Transposase with improved biosafety and efficiency

Abstract

The present invention relates to the field of transposases. In particular, it provides polypeptides comprising or comprising Tc1 / Mariner superfamily transposases, such as Sleeping Beauty transposase, which include nuclear export signals and chaperone-mediated autophagy signals. The transposases of the present invention have been found to exhibit reduced half-life, and therefore improved biosafety. The present invention further provides sequence-optimized RNAs comprising 5'UTR and 3'UTR that encode transposases having high transposition efficiency. The RNA-encoded transposases may be, for example, the transposases of the present invention characterized by a short half-life while maintaining complete integration efficiency.

Description

[Technical Field]

[0001] The present invention relates to the field of transposases. In particular, it provides polypeptides comprising or comprising Tc1 / Mariner superfamily transposases, such as Sleeping Beauty transposase, which include nuclear export signals and chaperone-mediated autophagy signals. The transposases of the present invention have been found to exhibit reduced half-life, and therefore improved biosafety. The present invention further provides sequence-optimized RNA comprising 5'UTR and 3'UTR encoding a transposase having high transposition efficiency. The RNA-encoded transposase may be, for example, the transposase of the present invention characterized by a short half-life while retaining complete integration efficiency. The present invention also provides vectors and cells for the expression of the transposase and / or RNA, kits and pharmaceutical compositions comprising the transposase and / or RNA for use in, for example, gene therapy, for example, adoptive T cell therapy, or uses and methods for gene delivery to the genome of cells. [Background technology]

[0002] Gene therapy is attracting great and growing attention due to its inherent potential to cure a wide range of diseases. The success of gene therapy depends not only on its simplicity, cost-effectiveness, and scalability, but also critically on the safety and efficiency of the gene transfer system used.

[0003] The nonviral Sleeping Beauty (SB) transposon system is relatively well-documented and consists of a transposon and a transposase. In its natural form, the gene encoding the transposase is located between the so-called "inverted terminal sequences" (ITRs) on the transposon. When expressed, the transposase can bind to these ITRs and catalyze the excision of the transposon from the donor DNA locus and its reintegration adjacent to a TA dinucleotide base pair in the recipient DNA sequence. By exchanging the transposase gene with a cassette containing the gene of interest (GOI) and providing the transposase in trans form as DNA, mRNA, or protein, the SB technology has become an efficient and universally applicable gene transfer system. The physical separation of the transposon and transposase offers a wide range of optimization possibilities, opening up numerous potential applications in various cell types of vertebrates (including humans). Indeed, the SB transposon system is increasingly being used in the development of gene therapies, with the first approaches already in clinical development. As a result, regulatory authorities are increasingly paying attention to the biological safety aspect.

[0004] To avoid undesirable genotoxic effects, gene transfer systems should not remain in the cell nucleus for longer than necessary; that is, if transposases are used, they should be removed as quickly as possible after successful transposition. Otherwise, in the presence of active transposases, already incorporated GOIs may be removed or reassembled, which is associated with an increased risk of genomic instability. Furthermore, SB transposases are sensitive to aggregation, and their accumulation, for example, due to overexpression, induces toxicity. Safety concerns associated with long-term transposition activity, and the importance of narrowing the gene delivery activity of transposon systems to a narrower timeframe, have already been recognized, for example, in Imre et al., 2023 or Bushman, 2023.

[0005] Therefore, there is a clear need to optimize the half-life of the transposase, but this problem has not yet been addressed by the means described in the prior art.

[0006] In light of these circumstances, the inventors addressed the problem of providing advantageous variants of transposases commonly used in gene therapy, characterized by a shortened half-life and the resulting improvement in biosafety.

[0007] This problem is solved by the present invention, particularly by the subject matter of the claims. [Overview of the project]

[0008] In particular, the present invention provides polypeptides that can recruit transposons, including nuclear export signals and chaperone-mediated autophagy signals.

[0009] Transposons (also referred to herein as transposition elements or simply TEs) are DNA sequences that have the ability to move their genetic information within the genome. TEs can be classified into two groups. Class I TEs are so-called retrotransposons that follow a copy-and-paste mechanism and use RNA intermediates for this process (Wicker et al. 2007). Class II transposons rely solely on DNA intermediates for their transposition process. In this category, subclass I transposons follow a cut-and-paste mechanism, during which the transposon is excised from one genomic location and reintegrated at a different location (Wicker et al. 2007).

[0010] In preferred embodiments, the polypeptide according to the present invention can recruit transposons of the Tc1 / Mariner superfamily, i.e., the polypeptide contains a Tc1 / Mariner superfamily transposase.

[0011] The Tc1 / Mariner superfamily of transposons follows the standard cut-and-paste mechanism of class II subclass I transposons. The TE of this superfamily is flanked by an inverted terminal sequence (ITR) and possesses a gene encoding a transposase, an enzyme factor that catalyzes the transposition reaction (Wicker et al. 2007). The transposase binds to the ITR, excises the TE from the donor locus, reintegrates it flanked by the TA target sequence, and ultimately results in duplication of the TA target site (Wicker et al. 2007). Thus, as used herein, the term “transposase” refers to an enzyme that can mediate a transposition reaction as a component of a functional nucleic acid-protein complex. As used herein, the term “transposition reaction” refers to the reaction in which a transposon is inserted into a target nucleic acid.

[0012] Preferably, the transposase is active in human cells. For example, it may be Sleeping Beauty, Frog Prince, Minos, ZB, Tdr1, or Passport transposase. In a preferred embodiment, the polypeptide of the present invention comprises Sleeping Beauty transposase.

[0013] The Sleeping Beauty (SB) transposon was the first DNA transposon to be reconstructed from fossil DNA sequences within fish genomes and shown to be active in vertebrates (Ivics et al., 1997). It has been widely used as a genetic engineering tool in various preclinical and clinical studies (Amberger et al., 2020). Since the discovery of the SB transposon, several mutations have been identified, resulting in higher overall integration efficiency. These mutations have led to the currently most active transposase variant of SB, called SB100X (Mates et al., 2009). The structural and biochemical features of the SB transposase that catalyze the transposition reaction are of particular interest because, based on the enzymatic activity of the transposase, the efficiency of SB transposon integration into target cell genomes can be enhanced. The SB transposase contains an N-terminal DNA-binding domain (amino acids [aa]1-110) and a C-terminal catalytic domain (DDE; aa 114-340) (Ivics et al., 1997). Both domains are connected by a flexible linker region containing a nuclear localization signal (NLS; aa 97-123) (Ivics et al., 1997). The DNA-binding domain consists of two subdomains, PAI and RED, each forming a helix-turn-helix (HTH) motif crucial for the recognition and binding of transposon DNA (Ivics et al., 1997, Izsvak et al., 2002). The catalytic domain has its three conserved amino acids (D153, D244, E279 (DDE)) at its catalytic center and catalyzes the DNA hydrolysis reaction necessary for excision and transesterification that occurs in the integration reaction (Izsvak et al., 2002, Montano et al., 2011, Yang et al., 2006).

[0014] Other transposases of the Tc1 / Mariner superfamily, such as Frog Prince, have a similar structure.

[0015] Therefore, preferably, the polypeptide according to the present invention comprises a Sleeping Beauty transposase having at least 80% amino acid identity with SEQ ID NO: 43 (SB100X), and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, only one, two, or three amino acid differences exist from the aforementioned sequence. The polypeptide according to the present invention may also comprise an amino acid sequence having 100% sequence identity with SEQ ID NO: 43. In the context of the present invention, the amino acid sequence of SEQ ID NO: 43 (SB100X) is considered a “wild-type” Sleeping Beauty transposase sequence, despite being a synthetic construct.

[0016] In another embodiment, the polypeptide according to the present invention does not contain the Sleeping Beauty transposase but contains a different member of the Tc1 / Mariner superfamily of transposases. This may include, for example, an FP (Frog Prince) transposase having at least 80% amino acid identity with SEQ ID NO: 44, and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, there may be only one, two, or three amino acid differences from the sequence. The polypeptide according to the present invention may also contain an amino acid sequence having 100% sequence identity with SEQ ID NO: 44.

[0017] Alternatively, the polypeptide according to the present invention may include a Minos transposase having at least 80% amino acid identity with SEQ ID NO: 45, and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, only one, two, or three amino acid differences exist from the sequence. The polypeptide according to the present invention may also include an amino acid sequence having 100% sequence identity with SEQ ID NO: 45.

[0018] Alternatively, the polypeptide according to the present invention may include a ZB transposase having at least 80% amino acid identity with SEQ ID NO: 46, and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, only one, two, or three amino acid differences exist from the sequence. The polypeptide according to the present invention may also include an amino acid sequence having 100% sequence identity with SEQ ID NO: 46.

[0019] Alternatively, the polypeptide according to the present invention may comprise a Tdr1 transposase having at least 80% amino acid identity with SEQ ID NO: 47, and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, only one, two, or three amino acid differences may exist from the sequence. The polypeptide according to the present invention may also comprise an amino acid sequence having 100% sequence identity with SEQ ID NO: 47.

[0020] In yet another embodiment, the polypeptide according to the present invention may comprise a Passport transposase having at least 80% amino acid identity with SEQ ID NO: 48, and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, only one, two, or three amino acid differences exist from the sequence. The polypeptide according to the present invention may also comprise an amino acid sequence having 100% sequence identity with SEQ ID NO: 48.

[0021] Each transposase is classified by its amino acid identity with its respective "wild-type" transposase sequence. That is, if a transposase has at least 80% amino acid identity with any of the listed sequences, and its sequence identity with one of those sequences is the highest, it is classified as a transposon of that class.

[0022] The polypeptides according to the present invention preferably include transposases containing highly active variants of transposases, i.e., transposases containing mutations that result in enhanced transposition activity (i.e., greater than 100%, preferably greater than 110%, greater than 120%, greater than 150%, or about 200% or more) compared to any “wild-type” version of any of the transposases described herein. In the context of the present invention, the terms “transposition efficiency” and “transposition activity” are used synonymously and refer to the activity of a given transposase that can be evaluated in a transposition reaction. Appropriate experimental setups may be described in the Experimental Section of this specification or may use the classic binary transposition assay described in Ivics et al., 1997.

[0023] Numerous mutations at various positions have been identified that can enhance the transposition activity of transposases, such as SB transposase. For example, International Publication No. 2009 / 003671, incorporated herein by reference, discloses highly active variants of transposases comprising one or more properties, in particular substitutions or groups of substitutions that enhance the transposition activity of the transposase. Accordingly, in one embodiment, the polypeptide according to the present invention comprises a highly active variant of any of the transposases described herein, preferably a highly active variant of SB transposase comprising at least one of the following substitutions or groups of substitutions. (1) K14R, K13D, K13A, K30R, K33A, T83A, I100L, R115H, R143L, R147E, A205K / H207V / K208R / D210E, H207V / K208R / D210E, R214D / K215A / E216V / N217Q; M243Q, E267D, T314N, and / or G317E; (2)K14R / / R214D / K215A / E216V / N217Q; (3)K33A / R115H / R214D / K215A / E216V / N217Q / M243H; (4)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (5)K13D / K33A / T83N / H207V / K208R / D210E / M243Q; (6)K13A / K33A / R214D / K215A / E216V / N217Q; (7)K33A / T83N / R214D / K215NE216V / N217Q / / G317E; (8)K14R / T83A / M243Q; (9)K14R / T83A / I100L / M243Q; (10)K14R / T83A / R143L / M243Q; (11)K14R / T83A / R147E / M243Q; (12)K14R / T83A / M243Q / E267D; (13)K14R / T83A / M243Q / T314N; (14)K14R / K30R / I110L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (15)K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H; (16)K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (17)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / E267D; (18)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H / T314N; (19)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / G317E; (20)K14R / K33A / R115H / R214D / K215A / E216V / N217Q / M243H; (21)K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / T314N; (22)K14R / K30R / R143U / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / E267D; (23)K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / T314N; (24)K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / G317E; (25)K14R / K33A / R115H / R143L / R214D / K215A / E216V / N217Q / M243H; (26)K14R / K33A / R115H / R147E / / R214D / K215A / E216V / N217Q / M243H; (27)K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / E267D; (28)K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / T314N; (29)K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / G317E; (30) K14R / T83A / M243Q / G317E; or (31)K13A / K33A / T83N / R214D / K215A / E216V / N217Q.

[0024] All amino acid “positions” used in the context of this invention refer to the amino acid sequence of the SB transposase of SEQ ID NO: 43 and are determined in relation only to the transposase portion of the polypeptide. Those skilled in the art will be able to easily determine, for example, by alignment, the corresponding positions of one or more of the outlined substitutions or groups of substitutions in the amino acid sequences of other naturally occurring transposases described herein.

[0025] The polypeptide according to the present invention may further include any of the substitutions disclosed in European Patent Application No. 22200543.1, i.e., a substitution at position 124. When the transposase is an SB or Tdr1 transposase such as SB100X, the original or "wild-type" amino acid residue at position 124 is Q. For ZB, it is N; for FP, it is S; for Minos, it is S; and for Passport, it is K. The amino acid that substitutes the wild-type amino acid residue at position 124 may be selected from the group consisting of C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In one embodiment, the substituted amino acid at position 124 is C, A, R, D, E, G, H, I, L, M, F, P, S, T, W, Y, and V. The substituted amino acid at position 124 can also be, for example, C, A, D, E, G, H, I, L, M, F, P, W, Y, and V.

[0026] In the context of the present invention, if the preferred transposase is an SB transposase such as SB100X, the substituted amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. The preferred amino acid in the above context is C, S, or M, most preferably C. The same applies to the Tdr1 transposase.

[0027] If the transposase is a ZB transposase, the substituted amino acid at position 124 may be C, A, R, D, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. The preferred amino acid in the above context is C, S, or M, most preferably C.

[0028] If the transposase is an FP transposase, the substituted amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, K, M, F, P, T, W, Y, and V. The preferred amino acid in the above context is C or M, most preferably C.

[0029] If the transposase is a Passport transposase, the substituted amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, M, F, P, S, T, W, Y, and V. The preferred amino acid in the above context is C or M, most preferably C.

[0030] As described in European Patent Application No. 22200543.1, a significant increase in transposition activity was achieved when the amino acid at position 124 was C. Therefore, in particularly advantageous embodiments, the polypeptide of the present invention comprises a transposase having a substitution that results in C at position 124.

[0031] If necessary, the polypeptide may include any of the transposases and their variants described herein, and further include the modifications disclosed in international patent application PCT / EP2022 / 075007, which is incorporated herein by reference.

[0032] As described in PCT / EP2022 / 075007, transposases of the Tc1 / Mariner superfamily containing a substitution at any of positions 187, 247, or 248, e.g., the K248R substitution, have an improved integration pattern compared to transposases without the substitution at the aforementioned position. They have a better safety profile because they reduce the integration of transposons into exons and transcriptional regulatory regions of genes in the human genome. Therefore, transposons are incorporated into the genetically safe harbor at a higher frequency. Accordingly, the polypeptides of the present invention may have increased specificity for integration into the genome and therefore may contain one or more of the following substitutions: a) The substituted amino acid at position 187 may be A, N, C, Q, G, I, L, M, S, V, W, K, R, E, P, T, and S, preferably V; b) The amino acid at the substituted position 247 may be R, C, A, or S; preferably R, and / or c) The amino acid at the substituted position 248 may be R, S, V, I, or C, preferably R.

[0033] For substitution at position 187 in SB transposase, substitutions of H187A, H187N, H187C, H187Q, H187G, H187I, H187L, H187M, H187S, H187V, H187W, H187K, H187R, H187E, H187P, H187T, and H187S are preferred, with H187V substitution being the most preferred.

[0034] For substitutions at position 247 in SB transposase, P247R, P247C, P247A, and P247S substitutions are preferred, with P247R substitution being the most preferred.

[0035] For substitution at position 248 in SB transposase, K248R, K248S, K248V, K248I, or K248C substitutions are preferred, with K248R substitution being the most preferred.

[0036] The polypeptides of the present invention may also include transposases having improved solubility in water compared to SB of SEQ ID NO: 43, e.g., high-solubility transposases described in Querques et al., 2009 or EP3673053A. To improve solubility, the transposase may further include, for example, at least one substitution at amino acid positions 176 and / or 212, where the amino acid at position 176 is not C and the amino acid at position 212 is not I, preferably the amino acids at positions 176 and / or 212 are S, and optionally both amino acids at positions 176 and 212 are S.

[0037] The relatively long half-lives of transposases such as SB100X and its variants constitute a biosafety issue because prolonged transposase activity can lead to, for example, the re-mobilization of incorporated transposons, thus causing genomic instability. Therefore, we have tested various strategies to shorten the half-life of transposases. Since transposases such as SB100X are DNA-modifying enzymes, they localize and act in the cell nucleus. Therefore, one of the main objectives was to (a) reduce the level of transposase protein expressed in the reaction space (nucleus) and (b) promote the degradation of enzymatically active transposase levels throughout the target cell.

[0038] As a first means to achieve this objective, the polypeptide according to the present invention contains a nuclear export signal. A nuclear export signal (NES) is a short peptide sequence of about 8-15 amino acid residues that can target cargo proteins transported from the nucleus to the cytoplasm of eukaryotic cells via the nuclear pore complex. Many different NESs have been identified in the past. However, conventional NESs are usually Φ1-X 2-3 -Φ2-X 2-3 The consensus amino acid sequence is -Φ3-X-Φ4, where Φn represents a hydrophobic amino acid selected from L, V, I, F, or M, and X can be any amino acid (Kutay and Guttinger, 2005; Xu et al., 2012). The hydrophobic amino acid residues of the NES mediate the translocation of proteins to the cytoplasm by establishing interactions with exportins such as CRM1. These exportins then directly interact with the building blocks of the nuclear pore complex, the so-called nucleoporin, which is embedded in the nuclear membrane and thereby delivers cargo proteins into the cytoplasm.

[0039] The NES of the polypeptide according to the present invention is Φ1-X 2-3 -Φ2-X 2-3- It may have an amino acid sequence corresponding to the consensus sequence of -Φ3-X-Φ4. Alternatively, the NES sequence may deviate from the consensus sequence. For example, it may include any of the extended consensus sequences described by Kosugi et al., 2008, such as Φ1-X3-Φ2-X2-Φ3-X-Φ4; Φ1-X2-Φ2-X2-Φ3-X-Φ4; Φ1-X3-Φ2-X3-Φ3-X-Φ4; Φ1-X2-Φ2-X3-Φ3-X-Φ4; Φ1-X-Φ2-X2-Φ3-X-Φ4; Φ1-X2-Φ2-X3-Φ3-X2-Φ4, where Φn is a hydrophobic residue selected from L, I, V, M, F, W, C, T or A, and W, C, T and A may only be tolerated at one of the four Φ positions. Since CRM1 provides five hydrophobic pockets for binding to the Φ positions of NES, the NES may also include a sequence containing five important hydrophobic positions instead of four. Such NES sequences are disclosed, for example, by Guttler et al., 2010 or Guttler and Gorlich, 2011, and include an additional hydrophobic residue Φ0 at the N-terminus. Thus, NES is Φ0-X 0,3 -Φ1-X3-Φ2-X 2,3 - It may have the amino acid sequence of -Φ3-X-Φ4, and Φ0 may be I, V, M, L, A, Y, F, W, or P. Φ1 may be L, I, V, M, F, A, or W; Φ2 may be F, M, L, I, V, Y, or W. Φ3 may be L, M, I, V, F, W, or A; Φ4 may be L, I, M, V, or F. Alternatively, the NES sequence may be Φ0-Φ1 pro -X1-Φ2-X2-Φ3-X-Φ4, and Pro is strongly preferred at the position of Φ1. NES may also be any of the consensus sequences first described by Xu et al., 2012, such as Φ1-X 1,2,3 -Φ2-[^W]2-Φ3-[^W]-Φ4, Φ1-X 2,3 -Φ2-[^W]3-Φ3-[^W]-Φ4 or Φ1-X2-Φ2-X[^W]2-Φ3-[^W]2-Φ4, where [^W] is any of the 20 amino acids other than W, and A and T residues may be used only once at either the position of Φ1 or Φ2.

[0040] Those skilled in the art can easily select a suitable NES sequence to achieve successful nuclear export of the polypeptide of the present invention. However, in preferred embodiments, the NES may comprise an amino acid sequence having at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity with SEQ ID NO: 30 (LALKLAGLDI). Most preferably, the NES comprises or consists of an amino acid sequence having 100% sequence identity with SEQ ID NO: 30.

[0041] The NES is preferably linked to the N-terminus of the polypeptide according to the present invention, i.e., to the N-terminus of the transposase. However, in another embodiment, it may be located at another suitable position within the polypeptide.

[0042] The polypeptide according to the present invention further comprises a proteolytic signal (also known as a degron), i.e., an amino acid sequence that can be recognized by a proteolytic machinery to mediate the degradation of the polypeptide. Proteins having a degradation signal are degraded by either the autophagy-lysosomal pathway or ubiquitin-dependent targeting to the proteasome.

[0043] The inventors have surprisingly found that the half-life of transposase polypeptides can be most effectively reduced when they are targeted for degradation via the autophagy-lysosome pathway rather than the ubiquitin-proteasome system (see Examples). Therefore, in preferred embodiments, the proteolytic signal of the polypeptide of the present invention is a signal that targets the polypeptide of the present invention for autophagy-lysosome degradation, i.e., a chaperone-mediated autophagy (CMA-) signal.

[0044] Autophagy refers to the process by which intracellular macromolecules, such as proteins, are transported to specialized single-membrane organelles known as lysosomes. The lysosomal lumen has an acidic pH and contains numerous different hydrolytic enzymes, i.e., proteases, lipases, glycosidases, and nucleases that can catalyze the degradation of macromolecular substrates. The autophagy-lysosomal pathway is normally a non-selective protein and organelle degradation process, but can become selective during starvation, for example, in a process called chaperone-mediated autophagy (CMA). During CMA, only a subset of cytosolically soluble proteins are degraded. The selectivity of CMA is based on the presence of specific motifs in the amino acid sequence of the substrate protein, which are recognized in the cytosol by chaperones and co-chaperones, such as hsc70 (a 70-kDa heat shock homologous protein). These chaperone proteins target CMA substrates to lysosomes and assist in the unfolding of the substrates. When substrate proteins are transported to the lysosomal membrane, they bind to the cytosolic tail of lysosomal-associated membrane protein 2A (LAMP-2A), which is a glycoprotein that subsequently polymerizes into a higher-order complex necessary for substrate transfer into the lysosomal lumen.

[0045] A specific protein motif or signal recognized by CMA typically consists of five amino acids and must satisfy the following characteristics: one amino acid in the sequence must be basic, another must be acidic, a third amino acid must be hydrophobic, and a fourth amino acid may be either basic or hydrophobic but must not be acidic. Furthermore, the exact order of these amino acids appears to be of little or no relevance (Orenstein and Cuervo, 2010). Therefore, it is preferable that the CMA signal of the polypeptide of the present invention also follows the above characteristics. The best-studied chaperone-mediated autophagy signal is KFERQ, which was found in ribonuclease A, the first CMA substrate identified to date. The inventors have found that the use of KFERQ as a CMA signal in combination with the addition of NES is suitable for significantly reducing the half-life of SB100X transposase. Therefore, in a preferred embodiment, the CMA signal of the polypeptide according to the present invention has the amino acid sequence KFERQ (SEQ ID NO: 31). However, it may also have any of the following amino acid sequences: KFSRK (SEQ ID NO: 32), VKKDQ (SEQ ID NO: 33), KFFEQ (SEQ ID NO: 34), KIREI (SEQ ID NO: 35), QVELR (SEQ ID NO: 36), QYFKS (SEQ ID NO: 37), LKSFQ (SEQ ID NO: 38), LKYFQ (SEQ ID NO: 39), EKFLQ (SEQ ID NO: 40), QREFK (SEQ ID NO: 41), or VDKFQ (SEQ ID NO: 42). In certain preferred embodiments, the CMA has an amino acid sequence selected from the group including KFERQ (SEQ ID NO: 31), KFFEQ (SEQ ID NO: 34), QVELR (SEQ ID NO: 36), LKSFQ (SEQ ID NO: 38), LKYFQ (SEQ ID NO: 39), and EKFLQ (SEQ ID NO: 40).

[0046] The polypeptides according to the present invention may include proteolytic signals other than CMA signals, and may include, for example, degradation signals that target the polypeptide to a ubiquitin ligase machine or proteasome.

[0047] The proteolytic signal, preferably the CMA signal, may be located at or near the N-terminus of the polypeptide, at or near the C-terminus of the polypeptide, or within the polypeptide. In preferred embodiments, both the NES and the proteolytic signal, e.g., the CMA signal, are located at the N-terminus of the polypeptide, i.e., they form a tag linked to the N-terminus of the transposase, and preferably the NES and the proteolytic signal are arranged such that the NES forms the N-terminus of the final polypeptide of the present invention. When the NES and / or proteolytic signal, e.g., the CMA signal, are linked to the N-terminus of the transposase, the amino acid sequence of the transposase may lack 1 to 10 N-terminal amino acids of the naturally occurring full-length transposase. Preferably, the amino acid sequence of the transposase lacks methionine at position M1. The amino acid sequence of the transposase may also lack 1 to 10 C-terminal amino acids of the naturally occurring full-length transposase if the NES and / or proteolytic signal, e.g., the CMA signal, are linked to the C-terminus of the transposase.

[0048] NES and proteolytic signals, such as the CMA signal, can be linked to the N-terminus of a transposase via a suitable linker sequence. The linker sequence should be selected so as not to interfere with the proper folding of the final polypeptide into a functional transposase. It may be, for example, 1 to 50 amino acids long. For example, the linker sequence may include the amino acid sequence of SEQ ID NO: 49 (KLGGGAPAVGGGPKAADK). However, those skilled in the art will be able to identify alternative suitable linker sequences different from those of SEQ ID NO: 49. In alternative embodiments, NES and proteolytic signals, such as the CMA signal, can be linked directly to the N-terminus of the polypeptide, i.e., no linker sequence is required.

[0049] In preferred embodiments, the polypeptide according to the present invention may include an amino acid sequence having 80% sequence identity with SEQ ID NO: 50, comprising an SB100X transposase and NES and CMA signals linked to its N-terminus via a linker sequence. It may optionally include an amino acid sequence having more than 80% sequence identity with SEQ ID NO: 50, for example, at least 85%, at least 90%, at least 91, at least 92, at least 93, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity with SEQ ID NO: 50. It may also include, or consist of, an amino acid sequence having 100% sequence identity with SEQ ID NO: 50.

[0050] As shown in the following examples, the inventors found that the addition of NES and CMA signal sequences significantly shortens the half-life of SB100X transposase by increasing the transposase degradation rate by at least five times. Therefore, the inventors succeeded in producing SB100X transposase (also referred to herein as "NES-CMA-SB100X") which is characterized in particular high biosafety because the modifications described herein minimize the risk of transposon reintegration and undesirable accumulation of transposase proteins.

[0051] However, the decrease in the transposase half-life was also associated with a decrease in transposition efficiency.

[0052] As shown in the following examples, NES-CMA-SB100X transposase exhibited approximately 25% lower transposition activity in mammalian cell lines and approximately 50% lower activity in primary human immune cells compared to conventional SB100X transposase.

[0053] To take this into consideration, a larger amount of the transposase can be used. However, maintaining transposition activity is preferred.

[0054] For example, the transposition efficiency of a transposase can be at least partially restored by introducing any of the amino acid substitutions disclosed herein that can enhance transposition efficiency, such as the 124-position amino acid substitution disclosed herein. As shown in the following examples, the Q124C substitution was sufficient to partially restore the transposition efficiency of NES-CMA-SB100X to that of conventional SB100X.

[0055] Furthermore, the inventors decided to optimize the transposase expression profile with the aim of restoring the transposition efficiency of NES-CMA-SB100X to that of SB100X. Typically, in vitro transcribed messenger RNA (mRNA) can be used as a source of transposases such as SB transposase to eliminate the risk of inadvertent integration of the transposase gene into the genome.

[0056] Accordingly, the present invention also provides polypeptides capable of recruiting transposons described herein, for example, RNA encoding a polypeptide according to the present invention, comprising a series of modifications that have a positive effect on the transposition efficiency of the encoded transposase. Strictly speaking, it is understood that modifications to the RNA itself do not result in a change in the amino acid sequence of the encoded polypeptide, and therefore do not change the transposition efficiency of the polypeptide itself. Rather, these RNA modifications can optimize polypeptide expression by, for example, achieving RNA stabilization and improving its ribosome binding and / or translation. The use of such modified RNA for optimized transposase expression can compensate, for example, for the loss of transposition efficiency of the polypeptide of the present invention due to the addition of NES and CMA signal sequences. Therefore, whenever it is mentioned in the context of the present invention that one or more of the RNA modifications disclosed herein affect the transposition efficiency of the encoded transposase, for example, by increasing, enhancing, or restoring it, or by bringing it to a level comparable to that of a reference transposase, this expression should be understood to mean that the use of such modified RNA in the present invention has a significant and favorable effect on transposition efficiency because it results in the optimized expression of a polypeptide capable of recruiting transposons.

[0057] As a first method, the RNA according to the present invention encodes a polypeptide capable of recruiting a transposon, and the RNA comprises a 5'UTR and a 3'UTR.

[0058] The eukaryotic 5'UTR is also known as a leader sequence and contains multiple cis-acting regulatory regions and binding sites for different translation initiation factors such as eIF4E and eIF4G. Therefore, the 5'UTR plays a crucial role in the regulation of mRNA translation.

[0059] Similarly, the 3'UTR contains regulatory sequences that influence mRNA polyadenylation, translation efficiency, localization, and stability. In particular, microRNAs preferentially bind to the 3'UTR to regulate gene expression post-transcriptionally.

[0060] The inventors tested various 5'UTRs and 3'UTRs from different sources for their ability to improve the transposition / incorporation efficiency of RNA-encoded transposons according to the present invention. The 5'UTR of β-globin was found to exhibit a particularly advantageous effect on transfection stability. Similar effects were further observed using synthetic 5'UTRs containing the elF4G aptamer, i.e., short regions of approximately 25–70 nt bound with high affinity by the translation elongation factor elF4G. Exemplary aptamers are disclosed, for example, in Miyakawa et al., 2006, the contents of which are incorporated herein by reference.

[0061] In contrast, we found that 5'UTRs containing the JK stem-loop region of encephalomyocarditis virus IRES, as described by Terenin et al., 2013, did not produce the same desired effect as 5'UTRs containing β-globin or elF4G aptamers.

[0062] Therefore, in a preferred embodiment, the RNA 5'UTR according to the present invention is a β-globin 5'UTR or a 5'UTR containing an elF4G aptamer.

[0063] For example, the RNA 5'UTR according to the present invention may be a β-globin 5'UTR containing a nucleic acid sequence having at least 80%, and optionally at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity with SEQ ID NO: 51. It may also contain, or consist of, a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 51.

[0064] Alternatively, the 5'UTR of RNA according to the present invention may be a 5'UTR containing an elF4G aptamer, and therefore may contain a nucleic acid sequence having at least 80%, and optionally at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 52. It may also contain, or consist of, a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 52.

[0065] Alternatively, the RNA 5'UTR according to the present invention may also be a synthetic 5'UTR containing a nucleic acid sequence having at least 80%, and optionally at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity with SEQ ID NO: 61, and containing a sequence that is less likely to form secondary structural elements such as hairpins. It may also contain, or consist of, a nucleic acid sequence having 100% sequence identity with SEQ ID NO: 61.

[0066] The RNA 3'UTR according to the present invention is preferably selected from the group consisting of one or more β-globin 3'UTRs, α-globin 2 3'UTRs, 3'UTRs containing woodchuck hepatitis virus (WHV) post-transcriptional regulatory elements (WPREs), βα-globin 3'UTRs, or cleaved β-globin 3'UTRs.

[0067] Accordingly, in one embodiment, the RNA according to the present invention comprises one or more β-globin 3'UTRs, for example two or three, preferably two, that contain a nucleic acid sequence having at least 80%, optionally at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 53. It may also contain a 3'UTR that contains one or more nucleic acid sequences having 100% sequence identity with or consisting of SEQ ID NO: 53.

[0068] In a second embodiment, the RNA according to the present invention comprises an α-globin 2 3'UTR containing a nucleic acid sequence having at least 80%, and optionally at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 54. It may also comprise a 3'UTR containing a nucleic acid sequence having or comprising 100% sequence identity with SEQ ID NO: 54.

[0069] In a third embodiment, the RNA according to the present invention comprises a 3'UTR containing a WPRE that includes a nucleic acid sequence having at least 80%, optionally at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 55. It may also comprise a 3'UTR containing a nucleic acid sequence having or comprising 100% sequence identity with SEQ ID NO: 55.

[0070] In a fourth embodiment, the RNA according to the present invention comprises a βα-globin 3'UTR. The β- and α-globin 3'UTRs are expected to regulate mRNA stability through distinct mechanisms and potentially have synergistic effects (Russell and Liebhaber, 1996). Thus, the 3'UTR may contain a nucleic acid sequence having at least 80%, and optionally at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 56. It may also contain a 3'UTR containing a nucleic acid sequence having or comprising 100% sequence identity with SEQ ID NO: 56.

[0071] In a fifth embodiment, the RNA according to the present invention comprises a cleaved β-globin 3'UTR, i.e., a β-globin 3'UTR lacking a signal sequence for native polyadenylation. Thus, the 3'UTR of the RNA according to the present invention may contain a nucleic acid sequence having at least 80%, optionally at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 57. It may also contain a 3'UTR comprising a nucleic acid sequence having or consisting of 100% sequence identity with SEQ ID NO: 57.

[0072] The inventors conducted various experiments to identify RNA encoding transposases that could express the NES-CMA-SB100X polypeptide according to the present invention, which contained specific combinations of 5'UTR and 3'UTR disclosed herein and resulted in increased transposition efficiency (i.e., due to improved expression), for example, transposition efficiency comparable to that of conventional SB100X (SEQ ID NO: 43). In other words, the inventors identified RNA constructs encoding the NES-CMA-SB100X polypeptide containing specific combinations of 5'UTR and 3'UTR that, when experimentally tested in cells, mediate transposition efficiency comparable to that of conventional SB100X. The most effective RNAs identified in these experiments contained the following combinations: (a) β-globin 5'UTR and two β-globin 3'UTRs, (b) β-globin 5'UTR and cleaved β-globin, (c)β-globin 5'UTR and α-globin 2 3'UTR, (d) β-globin 5'UTR and βα-globin 3'UTR, (e) elF4G aptamer and 5'UTR containing two β-globin 3'UTRs.

[0073] Therefore, the RNA of the present invention preferably comprises a combination of 5'UTR and 3'UTR according to any of (a) to (e).

[0074] The RNA of the present invention is preferably further codon-optimized for expression in target organisms and / or target tissues. To this end, isolated triplet codons in the nucleotide sequence encoding the transposase are replaced with synonymous codons during the in vitro mutagenesis process to match the transposase expression rate to the preferred codon usage frequency of the target organism or target tissue. Codon optimization results in improved expression of the encoded polypeptide in selected host organisms, e.g., human cells. Tables for appropriately adjusting nucleic acid sequences to the specific transcription / translation machinery of host cells are known to those skilled in the art. Generally, it is preferable to match the G / C content of the nucleotide sequence to specific host cell conditions. For expression in human cells, an increase in G / C content of at least 10%, more preferably at least 20%, 30%, 50%, 70%, and even more preferably 90% of the maximum G / C content (encoding each peptide variant of the present invention) is preferred. Preparation and purification of such nucleic acids and / or derivatives are usually carried out by standard procedures.

[0075] The RNA according to the present invention further comprises a poly(A) tail having a length of 50 to 150 nt. The poly(A) tail consists of a plurality of adenosine monophosphates that are added to the mRNA post-transcriptionally to protect the mRNA molecule from enzymatic degradation in the cytoplasm and to assist in transcription termination, export of mRNA from the nucleus, and translation. Preferably, the poly(A) tail of the RNA disclosed herein has a length of 75 to 100 nt, for example, 80 to 95 nt or 85 to 90 nt. Most preferably, the poly(A) tail has a length of 90 nt.

[0076] The RNA according to the present invention further comprises a 5' cap structure. The 5' cap structure may be a Cap0 structure, i.e., N7-methylguanosine bound to the 5' nucleotide of the RNA of the present invention via a 5'-to-5' triphosphate crosslink. Such a Cap0 structure is also commonly known as m7G cap or m7Gppp-. Alternatively, the 5' cap structure may be a Cap1 structure, i.e., in addition to the N7-methylguanosine bound to the 5' nucleotide of the RNA of the present invention as described above, the ribose sugar of the RNA's initial 5' nucleotide further comprises methylation at the 2'O position. The Cap1 structure is also commonly called an m7GpppNm structure. If necessary, the 5' cap structure may also be a Cap2 structure, which corresponds to the Cap1 structure but has methylated 2'-hydroxyl groups on the first two ribose sugars. The inventors have found that the presence of Cap1 in particular can have a positive effect on the expression of the encoded transposase. Therefore, in preferred embodiments, the RNA according to the present invention comprises a 5' Cap1 structure.

[0077] The RNA according to the present invention is preferably a nucleoside-modified RNA, that is, a synthetic RNA comprising one or more naturally modified nucleosides or synthetic nucleoside analogs. For example, the RNA according to the present invention may comprise one or more modified nucleosides selected from the group comprising 5-methyl-CTP, N4-acetyl-CTP, pseudo-UTP, N1-methyl pseudo-UTP, 6-methoxy-UTP, 2-thio-UTP, N6-methyl-ATP, or N1-methyl-ATP.

[0078] In the context of the present invention, RNA according to the present invention is considered nucleoside-modified if it contains at least one modified nucleoside at any position in the nucleic acid sequence. However, in embodiments in which RNA according to the present invention is nucleoside-modified RNA, preferably at least 50% of its uridine nucleoside, cytidine nucleoside, guanosine nucleoside and / or adenosine nucleoside are replaced with appropriate corresponding modified nucleosides. More preferably, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the uridine nucleoside, cytidine nucleoside, guanosine nucleoside and / or adenosine nucleoside present in the RNA are replaced with the corresponding modified nucleosides described herein. Most preferably, the nucleoside-modified RNA is completely replaced, i.e., 100% of the uridine nucleosides, cytidine nucleosides, guanosine nucleosides, and / or adenosine nucleosides present in the RNA are replaced by appropriate corresponding modified nucleosides. For example, if the RNA according to the present invention is a nucleoside-modified RNA containing pseudo-UTPs, then preferably at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or most preferably 100%, of the uridine nucleosides in the RNA are replaced by pseudo-UTPs. In the latter case, the RNA is considered to be completely replaced.

[0079] The inventors have found that the use of pseudouridine-5'-triphosphate (pseudo-UTP), 5-methoxyuridine-5'-triphosphate (5-methoxy-UTP), and 5-methylcytidine-5'-triphosphate (5-methyl-CTP) in the RNA according to the present invention encoding the NES-CMA-SB100X transposase resulted in stable transposon integration efficiency in primary T cells comparable to that of the SB100X transposase. Therefore, in preferred embodiments, the RNA of the present invention comprises one of the modified nucleosides selected from pseudo-UTP, 5-methoxy-UTP, or 5-methyl-CTP. The RNA may also comprise combinations of different types of modified nucleosides. For example, it may include combinations of pseudo-UTP and 5-methoxy-UTP, combinations of pseudo-UTP and 5-methyl-CTP, combinations of 5-methoxy-UTP and 5-methyl-CTP, or combinations of pseudo-UTP, 5-methoxy-UTP and 5-methyl-CTP.

[0080] Preferably, the RNA according to the present invention is transcribed in vitro. However, in alternative embodiments, the RNA may be transcribed from a suitable intracellular vector, as described below.

[0081] The inventors have optimized the expression of the NES-CMA-SB100X polypeptide using RNA containing the modifications described herein, thereby obtaining an improved biosafety transposase system with transposition efficiency comparable to or even higher than that of the conventional SB100X transposase. Therefore, the RNA preferably encodes a polypeptide according to the present invention, i.e., one of the transposases described herein linked to NES and a proteolytic signal, such as a CMA signal. Modifications to the RNA encoding the polypeptide of the present invention allow for compensation of the loss of transposition efficiency due to a reduction in the transposase half-life.

[0082] For example, in the first embodiment, the RNA according to the present invention comprises the nucleic acid sequence of Sequence ID No. 62 and is named A1K. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises the β-globin 5'UTR and 3'UTR containing the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE). The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides and thus may also consist of the nucleic acid sequence of Sequence ID No. 4.

[0083] In another embodiment, the RNA according to the present invention comprises the nucleic acid sequence of Sequence ID No. 63 and is referred to as A1H. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises a β-globin 5'UTR and a single β-globin 3'UTR. The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides and thus may also consist of the nucleic acid sequence of Sequence ID No. 6.

[0084] In another embodiment, the RNA according to the present invention comprises the nucleic acid sequence of Sequence ID No. 64 and is referred to as A1I. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises the β-globin 5'UTR and α-globin 2 3'UTR. The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides and thus may also consist of the nucleic acid sequence of Sequence ID No. 7.

[0085] In another embodiment, the RNA according to the present invention comprises the nucleic acid sequence of SEQ ID NO: 65 and is referred to as A1O. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises β-globin 5'UTR and βα-globin 3'UTR. The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides and thus may also consist of the nucleic acid sequence of SEQ ID NO: 8.

[0086] In another embodiment, the RNA according to the present invention comprises the nucleic acid sequence of Sequence ID No. 66 and is referred to as A1P. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises a β-globin 5'UTR and a cleaved β-globin 3'UTR. The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides and thus may also consist of the nucleic acid sequence of Sequence ID No. 9.

[0087] In another embodiment, the RNA according to the present invention is one comprising the nucleic acid sequence of SEQ ID NO: 67 and referred to as B1M, or one comprising the nucleic acid sequence of SEQ ID NO: 68 and referred to as B3M. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises an elF4G aptamer and a 5'UTR containing two β-globin 3'UTRs. The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides, and thus may consist of the nucleic acid sequences of SEQ ID NO: 10 (B1M) or 11 (B3M), respectively.

[0088] In a preferred embodiment, the RNA according to the present invention comprises the nucleic acid sequence of Sequence ID No. 69, referred to as A2M. Thus, it encodes the NES-CMA-SB100X transposase disclosed herein and comprises a β-globin 5'UTR and two β-globin 3'UTRs. The RNA may further comprise a poly(A)tail containing 90 adenine nucleotides, and thus may consist of the nucleic acid sequence of Sequence ID No. 5.

[0089] The RNA according to the present invention may also contain a nucleic acid sequence corresponding to SEQ ID NO: 69, or consist of a nucleic acid sequence corresponding to SEQ ID NO: 5, wherein each cytidine nucleoside (i.e., 100% cytidine nucleoside) of the RNA is substituted with 5-methyl-CTP. The RNA thus substituted is referred to as A2M_C1 in the context of the present invention.

[0090] Alternatively, the RNA may also contain a nucleic acid sequence corresponding to SEQ ID NO: 69, or consist of a nucleic acid sequence corresponding to SEQ ID NO: 5, wherein each uridine nucleoside (i.e., 100% uridine nucleoside) of the RNA is substituted with 5-methoxy-UTP. The RNA thus substituted is referred to as A2M_U3 in the context of this invention.

[0091] Alternatively, the RNA may also contain a nucleic acid sequence corresponding to Sequence ID No. 69, or consist of a nucleic acid corresponding to Sequence ID No. 5, wherein each uridine nucleoside in the RNA (i.e., 100% uridine nucleoside) is substituted with a pseudo-UTP. The RNA thus substituted is referred to as A2M_U1 in the context of the present invention.

[0092] The RNA according to the present invention does not necessarily have to encode the NES-CMA-SB100X polypeptide according to the present invention. Rather, the disclosed modifications in the RNA have the potential to optimize expression and thereby increase the transposition efficiency of any of the transposases and their variants described herein. Therefore, the RNA of the present invention may encode a polypeptide having at least 80% amino acid identity with any of SEQ ID NOs. 43-48, and optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. For example, the RNA of the present invention may encode a polypeptide having at least 80% amino acid identity with SEQ ID NOs. 43-48 and containing one or more of the advantageous amino acid substitutions described above. The RNA may also encode a polypeptide containing or consisting of an amino acid sequence having 100% sequence identity with SEQ ID NOs. 43-48.

[0093] In one embodiment, the RNA according to the present invention comprises, for example, the nucleic acid sequence of Sequence ID No. 3 and is named SB100X A8H. Thus, it encodes the SB100X transposase and comprises a poly(A) tail containing a β-globin 5'UTR and a single β-globin 3'UTR, as well as 90 adenine nucleotides. The RNA according to the present invention may also consist of the nucleic acid sequence of Sequence ID No. 3.

[0094] Another object of the present invention is a vector for introducing RNA or polypeptides according to the present invention into target cells, preferably mammalian cells. The vector may, for example, encode or contain RNA or polypeptides according to the present invention. In some embodiments, the vector may be, for example, a vector that, when expressed, results in the production of RNA or polypeptides according to the present invention. Thus, it may be an expression vector encoding RNA or polypeptides according to the present invention. Examples of such expression vectors include, but are not limited to, minicircles, plasmids, cosmids, phages, viruses, or artificial chromosomes. In particular, the vector can be used to transport RNA according to the present invention into suitable host cells. Once inside the host cell, the expression vector may replicate independently of or simultaneously with the host chromosomal DNA, producing several copies of the vector and its inserted DNA. If a non-replicating expression vector is used (often for safety reasons), the vector may not replicate and may simply direct the expression of RNA. Depending on the type of expression vector, the expression vector may be lost from the cell, i.e., it only transiently expresses RNA into a protein. It may be stable in the cell. The expression vector typically contains an expression cassette, i.e., the necessary elements that enable the transcription of nucleic acids into mRNA molecules.

[0095] Preferably, the vector is suitable for the expression of the RNA or polypeptide of the present invention in mammalian cells, such as human cells, such as stem cells, or lymphocytes, such as T lymphocytes. If the vector consists of DNA, it preferably includes at least a gene regulatory region. The regulatory region may be a transcriptional regulatory region selected from the group consisting of, for example, promoters, enhancers, silencers, locus regulatory regions, and boundary elements. The promoter or other expression regulatory region may be operably linked to the nucleic acid encoding the polypeptide of the present invention to regulate polypeptide / protein expression, for example, quantitatively or in a tissue-specific manner. The promoter may be a constitutive promoter or an inductive promoter.

[0096] The vector may also be suitable for introducing already transcribed RNA, such as the in vitro transcribed mRNA described herein, or the translated polypeptide according to the present invention, into host cells such as mammalian cells. Therefore, the vector according to the present invention does not need to be an expression vector encoding the RNA or polypeptide of the present invention. Rather, the vector may be a vehicle containing the RNA or polypeptide of the present invention, such as a nanoparticle, that can induce and release the RNA or polypeptide into target cells. For example, in a preferred embodiment, the vector may be a lipid nanoparticle containing the RNA according to the present invention. Those skilled in the art will recognize additional suitable vectors that can transport the RNA or polypeptide into target cells.

[0097] Another object of the present invention is cells comprising the RNA and / or polypeptides of the present invention. Typically, the cells include both. The cells may be bacterial cells, for example, in the context of plasmid proliferation, but are preferably eukaryotic cells, particularly mammalian cells. They may be, for example, human cells, mouse cells, rabbit cells, or rat cells. Human cells are preferred throughout the present invention. The cells may be stem cells, such as pluripotent stem cells (e.g., induced pluripotent stem cells), hematopoietic stem cells, or lymphocytes, such as T lymphocytes. For example, the cells are preferably human cells selected from the group including pluripotent stem cells or human lymphocytes, preferably human T lymphocytes. The cells may also be tumor cells.

[0098] Furthermore, the present invention is a) The polypeptide of the present invention, the RNA of the present invention, the vector of the present invention and / or the cell of the present invention, and b) Nucleic acids comprising cargo nucleic acids adjacent to the inverse terminal sequence (ITR) of a transposon that can be recruited by the transposase. We provide a kit that includes this.

[0099] Such kits may be used to deliver cargo nucleic acids, for example, the RNA of the present invention encoding a polypeptide according to the present invention, to the genome of a cell, such as a human cell. Cargo nucleic acids are typically exogenous nucleic acids, i.e., exogenous nucleic acids to the cell to which they will be delivered. Therapeutic nucleic acids may encode reduced or deficient proteins, tumor suppressor genes, immunomodulators, such as cytokines, antigens, antibodies, T cell receptors (TCRs), or chimeric antigen receptors (CARs), for example in metabolic diseases, for example in connection with gene therapy for said diseases. In preferred embodiments, therapeutic nucleic acids are useful in the treatment of cancer. The kit may be used for in vivo, ex vivo, or in vitro application, for use in medicine, such as in cancer treatment. Thus, it may be a pharmaceutical kit. Cargo nucleic acids may alternatively encode fluorescent proteins and / or other select markers. It may also encode proteins intended to be produced in cells, such as in cell cultures, or hormones such as insulin.

[0100] The present invention also provides pharmaceutical compositions comprising the polypeptides of the present invention, the RNA of the present invention, the vectors of the present invention, the cells of the present invention, and / or components of the kits of the present invention. Pharmaceutical compositions typically further comprise pharmaceutically acceptable carriers and / or excipients. The term “carrier” refers to a natural or synthetic organic or inorganic component to which an active ingredient is combined to facilitate, enhance, or enable application. According to the present invention, the term “carrier” also includes one or more suitable solid or liquid fillers, diluents, excipients, or encapsulants suitable for administration to a subject. Possible carrier materials (e.g., diluents) are, for example, sterile water, Ringer's solution, Ringer's lactate solution, physiological saline, bacteriostatic saline (e.g., physiological saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hanks’ solution, fixative oil, polyalkylene glycol, hydrogenated naphthalene, and biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the carrier is PBS. The resulting solution or suspension is preferably isotonic with the recipient's blood. The carrier may also be a volume extender, such as trehalose, for example, in the context of a lyophilized composition. Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.

[0101] The pharmaceutical composition is preferably intended for use in adoptive T-cell therapy or gene therapy. Gene therapy may be for the treatment of metabolic or immunological diseases. In one embodiment, gene therapy includes, but is not limited to, autologous or xenogeneic T-cell therapy, gene therapy targeting any cell type in the blood, hematopoietic stem cell therapy, liver gene therapy, central nervous system gene therapy, eye gene therapy, muscle gene therapy, skin gene therapy and / or gene therapy for the treatment of cancer.

[0102] The pharmaceutical compositions of the present invention may also be of interest in vaccination therapy for the incorporation of antigens into human professional antigen-presenting cells, such as dendritic cells, macrophages, or B cells or their precursors. For example, a specific tumor antigen, such as MAGE-1, may be encoded in cargo nucleic acids for tumor vaccination, or in pathological antigens for treating infectious diseases originating from pathogens, such as leprosy, tetanus, pertussis, typhoid fever, paratyphoid fever, cholera, plague, tuberculosis, meningitis, bacterial pneumonia, anthrax, botulism, bacterial dysentery, diarrhea, food poisoning, syphilis, gastroenteritis, trench fever, influenza, scarlet fever, diphtheria, gonorrhea, toxic shock syndrome, Lyme disease, typhus, listeriosis, peptic ulcer, and Legionnaires' disease; for example, acquired immunodeficiency syndrome, adenovirus infections, alphavirus infections, arbovirus infections, Borne's disease, bunyavirid infections, calicivirid infections, varicella, genital warts, coronavirus infections, coxsackievirus infections, and cytomegalovirus infections. For example, for the treatment of viral infections, such as viral infections, dengue fever, DNA virus infections, impetigo, infectious encephalitis (arboviral), Epstein-Barr virus infection, erythema infectiosum, hantavirus infection, viral hemorrhagic fever, human viral hepatitis, herpes simplex, herpes zoster, herpes zoster otophylaxis, herpesvirus infections, infectious mononucleosis, avian influenza, human influenza, Lassa fever, measles, molluscum contagiosum, mumps, paramyxoviridae infections, sandfly fever, polyomavirus infections, rabies, respiratory syncytial virus infections, Rift Valley fever, RNA virus infections, rubella, late-onset viral diseases, smallpox, subacute sclerosing panencephalitis, tumor virus infections, warts, West Nile fever, viral diseases, and yellow fever; for example, for the treatment of protozoan infections that cause malaria. The method of the subject can be used to deliver a wide variety of therapeutic nucleic acids.

[0103] According to the present invention, a pharmaceutical composition contains an effective amount of an active agent, such as a polypeptide, RNA, vector, or cell described herein, to produce a desired reaction or effect. The pharmaceutical composition according to the present invention is preferably sterile. The pharmaceutical composition can be provided in a uniform dosage form and can be prepared by methods known in itself. The pharmaceutical composition according to the present invention may be, for example, in the form of a solution or suspension.

[0104] pharmaceutically acceptable carriers, adjuvants, or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intrathecal, intrahepatic, intrafocal, and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition is administered orally, intraperitoneally, or intravenously. The sterile injectable form of the pharmaceutical composition of the present invention may be an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using appropriate dispersants or wetting and suspending agents. The sterile injection preparation may also be a sterile injection solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixative oils have conventionally been used as solvents or suspension media.

[0105] The pharmaceutical compositions of the present invention are preferably used to treat diseases, particularly those caused by gene deficiencies such as cystic fibrosis, hypercholesterolemia, hemophilia (e.g., A, B, C, or XIII), immunodeficiency including HIV, Huntington's disease, and α-antitrypsin deficiency, as well as colon cancer, melanoma, kidney cancer, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer, glioma, thyroid cancer, breast carcinoma, prostate tumor, hepatoma, and various virus-induced tumors, such as papillomavirus-induced carcinomas (e.g., cervical carcinoma), adenocarcinoma, and herpesvirus-induced tumors (e.g., Burkitt lymphoma, EBV-induced B-cell lymphoma). Lymphoma, hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2-induced lymphoma, acoustic neuroma, lung cancer, pharyngeal cancer, anal carcinoma, glioblastoma, lymphoma, rectal carcinoma, astrocytoma, brain tumor, gastric cancer, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, melanoma, bladder cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease It is suitable for the treatment of bronchial carcinoma, pituitary carcinoma, mycosis fungoides, esophageal cancer, breast cancer, schwannoma, squamous cell carcinoma, Burkitt lymphoma, laryngeal cancer, thymoma, endometrial carcinoma, bone cancer, non-Hodgkin lymphoma, urethral cancer, CUP syndrome, oligodendroglioma, vulvar cancer, intestinal cancer, esophageal carcinoma, small intestinal tumor, craniopharyngioma, ovarian carcinoma, ovarian cancer, liver cancer, leukemia, or skin or eye cancer; etc.

[0106] The pharmaceutical composition is preferably for the treatment of a human subject. The present invention also provides a method for treating a subject, for example, a human subject in need, for example, one of the diseases mentioned herein, comprising administering an effective amount of the pharmaceutical composition or components of the pharmaceutical kit of the present invention to the subject.

[0107]

[0108] The use of the polypeptide, RNA, vector, cells, or kit of the present invention for introducing exogenous nucleic acids into the genome of cells, such as in vitro use, is also described.

[0109] Furthermore, the present invention relates to a method for preparing cells having exogenous nucleic acids incorporated into the cell genome, and optionally an in vitro method. a) A step of providing the polypeptide of the present invention to cells, and b) Providing the cells with nucleic acids, including the exogenous nucleic acid adjacent to the terminal inverted repeat sequence (TIR) ​​of a transposon that can be recruited by the polypeptide. Methods that include...

[0110] If necessary, the polypeptide is provided to cells by introducing the RNA of the present invention into the cells. Alternatively, the polypeptide can be provided in polypeptide form, for example, as a transposase with a shortened half-life as described herein. In one embodiment, the exogenous nucleic acid is contained in the vector of the present invention. It may also be administered separately.

[0111] In one embodiment, the RNA and / or polypeptide and / or exogenous nucleic acid of the present invention is delivered into cells using a method selected from the group consisting of electroporation, microinjection, lipoprotein particles, and virus-like particles. Electroporation has been found to be particularly suitable for transfecting primary cells, such as T cells.

[0112] The present invention also provides a method for preparing a protein, comprising carrying out a step of a method for preparing a cell having an exogenous nucleic acid incorporated into the cell's genome, wherein the protein is encoded by the exogenous nucleic acid and is operably linked to an expression regulatory element such as a promoter suitable for expression in the cell.

[0113] Another object of the present invention is a method for preparing the polypeptide of the present invention, comprising culturing cells containing the RNA of the present invention and isolating the polypeptide.

[0114] Throughout this invention, the term “approximately” is intended to be understood as “+ / -10%”. When “approximately” refers to a range, it refers to both the lower and upper limits of the range. “A” is intended to mean “one or more” unless otherwise specified. All references cited herein are incorporated fully herein. This invention is further illustrated by, but is not limited to, the following embodiments. Table 1: Sequence Listing [Table 1-1] [Table 1-2] [Brief explanation of the drawing]

[0115] [Figure 1] Western blots (polyclonal anti-SB) from HeLa Tet-Off SB or HeLa Tet-Off SB100X strains after different incubation times with doxycycline to stop transposase expression. 1) No doxycycline was added. 2)-6) Cells were incubated in doxycycline for 1-4 days or 6) 1 week before samples were collected. Both SB ("wild-type" as described by Ivics et al., 1997) and SB100X transposase proteins persisted for several days.

[0116] [Figure 2]Targeting SB100X for ubiquitin-mediated proteasome degradation based on the N-terminal ordered proteolytic pathway, Strategy 1: Transposition activity is impaired when the amino acid at the second position (N2) of the transposase is modified to target ubiquitin-proteasome degradation. A representative transposition assay in HeLa cells grown on puromycin-containing medium is shown. Cells were exposed to a plasmid expressing the Sleeping Beauty transposon, which contains the puromycin resistance gene and the SB100X transposase with a G (glycine) to C (cysteine) modification at the N-terminal amino acid position 2. Left: Negative control D3 (catalyzably inactive mutant transposase d3SB), Middle: Transposase with G to C modification at N-terminal amino acid position 2, Right: Positive control (SB100X). Experiments were performed in pairs.

[0117] [Figure 3] Targeting of SB100X for ubiquitin-mediated proteasome degradation based on the N-terminal ordered proteolytic pathway. Strategy 2: (A) The SB100X gene was fused downstream of human ubiquitin in a strategy based on the N-terminal ordered proteolytic pathway (Mulder et al., 2000). A similar strategy was used to target and degrade HIV-1 integrase. Downward arrows indicate the location of post-translational cleavage between ubiquitin and SB100X. (B) A series of SB100X variants differing at the 1-position amino acid were generated, transfected into HeLa cells, and tested for both translocation activity and protein stability (cycloheximide assay, 1-6 hours); (-) untransfected; (+) untreated. Only mutants with relatively high translocation activity are shown. However, none of the tested mutants showed a decrease in half-life.

[0118] [Figure 4]Targeting of SB100X for ubiquitin-mediated proteasomal degradation based on the N-terminal ordered proteolytic pathway. Strategy 3: Use of the Shld1-dependent destabilization domain, as described by Banaszynski et al., 2006, to target SB100X to the ubiquitin-mediated proteasomal degradation pathway. This system is based on a modified FKBP(*) that provides strictly controllable heterodimerization of FKBP* / FRB in the presence of rapamycin. When fused to the Ubi subunit (UbN, UbC), proteasomal degradation of the target protein (Pro) can be controlled. (A) Schematic diagram of the TShld system described by Banaszynski et al., 2006. (B) Schematic diagram of a modified version of the system described in (A) for targeting SB100X for proteasomal degradation. (C) Two representative colony-transition assays. Top: Left: No transposase, Middle: Catalytically inactive mutant transposase D3SB (D3), Right: SB100X, Bottom: TShld-SB100X in the presence of ligand, rapamycin (RAPA). Left: No Rapa, Middle: RAPA1d, Right: RAPA5d. Controllable degradation of SB100X was expected, but no difference was observed.

[0119] [Figure 5] High-throughput transcriptome analysis suggests that Sleeping Beauty-mediated transposition is associated with the upregulated expression of several cathepsins known to be important in the overall degradation of lysosomal proteins. The volcano plot shows significantly dysregulated genes in response to SB100X protein expression in HeLa cells. Left: Downregulated, Right: Upregulated. Note that the upregulated genes belong to the gene ontology of lysosomal degradation (10-4).

[0120] [Figure 6] Inhibition of the lysosomal pathway enhances SB-mediated transposition. The graph shows quantification of colony formation-transposition assays in the presence of the B- and L-cathepsin inhibitor E64D.

[0121] [Figure 7] The NES-CMA-SB100X variant exhibits slightly reduced transposition activity compared to SB100X in an EGFP-based transposon assay. (A) FACS sorting of GFP-expressing HeLa cells after SB-mediated transposition. The NES-CMA-SB100X variant was compared to SB100X and a catalytically inactive mutant transposase (d3SB, negative control). The reporter plasmid was co-transfected with a construct encoding the SB transposase variant. The assay is based on the integration of the SB transposon marked by the EGFP reporter sequence. At integration, the measured GFP fluorescence signal reports on the catalytic activity of the transposase. (B) Quantification of the results from (A).

[0122] [Figure 8]The NES-CMA-SB100X variant exhibits significantly faster degradation compared to the SB100X transposase and the NES-UbiMet-SB100X control variant (A-B). To determine the half-life of the NES-CMA-SB100X variant, 500,000 HeLa cells were transfected with 450 ng plasmid constructs encoding either SB100X, NES-CMA-SB100X (targeting lysosomal degradation), or NES-UbiMet-SB100X (targeting ubiquitin-mediated degradation) variants. Two days after transfection, the cell medium was replaced with fresh medium supplemented with 100 mM cycloheximide to inhibit de novo protein production. After incubation with cycloheximide (1 and 2 hours), cells were harvested and fractionated, and different transposase protein variants were quantified by densitometry based on Western blotting. The amount of transposase protein molecules was normalized relative to cell fraction marker proteins. Histone H3 was used as the nuclear fraction marker protein, and HSP90 functioned as the cytosolic fraction marker. (A) Quantification of nuclear localization of the indicated SB100X variant with respect to histone H3 after 1 or 2 hours of exposure to 100 mM cycloheximide. Note that the NES-CMA-SB100X transposase showed 4,36-fold faster degradation compared to SB100X. (B) Quantification of cytoplasmic localization of the indicated SB100X variant with respect to HSP90 after 1 or 2 hours of exposure to 100 mM cycloheximide. Note that the level of SB transposase protein after 1 hour of cycloheximide treatment was set to 100%. The total SB protein depletion between the 1-hour and 2-hour cycloheximide treatment time points was SB100X-35%, NES-CMA-SB00X-75%, and 4%.

[0123] [Figure 9]The NES-CMA-SB100X variant exhibited significantly faster degradation compared to the SB100X transposase (measured between 1 hour and 24 hours of cycloheximide treatment). Three independent experiments were conducted. SB transposase levels were determined from whole cell lysates (no fractionation). The amount of each transposase variant was quantified by densitometry, and these amounts were normalized by total protein mass using the Chemi Doc® MP imaging system (BioRad).

[0124] [Figure 10] Transposition activity of NES-CMA-SB100X expressed from in vitro transcribed (ivt) mRNA. Ju76 cells were transfected with 50 μg / mL pT4-MP71-GFP plasmid DNA and 30–150 μg / mL ivt mRNA encoding either SB100X (A) or the NES-CMA-SB100X variant (B). The percentage of eGFP-positive cells was tracked for 15 days to measure stable integration. (C) The graph shows the correlation between integration efficiency observed by stable eGFP expression of the tested SB100X variants and mRNA concentration.

[0125] [Figure 11]Stable gene integration in Jurkat cells using a modified CMA-NES-SB100X construct. Results were normalized to SB100X mRNA (construct 1). 1:SB100X (SEQ ID NO: 1); 2:NES-CMA-SB100X (SEQ ID NO: 2); 3:SEQ ID NO: 3; 4:SEQ ID NO: 4; 5:SEQ ID NO: 5; 6:SEQ ID NO: 6; 7:SEQ ID NO: 7; 8:SEQ ID NO: 8; 9:SEQ ID NO: 9; 10:SEQ ID NO: 10; 11:SEQ ID NO: 11; 12:SEQ ID NO: 12; 13:SEQ ID NO: 13; 14:SEQ ID NO: 14; 15:A2M_U1 (SEQ ID NO: 5, pseudo-UTP substitution); 16:A2M_U2 (SEQ ID NO: 5, N1-methyl pseudo-UTP substitution); 17:A2M_U3 (Sequence No. 5, 5-methoxy-UTP substitution); 18:A2M_U4 (SEQ ID NO: 5, 2-thio-UTP substitution); 19:A2M_C1 (SEQ ID NO: 5, 5-methyl-CTP substitution); 20:A2M_C2 (SEQ ID NO: 5, N4-acetyl-CTP substitution); 21:A2M_A1 (SEQ ID NO: 5, N6-methyl-ATP substitution); 22:SEQ ID NO: 22; 23:SEQ ID NO: 23; 24:SEQ ID NO: 24; 25:SEQ ID NO: 25; 26:SEQ ID NO: 26; 27:SEQ ID NO: 27; 28:SEQ ID NO: 28; 29:SEQ ID NO: 29. The capping structure used was either the Cap0 structure (RNA constructs 1 and 2) or Cap1 (RNA constructs 3-29) to enhance translation efficiency and stability. Either a 5'UTR without any (RNA constructs 1 and 2), a 5'UTR of the human β-globin gene (RNA constructs 3-9, 15-29), an eIF4G aptamer (RNA constructs 10 and 11), a 5'UTR containing the JK stem-loop region of the encephalomyocarditis virus IRES (RNA construct 12), or a 5'UTR with no or weak predicted secondary structural elements such as a hairpin (RNA constructs 13 and 14) was used. One of the following RNA constructs was used: one without a CMA motif bound to NES (RNA constructs 1 and 3), KFERQ (RNA constructs 2, 4-21), VKKDQ (RNA construct 22), KFFEQ (RNA construct 23), KIREI (RNA construct 24), QVELR (RNA construct 25), QYFKS (RNA construct 26), LKSFQ (RNA construct 27), LKYFQ (RNA construct 28), or EKFLQ (RNA construct 29).Either the CDS of SB100x (RNA constructs 1 and 3) or the CDS of CMA-NES-SB100x (RNA constructs 2, 4-29) was used. One of the following was used: no 3'UTR (RNA constructs 1 and 2), human β-globin 3'UTR (RNA constructs 3 and 6), WPRE 3'UTR (RNA constructs 4, 12-14), two human β-globin 3'UTRs in succession (RNA constructs 5, 10-11, 15-29), human α-globin 3'UTR (RNA construct 7), a combination of human β-globin and α-globin 3'UTR (RNA construct 8), or a shortened version of the human β-globin 3'UTR (RNA construct 9). Either an enzymatically added poly(A) tail of 100-300 nucleotides (RNA constructs 1 and 2) or a 90-nucleotide transcribed poly(A) tail (RNA constructs 3-29) was used. Either an unmodified nucleotide (RNA constructs 1-14, 22-29), a pseudo-UTP (RNA construct 15, A2M_U1), an N1-methyl pseudo-UTP (RNA construct 16, A2M_U2), a UTP completely substituted with 5-methoxy-UTP (RNA construct 17, A2M_U3) or 2-thio-UTP (RNA construct 18, A2M_U4), a CTP completely substituted with 5-methyl-CTP (RNA construct 19, A2M_C1) or N4-acetyl-CTP (RNA construct 20, A2M_C2), or an ATP completely substituted with N6-methyl-ATP (RNA construct 21, A2M_A1) was used.

[0126] [Figure 12]Several variants were tested as mRNA in primary human CD3+ cells for (A) stable pT4-mediated integration of the reporter gene and (B) cell viability. (A) Stable integration of the reporter gene into human CD3+ cells 15 days after transfection. (B) Cell viability 15 days after transfection. 1:SB100X (SEQ ID NO: 1); 2:NES-CMA-SB100X (SEQ ID NO: 2); 3:SEQ ID NO: 3; 5:SEQ ID NO: 5; 6:SEQ ID NO: 6; 11:SEQ ID NO: 11; 15:A2M_U1 (SEQ ID NO: 5, pseudo-UTP substitution); 17:A2M_U3 (SEQ ID NO: 5, 5-methoxy-UTP substitution); 19:A2M_C1 (SEQ ID NO: 5, 5-methyl-CTP substitution).

[0127] [Figure 13] Intracellular concentrations of Sleeping Beauty transposase for constructs with different CMA motifs 4 days after transfection, as determined by Western blotting. The amounts are normalized to the intracellular transposase concentration 4 hours after transfection. 3:SEQ ID NO: 3; 17:A2M_U3 (SEQ ID NO: 5, 5-methoxy-UTP substitution); 22:SEQ ID NO: 22; 23:SEQ ID NO: 23; 24:SEQ ID NO: 24; 25:SEQ ID NO: 25; 26:SEQ ID NO: 26; 27:SEQ ID NO: 27; 28:SEQ ID NO: 28; 29:SEQ ID NO: 29.

[0128] [Figure 14] The Q124C mutation increases the translocation efficiency of NES-CMA-SB100X, as determined by a colony-forming translocation assay in human HeLa cells. Results were normalized to SB100X. [Examples]

[0129] Example 1 Improving the efficacy and safety of the SB100X transposon system: Development of a short-half-life version of the highly active Sleeping Beauty (SB) transposase SB100X. Introduction Improvements in biosafety for cell engineering are attracting attention from regulatory authorities. Gene delivery vectors should be removed from modified cells as quickly as possible to avoid undesirable genotoxicity.

[0130] The embedded nonviral Sleeping Beauty (SB) transposon system is a widely used genome engineering tool and is routinely used in a wide range of cell types [Ivics et al., 1997; Izsvak et al., 2000]. The highly active transposase variant (SB100X) has been demonstrated to efficiently integrate transgenes into various human cell types [Mates et al., 2009]. Currently, the SB100X system is a widely used therapeutic delivery and expression vector in humans [reviewed in Kebriaei et al., 2017; Narayanavari et al., 2017; Sandoval-Villegas et al., 2021]. Ongoing efforts are being made to improve the biosafety of the system.

[0131] The Sleeping Beauty (SB) transposon system consists of two components. The transposase is a recombinase enzyme that catalyzes the transposition of SB transposons. After expression, the transposase immediately catalyzes the transposition reaction, and its presence is no longer required. In fact, the continued presence of the transposase is a safety concern because it can induce re-recruitment events and lead to genomic instability. The SB100X transposase is predicted to have a relatively long half-life (>30 hours) [Voigt et al., 2016]. Transposition activity can be detected over several days after induction (Figures 1A and 1B). During this period, the transposase may support multiple undesirable transpositions. Furthermore, the transposase is sensitive to aggregation, and persistent transposase molecules contribute to nuclear aggregation, potentially causing cytotoxicity, especially if the transposase is over-administered. In particular, with currently available mammalian cloning vectors, the expressed protein is frequently overexpressed.

[0132] The objective of this study was to shorten the lifespan of transposase proteins, thereby eliminating toxicity issues and improving the biosafety of the system.

[0133] In prior art, the half-life of the transposase was not altered. The inventors tested the concept that it may be possible to guide the SB100X transposase into the proteolytic pathway by fusing it with a degradation signal.

[0134] In eukaryotic cells, damaged proteins or organelles can be removed by proteasomes or lysosomes [de Duve, 2005, Dikic, 2017]. The two pathways are independent but interconnected [Wang et al., 2014].

[0135] result SB100X transposase is a relatively stable protein and can be detected in cells [Mates et al., 2009] and Figure 1 for several days. To improve the safety of the SB100X transposon system for clinical application, the inventors targeted either (1) the ubiquitin-mediated proteasome (UPS) or (2) the autophagy-lysosome (ALP) degradation pathway with Sleeping Beauty transposase.

[0136] The ubiquitin-proteasome system (UPS) is one of the major pathways by which eukaryotic cells achieve selective proteolysis [Kleiger et al., 2014]. The UPS is the primary proteolysis pathway for small, short-lived, soluble, misfolded, and damaged proteins [reviewed in Dikic in 2010]. In particular, protein targeting to the ubiquitin degradation pathway has been successfully applied to many proteins [Zhao et al., 2022]. The first degron discovered was located at the N-terminus of a protein [Bachmair et al., 1986]. ALP recognizes and removes long-lived, large, potentially dangerous cellular components such as insoluble protein aggregates and dysfunctional or excess organelles [reviewed in Dikic in 2010]. The ALP process delivers cytoplasmic components to lysosomes for subsequent degradation.

[0137] In particular, UPS and ALP communicate across multiple points [Wang and Robbins 2014], influencing each other and mutually regulating the levels of their key components to regulate and balance their actions in proteostasis. In chaperone-mediated autophagy (CMA), proteins are selected by a chaperone, target lysosomes, and the complex transposes across the lysosomal membrane for degradation.

[0138] Because SB transposases are relatively small proteins, they initially attempted to target UPS (1a-c).

[0139] 1a. Amino acid modification at the second position (N2) of the transposase. N-terminal residues influence protein stability via the N-terminal ordered pathway. Calculation-based identification of destabilizing N-terminal motifs (degrons) in proteins revealed that the majority of the top 100 predicted destabilizing N-terminal motifs encoding either lysine (K2), arginine (R2), or cysteine ​​(C2) at the second position [Timms et al., 2019]. Therefore, we swapped the 2nd position of SB transposase from G (glycine) to C (cysteine). However, changing the amino acid at the second position of SB transposase (from G to C) impaired its activity (Figure 2).

[0140] 1b. Strategies for transposases to promote protein degradation by targeting the proteasomal degradation pathway HIV-1 integrase and SB transposase are enzymatically related [Voigt et al., 2016]. HIV-1 integrase has been shown to be a physiological substrate of the N-terminal ordered degradation pathway [Mulder et al., 2000]. By altering the N-terminal residue of HIV-1 integrase, it has been possible to generate various HIV-1 versions with different stabilities [Mulder et al., 2000].

[0141] In this approach, the SB100X gene was fused downstream of human ubiquitin, following the HIV-1 example [Mulder et al., 2000] (Figure 3). A series of SB100X variants with different amino acid sequences at position 1 were constructed and tested for both transposition activity and protein stability (Figure 3). Downward arrows indicate the location of post-translational cleavage between ubiquitin and the X-transposase (SB100X) (Figure 3).

[0142] A third approach, following the strategy suggested by 1c. [Banaszynski et al., 2006], was also unsuccessful (Figure 4). This strategy provides highly sensitive regulation of the stability of proteins that can target ubiquitin-mediated degradation pathways.

[0143] In summary (1a-c), the cycloheximide protein stability assay revealed that none of the generated mutations affected the half-life of SB100X transposase, suggesting that SB100X transposase itself cannot be targeted by the ubiquitin-mediated UPS degradation pathway.

[0144] 2. Strategies for promoting protein degradation by targeting the autophagy-mediated proteolytic pathway with transposases. To investigate the cellular response to the presence of SB transposase, we performed high-throughput transcriptome analysis. This approach revealed that SB transposition upregulates the expression of several cathepsins known to be important in the overall degradation of lysosomal proteins (Figure 5) and is involved in autophagy (reviewed by Yadati et al. in 2020). In particular, inhibiting the lysosomal pathway with the cathepsin (B,L) inhibitor E64D enhanced SB-mediated transposition (Figure 6). In summary, these observations suggest that SB100X transposase is sensitive to the "lysosomal" protein degradation pathway.

[0145] Following the failures of previous attempts (proteasome targeting strategies 1a-c), we decided to explore an alternative approach that targets the chaperone-mediated autophagy (CMA) proteolytic pathway, which utilizes transposases by selective subsets of proteins. In particular, unlike other targeted proteolytic strategies, this approach is not yet established and faces several unresolved challenges (reviewed by Zhao et al. in 2022).

[0146] The targeting strategy is based on a specific CMA signaling pathway involving a unique pentapeptide (KFERQ) [Dice, 1990; Wing et al., 1991]. In the CMA, the heat shock 70 (HSC70) chaperone recognizes protein substrates containing the KFERQ motif [Dice, 1990; Wing et al., 1991]. The HSC70-KFERQ protein-substrate complex then binds to lysosome-associated membrane protein 2A (LAMP2) and translocates to the lysosomal lumen for degradation [Gough et al., 1995]. Therefore, in principle, proteins containing the KFERQ motif can be used to degrade proteins, representing an alternative approach in targeted proteolysis [Fan et al., 2014].

[0147] To target SB100X with CMA, the transposase was fused with a specific autophagy-mediated proteolytic signal, a unique pentapeptide (KFERQ) [Dice, 1990; Wing et al., 1991]. Furthermore, since the SB100X transposase is a nuclear protein, the inventors hypothesized that transporting the expressed SB100X protein out of the nucleus in combination with the targeting strategy would accelerate the degradation rate of the SB100X transposase. Therefore, to promote the clearance of the SB100X transposase protein from nuclear space, a nuclear export signal (NES) derived from the protein kinase inhibitor-α (LALKLAGLDIKFERQ, SEQ ID NO: 60) was further fused to the N-terminus of the pentapeptide signal.

[0148] NES location: The functionality of the NES signal does not depend on its location within the protein sequence [https: / / doi.org / 10.1016 / C2010-0-64974-1], but SB transposases are sensitive to modifications that may impair activity or lead to structural instability [Narayanavari et al., 2017]. SB transposases can tolerate N-terminal tagging, but C-terminal tagging impairs activity. In principle, it is possible to embed targeted signals into proteins, but this is not trivial and requires careful design and optimization. Furthermore, it was important to use a linker between the transposase and the NES-CMA signal. Therefore, we ligated a binary signal sequence to the SB100X transposase using a linker peptide sequence (KLGGGAPAVGGGPKAADK, SEQ ID NO: 49) that is publicly available elsewhere [Kovac et al., 2020].

[0149] The activity of a novel NES-CMA-SB100X variant was measured using an EGFP-based transposon assay in HeLa cells. This assay is based on the incorporation of an SB transposon marked with an EGFP reporter sequence. The GFP fluorescence signal measured at incorporation reports on the catalytic activity of the transposase. Here, in human HeLa cells, the NES-CMA-SB100X variant was compared with SB100X and a catalytically inactive mutant transposase (d3SB, negative control) (Figure 7). The reporter plasmid was co-transfected with a construct encoding the SB transposase variant. Note that transfection efficiency (TE, %) was determined using the GFP signal on day 2 post-transfection. At day 14 post-transfection, background from the unincorporated background was barely detectable, and the GFP signal was attributed to the incorporated GFP-labeled transposon (Int). The transposase transposition efficiency was then normalized by TE (i.e., Int / TE). Therefore, the "activity of the NES-CMA-SB100X transposase version" is Int / TE as a percentage of SB100X. NES-CMA-SB100X has approximately 70% of the activity of conventional SB100X (Figure 7).

[0150] To determine the half-life of the NES-CMA-SB100X variant, 500,000 HeLa cells were transfected with a 450 ng plasmid construct encoding the SB variant. To ensure equivalent expression, the SB transposase gene was under the control of the CMV promoter in all constructs. The transposase versions tested were SB100X, NES-CMA-SB100X, and NES-UbiMet-SB100X. NES-CMA-SB100X and NES-UbiMet-SB100X both possess an NES signal but are transposase versions targeting either the CMA or ubiquitin-mediated degradation pathway, respectively. Two days after transfection, the cell medium was replaced with fresh medium supplemented with 30–100 mM cycloheximide (which inhibits de novo protein production). Following incubation with cycloheximide (1 and 2 hours), cells were collected and fractionated using the NE-PER kit (Thermo Scientific®) according to the manufacturer's recommendations. Cell extracts were loaded onto SDS-PAGE, and different transposase protein variants were quantified by densitometry. The amount of transposase molecules was normalized against cell fraction marker proteins. The nuclear fraction marker protein histone H3 was used for the nuclear fraction, and the cytoplasmic marker protein HSP90 was used for the cytoplasmic fraction. This experiment clearly demonstrated that the bound NES-CMA signal is functional and that in the nuclear fraction, the NES-CMA-SB100X version exhibits approximately 4.3 times faster degradation compared to the conventional SB100X transposase (Figure 8). Using the same experimental setup but without fractions, the total amount of transposase versions was analyzed by densitometry, and their amounts were normalized against total protein mass using the Chemi Doc® MP imaging system (BioRad). In these experiments, the NES-CMA-SB100X transposase showed statistically significant (**)5,4x faster degradation compared to the original SB100X (Figure 9).

[0151] 3. Strategies to increase translocation efficiency while maintaining a shorter SB100X half-life through mRNA modification. mRNA encoding SB100X was transcribed in vitro, and its efficiency was tested in a transposition assay in Jurkat cells (Figure 10A). Stable transposition was tracked by co-transfection with a plasmid containing a fluorescent reporter gene (enhanced green fluorescent protein) adjacent to the pT4 transposon sequence. Over time, only cells that stably transposed exhibited intracellular fluorescence as measured by flow cytometry. Compared to NES-CMA-SB100X (Figure 10B), conventional SB100X (Figure 10C) functioned approximately twice as well.

[0152] To enhance the efficiency of NES-CMA-SB100X while maintaining a short half-life, modifications were made to the capping structure of the transcribed mRNA, the 5' untranslated region (5'UTR), the NES-CMA-SB100x coding sequence, the 3' untranslated region (3'UTR), and the poly(A) tail, incorporating modified nucleosides and using additional CMA motifs. mRNA for all constructs was generated and tested at optimized concentrations in Jurkat cells (Figure 11). The capping structure used was either the Cap0 structure (RNA constructs 1 and 2) or Cap1 (RNA constructs 3-29) to enhance translation efficiency and stability. The inventors used one of the following: no 5'UTR (RNA constructs 1 and 2), a 5'UTR of the human β-globin gene (RNA constructs 3-9, 15-29), an eIF4G aptamer (RNA constructs 10 and 11), a 5'UTR containing the JK stem-loop region of the encephalomyocarditis virus IRES (RNA construct 12), or a 5'UTR with no or weak predicted secondary structural elements, such as a hairpin (RNA constructs 13 and 14). The inventors used either RNA constructs without a CMA motif bound to NES (RNA constructs 1 and 3), or one of the following NES-bound RNA constructs: KFERQ (RNA constructs 2, 4-21), VKKDQ (RNA construct 22), KFFEQ (RNA construct 23), KIREI (RNA construct 24), QVELR (RNA construct 25), QYFKS (RNA construct 26), LKSFQ (RNA construct 27), LKYFQ (RNA construct 28), or EKFLQ (RNA construct 29). They also used either CDS of SB100x (RNA constructs 1 and 3) or CDS of CMA-NES-SB100x (RNA constructs 2, 4-29). The inventors used one of the following: no 3'UTR (RNA constructs 1 and 2), 3'UTR of human β-globin (RNA constructs 3 and 6), 3'UTR of WPRE (RNA constructs 4, 12-14), two consecutive human β-globin 3'UTRs (RNA constructs 5, 10-11, 15-29), 3'UTR of human α-globin (RNA construct 7), a 3'UTR of a combination of human β-globin and α-globin (RNA construct 8), or a shortened version of the human β-globin 3'UTR (RNA construct 9).The inventors used either an enzymatically added poly(A) tail of 100-300 nucleotides (RNA constructs 1 and 2) or a 90-nucleotide transcribed poly(A) tail (RNA constructs 3-29). The inventors also used either an unmodified nucleotide (RNA constructs 1-14, 22-29) or a pseudo-UTP (RNA construct 15), an N1-methyl pseudo-UTP (RNA construct 16), a 5-methoxy-UTP (RNA construct 17) or a 2-thio-UTP (RNA construct 18), a CTP having a 5-methyl-CTP (RNA construct 19) or an N4-acetyl-CTP (RNA construct 20), or a UTP completely substituted with an ATP having N6-methyl-ATP (RNA construct 21).

[0153] In this initial screening, several mRNA variants encoding CMA-NES-SB100X were identified, and these exhibited similar or higher stable integration efficiencies in Jurkat cells under optimized conditions compared to both SB100X and unmodified CMA-NES-SB100X. Several variants were tested in primary human CD3+ cells for pT4-mediated stable integration of the reporter gene (Figure 12). RNA construct 17 was the best in terms of relative integration efficiency and was significantly better than the SB100X sequence in terms of cell viability after 15 days of transfection and culture growth.

[0154] To ensure that the beneficial features observed in Jurkat and CD3+ cells are not caused by the extended half-life of CMA-NES-SB100X via mRNA modification, we measured the relative abundance of the transposase 96 hours after transfection with several sequences (Figure 13). As expected, most CMA-containing constructs (RNA constructs 17, 23-29) showed significantly lower half-lives than SB100X with similar mRNA modifications. Interestingly, the VKKDQ CMA motif derived from the amyloid-beta precursor protein gene in RNA construct 22, derived from the amyloid-beta precursor protein gene, increased the transposase half-life.

[0155] A strategy to increase rearrangement efficiency while maintaining a shorter SB100X half-life by amino acid substitution at position 4,124. Previous studies have shown that Q124C substitution can improve the dislocation efficiency of SB100X (EP4353821A1). To determine whether this particular amino acid substitution in the SB100X coding sequence can at least partially restore the translocation efficiency of NES-CMA-SB100X to that of conventional SB100X, the inventors performed a colony-forming translocation assay by transfecting HeLa cells with expression plasmids encoding conventional SB100X (SB100X), the Q124C variant (SB100X Q124C), NES-CMA-SB100X (NES-CMA-SB100X), and NES-CMA-SB100X with the Q124C substitution (SB100X NES CMA variant Q124C) together with a puromycin-resistant (puro) gene-tagged SB vector (pT2-B-puro), and evaluated the relative efficiency of translocation by counting puromycin-resistant cell colonies after antibiotic selection. As expected, NES-CMA-SB100X exhibited significantly lower translocation efficiency than SB100X. Notably, the Q124C mutation, which resulted in high activity of SB100X by causing a twofold increase in translocation rate compared to SB100X, also partially restored the translocation activity of the NES-CMA-SB100X construct: the activity of the SB100X NES CMA mutant Q124C construct was approximately 2–3 times higher than the activity of NES-CMA-SB100X, and about 50% of the activity of SB100X (Figure 14). Therefore, these results clearly demonstrate that introducing the Q124C mutation into the SB100X coding sequence is an appropriate strategy for enhancing the translocation efficiency of CMA-NES-SB100X.

[0156] material and method Cell culture and cell transfection Human cervical cancer cells (HeLa) were cultured at 37°C and 5% CO2 in Duverco-modified Eagle medium (DMEM) containing 4.5 g / l D-glucose and 10% fetal bovine serum, supplemented with penicillin (100 μg / ml) and streptomycin (100 μg / ml). Generally, cells were seeded in 6-well plates and transfected using JetPrime® or Lipofectamine®-3000 (Thermo Fischer) reagents according to the recommended manufacturer's protocol. Jarcut cells were cultured at 37°C and 5% CO2 in RPMI medium supplemented with Glutamax, FBS, MEM, sodium pyruvate, penicillin (100 μg / ml), and streptomycin (100 μg / ml). CD3+ cells were cultured in interleukin-supplemented TexMACS medium (Miltenyi) at 37°C and 5% CO2. The cells were transfected using a 4D Nucleofector (Lonza). The cell cultures were analyzed by flow cytometry using MACSQuant (Miltenyi).

[0157] In vitro transcription mRNA Plasmids encoding different (NES-CMA)-SB100x variants were linearized before transcription and enzymatically polyadenylated as needed. All reagents were supplied by Jena Bioscience GmbH. mRNA was purified with Mag-Bind TotalPure NGS beads (Omegabiotek) and eluted in water.

[0158] Western blot Cells were harvested from 6-well plates for Western blot analysis. Subsequently, the cells were washed with phosphate-buffered saline (PBS) and lysed on ice for 40 minutes in a lysis buffer containing 50 mM TRIS HCl pH 8.0, 10 mM EDTA, 100 mM NaCl, 5% glycerol, 1% NP-40 and protease inhibitor mini-tablets, EDTA-free (Pierce®) and benzonase nuclease (NOVAGEN), as recommended by the manufacturer. The total lysates were separated by 8–12% or 4–20% Mini-Protean® TGX® SDS-PAGE gel and transferred to PVDF membranes using the Bio-Rad Trans-Blot® Turbo® transfer system. The membranes were blocked with TBS-T containing 5% skim milk powder (TBS supplemented with 0.05% Tween®-20) and incubated overnight at 4°C with appropriate dilutions of primary antibodies (aSB (R&D Systems AF2798), aHSP90 (Cell Signaling#4874), and histone H3 (Cell Signaling#9715)) in TBS-T containing 5% skim milk powder. The membranes were washed with TBS-T buffer and incubated at room temperature for 1 hour with alkaline phosphatase-conjugated (Sigma-Aldrich) or horseradish peroxidase-conjugated (Promega) secondary antibodies in TBS-T containing 5% skim milk powder. Subsequently, the blots were washed with TBS-T. Bands were detected by ECL® Prime Western Blotting Detection Reagent (Amersham), and images were then analyzed by Chemi Doc® MP Imaging System (Bio-Rad). Protein levels were determined using densitometry analysis software from the BioRad-ChemiDoc™ MP imaging system.

[0159] References [ka] [ka]

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Claims

1. A polypeptide comprising a Tc1 / Mariner superfamily transposase capable of recruiting transposons, wherein the polypeptide comprises a nuclear export signal and a chaperone-mediated autophagy signal, and the transposase is selected from the group comprising Sleeping Beauty, Frog Prince, Minos, ZB, and Passport transposases, preferably Sleeping Beauty transposase.

2. The polypeptide according to claim 1, wherein the polypeptide comprises an SB100X transposase having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 43, and preferably the polypeptide further has an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

50.

3. The polypeptide according to any one of the preceding claims, wherein the chaperone-mediated autophagy signal has an amino acid sequence selected from the group including SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

4. RNA encoding a polypeptide according to any one of the preceding claims, wherein the RNA comprises a 5'UTR and a 3'UTR, and the 5'UTR is selected from the group comprising a β-globin 5'UTR and a 5'UTR containing an elF4G aptamer.

5. The RNA according to claim 4, wherein the 3'UTR is selected from the group consisting of one or more β-globin 3'UTR, α-globin 2 3'UTR, 3'UTR containing WPRE, βα-globin 3'UTR, and cleaved β-globin 3'UTR.

6. The RNA mentioned above, a) β-globin 5'UTR and two β-globin 3'UTRs, b) β-globin 5'UTR and cleaved β-globin 3'UTR, c) β-globin 5'UTR and α-globin 2 3'UTR d) β-globin 5'UTR and βα-globin 3'UTR or e) 5'UTR containing elF4G aptamer and two β-globin 3'UTRs RNA according to any one of claims 4 or 5, comprising:

7. The RNA according to any one of claims 4 to 6, wherein the RNA is codon-optimized.

8. The RNA according to any one of claims 4 to 7, wherein the RNA includes a poly-A tail having a length of 50 to 150 nt, preferably 75 to 100 nt, for example 90 nt.

9. The RNA according to any one of claims 4 to 8, wherein the RNA includes a 5'Cap1 structure.

10. The RNA according to any one of claims 4 to 9, wherein the RNA comprises one or more modified nucleosides selected from the group consisting of 5-methyl-CTP, N4-acetyl-CTP, pseudo-UTP, N1-methyl pseudo-UTP, 6-methoxy-UTP, 2-thio-UTP, N6-methyl-ATP, and N1-methyl-ATP.

11. The RNA according to any one of claims 4 to 10, wherein the RNA comprises the nucleic acid sequence of SEQ ID NO: 62, 63, 64, 65, 66, 67, 68, or 69, preferably SEQ ID NO:

69.

12. A vector for introducing RNA according to any one of claims 4 to 11 and optionally a polypeptide according to claims 1 to 3 into target cells, wherein the vector comprises a nucleic acid encoding RNA according to any one of claims 4 to 11 or a polypeptide according to any one of claims 1 to 3, or the vector comprises RNA according to any one of claims 4 to 11 or a polypeptide according to any one of claims 1 to 3.

13. A cell comprising RNA and / or polypeptide according to any one of claims 4 to 11, wherein the cell is a human cell selected from the group including pluripotent stem cells and human lymphocytes, and optionally human T lymphocytes.

14. a) A polypeptide according to any one of claims 1 to 3, RNA according to any one of claims 4 to 11, a vector according to claim 12 and / or a cell according to claim 13, and b) Nucleic acids comprising cargo nucleic acids adjacent to the terminal inverted repeat sequence (TIR) ​​of a transposon that can be recruited by the transposase. A kit that includes this.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 3, RNA according to any one of claims 4 to 11, a vector according to claim 12, cells according to claim 13, and / or a kit according to claim 14, and optionally a pharmaceutically acceptable carrier and / or excipient, wherein the pharmaceutical composition is preferably for use in adoptive T cell therapy or gene therapy.

16. Use of a polypeptide according to any one of claims 1 to 3, RNA according to any one of claims 4 to 11, a vector according to claim 12, a cell according to claim 13, or a kit according to claim 14 for introducing an exogenous nucleic acid into the genome of a cell, wherein the use is, if necessary, an in vitro use.

17. A method for preparing cells having exogenous nucleic acids incorporated into the cell genome, and optionally an in vitro method, a) A step of providing the cells with a polypeptide according to any one of claims 1 to 3 or RNA according to any one of claims 4 to 11, and b) Providing the cells with a nucleic acid comprising the exogenous nucleic acid adjacent to a terminal inverted repeat sequence (TIR) ​​of a transposon that can be recruited by the polypeptide or the polypeptide encoded by the RNA. Methods that include...

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