Expression constructs for genetic modification of cells

Abstract

To provide improved means and methods for stable transfection of cells.SOLUTION: Provided is a polynucleotide comprising at least one promoter, at least one expressible construct, and an S / MAR element, wherein the polynucleotide is an integration construct or a non-integrative vector construct, wherein the S / MAR element is located downstream of the promoter and of the expressible construct, and wherein the S / MAR element is flanked by a splice donor and a splice acceptor. A composition and a host cell comprising the polynucleotide, as well as uses and methods related thereto, are also provided.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a polynucleotide comprising at least one promoter, at least one expressible construct, and an S / MAR element, wherein the polynucleotide is an integration construct or a non-integration vector construct, the S / MAR element is located downstream of the promoter and of the expressible construct, and the S / MAR element is adjacent to a splice donor and a splice acceptor. The invention also relates to a composition and a host cell comprising the polynucleotide, and to related uses and methods.

Background Art

[0002] Genetic modification of cells is routinely used in modern cell culture for scientific purposes. However, the use of the corresponding techniques in the treatment of genetic diseases caused by gene mutations, while highly desirable, is hampered by the fact that available methods typically provide only transient modifications, e.g., transient transfection protocols, while methods for providing stable modification of cells still rely on integration of the transgene into the genome of the host cell. However, integration of the transgene, even when targeting a specific locus, carries the risk of inducing harmful mutations, which can, for example, result in cancer as a side effect of treatment.

[0003] The scaffold / matrix attachment region (S / MAR), also known as the scaffold attachment region (SAR) or matrix attachment region (MAR), is known as a sequence in the eukaryotic genome that mediates the attachment of the nuclear matrix. Furthermore, S / MAR sequences have been found to have insulator properties and to block the extension of condensed chromatin domains into transcriptionally active regions and the interaction between distal enhancers and promoters (Yusufzai and Felsenfeld (2004), PNAS 101(23):8620). S / MARs are AT-rich sequences, and some AT-rich motifs have been found to be further concentrated (Liebeich et al., (2002), NAR 30(15):3433). Various vectors have been proposed for stable maintenance in cells based on the S / MAR motif, for example, in US Patent No. 6,410,314 and Haase et al., (2010), BMC Biotechnology 10:20. Furthermore, epigenetic effects that affect 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. SUMMARY OF THE INVENTION

[0004] Nevertheless, there is a need in the art for improved means and methods for the stable transfection of cells that use S / MAR elements while maintaining sufficient expression of the transgene, particularly over long periods of time. This problem is solved by the means and methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0005]

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Figure 4-1

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Mode for Carrying Out the Invention

[0006] Accordingly, the present invention relates to a polynucleotide comprising at least one promoter, at least one expressible construct, and an S / MAR element, wherein the polynucleotide is an integration construct or a non-integration vector construct, the S / MAR element is located downstream of the promoter and the expressible construct, and the S / MAR element is adjacent to a splice donor and a splice acceptor.

[0007] As used hereinafter, the terms "have", "comprise", or "include", or any grammatical variations thereof, are used in a non-exclusive sense. Thus, both of these terms can refer to situations where there are no additional features in the entity described in connection therewith other than the features introduced by these terms, as well as situations where one or more additional features are present. By way of example, the expressions "A has B", "A comprises B", and "A includes B" can all refer to situations where there are no other elements in A other than B (i.e., the situation where A consists solely and exclusively of B), as well as situations where there are one or more additional elements, such as element C, elements C and D, or additional elements, in the entity A other than B.

[0008] Furthermore, as used hereinafter, the terms "preferably", "more preferably", "most preferably", "in particular", "more particularly", "specifically", "more specifically", or similar terms are used with optional features without limiting further possibilities. Thus, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The present invention can be practiced, as will be recognized by those skilled in the art, by using alternative features. Similarly, features introduced by "in one embodiment of the present invention" or similar expressions do not limit in any way further embodiments of the present invention, do not limit in any way the scope of the present invention, and do not limit in any way the possibility of combining features introduced in this way with other optional or non-optional features of the present invention, and are intended to be optional features.

[0009] Further, unless otherwise indicated, the term "about" relates to an indicated value having the technical accuracy generally recognized in the relevant art, preferably to within ±20% of the indicated value, more preferably ±10%, and most preferably ±5%. Further, the term "essentially" indicates that there is no deviation that affects the indicated result or use, i.e., the potential deviation does not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Thus, "consisting essentially of" means excluding materials that are present as impurities, inevitable materials that result from the process used to provide the component, and other components other than those added for purposes other than achieving the technical effects of the present invention, while including the specified components. For example, a composition defined using the phrase "consisting essentially of" includes 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 "substantially identical" indicates an identity percentage 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 "substantially identical" includes 100% identity. The foregoing applies, with the necessary modifications, to the term "substantially complementary".

[0010] As used herein, the term "polynucleotide" refers to linear or circular nucleic acid molecules. This term encompasses single-stranded as well as partially or fully double-stranded polynucleotides. Preferably, the polynucleotide is RNA or DNA including cDNA, more preferably DNA. Further, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified derivatives, such as biotinylated polynucleotides. The polynucleotides of the present invention are preferably provided either as isolated polynucleotides (i.e., isolated from their natural context) or in a genetically modified form. The polynucleotides of the present invention include at least one promoter active in a host cell, at least one expressible construct, and an S / MAR element. Further, the polynucleotide has a biological activity that provides for the expression of the expressible construct in the host cell, as specified hereinbelow in all cases. Preferably, the polynucleotide has a length of up to 1 Mb, more preferably up to 500 kb, even more preferably up to 200 kb, and most preferably up to 100 kb. Preferably, the polynucleotide is a non-naturally occurring polynucleotide. Thus, preferably, the nucleotide is an artificial polynucleotide. Also preferably, the polynucleotide is a chimeric polynucleotide. More preferably, the polynucleotide contains at least one nucleic acid sequence that is heterologous to the remaining nucleic acid sequences it contains. Preferably, the polynucleotide is lacking any centromere and / or telomere sequences. Also preferably, the polynucleotide contains a transcriptional insulator element upstream of the above promoter. Preferred transcriptional insulator elements are element 40 and the S / MAR element as specified herein. Thus, preferably, the promoter and the expressible construct are isolated from the remaining sequences contained within the polynucleotide by the presence of at least one insulation element, more preferably by being flanked (sandwiched) by the insulation element.

[0011] Preferably, the polynucleotide comprises additional expression control sequences that enable the expression of genes in prokaryotes and / or eukaryotes, preferably eukaryotic host cells or their isolated fractions. The expression of the polynucleotide preferably comprises the transcription of the polynucleotide into translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably comprise regulatory sequences that ensure the initiation of transcription and, optionally, a polyA signal that ensures the termination of transcription and the stabilization of the transcript. Additional regulatory elements may include transcriptional enhancers as well as translational enhancers. Examples of regulatory elements that enable 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), the CMV enhancer, the SV40 enhancer, or the globin intron in mammalian cells and other animal cells. Regulatory sequences preferred for the expression of microRNA or siRNA are also known in the art. Furthermore, inducible or cell-type specific expression control sequences may be included in the polynucleotides of the present invention. Inducible expression control sequences may include the tet or lac operator sequences, or sequences that are inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to the elements involved in the initiation of transcription, such regulatory elements may also include transcription termination signals, such as the SV40-polyA site or the tk-polyA site, downstream of the polynucleotide.

[0012] As used herein, the term polynucleotide preferably includes variants of the specifically recited polynucleotide. More preferably, the term polynucleotide relates to the specific polynucleotide shown. The term "polynucleotide variant" as used herein includes polynucleotide variants related to the polynucleotides herein, which are nucleic acid sequences characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition and / or deletion, and which have the biological activity(ies) specified for the specific polynucleotide. Thus, it should be understood that polynucleotide variants referred to according to the present invention will have different nucleic acid sequences due to at least one nucleotide substitution, deletion and / or addition. Preferably, the polynucleotide variant includes an ortholog, paralog or other homolog of a specific polynucleotide or a functional subsequence thereof, such as an S / MAR element. Also preferably, the polynucleotide variant includes a naturally occurring allele of a specific polynucleotide or a functional subsequence thereof. Polynucleotide variants also preferably include polynucleotides comprising nucleic acid sequences that can hybridize to the aforementioned specific polynucleotide or a functional subsequence thereof 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 conditions in 6× sodium chloride / sodium citrate (= SSC) at about 45 °C, followed by one or more washing steps in 0.2× SSC, 0.1% SDS at 50 - 65 °C. Those skilled in the art know that these hybridization conditions vary depending on the type of nucleic acid, for example, with respect to the temperature and concentration of the buffer when an organic solvent is present. For example, under "standard hybridization conditions", the temperature varies depending on the type of nucleic acid between 42 °C and 58 °C in an aqueous buffer with a concentration of 0.1× - 5× SSC (pH 7.2).When an organic solvent is present in the aforementioned buffer (e.g., 50% formamide), the temperature under standard conditions is about 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 hybridization temperatures are determined, for example, for nucleic acids having a length of about 100 bp (= base pairs) and a 50% G+C content in the absence of formamide. Thus, other conditions that are more suitable for low G+C DNA, which are in principle known to those skilled in the art, can be found to be more appropriate by those skilled in the art. Those skilled in the art know how to determine the required hybridization conditions by referring to standard textbooks. Alternatively, polynucleotide variants can be obtained by PCR-based techniques, such as 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 sequence comparison of the nucleic acid sequences of the polynucleotides of the present invention or the amino acid sequences of the polypeptides of the present invention with the sequences of other organisms. As templates, DNA or cDNA derived from bacteria, fungi, plants, or preferably animals can be used. Furthermore, the variants include polynucleotides comprising nucleic acid sequences that are 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 shown nucleic acid sequence or its functional partial sequence. Furthermore, polynucleotides comprising nucleic acid sequences encoding amino acid sequences that are 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 shown amino acid sequence are also included. The percent identity values are preferably calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on various algorithms are available to those skilled in the art for comparing different sequences.In this regard, the algorithms of Needleman and Wunsch, or Smith and Waterman yield particularly reliable results. To perform sequence alignment, the program PileUp (J. Mol. Evolution., Vol. 25, pp. 351-360, 1987, Higgins et al., CABIOS, Vol. 5, 1989: pp. 151-153) or the programs Gap and BestFit (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)), Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))) are preferably used. Preferably, the above programs are used with their standard parameters. The value of the sequence identity as described above in percent (%) units is preferably determined using the following settings, which are normally used as the standard settings for sequence alignment, unless otherwise specified: gap weight: 50, length weight: 3, average match: 10.000 and average mismatch: 0.000, over the entire sequence region, using the program GAP.

[0013] A polynucleotide comprising a fragment of any of the specifically shown nucleic acid sequences, wherein the polynucleotide retains one or more of the shown activities, is also included as a variant polynucleotide of the present invention. As used herein, a fragment preferably comprises at least 200, preferably at least 300, more preferably at least 400 consecutive nucleotides of any one of the specific nucleic acid sequences, or at least 100, preferably at least 200, more preferably at least 300 consecutive amino acids of any one of the specific amino acid sequences, and encodes an amino acid sequence that still has the shown activity.

[0014] The polynucleotides of the present invention consist of, consist essentially of, or contain any of the aforementioned nucleic acid sequences. Thus, they may likewise contain additional nucleic acid sequences. Specifically, the polynucleotides of the present invention may encode, for example, a fusion protein or a selectable marker. Such a fusion protein may contain, as an additional moiety, a polypeptide for monitoring expression (e.g., a green, yellow, blue, or red fluorescent protein, alkaline phosphatase, etc.), or a so-called "tag" that can serve as a detectable marker or as an auxiliary means for purification purposes. Tags for various purposes are well known in the art and are described elsewhere in this specification.

[0015] The polynucleotide contains at least one expressible construct. As used herein, the term "expressible construct" relates to a nucleic acid sequence of interest that is introduced into a host cell and expressed therein. As used herein, the term expressible construct includes all nucleic acid sequences that cause at least one gene product to be produced in a host cell in the context of the polynucleotides specified herein. Thus, an expressible construct preferably does not require a secondary promoter in addition to that provided by the polynucleotide itself. However, it is also contemplated that an expressible construct may contain a secondary promoter, specified below herein, that induces or additionally induces transcription of one or more sequences of interest. Preferably, an expressible construct encodes multiple gene products. Thus, an expressible construct can encode, for example, two polypeptides, one of which can be a selectable marker as specified below herein. Preferably, the gene product is RNA, including microRNA, siRNA, and mRNA, preferably mRNA, and / or the gene product is a polypeptide as specified elsewhere herein. Thus, preferably, an expressible construct is a polynucleotide of interest, e.g., a nucleic acid sequence encoding RNA and / or a polypeptide of interest.

[0016] Preferably, the RNA to be encoded is an interfering, non-coding nucleic acid. Thus, the non-coding interfering nucleic acid expressed from the polynucleotide can typically be an antisense RNA, siRNA, microRNA or ribozyme. Thereby, by using the polynucleotides specified herein, gene expression in a host cell can be modified, i.e., down-regulated, and can also be used, for example, to treat a disease or disorder including those described elsewhere herein. The improved stability and expression characteristics of the polynucleotides specified herein can also improve gene silencing approaches. Thus, preferably, the RNA of interest is a therapeutic RNA. As used herein, the term "therapeutic RNA" relates to any RNA that mediates a change in the physiological and / or metabolic state of a host cell comprising the therapeutic RNA. Thus, preferably, the therapeutic RNA is an interfering non-coding RNA, particularly siRNA, microRNA, antisense RNA, or ribozyme, as specified above herein. Means and methods for designing interfering nucleic acids for use, for example, in gene silencing are known to those of skill in the art. More preferably, the therapeutic RNA is an mRNA encoding a therapeutic polypeptide, or a subunit or active fragment thereof, as specified below herein. Preferably, the therapeutic RNA mediates a change in the physiological and / or metabolic state of a host cell comprising the therapeutic RNA, thereby preferably contributing to the amelioration or treatment of a disease or disorder, as specified elsewhere herein.

[0017] The polypeptide of interest can in principle be any polypeptide whose overexpression in a host cell is desired. Polypeptides for which high and / or continuous expression is desired are preferred. Preferably, the polypeptide of interest is a therapeutic polypeptide. As used herein, the term "therapeutic polypeptide" relates to any polypeptide that mediates a change in the physiological and / or metabolic state of a host cell comprising the therapeutic polypeptide as described above, and / or a cell in direct or fluid contact with such a host cell (preferably via a body fluid, more preferably via blood, lymph, saliva, cerebrospinal fluid, and / or interstitial fluid). More preferably, the therapeutic polypeptide mediates a change in the physiological and / or metabolic state of a host cell and / or a cell in direct or fluid contact with such a host cell, thereby preferably contributing to the amelioration or treatment of a disease or disorder as specified elsewhere herein. Preferably, the therapeutic polypeptide is preferably an antibody as specified below herein. Also preferably, the therapeutic polypeptide is a T cell receptor (TCR), more preferably a human or chimeric T cell receptor, a chimeric antigen receptor (CAR), preferably a MART1 TCR, or a polypeptide that is absent in cells affected by a genetic disease as specified elsewhere herein. Thus, for example preferably, the polynucleotide comprises at least one expressible construct encoding a polypeptide that provides phenylalanine hydroxylase activity (EC 1.14.16.1) for the treatment of phenylketonuria, or encoding the REP1 gene for the treatment of choroideremia, or encoding the RPE65 gene for the treatment of Leber congenital amaurosis, or encoding factor VIII, factor IX, and / or factor X for the treatment of hemophilia, or encoding the USH2a gene for the treatment of Usher disease.

[0018] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, single-chain antibodies, single-domain antibodies (VHHs) also known as nanobodies, and antibody fragments as long as they exhibit the desired binding activity. Preferably, the antibody is a single-chain antibody or a VHH (nanobody). Preferably, the antibody is a therapeutic antibody, i.e., it has binding activity to a disease-related molecule associated with treatment, preferably a polypeptide, and contributes to the treatment of a disease or disorder caused or exacerbated by the disease-related molecule. "Antibody fragments" relevant to this specification include a portion of an intact antibody that contains its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment (the name reflects its ability to crystallize readily). Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigens. "Fv" is the minimal antibody fragment that contains a complete antigen-binding site. Preferably, the dimeric Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain held together by non-covalent bonds. In the single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can bind in a "dimeric" structure similar to that of the dimeric Fv species. In this configuration, the three hypervariable regions (HVRs, also called complementarity-determining regions (CDRs)) of each variable domain interact to define the antigen-binding site. Collectively, six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind the antigen.The term "diabody" means an antibody fragment having two antigen-binding sites, the fragments comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domain on the other chain, creating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described, for example, in EP 0 404 097; WO 93 / 1161; Hudson et al., Nat. Med. 9 (2003) 129-134; and Hollinger et al., PNAS USA 90 (1993) 6444-6448. Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9 (2003) 129-134. The term "single domain antibody" (VHH) or "nanobody" relates to an antibody fragment containing one variable antibody domain and is in principle known to the person skilled in the art. A review is provided, for example, by Muyldermanns et al. (2009), Vet Immunol Immunopathol. 128 (1-3):178. Preferably, the VHH comprises the CDRs of a heavy chain antibody obtained preferably from alpaca, dromedary, camel, llama, or shark immunized with a target polypeptide. Preferably, the antibody is an anti-tumor antigen antibody, more preferably an anti-tumor specific antigen antibody, even more preferably an anti-carcinoembryonic antigen (CEA) antibody, even more preferably a single chain anti-CEA antibody, and most preferably one encoded by SEQ ID NO: 16.

[0019] Preferably, the expressible construct comprises, or further comprises, a coding sequence encoding a selectable marker polypeptide, and preferably, the promoter and / or secondary promoter of the polynucleotide and the above 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 refers to a nucleic acid sequence that, when expressed in a host cell, confers resistance to at least one condition that mediates a selection pressure on the host cell when applied. Selectable markers are known in the art for both prokaryotic and eukaryotic cells. Preferably, the selectable marker is a selectable marker for eukaryotic cells. Preferably, the selectable marker is a selectable marker polypeptide having transporter activity and / or enzyme activity that removes or modifies the selectable compound from the host cell, more preferably a selectable marker polypeptide that modifies the selectable compound so as to inactivate it. Preferably, the selectable marker gene further encodes at least one intron upstream of the sequence encoding the selectable marker polypeptide, preferably. Preferably, the selectable marker is a marker that mediates resistance to puromycin, blasticidin, neomycin, and / or zeocin, more preferably a marker that mediates resistance to puromycin. Thus, preferably, the promoter and the selectable 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. Preferably, the selectable 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, preferably, the selectable marker is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), both of which are generally known in the art. Preferably, the TCR and / or CAR preferably have known specificities such that T cell signaling can be induced in host cells containing the TCR and / or CAR. Preferably, in such cases, the host cell is a T cell or an NK cell.Preferably, the selectable marker gene lacks a polyA signal and a transcription termination signal(s). Thus, preferably, the polynucleotide further comprises a coding sequence (selectable marker sequence) encoding a selectable marker, wherein the selectable marker sequence is included in an expressible construct, and preferably, the promoter and / or secondary promoter and the selectable marker sequence together constitute a selectable marker gene, and the selectable marker is a selectable marker for eukaryotic cells. Preferably, the selectable marker is puromycin acetyltransferase (Genbank Acc No. KX548903.1 (SEQ ID NO: 9)) encoded by nucleotides 535-1134 (SEQ ID NO: 10) of Genbank Acc No. KX548903.1. Thus, the selectable marker gene preferably comprises a nucleic acid sequence that a) causes the expression of a puromycin-resistant polypeptide comprising the sequence of SEQ ID NO: 9, b) causes the expression of a puromycin-resistant polypeptide comprising a sequence that is 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 that is at least 70% identical to the sequence of SEQ ID NO: 10, e) comprises a nucleic acid sequence encoding a puromycin-resistant polypeptide comprising, preferably consisting of, the sequence of SEQ ID NO: 9, and / or f) comprises a nucleic acid sequence encoding a puromycin-resistant polypeptide comprising a sequence that is at least 70% identical to the sequence of SEQ ID NO: 9, preferably consisting of.

[0020] As specified herein, an expressible construct is present 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 of a transcript containing the expressible construct. Splice donor and splice acceptor sites are known in the art. Preferably, the sequence encoding the expressible construct is present 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 of a transcript encoding a polypeptide, preferably a therapeutic polypeptide.

[0021] Preferably, the expressible construct contains a sequence that enables the expression of two (or more) polypeptides in a eukaryotic cell from one 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 derived from porcine teschovirus-1, intervening a selectable marker and a sequence encoding an RNA or polypeptide of interest. Suitable sequences are known in the art, for example, those described by Kim et al. (2011) PLoS ONE 6(4): e18556.

[0022] As used herein, the term "host cell" refers to any cell capable of receiving, integrating, or stably replicating a polynucleotide and expressing an expressible construct. 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, particularly a mouse cell or a rat cell. Even more preferably, the host cell is a mammalian cell, and most preferably a human cell. Preferably, 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, preferably a CD3+ cytotoxic T lymphocyte that is also positive for CD8 and CD45; a CD3+ helper T lymphocyte, preferably a CD3+ helper T lymphocyte that is also positive for CD4 and CD45; a CD3+ activated T lymphocyte, preferably a CD3+ activated T lymphocyte that is also positive for CD25 and CD45, a tumor-infiltrating lymphocyte, or a natural killer (NK) cell. Also preferably, the host cell is an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, an airway epithelial cell, a fibroblast, or a retinal epithelial cell. As will be understood by those skilled in the art, the polynucleotide may further have a sequence that enables replication in a bacterial cell, particularly an origin of bacterial replication. Preferably, the bacterial cell is a cell of a laboratory bacterial strain, more preferably an Escherichia coli cell.

[0023] The term "promoter" is generally known to those skilled in the art as a genetic element that, optionally in cooperation with further regulatory elements, induces the transcription level of a given gene. A promoter can be constitutive, i.e., it can provide a constant level of transcription essentially independent of the state of the host cell, or it can be regulated, i.e., it can provide a transcription level depending on the state of the host cell. Furthermore, a promoter can be cell type and / or tissue specific, i.e., it can provide a level of transcription detectable only in several or only one cell type. Preferably, the promoter according to the present invention is active in the host cell specified above herein. As will be understood by those skilled in the art, the choice of promoter can depend on the type of host cell to be targeted. Promoters suitable for a specific cell type as well as constitutive promoters are known in the art. Preferably, the promoter is a eukaryotic promoter, more preferably a constitutive eukaryotic promoter, even more preferably a strong 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" is used for the promoters specified above, while any other promoter potentially further present on the polynucleotide is referred to as a "secondary promoter". Thus, preferably, the promoter is a promoter that induces transcription to the S / MAR sequence in the host cell. Also preferably, for example, a promoter that does not induce transcription to the S / MAR sequence of the polynucleotide, which is a prokaryotic promoter, transcriptionally shielded from the S / MAR sequence, and / or induces transcription away from the S / MAR sequence, is a secondary promoter.Preferably, the promoter comprises less than 1000, more preferably less than 250, even more preferably less than 100, and most preferably less than 20 consecutive base pairs corresponding to the apolipoprotein B promoter. Thus, preferably, the polynucleotide does not contain the human apolipoprotein B promoter, and more preferably does not contain the apolipoprotein B promoter.

[0024] Preferably, the expressible construct is located immediately downstream of the promoter, and / or the S / MAR sequence is located immediately downstream of the expressible construct. Preferably, being located "immediately downstream" means lacking intervening transcription termination signals, and more preferably lacking intervening genes. Thus, preferably, the transcript initiated by the promoter and containing the expressible construct sequence preferably contains the transcribed S / MAR sequence, and more preferably contains the complete S / MAR sequence contained in the polynucleotide. As will be understood by those skilled in the art in view of the descriptions elsewhere in this specification, the polynucleotide preferably further comprises splicing sites that mediate excision of the S / MAR sequence from the primary transcript. Thus, more preferably, at least the primary transcript initiated by the promoter and containing the expressible marker sequence preferably contains the transcribed S / MAR sequence, and more preferably contains the complete S / MAR sequence contained in the polynucleotide. Also preferably, the term "immediately downstream" includes polynucleotides in which the promoter and the S / MAR sequence are separated by an extended nucleic acid sequence, provided that no transcription termination signal intervenes between the promoter and the S / MAR. Preferably, the sequence intervening between the promoter and the first nucleotide of the expressible construct, or between the last nucleotide of the expressible construct and the S / MAR sequence, has 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.

[0025] The term "S / MAR element", also known by the name "scaffold / matrix attachment region", is generally known to those skilled in the art to relate to the DNA sequences that mediate the attachment of the eukaryotic nuclear matrix to DNA. S / MAR sequences typically originate from sequences in the DNA of eukaryotic chromosomes. A variety of S / MAR sequences are available, and the sequences are available from public databases, for example, those described in Liebich et al. (2002), Nucleic Acids Res. 30, pp. 312-374. According to the present invention, the nucleic acid sequence of the S / MAR element (previously called the S / MAR sequence) preferably contains at least three sequence motifs ATTA (SEQ ID NO: 1) per 100 nucleotides over a stretch of up to 200 nucleotides. Thus, the motifs contained in the S / MAR sequence contain a large number of 4-nucleotide 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 at most 3 kb, more preferably at most 2 kb, even more preferably at most 1.5 kb, even more preferably at most 1 kb, even more preferably at most 0.5 kb, and most preferably at most 0.25 kb. Thus, 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. As will be understood, the expression "containing n sequence motifs per 100 nucleotides" relates to the average number of the sequence motifs calculated per 100 base pair sequences and can thus 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 nucleotides is determined over the entire length of the S / MAR sequence.If in doubt, for example, if the boundaries of the S / MAR array cannot be determined, the number of sequence motifs per 100 base pairs of the polynucleotide is preferably the maximum number determinable for any 200 bp window within the polynucleotide, more preferably the maximum number determinable for any 500 bp window within the polynucleotide. Preferably, the S / MAR array contains at least 4 sequence motifs ATTA per 100 nucleotides over a stretch of up to 200 nucleotides, more preferably at least 5 sequence motifs ATTA per 100 nucleotides over a stretch of up to 200 nucleotides, even more preferably at least 6 sequence motifs ATTA per 100 nucleotides over a stretch of up to 200 nucleotides. Also preferably, the S / MAR array contains at least 3 sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides, more preferably at least 4 sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides, even more preferably at least 5 sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides, most preferably at least 6 sequence motifs ATTA per 100 nucleotides over a stretch of up to 400 nucleotides. Also preferably, the S / MAR array contains at least 3 sequence motifs ATTA per 100 nucleotides over a stretch of up to 500 nucleotides, more preferably at least 4 sequence motifs ATTA per 100 nucleotides over a stretch of up to 500 nucleotides, even more preferably at least 5 sequence motifs ATTA per 100 nucleotides over a stretch of up to 500 nucleotides, most preferably at least 6 sequence motifs ATTA per 100 nucleotides over a stretch of up to 500 nucleotides.Thus, preferably, the S / MAR sequence contains at least 10 sequence motifs ATTA over a 500-nucleotide sequence, more preferably at least 20 sequence motifs ATTA over a 500-nucleotide sequence, and even more preferably at least 30 sequence motifs ATTA over a 500-nucleotide sequence. Preferably, at least 80%, more preferably at least 90%, and most preferably at least 95% of the ATTA motifs in the S / MAR sequence are separated by, respectively, 9 to 13, preferably 10 to 12, and most preferably 11 base pairs.

[0026] Preferably, the S / MAR element preferably contains additional sequence motifs within the sequence containing the above ATTA motif herein. Preferably, the sequence stretch of the S / MAR element containing the above ATTA sequence motif further contains 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. Also preferably, the sequence stretch of the S / MAR element containing the above ATTA sequence motif and optionally the above ATTTA motif(s) further contains at least one, preferably at least two, more preferably at least four, and most preferably at least six palindrome motifs, preferably the motif TAAATATTTTA (SEQ ID NO: 3). Preferably, the motif TAAATATTTTA is contiguous with at least one motif ATTA at the 5' end and / or 3' end. Also preferably, the sequence stretch of the S / MAR element containing the above ATTA sequence motif contains 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).

[0027] Also preferably, the S / MAR sequence has a low G+C content. Those skilled in the art know a method of calculating the C+G content of a known sequence by counting all guanine and cytosine 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 above 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, when the boundaries of the S / MAR element cannot be determined, the sequence used for the calculation of the G+C content is the same as that used for the calculation of the number of ATTA motifs per 100 base pairs, as specified above herein. Also preferably, the S / MAR sequence has a small number of CG dinucleotides. Preferably, the sequence stretch of the S / MAR element containing the above sequence motif contains at most 6, more preferably at most 4, even more preferably at most 2 sequence motifs CG, and most preferably does not contain the sequence motif CG.

[0028] Preferably, the S / MAR sequence comprises the 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. Preferably, the S / MAR sequence comprises a sequence that is at least 70% identical to the sequence of SEQ ID NO: 15, and more preferably, the S / MAR sequence comprises the sequence of SEQ ID NO: 15. Also preferably, the S / MAR sequence comprises the S / MAR sequence of the beta interferon gene, preferably the human beta interferon gene, more preferably the S / MAR sequence of the human beta interferon gene. Thus, preferably, the S / MAR sequence comprises a sequence that is at least 70% identical to the sequence of SEQ ID NO: 17. More preferably, the S / MAR sequence comprises the nucleic acid sequence of SEQ ID NO: 17.

[0029] Preferably, the polynucleotide contains a polyA signal downstream of the S / MAR element. More preferably, the polynucleotide contains a polyA signal and a transcription termination signal downstream of the S / MAR element. A splice donor and a splice acceptor are adjacent to the S / MAR element. Thus, preferably, a transcript is transcribed from the promoter, and the sequence of the S / MAR element is excised by splicing from the transcript. Also preferably, the S / MAR sequence is spliced out from a transcript encoding a selection marker, preferably after transcription. Also preferably, the polynucleotide further contains the (secondary) bacterial origin of replication and / or the bacterial selection marker gene specified above herein. Preferably, the promoter driving the expression of the bacterial origin of replication and the bacterial selection marker gene is prokaryote-specific, i.e., more preferably non-functional in the host cell. Also preferably, the bacterial origin of replication and / or the bacterial selection marker gene, preferably all elements active in the prokaryotic cell contained in the polynucleotide, are shielded from the remaining sequences contained in the polynucleotide by the presence of at least one insulation element, more preferably by the insulation element being adjacent. Preferably, the bacterial origin of replication and / or the bacterial selection marker gene, preferably all elements active in the prokaryotic cell, are shielded from the remaining sequences contained in the polynucleotide by the presence of at least one insulation element at the 5'-end and the presence of at least one insulation element at the 3'-end. More preferably, the bacterial origin of replication and / or the bacterial selection marker gene, preferably all elements active in the prokaryotic cell contained in the polynucleotide, are shielded from the promoter by the presence of at least one insulation element, more preferably by the insulation element being adjacent. Preferably, the insulation element(s) is / are the anti-suppressor element 40 element (SEQ ID NO: 11) or a variant thereof, and / or the S / MAR element.

[0030] Thus, preferably, the polynucleotide comprises the sequence of SEQ ID NO: 7 or 8, or a sequence that is at least 70% identical to the sequence of SEQ ID NO: 7 or 8, preferably the sequence of SEQ ID NO: 12, or a sequence that is at least 70% identical to the sequence of SEQ ID NO: 12, more preferably the sequence of SEQ ID NO: 13, or a sequence that is at least 70% identical to the sequence of SEQ ID NO: 13, most preferably the sequence of SEQ ID NO: 14, or a sequence that 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 is replaced by a nucleic acid sequence encoding a different polypeptide, preferably a therapeutic polypeptide, more preferably a human T cell receptor (TCR), a chimeric antigen receptor (CAR), preferably the MART1 TCR.

[0031] Preferably, the polynucleotide is an integration construct. As used herein, the term "integration construct" includes all polynucleotides that have the activity of becoming covalently integrated into the genome of the host cell at a detectable rate when introduced into the host cell. Preferably, the integration construct is at least 100 integration events per fmol of transfected polynucleotide, more preferably at least 1000 integration events per fmol of transfected polynucleotide, even more preferably at least 10 4 integration events, most preferably at least 10 5 integration events per fmol of transfected polynucleotide. Preferably, the integration construct lacks an origin of replication of eukaryotes, preferably lacks an origin of replication, more preferably lacks a nucleic acid sequence that replicates and stably maintains the polynucleotide. Thus, preferably, the integration construct does not replicate episomally, preferably does not replicate autonomously, in the host cell, preferably in mammalian cells. Preferably, the integration construct comprises at least one integration signal.

[0032] Integration signals generally include all kinds of signals known in the art that can induce a host cell or a recombinant system contained therein, and covalently integrate a polynucleotide containing the integration signal into the genome of the host cell. Therefore, preferably, the integration signal is the free end of a linear polynucleotide, preferably a double-stranded polynucleotide, which can induce VD(J) recombination or, preferably, homologous recombination when appropriate homologous sequences are included. Therefore, preferably, the polynucleotides specified herein are double-stranded linear polynucleotides, preferably double-stranded linear DNA. Also preferably, the integration signal is a recombinase recognition sequence, a viral integration signal, a transposable element, etc. Therefore, preferably, the integration signal is a cre recombination site or a lox recombination site, a lambda attachment site, a zinc finger recombinase recognition site, a transcription activator-like effector nuclease (TALEN) recognition site, or a serine integrase recognition site, such as the recognition site of PhiC31 integrase derived from Streptomyces phage φC31. Therefore, preferably, the integration mediated by the integration signal can be non-sequence-specific, essentially sequence-specific, or sequence-specific.

[0033] Preferably, the polynucleotide is a non-integrating vector construct. As used herein, the term "non-integrating vector construct" refers to a polynucleotide construct that is maintained within a host cell for a period of time without being integrated into the genome of the host cell. Preferably, the non-integrating vector construct is detectable in the host cell after an average of 50 cell divisions, more preferably after an average of 100 cell divisions, by methods known in the art or as described elsewhere herein, preferably by PCR. As used herein, the term non-integrating vector construct refers to a polynucleotide that includes at least one nucleic acid sequence that replicates the polynucleotide and stably maintains it for at least the aforementioned period. As referred to herein, the at least one nucleic acid sequence that replicates and stably maintains the polynucleotide is a sequence that is present in the polynucleotide in addition to the S / MAR sequence. Thus, preferably, the at least one nucleic acid sequence that replicates and stably maintains the polynucleotide is not the S / MAR sequence. Preferably, the non-integrating vector construct is an artificial chromosome, preferably including a centromere and a telomere. Preferably, the non-integrating vector construct is a human artificial chromosome. More preferably, the non-integrating vector construct is an episomally replicating construct, i.e., in circular form. Thus, preferably, the non-integrating vector construct is a non-integrating viral vector construct.

[0034] As used herein, the term "non-integrating viral vector construct" refers to a polynucleotide construct that contains a replication signal derived from a virus that is not integrated into the host cell genome and is maintained within the host cell for a period of time, preferably as specified above herein. Thus, the non-integrating viral vector construct is preferably based on a virus that has been modified to not contain integrase activity. Instead, the non-integrating viral vector construct preferably is derived from a virus that integrates into the host cell genome only at a very low frequency, preferably less than 1 integration in 10 5 infected cells, more preferably less than 1 integration in 10 6less than 1 integration in each infected cell, more preferably 10 7 less than 1 integration in each infected cell, most preferably 10 8 less than 1 integration in each infected cell. Thus, preferably, the non-integrating viral vector construct is a viral vector construct based on adeno-associated virus (AAV), herpes virus, simian virus 40 (SV40), or papillomavirus. Preferably, the non-integrating viral vector construct is a non-integrating lentiviral construct.

[0035] As used herein, the term "replicate" relates to the activity of the polynucleotide that induces the production of at least two replications of the polynucleotide in a host cell during the cell replication cycle. Thus, preferably, replication of the polynucleotide in the host cell is determined by determining the presence of the polynucleotide after a series of cell divisions where the non-replicating polynucleotide is expected to be diluted. Preferably, 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. Preferably, detection of the polynucleotide in the host cell population is performed by PCR under standard conditions.

[0036] The term "episomal" replication is generally known to those skilled in the art to relate to the replication of a polynucleotide that is not incorporated into the cell genome, i.e., does not become covalently incorporated into the cell genome. Thus, preferably, episomal replication of a polynucleotide is replication of the polynucleotide as an autonomous replication unit. Preferably, episomal replication is maintenance of the polynucleotide in a 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. Episomal maintenance of circular DNA is preferably verified by plasmid rescue procedures known to those skilled in the art, i.e., preferably by preparing a lysate of the host cell and transforming the DNA contained therein into a suitable bacterial cell, e.g., an E. coli cell. If a suitable number of bacterial colonies obtained by the above method contain circular DNA as a plasmid having the same restriction pattern and / or sequence as the original circular DNA, preferably, the circular DNA is presumed to be maintained episomally. A further method for confirming episomal maintenance, also known to those skilled in the art, is DNA / DNA blotting ("Southern blot" method). Thus, preferably, total DNA of the host cell is prepared and digested with one or more restriction enzymes (plural possible). In a Southern blot using the original plasmid as a probe, if only the band corresponding to the original circular DNA is visible, preferably, it is concluded that the plasmid is maintained episomally. More preferably, episomal maintenance is verified as described in the examples of this specification.

[0037] Accordingly, as used herein, the term "episomally replicating" relates to the activity of a polynucleotide that induces the production of at least two replications of the polynucleotide in a host cell during the cell replication cycle, while the polynucleotide exists in the cell as an autonomous 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, the aforementioned number of cell divisions is the average number of cell divisions for the cell population.

[0038] Advantageously, in the studies underlying the present invention, by combining the S / MAR element adjacent to the identified splicing site with an expressible construct and a promoter that reads the S / MAR element, it has been found that a polynucleotide can be obtained that provides high and sustained expression in a host cell even when the polynucleotide is integrated into the cell genome or maintained as a non-integrating viral vector.

[0039] The definitions made above apply hereinafter with the necessary modifications. The further definitions and explanations made hereinafter also apply with the necessary modifications to all embodiments described herein.

[0040] The present invention further relates to a composition comprising a polynucleotide according to the present invention.

[0041] As used herein, the term "composition" relates to a composition of matter comprising a specified compound and optionally one or more acceptable carriers. Preferably, the composition is a pharmaceutically acceptable composition. Accordingly, preferably, the carrier is a pharmaceutically acceptable carrier. The compounds of the present invention can preferably be formulated as pharmaceutically acceptable salts. Preferred salts include acetate, methyl ester, HCl, sulfate, chloride, and the like.

[0042] The carrier(s) must be acceptable in the sense that they are compatible with the other ingredients of the formulation and not harmful to its recipient. The carrier used can be, for example, either solid, gel or liquid. Examples of solid pharmaceutical carriers are lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, etc. 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, etc. Similarly, the carrier or diluent can contain a time-delay material well known in the art, such as glyceryl monostearate or glyceryl distearate, alone or together with waxes. The 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 / are selected so as not to affect the biological activity of the compounds in the composition. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. Furthermore, the pharmaceutical composition or formulation can also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0043] Preferably, the composition mediates the entry of the polynucleotide into the host cell. Thus, preferably, the composition comprises at least one transfection agent. The selection of a suitable transfection agent can depend on the target host cell as well as the particular application envisioned. Transfection agents, suitable transfection conditions, and their selection criteria are well known in the art. Also, preferably, the composition comprises virus-like particles. Thus, preferably, the polynucleotide is packaged in the virus-like particles, i.e., preferably, the polynucleotide is contained in the virus-like particles. Thus, more preferably, the composition comprises genetically engineered virus particles containing the specified polynucleotide. Preferably, the virus-like particles or virus particles are derived from the virus specified above herein.

[0044] The pharmaceutical composition is preferably administered locally or systemically. Suitable routes of administration conventionally used in drug administration are oral administration, intravenous administration, 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, the polynucleotide compound can be administered in a gene therapy approach using a viral vector or a virus or liposome as specified above herein. Further, the compound can be administered in combination with other drugs in a common pharmaceutical composition or as separate pharmaceutical compositions, and the separate pharmaceutical compositions can be provided in the form of a kit of 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 can include appropriately mixing the ingredients, granulating and compressing, or dissolving to obtain the desired preparation. It is understood that the form and characteristics of the pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient combined therewith, the route of administration, and other well-known variables.

[0045] The therapeutically effective amount of a pharmaceutical composition refers to the amount of a compound used in the pharmaceutical composition of the present invention that prevents, ameliorates, or treats the symptoms associated with the diseases or conditions referred to herein. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmacological procedures in cell cultures or experimental animals, such as, for example, ED50 (the therapeutically effective dose in 50% of the population) and LD50 (the lethal dose in 50% of the population). The dose ratio between the therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50.

[0046] The dosing regimen is preferably determined according to any one of the above methods by the attending physician and other clinical factors. As is well known in the pharmaceutical art, the dosage for any individual patient depends on a number of factors including the size, body surface area, age, particular compound being administered, sex, time and route of administration, general health status, and other drugs being administered concurrently. Progression can be monitored by periodic evaluation. Typical doses can be, for example, in the range of 1 to 1000 μg. However, taking into account the aforementioned factors in particular, doses below or above this exemplary range are envisioned. Generally, the regimen for the normal administration of a pharmaceutical composition should be within the range of 1 μg to 10 mg units per day. When the regimen is continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute. Progression can be monitored by periodic evaluation. However, depending on the subject and the method of administration, the dosage of the substance can vary over a wide range so as to provide from about 0.01 mg per kilogram of body weight to about 10 mg per kilogram of body weight. When administering a viral vector, particularly an adeno-associated viral vector, a preferred dose is 5×10 11 ~2×10 13 viral particles or viral genome / kg body weight. As will be understood, these exemplary doses can be modified depending on additional factors such as the type of virus, target organ, etc., in addition to the above factors.

[0047] The pharmaceutical compositions and formulations referred to in this specification are administered at least once to treat or ameliorate or prevent the diseases or conditions described herein. However, the above pharmaceutical compositions can be administered more than once, for example, 1 to 4 times a day, for a non-limiting number of days.

[0048] Certain pharmaceutical compositions are prepared by methods well known in the pharmaceutical art and contain, either by mixing at least one of the active compounds referred to above herein or otherwise in association with a pharmaceutically acceptable carrier or diluent. To produce these particular pharmaceutical compositions, the active compound(s) are usually admixed with a carrier or diluent or enclosed or encapsulated in a capsule, sachet, cachet, paper or other suitable container or vehicle. The resulting formulations are employed in a method of administration, i.e., in the form of tablets, capsules, suppositories, solutions, suspensions, etc. The recommended dosage is that indicated in the instructions for the prescriber or user to predict dosage adjustment according to the recipient being considered.

[0049] The invention also relates to host cells containing a polynucleotide according to the invention, preferably integrated into its genome.

[0050] The 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 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 genetic diseases.

[0051] As used herein, the term "genetic disease" refers to a disease causally related to one or more modifications, preferably mutations, in the genome of an individual. Thus, preferably, a genetic disease is causally related to one or more epigenetic changes, more preferably to one or more genetic mutations. As will be understood, the symptoms of genetic diseases are often caused by the expression of mutated genes and / or the lack of expression of genes that provide the normal function of gene products in one or more specific tissues and / or cell types. Thus, 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 change in one gene. More preferably, the genetic disease is a monogenic recessive disease, i.e., caused by genetic changes in both alleles of the gene. Thus, preferably, improvement of the symptoms is expected by providing at least one unmodified copy of the affected gene. Most preferably, the genetic disease is phenylketonuria, alkaptonuria, Leber congenital amaurosis, coloboma, hemophilia, Usher disease, or Stargardt disease. In a preferred embodiment, the genetic disease is cancer.

[0052] The terms "treating" and "treatment" mean an improvement to a significant extent of the disease or disorder referred to in this specification, or the symptoms associated therewith. The above treatment as used in this specification also includes a complete recovery of health with respect to the disease or disorder referred to in this specification. It should be understood that the term treatment as used in this specification may not be effective in all subjects being treated. However, this term preferably requires that a statistically significant proportion of the subjects suffering from the disease or disorder referred to in this specification can be successfully treated. Whether a proportion is statistically significant can be determined by those skilled in the art using various well-known statistical evaluation tools such as determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test, etc., without detailed description. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment is effective for at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

[0053] The term "subject" means a metazoan. Preferably, the subject is an animal, more preferably a mammal, and most preferably a human. Preferably, the subject is suffering from the genetic disease specified above in this specification.

[0054] The present invention further relates to an apparatus comprising a polynucleotide according to the present invention, a composition according to the present invention, and / or a host cell according to the present invention.

[0055] As used herein, the term "device" relates to a system of means including means operably linked to each other to enable administration of a compound or composition of the invention. Preferred means for administering polynucleotides, compositions, or host cells are well known in the art. The method of linking the means to operate depends on the type of means included in the device and the type of administration envisioned. Preferably, the means are, in such cases, constituted by a single device. Thus, the device described above may include a delivery unit for administering the compound or composition and a storage unit for storing the compound or composition until administration. However, the means of the invention may appear as separate devices in such embodiments and are preferably intended to be packaged together as a kit. One of ordinary skill in the art will understand how to link the means without further effort. Preferred devices are those that can be applied without the special knowledge of an expert. In a preferred embodiment, the device is a syringe containing a compound or composition of the invention, more preferably a syringe with a needle. In another preferred embodiment, the device is an intravenous (IV) device containing a compound or composition. In another preferred embodiment, the device is an endoscopic device containing a compound or agent for flushing the administration site or further containing a needle for topical 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.

[0056] This application also relates to a method for stably expressing a construct capable of being expressed in a host cell, comprising: a) contacting the host cell with a polynucleotide according to the invention and / or a composition according to the invention; and b) thereby stably expressing a construct capable of being expressed in the host cell. The method described above is related to the above method.

[0057] A method for stably expressing a construct capable of being expressed in a host cell of the present invention is preferably an in vitro method. Further, it may include steps in addition to those explicitly described above. For example, additional steps may relate to, for example, preparing a host cell or a sample containing it for step a), and / or applying a selective pressure to the contacted host cell. Further, one or more of the above steps may be performed by an automated device.

[0058] The term "stable expression" of a host cell refers to the introduction of a polynucleotide, preferably a heterologous polynucleotide, containing an expressible construct into a cell such that the expressible construct is stably expressed by the host cell as specified above herein. Preferably, stable transfection does not involve stable episomal replication of the polynucleotide. Preferably, stable expression involves applying a selection pressure to the host cell after contact to select for the presence of a selectable marker. The selection pressure is applied after contact, optionally excluding an initial time frame that allows the polynucleotide to establish within the host cell. The duration of the initial time frame that allows the polynucleotide to establish within the host cell depends largely on the type of host cell contacted and the type of selectable marker used. Preferably, the duration of the initial time frame that allows the polynucleotide to establish within the host cell is from 1 hour to 48 hours, more preferably from 2 hours to 24 hours, most preferably from 3 hours to 16 hours. However, the duration of the initial time frame that allows the polynucleotide to establish within the host cell can also be zero. That is, the selection pressure can be applied immediately after or even during contact. More preferably, the selection force can be applied continuously, i.e., at substantially all time points after the initial time frame that allows the polynucleotide to establish within the host cell, in order to prevent the growth of host cells that do not contain the polynucleotide. Alternatively, the selection pressure can be applied transiently, more preferably to remove cells that have not received the polynucleotide. Preferably, transient application of the selection pressure is used when the polynucleotide is an integration construct or when the cells are reintroduced into an organism after the above contact. However, it is also conceivable that the selection pressure is not applied, especially when it is known that the efficiency of introducing the polynucleotide into the target host cell is sufficiently high and / or when a pure population of transgenic host cells is not very important.In a preferred embodiment, a stably transfected cell population can be obtained by enabling the expression of an expressible construct encoding a detectable polypeptide as specified above herein, and by selecting cells expressing the cargo sequence, for example by cell sorting, preferably by FACS.

[0059] The term "contacting" as used in the context of the methods of the present invention is understood by those skilled in the art. Preferably, this term relates to bringing at least one polynucleotide, vector, and / or host cell of the present invention into contact, for example, physically contacting a host cell so as to enable an interaction of the host cell with a compound(s), for example. Preferably, contacting comprises delivering at least one polynucleotide of the present invention into the interior of a host cell, preferably via a delivery means as specified above.

[0060] The present invention also relates to a method of treating a genetic disease in a subject, comprising: a) contacting the subject with a polynucleotide according to the present invention, a composition according to the present invention, and / or a host cell according to the present invention, and b) thereby treating the genetic disease in the subject.

[0061] The method of treating a genetic disease of the present invention is preferably an in vivo method. Further, it may include steps in addition to those explicitly stated above. For example, further steps may relate to, for example, preparing a host cell or a sample containing it for step a), and / or readministering the sample or host cell to the subject. Thus, the method of treating a genetic disease includes the steps of the method of stably transfecting a host cell as specified above. Further, one or more of the above steps may be performed by an automated device.

[0062] Furthermore, the present invention relates to the use of a polynucleotide of the present invention for stably genetically modifying a host cell.

[0063] The present invention also relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for the manufacture of a medicament. And to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for the manufacture of a medicament for treating a genetic disease, preferably a monogenic disease, more preferably a monogenic recessive disease, and most preferably F. In a preferred embodiment, as specified above herein, the genetic disease is cancer.

[0064] The present invention also relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for the generation of induced pluripotent stem cells (IPSCs) for the genetic modification of primary cells, preferably primary dermal fibroblasts. Preferably, the primary cells are mouse or human primary cells.

[0065] The term "primary cell" is understood by those skilled in the art in contrast to the cells of a cultured cell line. Thus, preferably, a primary cell is a cell derived from an organism and cultured for a maximum of 20 passages, more preferably a maximum of 15 passages, even more preferably a maximum of 10 passages, and even more preferably a maximum of 5 passages. Most preferably, a primary cell is a cell directly derived from a tissue of an organism, preferably a mouse or human tissue.

[0066] The term "stem cell" is also understood by those skilled in the art to relate to undifferentiated or poorly differentiated cells having the potential to differentiate into at least two cell types, preferably at least five cell types, more preferably into 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" relates to pluripotent stem cells derived from differentiated cells, preferably differentiated primary cells. Methods for generating IPSCs are known in the art and preferably involve the expression of four transcription factors in the cell (e.g., according to Takahashi et al. (2006), Cell. 126(4):663).

[0067] The present invention also relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for the genetic modification of embryonic stem cells, preferably non-human embryonic stem cells. The present invention also relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for manufacturing a medicament for treating the genetic diseases specified above herein, preferably, the medicament includes host cells containing the polynucleotide of the present invention.

[0068] The present invention also relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for the genetic modification of stem cells for producing transgenic animals, preferably non-human animals. The present invention further relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for producing transgenic animals, preferably non-human animals.

[0069] As used herein, the term "transgenic animal" preferably relates to an animal containing at least one heterologous polynucleotide introduced into the animal by means of genetic engineering methods. Preferably, a transgenic animal contains at least one, more preferably at least 10, still more preferably at least 1000, and even more preferably at least 10000 cells containing at least one polynucleotide according to the present invention.

[0070] The present invention also relates to the use of the polynucleotide according to the invention, the composition according to the invention, and / or the host cell according to the invention for the genetic modification of single-cell embryos, preferably non-human single-cell embryos, by pronuclear injection.

[0071] As understood by those skilled in the art, the term "pronuclear injection" preferably relates to injecting genetic material, preferably the polynucleotide of the present invention, into the nucleus of a fertilized oocyte for producing a transgenic animal, preferably a non-human animal.

[0072] The present invention further relates to the use of the polynucleotide of the present invention or the composition of the present invention for modifying gene expression in a host cell.

[0073] The present invention also relates to a method for increasing the expression of a eukaryotic expression construct comprising a promoter and an expressible construct, the method comprising including an S / MAR element adjacent to a splice donor and a splice acceptor downstream of the expressible construct. The present invention also relates to a method for increasing the expression of a eukaryotic expression construct comprising a promoter, an expressible construct, and an S / MAR element, the method comprising including a splice donor and a splice acceptor adjacent to the S / MAR element.

[0074] The present invention further relates to the use of an S / MAR element adjacent to a splice donor and a splice acceptor for increasing the expression of a eukaryotic expression construct. The present invention also relates to the use of a splice donor and a splice acceptor for increasing the expression of a eukaryotic expression construct comprising an S / MAR element.

[0075] The methods and uses for increasing the expression of a eukaryotic expression construct are preferably in vitro methods or uses. However, they can also be in vivo methods or uses, for example, as part of a therapeutic method. The methods and uses can include additional steps in addition to those specifically mentioned. The additional steps can relate, for example, to providing a host cell in which improved expression is desired, providing a polynucleotide comprising the eukaryotic expression construct, preferably the polynucleotide of the present invention, contacting the host cell with the polynucleotide, incubating the host cell after the contact, and recovering the product of the eukaryotic expression construct. If the method or use is an in vivo method or use, it can include administering or readministering the contacted cells to a subject.

[0076] The term "eukaryotic expression construct" is understood by those skilled in the art to relate to a polynucleotide comprising at least an expressible construct and a promoter (both specified above herein) that causes the construct to be expressed in a eukaryotic cell, preferably a host cell. Preferably, the eukaryotic expression construct comprises additional expression control sequences, particularly a transcription terminator, specified above herein.

[0077] The term "increasing expression" relates to an increase in the expression of a eukaryotic expression construct comprising an S / MAR element adjacent to a splice donor and a splice acceptor as compared to an expression construct comprising only an S / MAR element, i.e., an S / MAR element not adjacent to a splice donor and a splice acceptor. Preferably, increasing expression means increasing transcription. More preferably, increasing expression is increasing the production of a polypeptide by increasing the transcription of an expressible construct comprising the sequence encoding the polypeptide.

[0078] In view of the above, the following embodiments are preferred:

[0079] 1. A polynucleotide comprising at least one promoter, at least one expressible construct, and an S / MAR element, wherein the polynucleotide is an integration construct or a non-integration vector construct, the S / MAR element is located downstream of the promoter and the expressible construct, and the S / MAR element is adjacent to a splice donor and a splice acceptor.

[0080] 2. The polynucleotide of embodiment 1, wherein the polynucleotide is an integration construct or a non-integration viral vector construct.

[0081] 3. The polynucleotide of embodiment 1 or 2, wherein the polynucleotide is an integration construct.

[0082] 4. A polynucleotide according to any one of embodiments 1 to 3, wherein the integration construct comprises at least one integration signal.

[0083] 5. A polynucleotide according to embodiment 4, wherein the integration signal is a linear polynucleotide, a recombinase recognition sequence, a viral integration signal, or the free end of a transposable element.

[0084] 6. A polynucleotide according to any one of embodiments 1 to 5, wherein the polynucleotide lacks an origin of replication of eukaryotes, preferably lacks an origin of replication.

[0085] 7. A polynucleotide according to any one of embodiments 1 to 6, wherein the polynucleotide does not replicate episomally in a host cell, preferably a mammalian cell.

[0086] 8. A polynucleotide according to embodiment 1 or 3, wherein the polynucleotide is a non-integrating viral vector construct.

[0087] 9. A polynucleotide according to embodiment 8, wherein the non-integrating viral vector construct is a non-integrating lentiviral construct, an adeno-associated virus construct, a simian virus 40 construct, a papillomavirus construct, an adenovirus construct, a hepatitis virus construct, or a herpes virus construct.

[0088] 10. A polynucleotide according to embodiment 8 or 9, wherein the polynucleotide replicates episomally in a host cell, preferably a mammalian cell.

[0089] 11. A polynucleotide according to any one of embodiments 1 to 10, wherein the expressible construct comprises at least one coding sequence encoding a polypeptide, a sequence encoding siRNA, a sequence encoding microRNA, a sequence encoding antisense RNA, and / or a sequence encoding a ribozyme.

[0090] 12. The polynucleotide according to any one of Embodiments 1 to 11, wherein the polypeptide is a therapeutic polypeptide, preferably a human T cell receptor (TCR), a chimeric antigen receptor (CAR), preferably a MART1 TCR.

[0091] 13. The polynucleotide according to any one of Embodiments 1 to 12, wherein the expressible construct comprises at least one coding sequence encoding a selection marker.

[0092] 14. The polynucleotide according to any one of Embodiments 1 to 13, wherein the expressible construct comprises at least two coding sequences, preferably one of which encodes a selection marker.

[0093] 15. The polynucleotide according to any one of Embodiments 1 to 14, wherein the expressible construct comprises a coding sequence (selection marker sequence) encoding a selection marker, and the promoter and the selection marker sequence together constitute a selection marker gene, and the selection marker is a selection marker for eukaryotic cells.

[0094] 16. The polynucleotide according to Embodiment 15, wherein the selection 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.

[0095] 17. The polynucleotide according to any one of Embodiments 1 to 16, wherein a transcript is transcribed from the promoter, and the sequence of the S / MAR element is excised by splicing from the transcript.

[0096] 18. The polynucleotide according to any one of Embodiments 1 to 17, wherein the polyA signal downstream of the S / MAR element is retained in the splicing.

[0097] 19. The polynucleotide according to any one of Embodiments 1 to 18, wherein the host cell is a mammalian cell, preferably a human cell.

[0098] 20. The polynucleotide according to any one of Embodiments 1 to 19, wherein the polynucleotide lacks a centromere and / or telomere sequence.

[0099] 21. The polynucleotide according to any one of Embodiments 1 to 20, wherein the polynucleotide contains a transcriptional insulator element upstream of the promoter.

[0100] 22. The polynucleotide according to Embodiment 21, wherein the insulator element is Element 40 and / or an S / MAR element.

[0101] 23. The polynucleotide according to any one of Embodiments 1 to 22, wherein the promoter and the expressible construct are isolated from the remaining sequences contained in the polynucleotide by the presence of at least one inslational element, more preferably by being adjacent to the inslational element.

[0102] 24. A composition comprising the polynucleotide according to any one of Embodiments 1 to 23.

[0103] 25. A host cell comprising the polynucleotide according to any one of Embodiments 1 to 23, preferably integrated into its genome.

[0104] 26. The host cell according to Embodiment 25, 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, preferably also positive for CD8 and CD45; a CD3+ helper T lymphocyte, preferably also positive for CD4 and CD45; a CD3+ activated T lymphocyte, preferably also positive for CD25 and CD45, a tumor-infiltrating lymphocyte, a natural killer (NK) cell, an embryonic stem (ES) cell, an induced pluripotent stem (IPS) cell, an airway epithelial cell, a fibroblast, or a retinal epithelial cell.

[0105] 27. The host cell according to embodiment 25 or 26, wherein the polynucleotide is covalently bound to the chromosome of the host cell.

[0106] 28. The polynucleotide according to any one of embodiments 1 to 23, the composition according to embodiment 24, and / or the host cell according to any one of embodiments 25 to 27 for use in a medicament.

[0107] 29. The polynucleotide according to any one of embodiments 1 to 23, the composition according to embodiment 24, and / or the host cell according to any one of embodiments 25 to 27 for use in the treatment of a genetic disease.

[0108] 30. An apparatus comprising the polynucleotide according to any one of embodiments 1 to 23, the composition according to embodiment 24, and / or the host cell according to any one of embodiments 25 to 27.

[0109] 31. A method for stably expressing a construct that can be expressed in a host cell, comprising: a) contacting the host cell with the polynucleotide according to any one of embodiments 1 to 23 and / or the composition according to embodiment 24; and b) thereby stably expressing a construct that can be expressed in the host cell.

[0110] 32. A method for treating a genetic disease in a subject, comprising: a) contacting the subject with the polynucleotide according to any one of embodiments 1 to 23, the composition according to embodiment 24, and / or the host cell according to any one of embodiments 25 to 27; and b) thereby treating the genetic disease of the subject. 33. Use of the polynucleotide according to any one of embodiments 1 to 23 and / or the composition according to embodiment 24 for stably genetically modifying a host cell.

[0111] 33. Use of the polynucleotide according to any one of embodiments 1 to 23 and / or the composition according to embodiment 24 for stably genetically modifying a host cell.

[0112] 34. Use of the polynucleotide according to any one of Embodiments 1 to 23, the composition according to Embodiment 24 and / or the host cell according to any one of Embodiments 25 to 27 for stably genetically modifying a host cell.

[0113] 35. Use of the polynucleotide according to any one of Embodiments 1 to 23, the composition according to Embodiment 24 and / or the host cell according to any one of Embodiments 25 to 27 for manufacturing a medicament.

[0114] 36. Use of the polynucleotide according to any one of Embodiments 1 to 23, the composition according to Embodiment 24 and / or the host cell according to any one of Embodiments 25 to 27 for manufacturing a medicament for treating a genetic disease, preferably a single-gene disease, more preferably a single-gene recessive genetic disease, most preferably phenylketonuria, alkaptonuria, Leber congenital amaurosis, coloboma, or Stargardt disease.

[0115] 37. A method for increasing the expression of a eukaryotic expression construct comprising a promoter and an expressible construct, the method comprising including an S / MAR element adjacent to a splice donor and a splice acceptor downstream of the expressible construct.

[0116] 38. The method according to Embodiment 37, wherein the eukaryotic expression construct further comprises a transcription terminator.

[0117] 39. The method according to the embodiment, wherein the S / MAR element is included between the expressible construct and the transcription terminator.

[0118] 40. The method according to any one of Embodiments 37 to 39, wherein the S / MAR element is excised by splicing from a primary transcript initiated by the promoter.

[0119] 41. A method according to any one of embodiments 37 to 40, comprising providing the polynucleotide according to any one of embodiments 1 to 23 and / or the composition according to embodiment 24.

[0120] 42. A method according to any one of embodiments 37 to 41, further comprising contacting a host cell with a polynucleotide comprising the expression construct, preferably the polynucleotide according to any one of embodiments 1 to 23 and / or the composition according to embodiment 24.

[0121] 43. Use of an S / MAR element adjacent to a splice donor and a splice acceptor for increasing the expression of a eukaryotic expression construct.

[0122] 44. Use according to embodiment 43, wherein the eukaryotic expression construct comprises a promoter and an expressible construct.

[0123] 45. Use according to embodiment 44, wherein the use comprises including the S / MAR element downstream of the expressible construct.

[0124] 46. Use according to embodiment 44 or 45, wherein the eukaryotic expression construct further comprises a transcription terminator.

[0125] 47. Use according to embodiment 46, wherein the use comprises including the S / MAR element between the expressible construct and the transcription terminator.

[0126] 48. Use according to any one of embodiments 43 to 47, wherein the use comprises providing the polynucleotide according to any one of embodiments 1 to 23 and / or the composition according to embodiment 24.

[0127] 49. Use according to any one of embodiments 43 to 48, wherein the use further comprises contacting a host cell with a polynucleotide comprising the expression construct, preferably a polynucleotide according to any one of embodiments 1 to 23, and / or the composition according to embodiment 24.

[0128] All the cited references in this specification are incorporated herein by reference in their entire disclosures and for the disclosures specifically mentioned herein. The present invention also provides the following aspects. [1] A polynucleotide comprising at least one promoter, at least one expressible construct, and an S / MAR element, wherein the polynucleotide is an integration construct or a non-integrating vector construct, the S / MAR element is located downstream of the promoter and the expressible construct, the S / MAR element is adjacent to a splice donor and a splice acceptor, and the polynucleotide is an integration construct. [2] The polynucleotide according to [1], wherein the polynucleotide is an integration construct comprising at least one integration signal, preferably the integration signal is a free end of a linear polynucleotide, a viral integration signal, or a transposable element. [3] The polynucleotide according to [1], wherein the polynucleotide is a non-integrating viral vector construct, preferably a non-integrating lentiviral construct, an adeno-associated virus construct, a simian virus 40 construct, a papillomavirus construct, an adenovirus construct, a hepatitis virus construct, or a herpesvirus construct. [4] The polynucleotide according to any one of [1] to [3], wherein the expressible construct comprises a coding sequence encoding at least one polypeptide, a sequence encoding siRNA, a sequence encoding miRNA, a sequence encoding antisense RNA, and / or a sequence encoding a ribozyme. [5] The polynucleotide according to [4], wherein the polypeptide is a therapeutic polypeptide, preferably a human T cell receptor (TCR), a chimeric antigen receptor (CAR), preferably a MART1 TCR. [6] The polynucleotide according to any one of [1] to [5], wherein a transcript is transcribed from the promoter, and the sequence of the S / MAR element is excised by splicing from the transcript. [7] A composition comprising the polynucleotide according to any one of [1] to [6]. [8] A host cell comprising the polynucleotide according to any one of [1] to [6], preferably integrated into its genome. [9] The polynucleotide according to any one of [1] to [6], the composition according to [7], and / or the host cell according to [8] for use in medicine.

[10] The polynucleotide according to any one of [1] to [6], the composition according to [7], and / or the host cell according to [8] for use in the treatment of genetic diseases.

[11] Use of the polynucleotide according to any one of [1] to [6] and / or the composition according to [7] for stably genetically modifying a host cell.

[12] A method for increasing the expression of a eukaryotic expression construct comprising a promoter and an expressible construct, the method comprising including an S / MAR element adjacent to a splice donor and a splice acceptor downstream of the expressible construct.

[13] Use of an S / MAR element adjacent to a splice donor and a splice acceptor for increasing the expression of a eukaryotic expression construct.

[14] The method according to

[12] or the use according to

[13] comprising providing the polynucleotide according to any one of [1] to [6].

[15] The method or use according to any one of

[12] to

[14] further comprising contacting a host cell with a polynucleotide comprising the eukaryotic expression construct. [Example]

[0129] The following examples are merely illustrative of the present invention. They should in no way be construed as limiting the scope of the present invention.

[0130] Example 1: The linear construct shown in Fig. 1 was purified and transfected into Hek293T cells. The cells were selected in a medium containing puromycin (0.5 μg / ml) for 35 days, and then the relative GFP expression (the amount of GFP RNA compared to GAPDH) and the mean fluorescence intensity (MFI) in FACS were measured. As shown in Fig. 2, the levels of the transgene mRNA and protein are increased by the splicing site adjacent to the S / MAR sequence.

[0131] [Sequence Listing] SEQUENCE LISTING <110> Deutsches Krebsforschungszentrum <120> Expression constructs for the genetic modification of Cells <130> PA25-139 <150> EP19163509.3 <151> 2019-03-18 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> DNA <213> Homo sapiens <400> 1 atta 4 <210> 2 <211> 5 <212> DNA <213> Homo sapiens <400> 2 attta 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 ataaaatatt taattataaa cattttaatt 300 ataaaatatt taattataaa tattttaatt ataaaatatt taattataaa tattttaatt 360 ataaaatatt taattataaa tattttaatt ataaaatatt taattataaa tactttaatt 420 ataaaatatt taattataaa tattttaatt ataaaatatt taattataaa tattttaatt 480 ataaatattt taattataaa atatttaatt ataaaaacac aatta 525 <210> 7 <211> 825 <212> DNA <213> Homo sapiens <400> 7 gcaggctgag tgaaataaag 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 aaatatttaa ttataaatat tttaattata aaatatttaa ttataaatat tttaattata 600 aaatatttaa ttataaatat tttaattata aaatatttaa ttataaatac tttaattata 660 aaatatttaa ttataaatat tttaattata aaatatttaa ttataaatat tttaattata 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 ttatttcatc tcgaggccta 60 ccggagagcc ttgccttgca aaggcagaca gtcagtgagg aagactatgt ggcacatgaa 120 gacaccagag gtgttcctca ggatcaaagt atgtacaagc ctttgtgaat attttttcct 180 tctcacttgg caaatacaat tcctgagatc aataacctcg tctttttaat tttttcctcg 240 tctttttaac tatttataaa atattgaatt ataaaatatg taattataaa tactttaatt 300 ataaaatatg taattataaa tactttaatt ataaaatatg taattataaa tactttataa 360 aatatgtaat tataaaatat gtaattataa acattttaat tataaaatat gtaattataa 420 acattttaat tataaaatat gtaattataa acattttaat tataaaatat gtaattataa 480 acattttaat tataaaatat gtaattataa acattttaat tataaaatat ttaattataa 540 acattttaat tataaaatat ttaattataa atattttaat tataaaatat ttaattataa 600 atattttaat tataaaatat ttaattataa atattttaat tataaaatat ttaattataa 660 atactttaat tataaaatat ttaattataa atattttaat tataaaatat 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 ctgtataaag taccggtggc 600 atgtgtatta gtggagatgc agcctgtgct ctgcagacag ggagtcacac agacactttt 660 ctataatttc ttaagtgctt tgaatgttca agtagaaagt ctaacattaa atttgattga 720 acaattgtat attcatggaa tattttggaa cggaatacca aaaaatggca atagtggttc 780 tttctggatg gaagacaaac ttttcttctt taaaataaat tttattttat atatttgagg 840 ttgaccacat gaccttaagg atacatatag acagtaaact ggttactaca gtgaagcaaa 900 ttaacatatc taccatcgta catagttaca tttttttgtg tgacaggaac agctaaaatc 960 tacgtattta acaaaactcc taaagacaat acatttttat taactatagc cctcatgatg 1020 tacattagat 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 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 aggtcttctc cactcctggc 600 aggctgagtg aaataaagga cttgttattt 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 ataaatattt taattataaa atatttaatt ataaatattt taattataaa 1200 atatttaatt ataaatattt taattataaa atatttaatt ataaatactt taattataaa 1260 atatttaatt ataaatattt taattataaa atatttaatt ataaatattt taattataaa 1320 tattttaatt ataaaatatt taattataaa aacacaatta cctcatcttt ttaaatattt 1380 ttgcaaaata tttccctcca taatttctcc gtttccattt ttattctgtt acttaaat 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 atatttaatt ataaatattt taattataaa atatttaatt ataaatattt taattataaa 1800 atatttaatt ataaatattt taattataaa atatttaatt ataaatactt taattataaa 1860 atatttaatt ataaatattt taattataaa atatttaatt ataaatattt taattataaa 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 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 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 gaacgggggc cccatgttcg ccttccgccg cgtggaggag 1440 ctgcacagca acaccgagct gggcatcgtg gagtaccagc acgccttcaa gacccccatc 1500 gccttcgcca gatctggcag cggcgccacc aacttcagcc tgctgaagca ggccggcgac 1560 gtggaggaaa 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 caaggttaca agacaggtcg acttctccac tcctggcagg ctgagtgaaa 2280 taaaggactt gttatttcat ctcgaggcct accggagagc cttgccttgc aaaggcagac 2340 agtcagtgag gaagactatg tggcacatga agacaccaga ggtgttcctc aggatcaaag 2400 tatgtacaag cctttgtgaa tattttttcc ttctcacttg gcaaatacaa ttcctgagat 2460 caataacctc gtctttttaa ttttttcctc gtctttttaa ctatttataa aatattgaat 2520 tataaaatat gtaattataa atactttaat tataaaatat gtaattataa atactttaat 2580 tataaaatat gtaattataa atactttata aaatatgtaa ttataaaata tgtaattata 2640 aacattttaa ttataaaata tgtaattata aacattttaa ttataaaata tgtaattata 2700 aacattttaa ttataaaata tgtaattata aacattttaa ttataaaata tgtaattata 2760 aacattttaa ttataaaata tttaattata aacattttaa ttataaaata tttaattata 2820 aatattttaa ttataaaata tttaattata aatattttaa ttataaaata tttaattata 2880 aatattttaa ttataaaata tttaattata aatactttaa ttataaaata tttaattata 2940 aatattttaa ttataaaata tttaattata aatattttaa ttataaatat tttaattata 3000 aaatatttaa ttataaaaac acaattacct catcttttta aatatttttg caaaatattt 3060 ccctccataa tttctccgtt tccattttta ttctgttact taaataattc tgcagtcgac 3120 ggttactgac atccactttg cctttctctc cacaggtgtc cactctaccg cgggcccggg 3180 atccaccgga tctagataac 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 cacaaaaatc gacgctcaag 3540 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 3600 cctcgtgcgc tctcctgttc cgaccctgcc gcttacggga tacctgtccg cctttctccc 3660 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 3720 cgttcgctcc aagctgggct 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 agatcctttg 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 gcccctgatg 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 cattaaattt gattgaacaa ttgtatattc atggaatatt ttggaacgga ataccaaaaa 5880 atggcaatag tggttctttc tggatggaag acaaactttt cttctttaaa ataaatttta 5940 ttttatatat ttgaggttga ccacatgacc ttaaggatac atatagacag taaactggtt 6000 actacagtga agcaaattaa catatctacc atcgtacata gttacatttt tttgtgtgac 6060 aggaacagct aaaatctacg tatttaacaa aactcctaaa gacaatacat ttttattaac 6120 tatagccctc atgatgtaca ttagatctgt acatttatat tggctcatgt ccaatatgac 6180 cgccatgtt 6189 <210> 15 <211> 248 <212> DNA <213> Homo sapiens <400> 15 atttaattat aaacatttta attataaaat atttaattat aaatatttta attataaaat 60 atttaattat aaatatttta attataaaat atttaattat aaatatttta attataaaat 120 atttaattat aaatacttta attataaaat atttaattat aaatatttta attataaaat 180 atttaattat aaatatttta attataaata ttttaattat aaaatattta attataaaaa 240 cacaatta 248 <210> 16 <211> 777 <212> DNA <213> Artificial <220> <223> anti-CEA single-chain antibody <400> 16 atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60 agaggtgtac actccgacta caaagacgag caggtccaac tgcaggagtc aggacctgac 120 ctggtgaaac cttctcagtc actttcactg acttgcactg tcactggcta ctccatcacc 180 agtggttata gctggcactg gattcggcag tttccaggaa acaaactgga atggatgggc 240 tacatacaat acagtggtat cactaactac aacccctctc tcaaaagtcg aatctctatc 300 acccgagaca catccaagaa ccagttcttc ctgcagttga attctgtgac tactgaggac 360 acagccacat attactgtgc aagagaagac tatgattacc actggtactt cgatgtctgg 420 ggccaaggga ccacggtcac cgtctcctca ggaggtggtg gatcgggcgg tggcgggtcg 480 ggtggcggcg gatctgacat ccagctgacc cagtctccag caatcatgtc tgcatctcta 540 ggggaggaga tcaccctaac ctgcagtgcc agctcgagtg taagttacat gcactggtac 600 cagcagaagt caggcacttc tcccaaactc ttgatttata gcacatccaa cctggcttct 660 ggagtccctt ctcgcttcag tggcagtggg tctgggacct tttattctct cacaatcagc 720 agtgtggagg ctgaagatgc tgccgattat tactgccatc agtggagtag ttatccc 777 <210> 17 <211> 1990 <212> DNA <213> Homo sapiens <400> 17 agatctaaat aaacttataa attgtgagag aaattaatga atgtctaagt taatgcagaa 60 acggagagac atactatatt catgaactaa aagacttaat attgtgaagg tatactttct 120 ttccacataa atttgtagtc aatatgttca ccccaaaaaa gctgtttgtt aacttgccaa 180 cctcattcta aaatgtatat agaagcccaa aagacaataa caaaaatatt cttgtagaac 240 aaaatgggaa agaatgttcc actaaatatc aagatttaga gcaaagcatg agatgtgtgg 300 ggatagacag tgaggctgat aaaatagagt agagctcaga aacagaccca ttgatatatg 360 taagtgacct atgaaaaaaa tatggcattt tacaatggga aaatgatgat ctttttcttt 420 tttagaaaaa cagggaaata tatttatatg taaaaaataa aagggaaccc atatgtcata 480 ccatacacac aaaaaaattc cagtgaatta taagtctaaa tggagaaggc aaaactttaa 540 atcttttaga aaataatata gaagcatgcc atcatgactt cagtgtagag aaaaatttct 600 tatgactcaa agtcctaacc acaaagaaaa gattgttaat tagattgcat gaatattaag 660 acttattttt aaaattaaaa aaccattaag aaaagtcagg ccatagaatg acagaaaata 720 tttgcaacac cccagtaaag agaattgtaa tatgcagatt ataaaaagaa gtcttacaaa 780 tcagtaaaaa ataaaactag acaaaaattt gaacagatga aagagaaact ctaaataatc 840 attacacatg agaaactcaa tctcagaaat cagagaacta tcattgcata tacactaaat 900 tagagaaata ttaaaaggct aagtaacatc tgtggcaata ttgatggtat ataaccttga 960 tatgatgtga tgagaacagt actttacccc atgggcttcc tccccaaacc cttaccccag 1020 tataaatcat gacaaatata ctttaaaaac cattacccta tatctaacca gtactcctca 1080 aaactgtcaa ggtcatcaaa aataagaaaa gtctgaggaa ctgtcaaaac taagaggaac 1140 ccaaggagac atgagaatta tatgtaatgt ggcattctga atgagatccc agaacagaaa 1200 aagaacagta gctaaaaaac taatgaaata taaataaagt ttgaacttta gtttttttta 1260 aaaaagagta gcattaacac ggcaaagcca ttttcatatt tttcttgaac attaagtaca 1320 agtctataat taaaaatttt ttaaatgtag tctggaacat tgccagaaac agaagtacag 1380 cagctatctg tgctgtcgcc taactatcca tagctgattg gtctaaaatg agatacatca 1440 acgctcctcc atgttttttg ttttcttttt aaatgaaaaa ctttattttt taagaggagt 1500 ttcaggttca tagcaaaatt gagaggaagg tacattcaag ctgaggaagt tttcctctat 1560 tcctagttta ctgagagatt gcatcatgaa tgggtgttaa attttgtcaa atgctttttc 1620 tgtgtctatc aatatgacca tgtgattttc ttctttaacc tgttgatggg acaaattacg 1680 ttaattgatt ttcaaacgtt gaaccaccct tacatatctg gaataaattc tacttggttg 1740 tggtgtatat tttttgatac attcttggat tctttttgct aatattttgt tgaaaatgtt 1800 tgtatctttg ttcatgagag atattggtct gttgttttct tttcttgtaa tgtcattttc 1860 tagttccggt attaaggtaa tgctggccta gttgaatgat ttaggaagta ttccctctgc 1920 ttctgtcttc tgaaagagat tgtagaaagt tgatacaatt tttttttctt taaatatctt 1980 gatagaattc 1990

Claims

1. A polynucleotide comprising at least one promoter, at least one expressible construct, and an S / MAR element, wherein the polynucleotide is an integration construct or a non-integration vector construct, the S / MAR element is located downstream of the promoter and the expressible construct, the S / MAR element is adjacent to a splice donor and a splice acceptor, and the polynucleotide is an integration construct.

2. The polynucleotide according to claim 1, wherein the polynucleotide is an integration construct comprising at least one integration signal, preferably, the integration signal is a free end of a linear polynucleotide, a viral integration signal, or a transposable element.

3. The polynucleotide according to claim 1, wherein the polynucleotide is a non-integration viral vector construct, preferably a non-integration lentiviral construct, an adeno-associated virus construct, a simian virus 40 construct, a papillomavirus construct, an adenovirus construct, a hepatitis virus construct, or a herpes virus construct.

4. The polynucleotide according to any one of claims 1 to 3, wherein the expressible construct comprises a coding sequence encoding at least one polypeptide, a sequence encoding siRNA, a sequence encoding miRNA, a sequence encoding antisense RNA, and / or a sequence encoding ribozyme.

5. The polynucleotide according to claim 4, wherein the polypeptide is a therapeutic polypeptide, preferably a human T cell receptor (TCR), a chimeric antigen receptor (CAR), preferably a MART1 TCR.

6. The polynucleotide according to any one of claims 1 to 5, wherein a transcript is transcribed from the promoter, and the sequence of the S / MAR element is excised by splicing from the transcript.

7. A composition comprising the polynucleotide according to any one of claims 1 to 6.

8. A host cell comprising the polynucleotide according to any one of claims 1 to 6, preferably integrated into its genome.

9. The polynucleotide according to any one of claims 1 to 6, the composition according to claim 7, and / or the host cell according to claim 8 for use in medicine.

10. The polynucleotide according to any one of claims 1 to 6, the composition according to claim 7, and / or the host cell according to claim 8 for use in the treatment of genetic diseases.

11. Use of the polynucleotide according to any one of claims 1 to 6, and / or the composition according to claim 7 for stably genetically modifying a host cell.

12. A method for increasing the expression of a eukaryotic expression construct comprising a promoter and an expressible construct, the method comprising including an S / MAR element adjacent to a splice donor and a splice acceptor downstream of the expressible construct.

13. Use of an S / MAR element adjacent to a splice donor and a splice acceptor for increasing the expression of a eukaryotic expression construct.

14. The method according to claim 12 or the use according to claim 13, comprising providing the polynucleotide according to any one of claims 1 to 6.

15. The method or use according to any one of claims 12 to 14, further comprising contacting a host cell with a polynucleotide comprising the eukaryotic expression construct.

Images