proteins p5 and p6 of phages of the Phi29 group from Bacillus subtilis.

14. The polynucleotide according to any one of claims 10 to 13, wherein the terminal protein expressed by the polynucleotide sequence comprises SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 and retains the ability to act as a terminal protein.

15. The polynucleotide according to claim 13 or claim 14, wherein the DNA-binding proteins p5 and p6 expressed by the polynucleotide sequence comprise SEQ ID NO: 14 and SEQ ID NO: 16, respectively, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 14 and SEQ ID NO: 16 and retains the ability to act as a DNA-binding protein.

16. The polynucleotide according to any one of the preceding claims, wherein the pore is TraB from a Streptomyces species.

17. The polynucleotide according to claim 16, wherein the pore expressed by the polynucleotide sequence comprises SEQ ID NO: 24, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 and retains the ability to act as a pore.

18. The polynucleotide according to claim 16 or claim 17, further comprising the clt locus.

19. The polynucleotide according to claim 18, wherein the clt locus comprises SEQ ID NO: 26, or a variant of SEQ ID NO: 26 that differs by 1, 2, 3, 4, or 5 nucleotides, and wherein the variant retains the ability to act as a clt locus.

20. The polynucleotide according to any one of claims 1 to 15, wherein the pore is TdtA from a Thermus species.

21. The polynucleotide according to claim 20, wherein the pore expressed by the polynucleotide sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 28 and retains the ability to act as a pore.

22. The polynucleotide according to any one of the preceding claims, further comprising: (a) a promoter; and / or (b) a payload sequence.

23. The polynucleotide according to claim 22, wherein the payload sequence is a therapeutic gene, a CRISPR RNA-guided nuclease, optionally comprising CRISPR donor DNA, a zinc finger nuclease, or a TALEN, an antigen gene, or a gene encoding an immunogenic protein or a protein from a pathogen or a tumor, or an antibiotic, an antifungal, or an antiviral compound, or an antibody, or a chimeric antigen or a T cell receptor, or a B cell receptor.

24. A circular or linear plasmid comprising the polynucleotide as defined in any one of the preceding claims.

25. A pharmaceutical composition comprising the polynucleotide as defined in any one of claims 1 to 23, or the plasmid as defined in claim 24, and a pharmaceutically acceptable excipient.

26. A method of treatment comprising administering to an individual in need thereof the polynucleotide as defined in any one of claims 1 to 23, the plasmid as defined in claim 24, or the pharmaceutical composition as defined in claim 25.

Images