Non-integrating DNA vectors for genetic modification of cells

Abstract

To provide improved means and methods for stable cell transfection using S / MAR elements and avoiding the risk involved in the integration of a transgene into the genome of a host cell.SOLUTION: The invention provides a polynucleotide comprising at least one promoter and a S / MAR element, where the S / MAR element is positioned downstream the promoter, and the nucleic acid sequence of S / MAR element (S / MAR sequence) contains at least three ATTA sequences per 100 nucleotides in a stretch of at most 200 nucleotides. The invention further provides a composition and a host cell comprising the polynucleotide as well as the polynucleotide for use in medicaments and for use in the treatment of genetic diseases. The present invention also relates to a kit and to a device comprising the polynucleotide, and to a method and use related to the polynucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to polynucleotides comprising at least one promoter and an S / MAR element, the S / MAR element being located downstream of the promoter, and the nucleic acid sequence of the S / MAR element (S / MAR sequence) spanning a stretch of up to 200 nucleotides and comprising at least three sequence motifs ATTA (SEQ ID NO: 1) per 100 nucleotides. The present invention further relates to compositions and host cells comprising the polynucleotides, as well as polynucleotides for use in medicine and in the treatment of genetic diseases. The present invention also relates to kits and devices comprising the polynucleotides, as well as methods and uses related to the polynucleotides. [Background technology]

[0002] Genetic modification of cells is routinely used in modern cell cultures for scientific purposes.However, the use of corresponding technology in the treatment of genetic diseases caused by gene mutations is highly desirable, but available methods still usually only provide transient modification, such as transient transfection protocols, while methods that provide stable cell modification are usually hindered by the problem that they rely on the integration of transgenes into the genome of host cells.However, the integration of transgenes, even if targeted to specific gene loci, carries the risk of inducing harmful mutations, which may result in, for example, cancer as a side effect of treatment.

[0003] Scaffold / matrix attachment regions (S / MARs), also known as scaffold attachment regions (SARs) or matrix-associated regions (MARs), are known to mediate the attachment of the nuclear matrix in eukaryotic genomes. S / MARs are AT-rich sequences, and some AT-rich motifs have been found to be further enriched (Liebeich et al., (2002), NAR 30(15):3433). Various vectors have been proposed for stable maintenance in cells based on S / MAR motifs, for example, in U.S. Patent No. 6,410,314 and Haase et al., (2010), BMC Biotechnology 10:20. Furthermore, epigenetic effects affecting the replication of such vectors have been identified (Haase et al., (2013), PLOS One 8(11):e79262). Nevertheless, S / MAR-based vectors that are sufficiently stable for use in gene therapy are still not available. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 6,410,314 [Non-patent literature]

[0005] [Non-Patent Document 1] Liebeich et al. (2002), NAR 30(15):3433 [Non-patent document 2] Haase et al. (2010) BMC Biotechnology 10:20 [Non-patent document 3] Haase et al. (2013), PLOS One 8(11):e79262 Summary of the Invention [Problem to be solved by the invention]

[0006] Nevertheless, there is a need in the art for improved means and methods for stable transfection of cells, particularly using S / MAR elements, which avoid the risks associated with integration of the transgene into the genome of the host cell. This need is met by the means and methods disclosed herein.

[0007] Thus, the present invention relates to a polynucleotide comprising at least one promoter and an S / MAR element, said S / MAR element being located downstream of said promoter, and the nucleic acid sequence of said S / MAR element (S / MAR sequence) spanning a stretch of up to 200 nucleotides and comprising at least three sequence motifs ATTA per 100 nucleotides.

[0008] When used below, the terms "have", "comprise" or "include", or any grammatical variations thereof, are used in a non-exclusive manner. Thus, both of these terms can refer to a situation in which no further features are present in the entity described in this connection other than the features introduced by these terms, as well as to a situation in which one or more further features are present. For example, the expressions "A has B", "A comprises B" and "A includes B" can both refer to a situation in which no other elements are present in A other than B (i.e., a situation in which A consists solely and exclusively of B), and to a situation in which one or more further elements are present other than B, such as element C, elements C and D, or further elements.

[0009] Furthermore, as used hereinafter, the terms "preferably," "more preferably," "most preferably," "particularly," "more particularly," "particularly," "more particularly," or similar terms are used in conjunction with any term without further limiting possibilities. Features introduced by these terms are therefore optional features and are not intended to limit the scope of the claims in any way. The present invention may be implemented by using alternative features, as recognized by those skilled in the art. Similarly, features introduced by "in one embodiment of the present invention" or similar expressions are intended to be optional features, without any limitations on further embodiments of the invention, any limitations on the scope of the invention, and any limitations on the possibility of combining features introduced in this way with other optional or non-optional features of the invention.

[0010] Furthermore, unless otherwise indicated, the term "about" refers to the indicated value with a technical precision generally accepted in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5% of the indicated value. Furthermore, the term "essentially" indicates that there is no variation that affects the indicated result or use, i.e., potential variations do not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Thus, "essentially consisting of" means including the specified components, but excluding materials present as impurities, unavoidable materials present as a result of the process used to provide the component, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "essentially consisting of" encompasses any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a set of components contains less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of unspecified component(s). In the context of nucleic acid sequences, the term "essentially identical" indicates a percent identity value of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The foregoing applies mutatis mutandis to the term "essentially complementary."

[0011] The term "polynucleotide" as used herein refers to a linear or circular nucleic acid molecule. This term encompasses single-stranded and partially or completely double-stranded polynucleotides. Preferably, the polynucleotide is RNA or DNA, including cDNA. Furthermore, naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or chemically modified polynucleotides, including artificially modified derivatives, such as biotinylated polynucleotides, are also included. The polynucleotides of the present invention are preferably provided either as isolated polynucleotides (i.e., isolated from their natural context) or in genetically modified form. The polynucleotides of the present invention comprise at least one promoter and S / MAR element active in a host cell. Furthermore, the polynucleotides have the biological activity of episomally replicating in a host cell, all as specified herein below. Preferably, the polynucleotides have a length of at most 1 Mb, more preferably at most 500 kb, even more preferably at most 200 kb, and most preferably at most 100 kb. Preferably, the polynucleotides are non-naturally occurring polynucleotides. Thus, preferably, the polynucleotide is an artificial polynucleotide. Also preferably, the polynucleotide is a chimeric polynucleotide. More preferably, the polynucleotide comprises at least one nucleic acid sequence that is heterologous to the rest of the nucleic acid sequences that it comprises.

[0012] As used herein, the term polynucleotide preferably includes variants of a specifically designated polynucleotide. More preferably, the term polynucleotide relates to a designated specific polynucleotide. The term "polynucleotide variant" as used herein refers to a variant of a polynucleotide related herein, including a nucleic acid sequence characterized in that the sequence is derivable from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition, and / or deletion, and the polynucleotide variant has a biological activity or activities as identified for the specific polynucleotide. Thus, the polynucleotide variant referred to in accordance with the present invention is one that has a different nucleic acid sequence due to at least one nucleotide substitution, deletion, and / or addition. Preferably, the polynucleotide variant comprises an ortholog, paralog, or other homolog of a specified polynucleotide or a functional subsequence thereof, such as an S / MAR element. Also preferably, the polynucleotide variant comprises a naturally occurring allele of the specified polynucleotide or a functional subsequence thereof. Polynucleotide variants also encompass polynucleotides comprising a nucleic acid sequence capable of hybridizing to the aforementioned specific polynucleotide or a functional subsequence thereof, preferably under stringent hybridization conditions. These stringent conditions are known to those skilled in the art and can be found in standard textbooks. A preferred example of stringent hybridization conditions is hybridization in 6x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by one or more wash steps in 0.2x SSC, 0.1% SDS at 50-65°C. Those skilled in the art will recognize that these hybridization conditions vary depending on the type of nucleic acid, for example, with regard to the temperature and concentration of the buffer when an organic solvent is present. For example, under "standard hybridization conditions," the temperature is between 42°C and 58°C in an aqueous buffer with a concentration of 0.1x to 5x SSC (pH 7.2), depending on the type of nucleic acid. When an organic solvent is present in the aforementioned buffer, for example, in 50% formamide, the temperature under standard conditions is approximately 42°C.The hybridization conditions for DNA-DNA hybrids are preferably, for example, 0.1×SSC and 20°C to 45°C, preferably 30°C to 45°C. The hybridization conditions for DNA-RNA hybrids are preferably, for example, 0.1×SSC and 30°C to 55°C, preferably 45°C to 55°C. The above-mentioned hybridization temperatures are determined, for example, for a nucleic acid approximately 100 bp (= base pairs) in length and having a 50% G+C content in the absence of formamide. Therefore, other conditions more suitable for low G+C DNA, known in principle to those skilled in the art, may be found to be more appropriate. Those skilled in the art will know how to determine the required hybridization conditions by referring to standard textbooks. Alternatively, polynucleotide variants can be obtained by PCR-based techniques, for example, amplification of DNA based on mixed oligonucleotide primers, i.e., using degenerate primers for the conserved domains of the polypeptides of the present invention. The conserved domains of the polypeptides can be identified by comparing the nucleic acid sequence of the polynucleotide or the amino acid sequence of the polypeptides of the present invention with sequences from other organisms. DNA or cDNA of bacterial, fungal, plant, or preferably animal origin can be used as a template. Furthermore, variants include polynucleotides comprising a nucleic acid sequence or functional subsequence thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the specifically designated nucleic acid sequence. Furthermore, polynucleotides comprising a nucleic acid sequence encoding an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the specifically designated amino acid sequence are also encompassed. Percent identity values are preferably calculated over the entire amino acid or nucleic acid sequence region. A range of programs based on various algorithms are available to those skilled in the art for comparing different sequences. In this regard, the Needleman and Wunsch or Smith and Waterman algorithms provide particularly reliable results.To perform sequence alignment, the programs PileUp (J. Mol. Evolution., Vol. 25, pp. 351-360, 1987; Higgins et al., CABIOS, Vol. 5, 1989, pp. 151-153) or Gap and BestFit (Needleman and Wunsch, J. Mol. Biol., Vol. 48, pp. 443-453 (1970); Smith and Waterman, Adv. Appl. Math., Vol. 2, pp. 482-489 (1981)) are preferably used. Preferably, the above programs are used with their standard parameters. The above-mentioned sequence identity values in percent (%) should preferably be determined using the program GAP over the entire sequence region, unless otherwise specified, using the following settings which are usually used as standard settings for sequence alignment: gap weight: 50, length weight: 3, average match: 10.000 and average mismatch: 0.000.

[0013] Polynucleotides comprising fragments of any of the specifically indicated nucleic acid sequences, which retain one or more of the indicated activities, are also encompassed as variant polynucleotides of the invention. A fragment, as referred to herein, preferably comprises at least 200, preferably at least 300, more preferably at least 400 contiguous nucleotides of any one of the specified nucleic acid sequences, or at least 100, preferably at least 200, more preferably at least 300 contiguous amino acids of any one of the specified amino acid sequences, and still encode an amino acid sequence having the indicated activity.

[0014] The polynucleotides of the present invention either consist of, consist essentially of, or comprise the aforementioned nucleic acid sequences. Thus, they may also contain additional nucleic acid sequences. Specifically, the polynucleotides of the present invention may encode, for example, a fusion protein or a selectable marker. Such fusion proteins may contain as additional moieties polypeptides for monitoring expression (e.g., green, yellow, blue, or red fluorescent protein, alkaline phosphatase, etc.), or so-called "tags" that can serve as detectable markers or auxiliary means for purification purposes. Tags for different purposes are well known in the art and are described elsewhere herein.

[0015] Also preferably, the polynucleotide comprises at least one cargo sequence. As used herein, the term "cargo sequence" relates to a nucleic acid sequence of interest that is introduced into and stably maintained in a host cell. Preferably, the cargo sequence is a nucleic acid sequence, e.g., RNA, encoding the polynucleotide, and / or a polypeptide of interest. Preferably, the polypeptide of interest is a therapeutic polypeptide, more preferably a T cell receptor (TCR), even more preferably a human or chimeric T cell receptor, a chimeric antigen receptor (CAR), preferably a MART1 TCR, or a polypeptide that is lacking in cells affected by a genetic disease as specified elsewhere herein. Thus, for example, preferably, the polynucleotide comprises at least one cargo sequence encoding a polypeptide that provides phenylalanine-hydroxylase activity (EC 1.14.16.1) for the treatment of phenylketonuria. In a preferred embodiment, the cargo sequence is located between at least one promoter and an S / MAR element. In a further preferred embodiment, the cargo sequence is located between at least one promoter and an S / MAR element, and the S / MAR element is adjacent to (suffixed with) a splice donor and a splice acceptor; thus, preferably, the S / MAR element is spliced out from the transcript encoding the cargo sequence. Thus, in a preferred embodiment, the polynucleotide comprises a coding sequence encoding a polypeptide, and the coding sequence encoding the polypeptide is located between the promoter and the S / MAR element. In a further preferred embodiment, the sequence encoding the polypeptide is located between at least one promoter and an S / MAR element, and the S / MAR element is adjacent to a splice donor and a splice acceptor; thus, preferably, the S / MAR element is spliced out from the transcript encoding the polypeptide.

[0016] Preferably, the sequence encoding the selectable marker and the cargo sequence are interrupted by a sequence that allows expression of two (or more) polypeptides in eukaryotic cells from a single mRNA, such as an internal ribosome entry sequence (IRES), or more preferably a self-cleaving peptide sequence, such as, most preferably, the peptide 2A (P2A) sequence from porcine teschovirus-1. Suitable sequences are known in the art, for example, those described in Kim et al. (2011) PLoS ONE 6(4): e18556.

[0017] Preferably, the polynucleotide is DNA. Preferably, the polynucleotide comprises additional expression control sequences that allow expression of the gene in prokaryotes and / or eukaryotes, preferably eukaryotic host cells or isolated fractions thereof. Expression of the polynucleotide preferably involves transcription of the polynucleotide into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably include regulatory sequences ensuring transcription initiation and, optionally, a polyA signal ensuring transcription termination and transcript stabilization. Additional regulatory elements may include transcriptional and translational enhancers. Examples of regulatory elements allowing expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the SMVP-, U6-, H1-, 7SK-, CMV-, EFS-, SV40-, or RSV-promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. Furthermore, inducible or cell-type-specific expression control sequences can be included in the polynucleotides of the present invention. Inducible expression control sequences can include tet or lac operator sequences, or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to elements involved in transcription initiation, such regulatory elements can also include transcription termination signals downstream of the polynucleotide, such as the SV40 polyA site or tk polyA site.

[0018] The term "host cell" as used herein relates to any cell that can accept and stably replicate a polynucleotide. Preferably, the host cell is a eukaryotic cell, preferably a plant cell or a yeast cell, such as a cell of a strain of baker's yeast, or an animal cell. More preferably, the host cell is an insect cell or a mammalian cell, in particular a mouse cell or a rat cell. Even more preferably, the host cell is a mammalian cell, most preferably a human cell. Preferably, the host cells are CD34+ progenitor cells; CD61+ platelets; CD19+ B lymphocytes; CD14+ monocytes; CD15+ granulocytes; CD3+ cytotoxic T lymphocytes, preferably CD3+ cytotoxic T lymphocytes that are also positive for CD8 and CD45; CD3+ helper T lymphocytes, preferably CD3+ helper T lymphocytes that are also positive for CD4 and CD45; CD3+ activated T lymphocytes, preferably CD3+ activated T lymphocytes that are also positive for CD25 and CD45, tumor-infiltrating lymphocytes, or natural killer (NK) cells. As will be understood by those skilled in the art, the polynucleotide may further comprise sequences that enable replication in bacterial cells, particularly a bacterial replication origin. Preferably, the bacterial cells are cells of a laboratory bacterial strain, more preferably Escherichia coli cells.

[0019] The term "promoter" is known to those skilled in the art as a genetic element that, in principle, directs the transcription level of a given gene, optionally in cooperation with further regulatory elements. A promoter can be constitutive, i.e., provide a constant level of transcription essentially independent of the state of the host cell, or regulated, i.e., provide a transcription level dependent on the state of the host cell. Furthermore, a promoter can be cell type and / or tissue specific, i.e., provide a detectable level of transcription only in a few or only one cell type. Preferably, the promoter according to the present invention is active in the host cells specified herein above. As will be understood by those skilled in the art, the selection of a promoter can depend on the type of host cell to be targeted. Promoters suitable for specific cell types as well as constitutive promoters are known in the art. Preferably, the promoter is a eukaryotic promoter, more preferably a constitutive eukaryotic promoter, and even more preferably a stronger eukaryotic promoter. Preferably, the promoter is an EF1 alpha (elongation factor 1 alpha) promoter, a UbiC (ubiquitin C) promoter, a ROSA26 promoter, a PGK (phosphoglycerate kinase) promoter, and / or a CAG (chicken alpha-actin) promoter, more preferably an EF1 alpha promoter. Also preferably, the promoter is a cell- and / or tissue-specific eukaryotic promoter. As used herein, the term "promoter" refers to the promoters identified above, while any other promoter potentially present on the polynucleotide is referred to as a "secondary promoter." Thus, preferably, the promoter directs transcription to the S / MAR sequence in the host cell. Also preferably, a promoter that does not direct transcription to the S / MAR sequence of the polynucleotide is a secondary promoter, e.g., a prokaryotic promoter, a promoter that is transcriptionally shielded from the S / MAR sequence and / or a promoter that directs transcription away from the S / MAR sequence.Preferably, the promoter comprises fewer than 1000, more preferably fewer than 250, even more preferably fewer than 100, and most preferably fewer than 20 contiguous base pairs corresponding to the apolipoprotein B promoter. Thus, preferably, the polynucleotide does not comprise the human apolipoprotein B promoter, and more preferably does not comprise the apolipoprotein B promoter.

[0020] Preferably, the S / MAR sequence is located immediately downstream of the promoter and, if present, the selectable marker gene identified herein below. Preferably, "immediately downstream" refers to the absence of an intervening transcription termination signal, more preferably the absence of an intervening gene. Thus, preferably, when initiated and encoded by a promoter, the transcript containing the detectable marker sequence preferably contains the transcribed S / MAR sequence, more preferably the complete S / MAR sequence contained in the polynucleotide. As will be understood by those skilled in the art in light of the descriptions elsewhere herein, the polynucleotide may further contain splicing sites that mediate the excision of the S / MAR sequence from the primary transcript. Thus, more preferably, when initiated and encoded by a promoter, at least the primary transcript containing the detectable marker sequence preferably contains the transcribed S / MAR sequence, more preferably the complete S / MAR sequence contained in the polynucleotide. Also preferably, the term "immediately downstream" includes polynucleotides in which the promoter and S / MAR sequence are separated by an extended nucleic acid sequence, provided that a transcription termination signal is not mediated by the promoter and S / MAR. Preferably, the sequences intervening the promoter, or, if present, the stop codon of the selectable marker gene and the S / MAR sequence, have a length of at most 2 kb, more preferably at most 0.5 kb, even more preferably at most 0.2 kb, even more preferably at most 0.1 kb, and most preferably at most 50 bp.

[0021] Those skilled in the art are aware that the term "S / MAR element," also known as "scaffold / matrix attachment region," essentially refers to a DNA sequence that mediates the attachment of the nuclear matrix of eukaryotic cells to DNA. S / MAR sequences are typically derived from sequences in eukaryotic chromosomal DNA. A variety of S / MAR sequences are available, and sequences are available from public databases, such as those described in Liebich et al. (2002), Nucleic Acids Res. 30, 312-374. According to the present invention, the nucleic acid sequence of the S / MAR element (previously referred to as the S / MAR sequence) spans a stretch of up to 200 nucleotides and contains at least three sequence motifs ATTA per 100 nucleotides. Thus, the motif contained in the S / MAR sequence includes multiple tetranucleotide motifs, 5'-ATTA-3'. Preferably, the S / MAR sequence has a length of at least 200 nucleotides, more preferably at least 300 nucleotides, even more preferably at least 400 nucleotides, and most preferably at least 500 nucleotides. Preferably, the S / MAR sequence has a length of up to 3 kb, more preferably up to 2 kb, even more preferably up to 1.5 kb, even more preferably up to 1 kb, and most preferably up to 0.9 kb. In a preferred embodiment, the S / MAR sequence has a length of up to 0.7 kb, more preferably up to 500 bp, and most preferably up to 250 bp. Therefore, preferably, the S / MAR sequence has a length of 0.2 kb to 3 kb, more preferably 0.3 kb to 2 kb, even more preferably 0.4 kb to 1.5 kb, and most preferably 0.5 kb to 1 kb. It should be understood that the expression "containing n sequence motifs per 100 nucleotides" refers to the average number of such sequence motifs calculated per 100 base pairs of sequence and may therefore be a fraction. For example, the number of ATTA sequence motifs per 100 base pairs in SEQ ID NO: 6 is 34 / 525 base pairs × 100 base pairs = 6.5. Preferably, the number of sequence motifs per 100 base pairs is determined over the entire length of the S / MAR sequence. In case of doubt, for example, when the boundaries of the S / MAR sequence cannot be determined, the number of sequence motifs per 100 base pairs of the polynucleotide is determined.The number of sequence motifs is preferably the maximum number determinable for any 200 bp window within said polynucleotide, more preferably the maximum number determinable for any 500 bp window within said polynucleotide. Preferably, the S / MAR sequence comprises at least four sequence motifs ATTA per 100 nucleotides over a stretch of up to 200 nucleotides, more preferably at least five sequence motifs ATTA per 100 nucleotides over a stretch of up to 200 nucleotides, even more preferably at least six sequence motifs ATTA per 100 nucleotides over a stretch of up to 200 nucleotides. Also preferably, the S / MAR sequence comprises at least three sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides, more preferably at least four sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides, even more preferably at least five sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides, and most preferably at least six sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides. Also preferably, the S / MAR sequence comprises at least three ATTA sequence motifs per 100 nucleotides over a stretch of up to 500 nucleotides, more preferably at least four ATTA sequence motifs per 100 nucleotides over a stretch of up to 500 nucleotides, even more preferably at least five ATTA sequence motifs per 100 nucleotides over a stretch of up to 500 nucleotides, and most preferably at least six ATTA sequence motifs per 100 nucleotides over a stretch of up to 500 nucleotides. Thus, preferably, the S / MAR sequence comprises at least 10 ATTA sequence motifs over a 500 nucleotide sequence, more preferably at least 20 ATTA sequence motifs over a 500 nucleotide sequence, and even more preferably at least 30 ATTA sequence motifs over a 500 nucleotide sequence. Preferably, at least 80% of the ATTA motifs in the S / MAR sequence. More preferably at least 90%, and most preferably at least 95%, are separated by 9-13, preferably 10-12, and most preferably 11 base pairs, respectively.

[0022] Preferably, the S / MAR element comprises additional sequence motifs, preferably within the sequence comprising the ATTA motif described herein above. Preferably, the sequence stretch of said S / MAR element comprising said ATTA sequence motif further comprises at least one sequence motif ATTTA (SEQ ID NO: 2), preferably at least two sequence motifs ATTTA, more preferably at least four sequence motifs ATTTA, most preferably at least eight sequence motifs ATTTA. Also preferably, the sequence stretch of said S / MAR element comprising said ATTA sequence motif and optionally said ATTTA motif(s) further comprises at least one, preferably at least two, more preferably at least four, most preferably at least six palindromic motifs, preferably the motif TAAATAATTTTA (SEQ ID NO: 3). Preferably, said motif TAAATAATTTTA is contiguous with at least one motif ATTA at the 5'-end and / or the 3'-end. Also preferably, said sequence stretch of the S / MAR element comprising an ATTA sequence motif comprises at least one, preferably at least two, more preferably at least three, even more preferably at least four and most preferably at least five sequence motifs ATTATAAATATTTTAATTA (SEQ ID NO: 4), more preferably the sequence motif ATTTAATTATAAATATTTTAATTA (SEQ ID NO: 5).

[0023] Also preferably, the S / MAR sequence has a low G+C content. Those skilled in the art know how to calculate the C+G content of a known sequence by counting all guanine and cytidine bases in the sequence and dividing the cumulative result by the number of nucleotides in the sequence. Preferably, the sequence stretch of the S / MAR element containing the sequence motif ATTA has a G+C content of at most 30%, more preferably at most 20%, even more preferably at most 15%, even more preferably at most 10%, and most preferably at most 5%. Preferably, if the boundaries of the S / MAR element cannot be determined, the sequence used to calculate the G+C content is the same as that used to calculate the number of ATTA motifs per 100 base pairs, as specified herein above. Also preferably, the S / MAR sequence has a small number of CG dinucleotides. Preferably, the sequence stretch of the S / MAR element containing the sequence motif contains at most 6, more preferably at most 4, even more preferably at most 2 CG motifs, and most preferably does not contain any CG motif.

[0024] Preferably, the S / MAR sequence comprises an S / MAR sequence of the apolipoprotein B gene, preferably the human apolipoprotein B gene, more preferably the 3' S / MAR sequence of the human apolipoprotein B gene. More preferably, the S / MAR sequence comprises a variant of the human apolipoprotein B gene, more preferably a variant of the 3' S / MAR sequence of the human apolipoprotein B gene. Thus, preferably, the S / MAR sequence comprises a sequence that is at least 70% identical to the sequence of SEQ ID NO: 6, preferably SEQ ID NO: 7 or 8. More preferably, the S / MAR sequence comprises the nucleic acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 7, more preferably SEQ ID NO: 8. In a preferred embodiment, the S / MAR sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 15, more preferably the S / MAR sequence comprises the sequence of SEQ ID NO: 15.

[0025] Preferably, the polynucleotide comprises a polyA signal downstream of the S / MAR element. More preferably, the polynucleotide comprises a polyA signal and a transcription termination signal downstream of the S / MAR element. Also preferably, the S / MAR element is flanked by a splice donor and a splice acceptor, and thus, in a preferred embodiment, a transcript is transcribed from the promoter and the sequence of the S / MAR element is spliced out from the transcript by splicing. Also preferably, the S / MAR sequence is preferably spliced out from the transcript encoding the selectable marker after transcription. Also preferably, the polynucleotide further comprises a (secondary) bacterial origin of replication and / or a bacterial selectable marker gene as specified herein above. Preferably, the promoters driving expression of the bacterial origin of replication and the bacterial selectable marker gene are prokaryote-specific, i.e., more preferably, non-functional in the host cell. Also preferably, the bacterial replication origin and / or bacterial selectable marker gene, preferably all elements active in prokaryotic cells contained in the polynucleotide, are shielded from the remaining sequences contained in the polynucleotide by the presence of at least one shielding element, more preferably by flanking shielding elements. Preferably, the bacterial replication origin and / or bacterial selectable marker gene, preferably all elements active in prokaryotic cells, are shielded from the remaining sequences contained in the polynucleotide by the presence of at least one shielding element at the 5' end and at least one shielding element at the 3' end. More preferably, the bacterial replication origin and / or bacterial selectable marker gene, preferably all elements active in prokaryotic cells contained in the polynucleotide, are shielded from the promoter by the presence of at least one shielding element, more preferably by flanking shielding elements. Preferably, the shielding element(s) is / are an antirepression element 40 element (SEQ ID NO: 11) or a variant thereof, and / or an S / MAR element.

[0026] Thus, preferably the polynucleotide comprises the sequence of SEQ ID NO: 7 or 8, or a sequence which is at least 70% identical to the sequence of SEQ ID NO: 7 or 8, preferably SEQ ID NO: 12, or a sequence which is at least 70% identical to the sequence of SEQ ID NO: 12, more preferably SEQ ID NO: 13, or a sequence which is at least 70% identical to the sequence of SEQ ID NO: 13, most preferably SEQ ID NO: 13, or a sequence which is at least 70% identical to the sequence of SEQ ID NO: 14. Preferably, the polynucleotide comprises the sequence of SEQ ID NO: 14 in which the nucleic acid sequence encoding GFP has been replaced by a nucleic acid sequence encoding a different polypeptide, preferably a therapeutic polypeptide, more preferably a human T cell receptor (TCR), chimeric antigen receptor (CAR), preferably MART1 TCR.

[0027] Preferably, the polynucleotide further comprises a coding sequence encoding a selectable marker polypeptide interposed between the promoter of the polynucleotide and the S / MAR element of the polynucleotide; preferably, the promoter and the selectable marker sequence together constitute a selectable marker gene. As used herein, the term "selectable marker sequence" is used as an abbreviation for the expression "coding sequence encoding a selectable marker polypeptide." The term "selectable marker" is generally understood by those skilled in the art and relates to a nucleic acid sequence that, when expressed in a host cell, confers resistance to at least one condition that, when applied, mediates selective pressure on the host cell. Selectable markers are known in the art for prokaryotic and eukaryotic cells. Preferably, the selectable marker is a eukaryotic selectable marker. Preferably, the selectable marker is a selectable marker polypeptide, more preferably a selectable marker polypeptide having transporter and / or enzymatic activity that removes or inactivates a selection compound from hot cells. Preferably, the selectable marker gene further encodes at least one intron, preferably upstream of the sequence encoding the selectable marker polypeptide. Preferably, the selection marker is a marker that mediates resistance to puromycin, blasticidin, neomycin, and / or zeocin, more preferably puromycin. Thus, preferably, the promoter and selection marker together constitute a puromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, or a zeocin resistance gene, more preferably a puromycin resistance gene. In a preferred embodiment, the selection marker is a polypeptide that confers resistance to a specific set of growth conditions, preferably the presence and / or absence of a growth signal. Thus, in a preferred embodiment, the selection marker is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), both of which are commonly known in the art. Preferably, the TCR and / or CAR have a known specificity, such that T cell signaling can be induced in host cells containing said TCR and / or CAR.In such cases, the host cell is preferably a T cell or an NK cell. Preferably, the selection marker lacks a polyA signal and one or more transcription termination signals. Thus, in a preferred embodiment, the polynucleotide further contains a coding sequence encoding a selection marker (selection marker sequence), which is located between the promoter and the S / MAR element, and the promoter and the selection marker sequence together constitute a selection marker gene, and further, the selection marker is a eukaryotic selection marker.

[0028] Preferably, the selectable marker is puromycin acetyltransferase (Genbank Accession No. KX548903.1 (SEQ ID NO: 9), encoded by nucleotides 535 to 1134 (SEQ ID NO: 10) of Genbank Accession No. KX548903.1). Thus, the selectable marker gene preferably comprises a nucleic acid sequence that: a) causes expression of a puromycin resistance polypeptide comprising the sequence of SEQ ID NO: 9; b) causes expression of a puromycin resistance polypeptide comprising a sequence at least 70% identical to the sequence of SEQ ID NO: 9; c) comprises the sequence of SEQ ID NO: 10; d) comprises a sequence at least 70% identical to the sequence of SEQ ID NO: 10; e) comprises a nucleic acid sequence encoding a puromycin resistance polypeptide comprising, preferably consisting of, the sequence of SEQ ID NO: 9; and / or f) comprises a nucleic acid sequence encoding a puromycin resistance polypeptide comprising, preferably consisting of, a sequence at least 70% identical to the sequence of SEQ ID NO: 9.

[0029] As used herein, the term "replicate" refers to the activity of the polynucleotide to induce the production of at least two copies of the polynucleotide in host cells during the cell replication cycle.Therefore, preferably, the replication of polynucleotide in host cells is determined by determining the presence of polynucleotide after a series of cell divisions, during which non-replicated polynucleotides are expected to be diluted.Preferably, the replication is stable replication, i.e., replication to the extent that the polynucleotide is still detectable in the host cell population after an average of 50 cell divisions, more preferably after an average of 100 cell divisions, and most preferably after an average of 250 cell divisions.Preferably, the detection of polynucleotide in the host cell population is carried out by PCR under standard conditions.

[0030] The term "episomal" replication is known to those skilled in the art to refer to the replication of a polynucleotide that is, in principle, not integrated into the cellular genome, i.e., not covalently attached to the cellular genome. Therefore, preferably, episomal replication of a polynucleotide refers to the replication of the polynucleotide as an autonomous replicating unit. Preferably, episomal replication refers to the maintenance of the polynucleotide in the host cell in the form of a closed circular double-stranded DNA molecule. As will be understood by those skilled in the art, the actual replication of the polynucleotide may involve other forms, such as rolling circle replication. The episomal maintenance of the circular DNA is preferably verified by a plasmid rescue procedure known to those skilled in the art, i.e., by preparing a lysate of the host cell and transforming the DNA contained therein into suitable bacterial cells, such as E. coli cells. If a suitable number of bacterial colonies obtainable by the above method contain the circular DNA as a plasmid with the same restriction pattern and / or sequence as the original circular DNA, it is assumed that the circular DNA is preferably maintained episomally. Another method for confirming episomal maintenance, known to those skilled in the art, is DNA / DNA blotting ("Southern blot" method). Therefore, preferably, the total DNA of the host cell is prepared and digested with one or more restriction enzymes. If only the band corresponding to the original circular DNA is visible in the Southern blot using the original plasmid as a probe, it is preferably concluded that the plasmid is maintained episomally. More preferably, episomal maintenance is verified as described in the examples herein.

[0031] Therefore, the term "episomal replication" as used herein refers to the activity of a polynucleotide that induces the production of at least two copies of the polynucleotide in a host cell during a cell replication cycle, while the polynucleotide exists in the cell as an autonomously replicating entity. Stable episomal replication is episomal replication to the extent that the polynucleotide is still detectable in the host cell after at least 50 cell divisions, preferably at least 100 cell divisions, more preferably at least 250 cell divisions, and most preferably at least 500 cell divisions. Preferably, the aforementioned number of cell divisions is the average number of cell divisions for a cell population.

[0032] The polynucleotide of the present invention preferably lacks the simian virus 40 (SV40) origin of replication, the bovine papillomavirus (BPV) origin of replication, and the Epstein-Barr virus (EBV) origin of replication, preferably the polyomavirus origin of replication, the papillomavirus origin of replication, and the herpesvirus origin of replication, and more preferably the eukaryotic infectious virus origin of replication. More preferably, the vector lacks a known eukaryotic origin of replication. However, preferably, the polynucleotide further comprises a prokaryotic, preferably a bacterial, origin of replication, particularly an E. coli origin of replication. Preferably, the prokaryotic origin of replication is the only origin of replication contained in the polynucleotide.

[0033] Advantageously, in the research underlying the present invention, it was found that combining a specified S / MAR element with a promoter that reads said S / MAR element results in a polynucleotide that is highly stable in episomal form in the host cell, even in the absence of a dedicated replication origin. Furthermore, it was found that the efficiency of polynucleotide establishment can be further improved by using a puromycin resistance gene, ensuring transcription to the S / MAR element via the resistance gene, and transcriptionally isolating the promoter-S / MAR combination from other promoters potentially present in the polynucleotide.

[0034] The definitions made above apply mutatis mutandis below. The further definitions and explanations made further below also apply to all embodiments described herein mutatis mutandis.

[0035] The present invention further relates to a composition comprising a polynucleotide according to the invention.

[0036] The term "composition" as used herein refers to a composition of matter comprising a specified compound and, optionally, one or more acceptable carriers. Preferably, the composition is a pharmaceutically acceptable composition. Thus, preferably, the carrier is a pharmaceutically acceptable carrier. The compounds of the present invention can be preferably formulated as pharmaceutically acceptable salts. Preferred salts include acetate, methyl ester, HCl, sulfate, chloride, etc.

[0037] The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The carriers employed can be, for example, solid, gel, or liquid. Examples of solid pharmaceutical carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Examples of liquid carriers are phosphate buffered saline, syrup, oils such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions, and the like. Similarly, the carrier or diluent can include time-delay material well known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. Suitable carriers include those described above and others well known in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The diluent(s) is selected so as not to affect the biological activity of the compound in the composition. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0038] Preferably, the composition mediates the entry of polynucleotide into host cells.Therefore, preferably, the composition comprises at least one transfection agent.The selection of an appropriate transfection agent may depend on the target host cell and the intended specific application.Transfection agents, appropriate transfection conditions, and their selection criteria are well known in the art.Also, preferably, the composition comprises virus-like particles.Therefore, preferably, the polynucleotide is packaged in virus-like particles, i.e., preferably, the polynucleotide is contained in virus-like particles.

[0039] The pharmaceutical composition is preferably administered locally or systemically. Suitable administration routes conventionally used for drug administration are oral, intravenous, or parenteral administration, as well as inhalation. However, depending on the nature and mode of action of the compound, the pharmaceutical composition can also be administered by other routes. For example, polynucleotide compounds can be administered in gene therapy approaches by using viral vectors or viruses or liposomes, as specified herein above. Furthermore, the compound can be administered in combination with other drugs in a common pharmaceutical composition or as separate pharmaceutical compositions, which can be provided in the form of kit parts. The compound is preferably administered in a conventional dosage form prepared by combining the drug with a standard pharmaceutical carrier according to conventional procedures. These procedures may include mixing, granulating and compressing, or dissolving the ingredients as appropriate for the desired preparation. It is understood that the form and characteristics of the pharmaceutically acceptable carrier or diluent are determined by the amount of active ingredient to be combined therewith, the route of administration, and other well-known variables.

[0040] The therapeutically effective amount of a pharmaceutical composition refers to the amount of compound used in the pharmaceutical composition of the present invention that prevents, improves or treats the symptoms associated with the disease or condition referred to herein.The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population).The dose ratio between therapeutic effect and toxic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50.

[0041] The dosing regimen will be determined by the attending physician and other clinical factors, preferably according to one of the methods described above. As is well known in the pharmaceutical arts, the dosage for any single patient depends on numerous factors, including the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other concurrently administered medications. Progress can be monitored by periodic assessment. Typical doses can range, for example, from 1 to 1,000 μg. However, doses below or above this exemplary range are contemplated, particularly considering the aforementioned factors. Generally, the usual administration regimen for pharmaceutical compositions should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. However, depending on the subject and method of administration, the substance dosage can vary over a wide range, providing from about 0.01 mg per kg of body weight to about 10 mg per kg of body weight. When administering a viral vector, particularly an adeno-associated viral vector, a preferred dose is 5 x 10 to 2 x 10 viral particles or viral genomes / kg body weight. As will be appreciated, these exemplary doses may vary depending on the factors described above as well as additional factors such as the type of virus, target organ, etc.

[0042] The pharmaceutical compositions and formulations referred to herein are administered at least once to treat, ameliorate, or prevent the diseases or conditions described herein. However, the pharmaceutical compositions can be administered more than once, for example, from 1 to 4 times daily for an unlimited number of days.

[0043] Certain pharmaceutical compositions are prepared by methods well known in the pharmaceutical arts and contain at least one active compound as mentioned hereinabove in admixture or otherwise associated with a pharmaceutically acceptable carrier or diluent.To prepare these particular pharmaceutical compositions, the active compound(s) are usually mixed with a carrier or diluent, or enclosed or encapsulated in a capsule, sachet, cachet, paper, or other suitable container or vehicle.The resulting formulation is adapted for administration, i.e., in the form of a tablet, capsule, suppository, liquid, suspension, etc.Dosage recommendations are provided in the prescriber's or user's instructions to anticipate dosage adjustments depending on the intended recipient.

[0044] The present invention also relates to a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for use in medicine.The present invention further relates to a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for use in the treatment of a genetic disease.

[0045] The term "genetic disease" as used herein refers to a disease causally related to one or more modifications, preferably mutations, in an individual's genome. Thus, preferably, the genetic disease is causally related to one or more epigenetic changes, more preferably, one or more genetic mutations. As will be appreciated, the symptoms of a genetic disease are often caused by the expression of a mutated gene and / or the lack of expression of a gene that provides the normal function of the gene product in one or more specific tissue(s) and / or cell type(s). Therefore, it may be preferable to treat the genetic disease only in those cells in which the mutation contributes to the disease. Preferably, the genetic disease is a monogenic disease, i.e., caused by a genetic alteration in one gene. More preferably, the genetic disease is a monogenic recessive disease, i.e., caused by a genetic alteration in both alleles of a gene. Therefore, preferably, amelioration of symptoms is expected by providing at least one unaltered copy of the affected gene. Most preferably, the genetic disease is phenylketonuria, alkaptonuria, congenital asthenia of liver, choroideremia, or Stargardt's disease. In a preferred embodiment, the genetic disease is cancer.

[0046] The present invention also relates to a kit comprising a polynucleotide according to the invention and a compound that mediates cell entry.

[0047] The term "kit" as used herein refers to a collection of the aforementioned compounds, means, or reagents of the present invention, which may or may not be packaged together. The components of the kit may be constituted by separate vials (i.e., as separate parts of the kit) or may be provided in a single vial. Furthermore, it should be understood that the kit of the present invention can preferably be used to carry out the methods referred to herein above. It is envisaged that preferably, all components are provided in a ready-to-use manner for carrying out the methods referred to above. Furthermore, the kit preferably comprises instructions for carrying out said methods. The instructions may be provided by a user manual in paper or electronic form. Furthermore, the manual may comprise instructions for interpreting the results obtained when carrying out the aforementioned methods using the kit of the present invention. As will be understood from the above, the description of a kit comprising a polynucleotide preferably relates to a kit comprising, mutatis mutandis, the corresponding vector.

[0048] Preferably, the kit further comprises at least one compound that mediates cell entry for the polynucleotide contained therein, and the term "compound that mediates cell entry" refers to any means suitable for allowing the polynucleotide of the kit to enter the interior of a host cell, preferably into the host cell. Suitable compounds that mediate cell entry (delivery means) are known in the art and include, in particular, transfection means, packaging compositions, etc. Preferably, the polynucleotide of the present invention is pre-packaged in a delivery means, for example, in a viral particle, more preferably in a replication-defective viral particle, most preferably in a virus-like particle (VLP). Those skilled in the art are aware of delivery means that offer different specificities for cell receptors, so that a suitable delivery means can be selected for a given target host cell.

[0049] The present invention further relates to a device comprising a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention.

[0050] The term "device" as used herein refers to a system of devices comprising at least one device operatively linked to each other to allow administration of the compound or composition of the present invention. Preferred devices for administering polynucleotides, compositions, and host cells are well known in the art. The method of operatively linking the devices depends on the type of device included in the device and the type of administration envisioned. Preferably, the device in such a case is constituted by a single device. Thus, the device may include a delivery unit for administering the compound or composition and a storage unit for storing the compound or composition until administration. However, it is also contemplated that the device of the present invention may appear as separate devices in such an embodiment, preferably packaged together as a kit. Those skilled in the art will understand how to link the devices without further ado. Preferred devices are those that can be applied without the special knowledge of a specialized technician. In a preferred embodiment, the device is a syringe, more preferably a syringe with a needle, containing the compound or composition of the present invention. In another preferred embodiment, the device is an intravenous infusion (IV) device containing the compound or composition. In another preferred embodiment, the device contains a compound or agent for flushing the administration site, or is an endoscopic device containing a needle for local application of the compound or composition, e.g., to a tumor. In yet another preferred embodiment, the device is an inhaler containing a compound of the invention, more preferably, the compound is formulated for administration as an aerosol.

[0051] The present application also provides a method of stably transfecting a host cell, comprising: a) contacting said host cell with a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention; and b) stably transfecting a host cell therewith The present invention relates to a method comprising:

[0052] The method for stably transfecting a host cell of the present invention is preferably an in vitro method. Furthermore, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may involve, for example, providing the host cell or a sample containing it for step a) and / or applying selective pressure to the contacted host cell. Furthermore, one or more of the above steps may be performed by an automated device.

[0053] The term "stably transfecting a host cell" refers to introducing a polynucleotide, preferably a heterologous polynucleotide, into a cell such that the polynucleotide is stably replicated by the host cell identified herein above. Preferably, stable transfection involves stable episomal replication of the polynucleotide. Preferably, stable transfection involves applying selective pressure to the host cell after contact to select for the presence of a selectable marker. The selective pressure is applied after contact, optionally excluding an initial time window during which the polynucleotide is allowed to establish within the host cell. The duration of the initial time window during which the polynucleotide is allowed to establish within the host cell largely depends on the type of host cell contacted and the type of selectable marker used. Preferably, the duration of the initial time window during which the polynucleotide is allowed to establish within the host cell is 1 hour to 48 hours, more preferably 2 hours to 24 hours, and most preferably 3 hours to 16 hours. However, the duration of the initial time window during which the polynucleotide is allowed to establish within the host cell can also be zero. That is, selective pressure can be applied immediately after contact or even during contact. More preferably, selective pressure can be applied continuously, i.e., at substantially all times after the initial time frame allowing the polynucleotide to become established in the host cells, to prevent the proliferation of host cells that do not contain the polynucleotide. Alternatively, selective pressure can be applied transiently to remove cells that have not received the polynucleotide. Preferably, transient application of selective pressure is used when the cells are introduced back into the organism after the contact. However, it is also conceivable that selective pressure is not applied, particularly when the efficiency of polynucleotide introduction into the target host cells is known to be sufficiently high and / or when a pure population of transgenic host cells is not of great importance. In a preferred embodiment, a stably transfected cell population can be obtained by allowing a cargo sequence encoding a detectable polypeptide to be expressed as described above, and selecting cells expressing the cargo sequence, for example, by cell sorting, preferably FACS.In a preferred embodiment, stably transfecting includes stable episomal replication of the polynucleotide, preferably such that the polynucleotide remains detectable in the host cell population after an average of 50 cell divisions.

[0054] The term "contacting" used in the context of the methods of the present invention is understood by those skilled in the art. Preferably, the term relates to bringing at least one polynucleotide, vector, and / or host cell of the present invention into physical contact with the host cell, e.g., to allow interaction of the compound(s) with the host cell. Preferably, contacting comprises delivering at least one polynucleotide of the present invention into the interior of the host cell, preferably via a delivery means as specified above.

[0055] The present invention also provides a method of treating a genetic disease in a subject, comprising: a) contacting said subject with a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention; and b) thereby treating the genetic disease in said subject. The present invention also relates to a method comprising:

[0056] The method for treating a genetic disease of the present invention is preferably an in vivo method. Moreover, it may include steps in addition to those explicitly mentioned above. For example, further steps may involve, for example, providing host cells or a sample containing the same for step a) and / or re-administering the sample or host cells to a subject. Thus, the method for treating a genetic disease may include steps of the method for stably transfecting host cells identified above. Furthermore, one or more of the steps may be performed by an automated device.

[0057] Furthermore, the present invention relates to the use of the polynucleotides of the present invention for the stable genetic modification of a host cell.

[0058] The present invention also relates to the use of a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for the manufacture of a medicament, and to the use of a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for the manufacture of a medicament for the treatment of a genetic disease, preferably a monogenic disease, more preferably a monogenic recessive disease, most preferably phenylketonuria, alkaptonuria, congenital asthenia of the liver, choroideremia, or Stargardt's disease. In a preferred embodiment, the genetic disease is cancer, as described herein above.

[0059] The present invention also relates to the use of a polynucleotide according to the invention, a composition according to the invention and / or a host cell according to the invention for the genetic modification of primary cells, preferably primary skin fibroblasts, for the generation of induced pluripotent stem cells (IPSCs), preferably wherein said primary cells are mouse or human primary cells.

[0060] The term "primary cells" is understood by those skilled in the art to be in contrast to cells of cultured cell lines. Thus, preferably, primary cells are cells derived from an organism and have been cultured for up to 20 passages, more preferably up to 15 passages, even more preferably up to 10 passages, and even more preferably up to 5 passages. Most preferably, primary cells are cells directly derived from the tissue of an organism, preferably from mouse or human tissue.

[0061] The term "stem cell" will also be understood by those skilled in the art to refer to undifferentiated or poorly differentiated cells that have the potential to differentiate into at least two cell types, preferably at least five cell types, more preferably at least one complete cell lineage. Preferably, the stem cell is a totipotent stem cell, more preferably a pluripotent stem cell. The term "induced pluripotent stem cell" or "IPSC" refers to a pluripotent stem cell derived from a differentiated cell, preferably a differentiated primary cell. Methods for generating IPSCs are known in the art and preferably include the expression of four transcription factors in the cells (e.g., by Takahashi et al. (2006), Cell. 126(4):663).

[0062] The present invention also relates to the use of a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for the genetic modification of embryonic stem cells.The present invention also relates to the use of a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for the manufacture of a medicament for the treatment of a genetic disease, preferably a monogenic disease, more preferably a monogenic recessive disease, most preferably phenylketonuria, alkaptonuria, congenital asthenia of liver, choroideremia, or Stargardt's disease, wherein said medicament comprises a polynucleotide of the invention.

[0063] The present invention also relates to the use of a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention for the genetic modification of stem cells to generate transgenic animals.The present invention further relates to the use of a polynucleotide according to the invention, a composition according to the invention, and / or a host cell according to the invention to generate transgenic animals.

[0064] The term "transgenic animal" as used herein relates to said animals comprising at least one heterologous polynucleotide, preferably introduced into the animal by genetic engineering methods. Preferably, the transgenic animal comprises at least 1, more preferably at least 10, even more preferably at least 1000, even more preferably at least 10000 cells comprising at least one polynucleotide according to the invention.

[0065] The present invention also relates to the use of a polynucleotide according to the invention, a composition according to the invention and / or a host cell according to the invention for the genetic modification of a single-cell embryo by pronuclear injection.

[0066] As will be understood by those skilled in the art, the term "pronuclear injection" relates to the injection of genetic material, preferably a polynucleotide of the present invention, into the nucleus of a fertilized oocyte, preferably to generate a transgenic animal.

[0067] The present invention further relates to the use of the polynucleotides of the present invention or compositions of the present invention to modify gene expression in host cells. For this purpose, a polynucleotide sequence encoding an interfering non-coding nucleic acid can be included in an expressible form. Thus, the non-coding interfering nucleic acid expressed from the polynucleotide can typically be an antisense RNA, siRNA, microRNA, or ribozyme. As a result, gene expression can be modified, i.e., downregulated, by using the polynucleotides or compositions of the present invention. Expression vectors for interfering non-coding nucleic acids containing the polynucleotides of the present invention have improved stability and expression capabilities, but may simultaneously express functional non-coding interfering nucleic acids in host cells or host organisms. Therefore, the polynucleotides of the present invention and expression constructs containing expressible polynucleotides encoding the above-mentioned non-coding interfering nucleic acids can also be used for gene silencing in clinical settings, i.e., to treat diseases and disorders, including those described elsewhere herein. Improved stability and expression characteristics can also improve gene silencing approaches.

[0068] In view of the above, the following embodiments are preferred: 1. A polynucleotide containing at least one promoter and an S / MAR element, wherein the S / MAR element is located downstream of the promoter and the nucleic acid sequence of the S / MAR element (S / MAR sequence) contains at least three sequence motifs ATTA (SEQ ID NO: 1) per 100 nucleotides over a continuous stretch of up to 200 nucleotides.

[0069] 2. The polynucleotide of embodiment 1, wherein the S / MAR sequence comprises at least 4 sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 200 nucleotides, preferably at least 5 sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 200 nucleotides, more preferably at least 6 sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 200 nucleotides.

[0070] 3. The polynucleotide of embodiment 1 or 2, wherein the S / MAR sequence comprises at least three sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 400 nucleotides, preferably at least four sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 400 nucleotides, more preferably at least five sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 400 nucleotides, and most preferably at least six sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 400 nucleotides.

[0071] 4. The polynucleotide of any one of embodiments 1 to 3, wherein the S / MAR sequence comprises at least three sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 500 nucleotides, preferably at least four sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 500 nucleotides, more preferably at least five sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 500 nucleotides, and most preferably at least six sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 500 nucleotides.

[0072] 5. The polynucleotide of any one of embodiments 1 to 4, wherein the S / MAR sequence comprises at least 10 sequence motifs ATTA over a sequence of 500 nucleotides, preferably at least 20 sequence motifs ATTA over a sequence of 500 nucleotides, more preferably at least 30 sequence motifs ATTA over a sequence of 500 nucleotides.

[0073] 6. The polynucleotide of any one of embodiments 1 to 5, wherein the sequence section of the S / MAR element containing the ATTA sequence motif further comprises at least one sequence motif ATTTA (SEQ ID NO: 2), preferably at least two sequence motifs ATTTA, more preferably at least four sequence motifs ATTTA, and most preferably at least eight sequence motifs ATTTA.

[0074] 7. The polynucleotide of any one of embodiments 1 to 6, wherein the ATTA sequence motif and, optionally, the sequence section of the S / MAR element containing the ATTTA motif(s) further comprises at least one, preferably at least two, more preferably at least four, and most preferably at least six motifs TAAATATTTA (SEQ ID NO: 3).

[0075] 8. The polynucleotide of embodiment 7, wherein the motif TAAATATTTA is flanked at the 5' or 3' end by at least one motif ATTA.

[0076] 9. The polynucleotide of any one of embodiments 1 to 8, wherein the sequence section of the S / MAR element containing the ATTA sequence motif comprises at least one, preferably at least two, more preferably at least three, even more preferably at least four, and most preferably five sequence motifs ATTATAAATATTTTAATTA (SEQ ID NO: 4), more preferably the sequence motif ATTTAATTATAAATATTTTAATTA (SEQ ID NO: 5).

[0077] 10. The polynucleotide of any one of embodiments 1 to 9, wherein the sequence section of the S / MAR element containing the sequence motif has a G+C content of at most 30%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, and most preferably at most 5%.

[0078] 11. The polynucleotide of any one of embodiments 1 to 10, wherein the sequence section of the S / MAR element containing said sequence motif comprises at most six sequence motifs CG, more preferably at most four sequence motifs CG, even more preferably at most two sequence motifs GC, and most preferably no sequence motif CG.

[0079] 12. The polynucleotide of any one of embodiments 1 to 11, wherein the S / MAR sequence comprises an S / MAR sequence of the apolipoprotein B gene, preferably an S / MAR sequence of the human apolipoprotein B gene, more preferably the 3' S / MAR sequence of the human apolipoprotein B gene.

[0080] 13. The polynucleotide of any one of embodiments 1 to 12, wherein the S / MAR sequence comprises a sequence that is at least 70% identical to the sequence of SEQ ID NO: 6, preferably SEQ ID NO: 8 or SEQ ID NO: 7, more preferably SEQ ID NO: 8.

[0081] 14. The polynucleotide of any one of embodiments 1 to 13, wherein said S / MAR sequence comprises the nucleic acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 7.

[0082] 15. The polynucleotide of any one of embodiments 1 to 14, wherein the polynucleotide further comprises a coding sequence encoding a polypeptide, preferably a selectable marker, wherein the coding sequence encoding the polypeptide is between the promoter and the S / MAR element; preferably, the polynucleotide comprises a coding sequence encoding a selectable marker (selectable marker sequence), preferably a coding sequence encoding a selectable marker polypeptide, wherein the selectable marker sequence is between the promoter and the S / MAR element, preferably the promoter and the selectable marker sequence together constitute a selectable marker gene, and preferably the selectable marker is a selectable marker for eukaryotic cells.

[0083] 16. The polynucleotide of any one of embodiments 1 to 15, wherein the polynucleotide replicates episomally in the host cell, preferably wherein this episomal replication is stable episomal replication, preferably stable for at least 50, preferably at least 100, more preferably at least 250 cell divisions.

[0084] 17. The polynucleotide of any one of embodiments 1 to 16, wherein said S / MAR sequence is located immediately downstream of said promoter and, optionally, immediately downstream of said selectable marker gene.

[0085] 18. The polynucleotide of embodiment 17, wherein being located immediately downstream means that there is no transcription termination signal in between, and more preferably, there is no gene in between.

[0086] 19. The polynucleotide of embodiment 17 or 18, wherein being located immediately downstream means that the transcript of the selectable marker sequence is located so close that it includes the transcribed S / MAR sequence.

[0087] 20. The polynucleotide of any one of embodiments 17 to 19, wherein the immediate downstream is at most 250 bp, preferably at most 100 bp, more preferably at most 50 bp downstream from the last nucleotide of the stop codon of the selectable marker gene.

[0088] 21. The polynucleotide of any one of embodiments 16 to 20, wherein the promoter is active in the host cell and is preferably a constitutive eukaryotic promoter, preferably the EF1α (elongation factor 1α) promoter, the UbiC (ubiquitin C) promoter, the ROSA 26 promoter, the PGK (phosphoglycerate kinase) promoter, and / or the CAG (chicken alpha actin) promoter.

[0089] 22. The polynucleotide of any one of embodiments 1-21, wherein the promoter is a cell-specific and / or tissue-specific eukaryotic promoter.

[0090] 23. The polynucleotide of any one of embodiments 15 to 22, wherein the selectable marker gene further encodes at least one intron, preferably upstream of the sequence encoding the selectable marker polypeptide.

[0091] 24. The polynucleotide of any one of embodiments 15 to 23, wherein the selectable marker gene is a puromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, or a zeocin resistance gene, preferably a puromycin resistance gene.

[0092] 25. Puromycin resistance gene a) causing the expression of a puromycin resistance polypeptide containing the sequence of SEQ ID NO:9; b) causing the expression of a puromycin resistance polypeptide containing a sequence that is at least 70% identical to the sequence of SEQ ID NO:9; c) containing the sequence of SEQ ID NO: 10; d) contains a sequence that is at least 70% identical to the sequence of SEQ ID NO: 10; e) contains a nucleic acid sequence encoding a puromycin resistance polypeptide, which comprises, and preferably consists of, the sequence of SEQ ID NO: 9; and / or f) containing a nucleic acid sequence encoding a puromycin resistance polypeptide, which contains, and preferably consists of, a sequence at least 70% identical to the sequence of SEQ ID NO: 9; 25. The polynucleotide of embodiment 24.

[0093] 26. The polynucleotide of any one of embodiments 1 to 25, wherein the polynucleotide is free of the simian virus 40 (SV40) origin of replication, the bovine papillomavirus (BPV) origin of replication, and the Epstein-Barr virus (EBV) origin of replication, preferably free of the polyomavirus origin of replication, the papillomavirus origin of replication, and the herpesvirus origin of replication; more preferably free of the origin of replication of a eukaryotic infectious virus.

[0094] 27. The polynucleotide of any one of embodiments 1 to 26, wherein the vector contains a bacterial origin of replication as the sole origin of replication.

[0095] 28. The polynucleotide of any one of embodiments 1 to 27, wherein the polynucleotide contains a polyA signal downstream of the S / MAR element.

[0096] 29. The polynucleotide of any one of embodiments 1-28, wherein said S / MAR elements are flanked by a splice donor and a splice acceptor.

[0097] 30. The polynucleotide of any one of embodiments 15 to 29, wherein the selectable marker gene does not have a polyA signal.

[0098] 31. The polynucleotide of any one of embodiments 15 to 30, wherein a transcript is transcribed from the selectable marker gene and the sequence of the S / MAR element is spliced out from the transcript.

[0099] 32. The polynucleotide of embodiment 31, wherein the polyA signal downstream of the S / MAR element is retained in said splicing.

[0100] 33. The polynucleotide of any one of embodiments 1 to 20, wherein the host cell is a mammalian cell, preferably a human cell.

[0101] 34. The polynucleotide of any one of embodiments 1 to 33, wherein said polynucleotide further contains a bacterial origin of replication, and / or a bacterial selectable marker gene, and / or a cargo sequence.

[0102] 35. The polynucleotide of embodiment 34, wherein said bacterial selectable marker gene comprises a prokaryote-specific promoter.

[0103] 36. The polynucleotide of embodiment 34 or 35, wherein the bacterial origin of replication and / or bacterial selectable marker gene, preferably any element active in a prokaryotic cell, is separated from the rest of the sequences contained within the polynucleotide by the presence of at least one insulation element, more preferably by being flanked by multiple insulation elements.

[0104] 37. The polynucleotide of embodiment 36, wherein the insulation element is element 40 and / or an S / MAR element.

[0105] 38. The polynucleotide of embodiment 36 or 37, wherein the bacterial origin of replication and / or bacterial selectable marker gene, preferably any elements active in prokaryotic cells, are separated from the rest of the sequences contained within the polynucleotide by the presence of at least one insulation element at the 5' end and at least one insulation element at the 3' end.

[0106] 39. The polynucleotide of any one of embodiments 1 to 38, wherein the polynucleotide comprises the sequence of SEQ ID NO: 12 or a sequence at least 70% identical to the sequence of SEQ ID NO: 12; preferably the sequence of SEQ ID NO: 13 or a sequence at least 70% identical to the sequence of SEQ ID NO: 13, more preferably the sequence of SEQ ID NO: 14 or a sequence at least 70% identical to the sequence of SEQ ID NO: 14.

[0107] 40. The polynucleotide of any one of embodiments 1 to 39, wherein the polynucleotide comprises the sequence of SEQ ID NO: 14, in which the nucleic acid encoding GFP is replaced with nucleotides encoding another polypeptide, preferably a therapeutic polypeptide, more preferably a human T cell receptor (TCR), a chimeric antigen receptor (CAR), preferably a MART1 TCR.

[0108] 41. The polynucleotide of any one of embodiments 1 to 40, wherein the polynucleotide comprises the sequence of SEQ ID NO: 12; preferably the sequence of SEQ ID NO: 13; more preferably the sequence of SEQ ID NO: 14.

[0109] 42. A composition comprising a polynucleotide according to any one of embodiments 1 to 41.

[0110] 43. The composition of embodiment 42, wherein the composition mediates the transfer of the polynucleotide into a host cell.

[0111] 44. The composition of embodiment 31 or 32, wherein the composition comprises at least one transfection agent.

[0112] 45. The composition of any one of embodiments 31 to 33, wherein the composition comprises a virus-like particle, and preferably the polynucleotide is comprised in the virus-like particle.

[0113] 46. A host cell containing a polynucleotide according to any one of embodiments 1 to 41, wherein the host cell is preferably a CD34+ progenitor cell; a CD61+ platelet; a CD19+ B lymphocyte; a CD14+ monocyte; a CD15+ granulocyte; a CD3+ cytotoxic T lymphocyte, preferably also CD8 and CD45 positive; a CD3+ helper T lymphocyte, preferably also CD4 and CD45 positive; a CD3+ activated T lymphocyte, preferably also CD25 and CD45 positive, a tumor-infiltrating lymphocyte, or a natural killer (NK) cell.

[0114] 47. A polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46, for use in medicine.

[0115] 48. A polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46 for use in treating a genetic disease, preferably a monogenic disease, more preferably a monogenic recessive genetic disease, most preferably phenylketonuria, alkaptonuria, Leber's congenital amaurosis, choroideremia, or Stargardt's disease.

[0116] 49. A kit comprising a polynucleotide according to any one of embodiments 1 to 41 and a compound that mediates intracellular internalization.

[0117] 50. The kit of embodiment 49, wherein the compound that mediates intracellular internalization is a transfection means.

[0118] 51. The kit of embodiment 49 or 50, wherein the compound that mediates intracellular internalization is a packaging composition for packaging the polynucleotide into a virus-like particle.

[0119] 52. A device comprising a polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46.

[0120] 53. A method for stably transfecting a host cell, comprising: a) contacting said host cell with a polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46; and b) thereby stably transfecting host cells The method comprising:

[0121] 54. The method of embodiment 53, wherein the method further comprises applying selective pressure to the host cells to select for the presence of the selectable marker gene.

[0122] 55. The method of embodiment 53 or 54, wherein the selective pressure is applied continuously or, preferably, transiently.

[0123] 56. A method for treating a genetic disease in a subject, comprising: a) contacting the subject with a polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46; and b) thereby treating a genetic disease in said subject. The method comprising:

[0124] 57. Use of a polynucleotide according to any one of embodiments 1 to 41 for stably genetically modifying a host cell.

[0125] 58. Use of a polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46 for the manufacture of a medicament.

[0126] 59. Use of a polynucleotide according to any one of embodiments 1 to 41, a composition according to any one of embodiments 42 to 45, and / or a host cell according to embodiment 46 for the manufacture of a medicament for treating a genetic disease, preferably a monogenic disease, more preferably a monogenic recessive genetic disease, most preferably phenylketonuria, alkaptonuria, Leber's congenital amaurosis, choroideremia, or Stargardt's disease.

[0127] All references cited herein are hereby incorporated by reference for their entire disclosure content as well as for the disclosure content individually mentioned herein. [Brief explanation of the drawings]

[0128] [Figure 1] Establishment efficiency and analysis of genetically modified cell populations: A) Cell culture plate with crystal violet-stained colonies formed after 4 weeks of selection with puromycin; vector establishment efficiency was approximately 40%; B) FACS detection of GFP fluorescence in puromycin-selected cells; fluorescence was highly uniform, with very few non-fluorescent cells; 1 = pEPI, 2 = pSMARt. [Figure 2-1] Plasmid rescue results of the pS / MARt vector from established cell populations. Plasmid rescue and restriction enzyme analysis after bacterial transformation of total DNA from Hek293T cells established with pS / MARt and pEPI plasmid DNA. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3] Southern blot of pSMARt vector maintained in selected cells: An oligonucleotide hybridizing to the GFP gene of pS / MARt was used as a probe to detect vector DNA restricted with BamHI in extracts from host cells (pS / MARt 1-3); untransfected vector was used as a control ("pS / MARt(+)"). [Figure 4]Vector map of pS / MARt; ori: bacterial replication origin, P2A: sequence encoding the self-cleaving 2A peptide derived from porcine teschovirus-1, apolipoB MAR: S / MAR sequence derived from the apolipoprotein B gene. [Figure 5] Colony formation assay using various versions of pSMARt in Hek293T and HeLa cells. [Figure 6] pSMRt is efficiently maintained in dividing cells even in the absence of continuous selection. [Figure 7] The establishment efficiency of pSMARt is independent of the selectable marker (A), but the presence of an insulator element in front of the promoter increases its establishment efficiency (B); Figure 7A: 1 = pEPI, 2 = pSMARt-Ele40-GFP-2A-Puro, 3 = pSMARt-Ele40-GFP-2A-G418; B: 1 = pSMARt-UCOE, 2 = pSMARt-Ele40, 3 = pSMARt, 4 = pEPI. [Figure 8] The pSMARt vector is efficiently maintained in primary human CD3+ cells for over a month. [Figure 9] Introduction of splice junctions adjacent to SMARs improves vector establishment in dividing cells. (1) pEPI, (2) pS / MARt, (3) NP, (4) NP-SPlice (all 1, 2, 3, and 4 contain β-inf MAR), (5) pS / MARt, and (6) NPsplice (5 and 6 contain ApoLMAR). [Figure 10] pSMARt is maintained in mouse embryonic stem cells (mESCs), and the presence of the vector does not affect cell behavior. [Figure 11] The pSMARt vector is active during goblet formation and differentiation and is resistant to epigenetic silencing. [Figure 12] pSMARt sustains transgene GFP expression during stem cell differentiation. [Figure 13]Comparison of pSMARt vector and lentiviral modified CAR-T cells; real-time tumor killing, interferon production, and cytotoxicity analysis. [Figure 14] Analysis of in vivo tumor killing [Figure 15] Analysis of tumor samples and in vivo maintenance of pSMARt and lentiviral-modified CAR-T cells; 1 = isotype control, 2 = untreated, 3 = treated with mock T cells, 4 = T cells transfected with lentivirus encoding for CAR anti-human CEA, 5 = T cells transfected with pSMARt encoding for CAR anti-human CEA. [Figure 16] Expression data from pS / MARt and Nano vectors containing splicing sequences. Hek293T cell populations established with various versions of pS / MARt were analyzed for transgene expression 35 days after DNA delivery and selection in puromycin (0.5 μg / ml) by flow cytometry (A). Relative expression of the transgene GFP was assessed relative to the expression of the housekeeping gene GAPDH (B). This figure demonstrates that the introduction of splicing sequences improves transgene expression by increasing the amount of RNA in the cells. Figure (A): (1) pS / MARt, (2) Nano-S / MARt, (3) Nano-S / MARt-splice; Figure (B): (1) pS / MARt, (2) Nano-S / MARt, (3) Nano-S / MARt-splice.

[0129] The following examples are merely illustrative of the present invention and should not be construed as limiting the present invention in any way.

[0130] (Example) Example 1: Efficiency and analysis of establishment of genetically modified cell populations (Figure 1) The efficiency of generating stably expressing cells was evaluated in a colony formation assay using pS / MARt (Figure 4, SEQ ID NO: 14) compared with pEPI. Upon DNA delivery, cells positive for GFP transgene expression were isolated via FACS sorting (FACS Aria II), and 100 cells were seeded onto a 6-cm cell culture dish. They were then cultured for 4 weeks in the presence of 0.5 μg / ml puromycin. After 4 weeks, the cells were fixed with PFA, and colonies were stained with crystal violet. The colony count was considered to be the efficiency of vector establishment, i.e., the number of colonies formed per number of FACS-sorted cells seeded. The generation of stable cell lines was highly efficient, with over 40% of transfected cells becoming established (Figure 1A). The number of transgene (GFP)-expressing cells was estimated by flow cytometry. As shown in Figure 1B, pS / MARt generates a modified population in which transgene expression is homogeneous without a significant number of negative cells.

[0131] The efficiency of generating stable transgenes was evaluated by flow cytometry. Hek293T cells were transfected with either pEPI or pS / MARt. After 35 days of culture, the cells were subjected to FACS analysis to measure the percentage of cells that continued to express the transgene (GFP) and their mean fluorescence intensity. The upper panel in Figure 1B shows that pS / MARt produces a population of genetically modified cells with robust and uniform transgene expression. This is in contrast to cells transfected with pEPI, which show heterogeneous and low levels of transgene expression. The bar graph shows that in the above cell pool, there is no significant negative cell population with pS / MARt, whereas the pEPI lineage almost silences or loses transgene activity. In addition, the average transgene expression is significantly higher in the pS / MARt lineage. Experimental details: Cells were transfected with JetPEI and selected with 0.5 μg / ml puromycin and 1 mg / ml G418 for pEPI.

[0132] Example 2 - Plasmid rescue of pS / MARt vector from established cell populations (Figure 2) To determine the episomal status and molecular integrity of the vector in mammalian cells, DNA rescue experiments were performed.

[0133] Vector DNA was isolated from cells established with either the pS / MARt or pEPI plasmid. The cells were grown for 1 week in the presence of the antibiotic puromycin (0.5 μg / ml) and then for at least 30 days without antibiotics. For plasmid rescue, gDNA was extracted from established cells using the Blood & Tissue DNAeasy kit (Qiagen) and transformed into DH10B Escherichia coli (E. coli). Bacteria were grown on LB agar plates containing kanamycin (50 μg / ml). Twelve colonies were cultured overnight in liquid LB medium containing kanamycin (50 μg / ml), and plasmid DNA was extracted using a Miniprep kit (Qiagen). For analysis, DNA minipreps were digested with the restriction enzyme BamHI (Thermo Fisher) for 10 minutes at 37°C, and the restriction patterns were examined on a 1% agarose gel. As a control, the DNA used to transfect cells at the beginning of the establishment procedure was digested with the same enzymes and run as a standard. (A) Analysis of 12 representative samples isolated from pS / MARt demonstrated molecular equivalence to the original DNA vector, whereas (B) DNA rescue from cells established with pEPI was shown to be molecularly distinct from the original vector, with the smeared bands clearly indicating reconstitution of the original DNA.

[0134] Example 3 - pS / MARt Vectors are Maintained Episomal in Engineered Cells (Figure 3) To further verify that the pS / MARt vector episomally modifies mammalian cells, we physically characterized its structure by Southern blot analysis. We analyzed Hek293T cell populations cultured for at least 30 days after DNA transfection. Genomic DNA was extracted using the Blood & Tissue DNAeasy kit (Qiagen) and digested with the restriction enzyme BamHI (NEB) overnight at 37°C. Total cellular DNA was then separated on a 1% agarose gel and transferred to a nylon membrane. An oligonucleotide corresponding to the vector's GFP gene was used to generate a radioactive probe used to detect pS / MARt DNA in cellular DNA. The presence of a single band of the same size as the control vector in a sample indicates the episomal state of pS / MARt in the established mammalian cell population. The absence of a smear and / or additional bands indicates that the vector has not integrated or rearranged into the cellular genome.

[0135] Example 3 - Efficiency of generating stable expressing cells (Figure 5) The efficiency of generating stable-expressing cells using a range of pS / MARt DNA vectors carrying ApoL-MAR and beta-IFN-MAR components was evaluated using a colony formation assay. After DNA delivery, cells positive for the expression of the reporter gene GFP were isolated by FACS sorting (FACS Aria II) and 100 were seeded onto 6-cm cell culture dishes. The cells were then cultured for 4 weeks in the presence of 0.5 g / ml puromycin. After 4 weeks, the cells were fixed with PFA, and colonies were stained with crystal violet and quantified. The number of colonies was determined as the vector establishment efficiency. The assay showed that vectors engineered with ApoL MAR, core sequence, or fragment 2 were the most efficient in generating genetically modified cells.

[0136] Example 4 - Stability without selection (Figure 6) To assess whether the insulator sequence prevents silencing or loss of the DNA vector, we measured GFP transgene expression without antibiotic selection in cells transfected with (A) pSMARt-insulator-GFP-2a-Puro β-interferon MAR or (B) pSMARt-insulator-GFP-2a-Puro ApoL MAR. Cells were transfected with the DNA vector and cultured in 1 μg / ml puromycin. After 1 week, cells were split and the drug was removed from a group of cells representative of each transfection.

[0137] (1) pSMARt-insulator-GFP-2a-Puro β interferon MAR - cultured under continuous selection (2) pSMARt-insulator-GFP-2A-Puro β interferon MAR - deselected (3) pSMARt-insulator-GFP-2a-Puro ApoL MAR - cultured under continuous selection (4) pSMARt-insulator-GFP-2A-Puro ApoL MAR - deselected

[0138] Example 5 - Establishment efficiency (Figure 7) To evaluate the effect of the selection marker on establishment efficiency, colony formation assays were performed as shown in Figure 5. Figure 7 (A) shows that establishment efficiency with the DNA vector pS / MARt is independent of the antibiotic selection marker. The selection marker improves establishment efficiency when transcriptionally linked to the S / MAR motif. Figure 7 (B) shows that the incorporation of an additional insulation element between the bacterial backbone and the eukaryotic promoter improves establishment efficiency.

[0139] Example 6 - Primary Cell Stability (Figure 8) To evaluate the transfection efficiency of the DNA vector system of the present invention in primary human CD3+ cells and measure their expression profiles, we transfected three variants of the pS / MARt vector system and pERI encoding GFP and examined their ability to induce sustained expression of the reporter gene GFP in human T cells. The DNA vectors were introduced into freshly isolated PBMCs by electrotransfer (Nucleofector Device Y, Lonza), and the cells were cultured for 35 days in the presence of IL-2 (5 μg / ml, Biolegend). Every 7 days, the cells were checked for transgene expression, and their proliferation was stimulated by adding antibodies, anti-CD28 (Biolegend) and anti-CD3 (Biolegend), to the medium. pS / MARt carrying the core version of ApoLMAR can generate a higher number of transgene-expressing cells than pS / MARt carrying the full-length ApoL-MAR sequence, βIHN MAR, and pERI.

[0140] Example 7 - MAR splicing (Figure 9) To demonstrate that the introduction of splicing sequences can improve establishment efficiency, we constructed a series of DNA vectors with and without splice donor and acceptor sites flanking the MAR element. To assess the effect of splicing elements on establishment efficiency, we performed colony formation assays as shown in Figure 5.

[0141] The establishment efficiency of DNA vectors can be significantly improved by improving and minimizing the bacterial backbone. Due to the aforementioned improvements, i.e., the selection marker and insulator sequences, as well as the latest improvements to its backbone, pS / MARt establishes more efficiently than pERI. The backbone minimization shown in Figure 9 (3) further improves this. The introduction of a splicing sequence to remove the S / MAR element from the mRNA almost doubles the efficiency of this type of vector. This effect can be reproduced for similar vector compositions containing different S / MAR elements (5-6).

[0142] Example 8 - pS / MARt in stem cells (Figure 10) To evaluate whether pS / MARt can efficiently modify stem cells without molecular damage, stable stem cell lines were generated. E14 mouse embryonic stem cell (mESC) lines were established using the pS / MARt-GFP vector. One month after DNA transfer, expression of the reporter gene GFP was measured by fluorescence microscopy (A). pS / MARt-GFP-modified mESCs were stained for most common pluripotency markers, indicating that the presence of the episomal vector does not alter the pluripotency of the cells.

[0143] Example 9 - pS / MARt in cup formation (Figures 11 and 12) pS / MARt-GFP-labeled mESCs were injected into blastocysts of C57BL / 6 mice, resulting in the formation of chimeras. The hematopoietic organs of these chimeras, such as the spleen and bone marrow, were stained with a pan-blood surface marker (CD45), and their fluorescence was analyzed by flow cytometry. C57BL / 6 mice and constitutively expressing UBC::GFP mice were used as negative and positive controls, respectively (Figure 11).

[0144] pS / MARt results in sustained transgene expression during hematopoietic differentiation. mESCs were differentiated into HSCs and analyzed by flow cytometry before (day 0) and after (day 6) the differentiation process. Both parental and labeled cells successfully differentiated into HSCs, and cells labeled with pS / MARt-GFP maintained reporter gene expression throughout the process (Figure 12).

[0145] Example 10 - Comparison with lentivirus (Figure 13) (A) CD3+ cells sorted from two different healthy donors were modified with a CAR272-expressing S / MARt DNA vector targeting the human CEA epitope, and their cytolytic activity was confirmed by cytotoxicity and interferon-γ release assays in MCF-7 cells (a breast cancer cell line).

[0146] (B) T cells engineered with the CAR272-expressing S / MARt DNA vector system exhibit improved killing activity compared to CD3+ cells modified with lentivirus harboring the same expression cassette.

[0147] Example 11 - Tumor Killing (Figures 14 and 15) In vivo analysis of CAR-T cells generated by the S / MARt DNA vector system, lentivirus, and next-generation NanoS / MARt DNA vector (Figure 14).

[0148] 2x10 6 HT29 tumor cells were inoculated subcutaneously. 3x10 cells were generated using S / MARt DNA vectors, lentivirus, and next-generation vectors. 5 CAR+ T cells were injected into the tail vein of each mouse 7 days after tumor cell injection, without prior chemotherapy or radiation therapy. The tumor-targeting efficacy of the engineered cells was compared to mock-electroporated CD3+ T cells and recorded as tumor growth (A) and mouse survival (B).

[0149] The grown tumors were removed, cultured, lysed, and analyzed for the presence of CAR targets (Figure 15). The frequency of CAR+ CD3 in the tumor mass, as well as the presence of CAR-expressing CD3 in the spleen, was analyzed.

[0150] Residual tumors contained only tumor cells lacking the targeted epitope, indicating that S / MARt-treated T cells were effective in killing and eliminating tumor cells at levels at least comparable to lentiviral controls. In addition, CAR-T-positive cells were still detectable infiltrating the tumor and in the spleen, indicating that the DNA vector was still actively expressed in the transfected T cells. The present disclosure includes the following embodiments. [1] A polynucleotide containing at least one promoter and an S / MAR element, wherein the S / MAR element is located downstream of the promoter, and the nucleic acid sequence of the S / MAR element (S / MAR sequence) contains at least three sequence motifs ATTA (SEQ ID NO: 1) per 100 nucleotides over a continuous stretch of up to 200 nucleotides. [2] The polynucleotide of embodiment 1, wherein the S / MAR element is flanked by a splice donor and a splice acceptor. [3] The polynucleotide of embodiment 1 or 2, wherein the polynucleotide further contains a coding sequence encoding a polypeptide, preferably a selectable marker, and the coding sequence encoding the polypeptide is located between the promoter and the S / MAR element. [4] The polynucleotide described in any one of embodiments 1 to 3, wherein the polynucleotide further contains a coding sequence encoding a selection marker (selection marker sequence), the selection marker sequence is located between the promoter and the S / MAR element, the promoter and the selection marker as a whole constitute a selection marker gene, and the selection marker is a selection marker for eukaryotic cells. [5] The polynucleotide of embodiment 4, wherein the selectable marker gene is a puromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, or a zeocin resistance gene, preferably a puromycin resistance gene. [6] The polynucleotide according to any one of embodiments 2 to 5, wherein a transcript is transcribed from the promoter, and the sequence of the S / MAR element is excised from the transcript by splicing. [7] The polynucleotide of any one of embodiments 1 to 6, wherein the polynucleotide further contains a bacterial origin of replication and / or a bacterial selectable marker gene, and the bacterial origin of replication and / or the bacterial selectable marker gene is separated from the remaining sequences contained within the polynucleotide by the presence of at least one insulation element. [8] The polynucleotide of any one of embodiments 1 to 6, wherein the polynucleotide does not have a simian virus 40 (SV40) replication origin, a bovine papillomavirus (BPV) replication origin, or an Epstein-Barr virus (EBV) replication origin. [9] The polynucleotide of any one of embodiments 1 to 8, wherein the polynucleotide replicates episomally in the host cell, preferably the episomal replication is stable episomal replication, preferably in mammalian cells.

[10] A composition comprising the polynucleotide according to any one of embodiments 1 to 9, wherein the composition is preferably a pharmaceutical composition.

[11] A host cell containing the polynucleotide of any one of embodiments 1 to 9, wherein the host cell is preferably a CD34+ progenitor cell; a CD61+ platelet; a CD19+ B lymphocyte; a CD14+ monocyte; a CD15+ granulocyte; a CD3+ cytotoxic T lymphocyte, preferably also CD8 and CD45 positive; a CD3+ helper T lymphocyte, preferably also CD4 and CD45 positive; a CD3+ activated T lymphocyte, preferably also CD25 and CD45 positive; a tumor-infiltrating lymphocyte; or a natural killer (NK) cell.

[12] A polynucleotide according to any one of embodiments 1 to 9, a composition according to embodiment 10, and / or a host cell according to embodiment 11 for use in medicine, preferably for use in the treatment of a genetic disease.

[13] A kit comprising the polynucleotide according to any one of embodiments 1 to 9 and a compound that mediates intracellular internalization.

[14] A device comprising the polynucleotide of any one of embodiments 1 to 9, the composition of embodiment 10, and / or the host cell of embodiment 11.

[15] A method for stably transfecting a host cell, comprising: a) contacting said host cell with a polynucleotide according to any one of embodiments 1 to 9, and / or a composition according to embodiment 10, and / or a host cell according to embodiment 11; and b) thereby stably transfecting host cells The method comprising:

[16] The method of embodiment 15, wherein the stable transfection comprises stable episomal replication of the polynucleotide, preferably such that the polynucleotide remains detectable in the host cell population after an average of 50 cell divisions.

[17] Use of a polynucleotide according to any one of embodiments 1 to 8 for stably genetically modifying a host cell.

[18] The use of embodiment 17, wherein the stable genetic modification of the host cell comprises stable episomal replication of the polynucleotide in the host cell, preferably such that the polynucleotide remains detectable in the host cell population after an average of 50 cell divisions.

[0151] The present disclosure includes the following sequence information: SEQUENCE LISTING <110> Deutsches Krebsforschungszentrum <120> Non-Integrating DNA Vectors for the genetic modification of Cells <130> PA23-177 <150> EP17191829.5 <151> 2017-09-19 <160> 15 <170> PatentIn version 3.3 <210> 1 <211> 4 <212> DNA <213> Homo sapiens <400> 1 atta 4 <210> 2 <211> 5 <212> DNA <213> Homo sapiens <400> 2 atta 5 <210> 3 <211> 11 <212> DNA <213> Homo sapiens <400> 3 taaatatttt a 11 <210> 4 <211> 19 <212> DNA <213> Homo sapiens <400> 4 attataaata ttttaatta 19 <210> 5 <211> 24 <212> DNA <213> Homo sapiens <400> 5 atttaattat aaatatttta atta 24 <210> 6 <211> 525 <212> DNA <213> Homo sapiens <400> 6 atttataaaa tattgaatta taaaatatgt aattataaat actttaatta taaaatatgt 60 aattataaat actttaatta taaaatatgt aattataaat actttataaa atatgtaatt 120 ataaaatatg taattataaa cattttaatt ataaaatatg taattataaa cattttaatt 180 ataaaatatg taattataaa cattttaatt ataaaatatg taattataaa cattttaatt 240 ataaaatatg taattataaa cattttaatt ataaaatt taattataaa cattttaatt 300 ataaaatt taattatt taattatt ataaaatt taattatt tatttatt 360 ataaaatt taattatt tattatt ataaaatt taattatt tactttatt 420 ataaaatt taattatt taattatt ataaaatt taattatt taattatt 480 ataaattt taattataaa attttaatt ataaaaacac aatta 525 <210> 7 <211> 825 <212> DNA <213> Homo sapiens <400> 7 gcaggctgag tgaaaataag gacttgttat ttcatctcga ggcctaccgg agagccttgc 60 cttgcaaagg cagacagtca gtgaggaaga ctatgtggca catgaagaca ccagaggtgt 120 tcctcaggat caaagtatgt acaagccttt gtgaatattt tttccttctc acttggcaaa 180 tacaattcct gagatcaata acctcgtctt tttaattttt tcctcgtctt tttaactatt 240 tataaaatat tgaattataa aatatgtaat tataaatact ttaattataa aatatgtaat 300 tataaatact ttaattataa aatatgtaat tataaatact ttataaaata tgtaattata 360 aaatatgtaa ttataaacat tttaattata aaatatgtaa ttataaacat tttaattata 420 aaatatgtaa ttataaacat tttaattata aaatatgtaa ttataaacat tttaattata 480 aaatatgtaa ttataaacat tttaattata aaatatttaa ttataaacat tttaattata 540 aaatattaa ttataaattat ttaataatta aaatattaa ttataaattat ttaataatta 600 aaatattaa ttataaatat ttaattaata aaatattaa ttataaatac ttaattaata 660 aaatattaa ttataaattat ttaataatta aaatattaa ttataaattat ttaataatta 720 aatattttaa ttataaaata tttaattata aaaacacaat tacctcatct ttttaaatat 780 ttttgcaaaa tatttccctc cataatttct ccgtttccat tttta 825 <210> 8 <211> 854 <212> DNA <213> Homo sapiens <400> 8 cttctccact cctggcaggc tgagtgaaat aaaggacttg ttattcatc tcgaggccta 60 ccggagagcc ttgccttgca aaggcagaca gtcagtgagg aagactatgt ggcacatgaa 120 gacaccagag gtgttcctca ggatcaaagt atgtacaagc ctttgtgaat atttttcct 180 tctcacttgg caaatacaat tcctgagatc aataacctcg tctttttaat tttttcctcg 240 tctttttaac tatttaaa atattgaatt ataaaatatg taattataaa tactttaatt 300 ataaaatatg taattataaa tactttaatt ataaaatatg taattataaa tactttataa 360 aatatgtaat tataaaat gtaattaa acattttaat tataaaat gtaattaa 420 acattttaat tataaaatat gtaattataa acattttaat tataaaatat gtaattaa 480 acattttaat tataaaatat gtaattataa acattttaat tataaaat ttaattataa 540 acattttaat tataaaat ttaattataa attttaat tataaaat ttaattataa 600 atttttaat tataaaat ttaattaataa atttttaat tataaaat ttaattaataa 660 atactttaat tataaaat ttaattataa atatttaat tataaaat ttaattataa 720 atattttaat tataaatatt ttaattataa aatatttaat tataaaaaca caattacctc 780 atctttttaa atatttttgc aaaatatttc cctccataat ttctccgttt ccatttttat 840 tctgttactt aaat 854 <210> 9 <211> 199 <212> PRT <213> Artificial <220> <223> puromycin acetyltransferase, synthetic construct <400> 9 Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val 1 5 10 15 Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala 20 25 30 Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu 35 40 45 Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val 50 55 60 Trp Val Ala Asp Asp Gly Ala Ala Val Ala Val Trp Thr Thr Pro Glu 65 70 75 80 Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala 85 90 95 Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu 100 105 110 Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val 115 120 125 Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val 130 135 140 Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu 145 150 155 160 Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe 165 170 175 Thr Val Thr Ala Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys 180 185 190 Met Thr Arg Lys Pro Gly Ala 195 <210> 10 <211> 600 <212> DNA <213> Artificial <220> <223> puromycin acetyltransferase encoding sequence, synthetic construct <400> 10 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta 60 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac 120 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 240 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 420 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600 <210> 11 <211> 1031 <212> DNA <213> Artificial <220> <223> anti-repressive element40 <400> 11 gatcaagaaa gcactccggg ctccagaagg agccttccag gccagctttg agcataagct 60 gctgatgagc agtgagtgtc ttgagtagtg ttcagggcag catgttacca ttcatgcttg 120 acttctagcc agtgtgacga gaggctggag tcaggtctct agagagttga gcagctccag 180 ccttagatct cccagtctta tgcggtgtgc ccattcgctt tgtgtctgca gtcccctggc 240 cacacccagt aacagttctg ggatctatgg gagtagcttc cttagtgagc tttcccttca 300 aatactttgc aaccaggtag agaagtttgg agtgaaggtt ttgttcttcg tttcttcaca 360 atatggatat gcatcttctt ttgaaaatgt taaagtaaat tacctctctt ttcagatact 420 gtcttcatgc gaacttggta tcctgtttcc atcccagcct tctataaccc agtaacatct 480 tttttgaaac cagtgggtga gaaagacacc tggtcaggaa cgcggaccac aggacaactc 540 aggctcaccc acggcatcag actaaaggca aacaaggact ctgtataag taccggtggc atgtgtatta gtggagatgc agcctgtgct ctgcagacag ggagtcacac agacactttt ctataatttc ttaagtgctt tgaatgttca agtagaaagt ctaacattaa atttgattga acaattgtat attcatgga tattttgga cggaatacca aaaaatggca atagtggttc 840. tttctggatg gaagacaaac ttttcttctt taaataaat tttattttat atatttgagg 900. ttgaccacat gaccttaagg atacatatag acagtaaact ggttactaca gtgaagcaaa ttaacatatc taccatcgta catagttac tttttttgtg tgacaggac agctaaaatc takgtattta acaaaactcc taaagacaat acatttttat they cctcatgatg present c 1031 <210> 12 <211> 1438 <212> DNA <213> Artificial <220> <223> CMV Promoter - S / MAR sequence <400> 12 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt ccgccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 420 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa 540 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtctttc cactcctggc 600 aggctgagtg aaataagga cttgttatt catctcgagg cctaccggag agccttgcct 660 tgcaaaggca gacagtcagt gaggaagact atgtggcaca tgaagacacc agaggtgttc 720 ctcaggatca aagtatgtac aagcctttgt gaatattttt tccttctcac ttggcaaata 780 caattcctga gatcaataac ctcgtctttt taattttttc ctcgtctttt taactattta 840 taaaatattg aattataaaa tatgtaatta taaatacttt aattataaaa tatgtaatta 900 taaatacttt aattataaaa tatgtaatta taaatacttt ataaaatatg taattataaa 960 atatgtaatt ataaacattt taattataaa atatgtaatt ataaacattt taattataaa 1020 atatgtaatt ataaacattt taattataaa atatgtaatt ataaacattt taattataaa 1080 atatgtaatt ataaacattt taattataaa atatttaatt ataaacattt taattataaa 1140 atatttaatt ataaaaatt taattattaaaa atatttaatt ataaaaatt taattattaaaa 1200 atatttaatt ataaaattt taattattaaaa atatttaatt ataaatactt taattattaaaa 1260 attttaatt attaaattt taattattaaaa attttaatt attaaattt taattattaaaa 1320 tattttaatt ataaaatatt taattataaa aacacaatta cctcatcttt ttaaatattt 1380 ttgcaaaata tttccctcca taatttctcc gtttccattt ttatctgtt actaaat 1438 <210> 13 <211> 2038 <212> DNA <213> Artificial <220> <223> CMV Promoter - Puromycin - S / MAR sequence <400> 13 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 420 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa 540 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtatgacc gagtacaagc 600 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg 660 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg 720 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg 780 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg 840 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc 900 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg 960 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc 1020 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc 1080 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg 1140 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacttctc cactcctggc 1200 aggctgagtg aaataaagga cttgttattt catctcgagg cctaccggag agccttgcct 1260 tgcaaaggca gacagtcagt gaggaagact atgtggcaca tgaagacacc agaggtgttc 1320 ctcaggatca aagtatgtac aagcctttgt gaatattttt tccttctcac ttggcaaata 1380 caattcctga gatcaataac ctcgtctttt taattttttc ctcgtctttt taactattta 1440 taaaatattg aattataaaa tatgtaatta taaatacttt aattataaaa tatgtaatta 1500 taaatacttt aattataaaa tatgtaatta taaatacttt ataaaatatg taattataaa 1560 atatgtaatt ataaacattt taattataaa atatgtaatt ataaacattt taattataaa 1620 atatgtaatt ataaacattt taattataaa atatgtaatt ataaacattt taattataaa 1680 atatgtaatt ataaacattt taattataaa atatttaatt ataaacattt taattataaa 1740 attttaatt attaaattt taattattaaaa attttaatt attaaattt taattattaaaa 1800 atatttaatt ataaaattt taattataaa atatttaatt ataaatactt taattataaa 1860 atatttaatt ataataatt taattaataa atatttaatt ataataatt taattaataa 1920 tattttaatt ataaaatatt taattataaa aacacaatta cctcatcttt ttaaatattt 1980 ttgcaaaata tttccctcca taatttctcc gtttccattt ttattctgtt acttaaat 2038 <210> 14 <211> 6189 <212> DNA <213> Artificial <220> <223> Element40-GPF-P2A-Puromycin-S / MAR <400> 14 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt ccgccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 420 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa 540 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc 600 agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg 660 ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa 720 ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact 780 gacatccact ttgcctttct ctccacaggg gtaccgaagc cgctagcgct accggtcgcc 840 accatgcccg ccatgaagat cgagtgccgc atcaccggca ccctgaacgg cgtggagttc 900 gagctggtgg gcggcggaga gggcaccccc gagcagggcc gcatgaccaa caagatgaag 960 agcaccaaag gcgccctgac cttcagcccc tacctgctga gccacgtgat gggctacggc 1020 ttctaccact tcggcaccta ccccagcggc tacgagaacc ccttcctgca cgccatcaac 1080 aacggcggct acaccaacac ccgcatcgag aagtacgagg acggcggcgt gctgcacgtg 1140 agcttcagct accgctacga ggccggccgc gtgatcggcg acttcaaggt ggtgggcacc 1200 ggcttccccg aggacagcgt gatcttcacc gacaagatca tccgcagcaa cgccaccgtg 1260 gagcacctgc accccatggg cgataacgtg ctggtgggca gcttcgcccg caccttcagc 1320 ctgcgcgacg gcggctacta cagcttcgtg gtggacagcc acatgcactt caagagcgcc 1380 atccacccca gcatcctgca gaacggggc cccatgttcg ccttccgccg cgtgggaggag 1440 ctgcacagca acaccgagct gggcatcgtg gagtaccagc acgccttcaa gacccccatc 1500 gccttcgcca gatctggcag cggcgccacc aacttcagcc tgctgaagca ggccggcgac 1560 gtgggaaa accctgggcc catgaccgag tacaagccca cggtgcgcct cgccacccgc 1620 gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 1680 cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 1740 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 1800 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 1860 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg 1920 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 1980 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc 2040 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 2100 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 2160 acccgcaagc ccggtgcctg aagatctatg catgcagaag ttggtcgtga ggcactgggc 2220 aggtaagtat caagggttaca agacaggtcg acttctccac tcctggcagg ctgagtgaaa 2280 taaggactt gttatttcat ctcgaggcct accggagagc cttgccttgc aaaggcagac 2340 agtcagtgag gaagactatg tggcacatga agacaccaga ggtgttcctc aggatcaaag 2400 tatgtacaag cctttgtgaa tattttttcc ttctcacttg gcaaatacaa ttcctgagat 2460 caataacctc gtctttttaa ttttttcctc gtctttttaa ctatttaa aatattgaat 2520 tataaaatat gtaattataa atactttaat tataaaatat gtaattataa atactttaat 2580 tataaaatat gtaattataa atacttata aaatatgta ttataaaaata tgtaattata 2640 acattttaa ttataaaaata tgtaattaa acattttaa ttataaaaata tgtaattaa 2700 acattttaa ttataaaaata tgtaattaa acattttaa ttataaaaata tgtaattaa 2760 aacattttaa ttaaaaaaata ttaaattata aacattttaa ttaaaaaaata ttaaattata 2820 aatattttaa ttataaaata tttaattata aatattttaa ttataaaata tttaattata 2880 aatattttaa ttataaaata tttaattata aatactttaa ttataaaata tttaattata 2940 aatattttaa ttataaaata tttaattata aatattttaa ttataaatat tttaattata 3000 aaatatttaa ttataaaac acaattacct catcttttta aatatttttg caaaatattt 3060 ccctccataa tttctccgtt tccattttta ttctgttact taaataattc tgcagtcgac 3120 ggttactgac atccactttg cctttctctc cacaggtgtc cactctaccg cgggcccggg 3180 atccaccgga tctatagataac tgatcataat cagccatacc acatttgtag aggttttact 3240 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt 3300 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 3360 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 3420 tgtatcttac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 3480 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaaatc gacgctcaag 3540 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 3600 cctcgtgcgc tctcctgttc cgaccctgcc gcttacggga tacctgtccg cctttctccc 3660 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 3720 cgttcgctcc aagctggggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 3780 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 3840 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 3900 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 3960 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4020 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4080 agatccttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4140 gattttggtc atgccgtctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg 4200 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag 4260 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag 4320 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca 4380 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc 4440 gaacagttcg gctggcgcga gccctgatg ctcttcgtcc agatcatcct gatcgacaag 4500 accggcttcc atccgagtac gtgctctctc gatgcgatgt ttcgcttggt ggtcgaatgg 4560 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 4620 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 4680 ccagtccctt cccgcttcag tgacaacgtc gagtacagct gcgcaaggaa cgcccgtcgt 4740 ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc 4800 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 4860 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 4920 agaacctgcg tgcaatccat cttgttcaat cataatatta ttgaagcatt tatcagggtt 4980 cgtctcgtcc cggtctcctc ccatgcatgt caatattggc cattagccat attattcatt 5040 ggttatatag cataaatcaa tattggctat tggccattgc atacgttgta tctatatcat 5100 aatatgtaca aagcttgatc aagaaagcac tccgggctcc agaaggagcc ttccaggcca 5160 gctttgagca taagctgctg atgagcagtg agtgtcttga gtagtgttca gggcagcatg 5220 ttaccattca tgcttgactt ctagccagtg tgacgagagg ctggagtcag gtctctagag 5280 agttgagcag ctccagcctt agatctccca gtcttatgcg gtgtgcccat tcgctttgtg 5340 tctgcagtcc cctggccaca cccagtaaca gttctgggat ctatgggagt agcttcctta 5400 gtgagctttc ccttcaaata ctttgcaacc aggtagagaa gtttggagtg aaggttttgt 5460 tcttcgtttc ttcacaatat ggatatgcat cttcttttga aaatgttaaa gtaaattacc 5520 tctcttttca gatactgtct tcatgcgaac ttggtatcct gtttccatcc cagccttcta 5580 taacccagta acatcttttt tgaaaccagt gggtgagaaa gacacctggt caggaacgcg 5640 gaccacagga caactcaggc tcacccacgg catcagacta aaggcaaaca aggactctgt 5700 ataaagtacc ggtggcatgt gtattagtgg agatgcagcc tgtgctctgc agacagggag 5760 tcacacagac acttttctat aatttcttaa gtgctttgaa tgttcaagta gaaagtctaa 5820 cattaattt gattgaacaa ttgtatattc atggaattt ttggaacgga ataccaaaaa atggcaatag tggttctttc tggatggaag acaaactttt cttctttaaa ataaattttta ttttatatat ttgaggttga ccacatgacc ttaaggatac atatagacag taaactggtt actacagtga agcaaattaa catatctacc atcgtacata gttacatttt tttgtgtgac aggaacagct aaaatctacg tatttaacaa aactcctaaa gacaatacat ttttattaac fatherccctc atgatgtaca ttagatctgt acatttatat tggctcatgt ccaatatgac cgccatgtt 6189 <210> 15 <211> 248 <212> DNA <213> Homo sapiens <400> 15 attachment aaacatttta attachment aattatt attachment attachment attachment attachment attachment attachment aaatacttta attachment aattatt attachment aattattat aaatatttta attataatta ttttaattat aaaatattta attataaaaaa cacaatta 248

Claims

1. A polynucleotide comprising at least one promoter and an S / MAR element, wherein the S / MAR element is located downstream of the promoter, the nucleic acid sequence of the S / MAR element (S / MAR sequence) contains at least three sequence motifs ATTA per 100 nucleotides over a continuous stretch of up to 200 nucleotides, the S / MAR sequence comprising a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 6 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 15, and is up to 1 kb in length.

2. The polynucleotide of claim 1, wherein the S / MAR sequence comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO:

7.

3. (i) the polynucleotide further comprises a cargo sequence, wherein the cargo sequence encodes a therapeutic polypeptide; or (ii) the polynucleotide further comprises a cargo sequence, the cargo sequence encoding a therapeutic polypeptide, and the therapeutic polypeptide is a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a polypeptide that is lacking in cells affected by a genetic disease; A polynucleotide according to claim 1 or 2.

4. The polynucleotide according to any one of claims 1 to 3, wherein the polynucleotide further contains a coding sequence encoding a selectable marker (selectable marker sequence), the selectable marker sequence being present between the promoter and the S / MAR element, the promoter and the selectable marker as a whole constituting a selectable marker gene, and the selectable marker is a selectable marker for eukaryotic cells.

5. 5. The polynucleotide of any one of claims 1 to 4, wherein the polynucleotide further comprises a bacterial origin of replication and / or a bacterial selectable marker gene, wherein the bacterial origin of replication and / or the bacterial selectable marker gene is separated from the rest of the sequences contained within the polynucleotide by the presence of at least one insulation element.

6. The polynucleotide of any one of claims 1 to 5, wherein the polynucleotide does not have a simian virus 40 (SV40) origin of replication, a bovine papillomavirus (BPV) origin of replication, or an Epstein-Barr virus (EBV) origin of replication.

7. (i) the polynucleotide replicates episomally in the host cell; or (ii) the polynucleotide replicates episomally in the host cell, and the episomal replication is stable episomal replication; or (iii) the polynucleotide replicates episomally in the host cell, and the episomal replication is stable episomal replication and is episomal replication in mammalian cells; A polynucleotide according to any one of claims 1 to 6.

8. A host cell comprising the polynucleotide of any one of claims 1 to 7, wherein the host cell is a CD34+ progenitor cell; a CD61+ platelet; a CD19+ B lymphocyte; a CD14+ monocyte; a CD15+ granulocyte; a CD3+ cytotoxic T lymphocyte, a CD3+ cytotoxic T lymphocyte that is also positive for CD8 and CD45; a CD3+ helper T lymphocyte, a CD3+ helper T lymphocyte that is also positive for CD4 and CD45; a CD3+ activated T lymphocyte, a CD3+ activated T lymphocyte that is also positive for CD25 and CD45, a tumor-infiltrating lymphocyte, or a natural killer (NK) cell.

9. A composition comprising a polynucleotide according to any one of claims 1 to 7 and / or a host cell according to claim 8 for use in treating a genetic disease.

10. 10. The composition of claim 9, wherein the genetic disease is cancer, phenylketonuria, alkaptonuria, congenital asthenia of the liver, choroideremia, or Stargardt's disease.

11. (i) A method for stably transfecting a host cell, comprising: a) contacting said host cell with a polynucleotide according to any one of claims 1 to 7; and b) thereby stably transfecting host cells wherein the stable transfection comprises stable episomal replication of the polynucleotide. The method, or (ii) a method for stably transfecting a host cell, comprising: a) contacting said host cell with a polynucleotide according to any one of claims 1 to 7; and b) thereby stably transfecting host cells wherein the stable transfection comprises stable episomal replication of the polynucleotide, and wherein the stable transfection comprises episomal replication such that the polynucleotide remains detectable in the host cell population after an average of 50 cell divisions. The method.

12. Use of a polynucleotide according to any one of claims 1 to 7 for the stable genetic modification of a host cell, comprising: (i) the stably genetically modifying comprises stable episomal replication of the polynucleotide in the host cell; or (ii) the stably genetically modifying comprises stable episomal replication of the polynucleotide in the host cell, and the stably genetically modifying comprises episomal replication such that the polynucleotide remains detectable in the host cell population after an average of 50 cell divisions. The above use.

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