Use of transposase to improve expression and nuclear localization of introduced genes

Abstract

The present invention relates to a method for increasing the expression of a transgene of interest in a cell population and / or increasing the nuclear localization of the transgene of interest in a cell population using a transposase.

Description

Technical Field

[0001] The present invention relates to a method for improving the expression of a transgene in a cell population.

Background Art

[0002] Having an efficient method for introducing nucleic acids or proteins into cells and tissues is important for genetic manipulation. For this purpose, various methods have been developed, such as viral infection, electroporation, transfection by polymers and nanoparticles. These methods enable high efficiency in transfection, but the introduction of the target DNA into the nucleus remains a major challenge.

[0003] In fact, after entering the cell, foreign DNA faces various obstacles before reaching the nucleus. First, mammalian cells have defense mechanisms aimed at detecting and controlling foreign DNA, such as the interferon system. Semenova et al. (Nucleic acids research 2019, vol. 47, 19) reported that cytoplasmic DNA binds to transfected DNA within several hours after transfection, resulting in an inflammatory response. Second, foreign DNA molecules must pass through the barrier of the nuclear membrane. Vandenbroucke et al. (Nucleic acids research 2007, vol. 35, 12) proposed regulating the activity of the nuclear pore complex (NPC) by amphiphilic molecules to increase nucleocytoplasmic transport; however, amphiphilic molecules are associated with a certain level of toxicity and they do not seem to enable an increase in nuclear uptake for specific DNA carriers.

[0004] Other additional obstacles to transferring foreign DNA into the nucleus can be transport, cell type specificity, and cell division (see, for example, Bai et al. “Cytoplasmic transport and nuclear import of plasmid DNA.” Bioscience reports, 2017, vol. 37, 6 for a review).

[0005] Thus, the difficulty for foreign DNA to reach the nucleus of eukaryotic cells (both in vivo and in vitro) is a problem that many transfection methods have tried to overcome. The present invention provides a solution to help localize this foreign DNA into the nucleus by surrounding the gene of interest with ITRs and adding transposase to the transfection mixture.

[0006] Transposase is an enzyme that has naturally evolved to “cut and paste” or “copy and paste” DNA fragments into prokaryotic genomic DNA (such as Tn3, Tn5, and Tn7) or eukaryotic genomic DNA (such as Sleeping Beauty and PiggyBac). These enzymes are found in specific species' genomes with low transfer efficiency to avoid genomic toxicity. Transposase binds to payload DNA containing specific terminal inverted repeats (ITRs) called transposons, copies or cuts this sequence, and pastes it into random or semi-random genomic sites. Similar to most DNA-binding proteins, transposase is naturally directed to the nucleus by a nuclear localization signal (see, for example, Keith et al. “Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues.” BMC Molecular Biology 2008, vol. 9, 72).

[0007] The state-of-the-art technologies include different transposases, such as SB100 or highly active PiggyBac, which have been modified to increase their efficiency and create tools for mammalian genome editing. The transposases are also fused to DNA binding domains, such as Cas9 (see, for example, International Patent Publication No. WO 2022 / 129438 or WO 2020 / 243085) or dead Cas9 (see, for example, Hew et al., “RNA-guided piggyBac transposition in human cells.” Synthetic Biology 2019, vol. 4,1), to specifically target and edit specific genomic DNA positions.

[0008] However, although transposases are described as tools for genome editing (see, for example, Zhao et al., “PiggyBac transposon vectors: the tools of the human gene encoding.” Translational Lung Cancer Research 2016, vol. 5,1), their application relies on their insertion activity to insert genes into or disrupt genes in the host cell's DNA, which also presents the problem of insertional mutagenesis.

[0009] Furthermore, since actively dividing (proliferating) cells internalize nucleic acids much better than non-dividing quiescent cells (Bai et al. 2017), transfection is usually performed in actively dividing (proliferating) cells, and in particular, it is particularly difficult to transfect confluent cells that do not divide in culture or specific cell types. Therefore, it is necessary to improve the transfection efficiency in non-dividing cells.

[0010] Thus, the present invention provides evidence that by using a transposase together with a vehicle that promotes cytoplasmic transfection of nucleic acids, nuclear localization or nuclear uptake of a DNA of interest can be increased, whereupon this can be expressed (i.e., transiently expressed, transfected) by cells or inserted into genomic DNA. The method of the present invention also enables efficient transfection of quiescent cells. Finally, the method of the present invention also enables efficient transfection without insertion activity when using a catalytically inactive transposase. SUMMARY OF THE INVENTION

[0011] An object of the present invention is a method for increasing the expression of a nucleic acid molecule encoding at least one transgene of interest in a cell population, the method comprising contacting the cell population with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same, and a nucleic acid molecule encoding at least one transgene of interest in a step.

[0012] Another object of the present invention is a method for increasing the nuclear localization of a nucleic acid molecule encoding at least one transgene of interest in a cell population, the method comprising contacting the cell population with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same, and a nucleic acid molecule encoding at least one transgene of interest in a step.

[0013] Another object of the present invention is a method for editing the genome of a cell population, the method comprising contacting the cell population with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same, and a nucleic acid molecule encoding at least one transgene of interest A method comprising the step of contacting.

[0014] Another object of the present invention is a method for treating a genetic disease of a subject in need thereof, the method comprising administering to the subject a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule, and a nucleic acid molecule encoding at least one transgene of interest in a therapeutically effective amount, wherein expression of the transgene of interest in at least one cell of the subject in need thereof compensates for a gene deficiency that is the cause of the genetic disease. A method.

[0015] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is a deoxyribonucleic acid (DNA) molecule. In some embodiments, the DNA molecule is a complementary DNA (cDNA) molecule. In some embodiments, the DNA molecule is a genomic DNA (gDNA) molecule.

[0016] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest further comprises at least one inverted terminal repeat (ITR). In some embodiments, the nucleic acid molecule encoding at least one transgene of interest further comprises two ITR sequences. In some embodiments, at least one ITR sequence is adjacent to the transgene of interest. In some embodiments, the nucleic acid molecule encoding at least one transgene of interest further comprises two ITR sequences present at both ends of the transgene of interest. In some embodiments, at least one ITR sequence interacts with or binds non-covalently to the transposase.

[0017] In some embodiments, the DNA molecule is inserted into the genome of the cell population. In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is stably expressed by the cell population.

[0018] In some embodiments, the transgene of interest is an exogenous gene. In some embodiments, the transgene of interest is an endogenous gene.

[0019] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is contained in a plasmid, fosmid, cosmid, artificial chromosome, or viral vector. In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is contained in a plasmid. In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is contained in a viral vector. In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is contained in a DNA virus-based vector selected from the group consisting of viruses derived from the realms of Duplodnaviria, Monodnaviria, and Varidnaviria.

[0020] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof increases the nuclear localization of a nucleic acid molecule encoding at least one transgene of interest. In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof transfers a nucleic acid molecule encoding at least one transgene of interest to the nucleus or promotes the transfer of a nucleic acid molecule encoding at least one transgene of interest to the nucleus.

[0021] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof is a fusion protein comprising the transposase or a fragment thereof and at least one additional polypeptide or protein. In some embodiments, the at least one additional polypeptide or protein is a nuclease. In some embodiments, the at least one additional polypeptide or protein is an RNA-guided nuclease. In some embodiments, the at least one additional polypeptide or protein is a Cas nuclease. In some embodiments, the at least one additional polypeptide or protein is a Cas9 nuclease.

[0022] In some embodiments, the fusion protein further comprises a linker.

[0023] In some embodiments, the fusion protein has at least 75% amino acid sequence identity with SEQ ID NO: 2. In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0024] In some embodiments, the at least one additional polypeptide or protein is an aptamer-binding protein. In some embodiments, the at least one additional polypeptide or protein is an MS2 bacteriophage coat protein (MCP). In some embodiments, the fusion protein is capable of interacting covalently or non-covalently with a gRNA molecule comprising at least one MS2 aptamer via the MCP. In some embodiments, the gRNA molecule is capable of interacting covalently or non-covalently with an RNA-guided nuclease via at least one MS2 aptamer.

[0025] In some embodiments, the transposase is selected from the group consisting of hyperactive PiggyBac transposase, PiggyBac transposase, Sleeping Beauty transposase, SB11 transposase, Tol2 transposase, Mos1 transposase, and Frog Prince transposase. In some embodiments, the transposase is selected from the group consisting of hyperactive PiggyBac transposase and Sleeping Beauty transposase. In some embodiments, the transposase is hyperactive PiggyBac transposase. In some embodiments, the transposase is hyperactive PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the transposase is a modified hyperactive PiggyBac transposase that contains at least one amino acid mutation as compared to the amino acid sequence of the hyperactive PiggyBac transposase of SEQ ID NO: 1. In some embodiments, the transposase is Sleeping Beauty transposase. In some embodiments, the transposase or a fragment thereof has low catalytic activity. In some embodiments, the transposase or a fragment thereof has no catalytic activity. In some embodiments, the transposase having no catalytic activity has at least 75% amino acid sequence identity with SEQ ID NO: 3. In some embodiments, the transposase having no catalytic activity comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0026] In some embodiments, the protein or polypeptide comprises a transposase fragment. In some embodiments, the transposase fragment comprises or consists of at least one functional domain of a transposase. In some embodiments, the transposase fragment comprises or consists of an ITR binding domain. In some embodiments, the transposase fragment is a fragment of a hyperactive PiggyBac transposase or a Sleeping Beauty transposase. In some embodiments, the transposase fragment is a fragment of a hyperactive PiggyBac transposase, preferably comprising an ITR binding domain. In some embodiments, the transposase fragment is a fragment of a Sleeping Beauty transposase, preferably selected from the group consisting of the SB100 domain and the N57 domain of the Sleeping Beauty transposase.

[0027] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is a ribonucleic acid (RNA) molecule or a deoxyribonucleic acid (DNA) molecule. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is an RNA molecule. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is a messenger RNA (mRNA) molecule. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is a cDNA molecule or a plasmid. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population by transfection or transformation. In some embodiments, transfection is selected from the group consisting of lipofection, electroporation, sonication, nanoparticles, microinjection, and viral vector infection including non-integrative viral vector infection and integrative viral vector infection. In some embodiments, transformation comprises using an integrative viral vector or a modified integrative virus.

[0028] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population together with a nucleic acid molecule encoding at least one transgene of interest. In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population prior to a nucleic acid molecule encoding at least one transgene of interest. In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population 1 hour to 72 hours prior to a nucleic acid molecule encoding at least one transgene of interest. In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population approximately 4 hours prior to a nucleic acid molecule encoding at least one transgene of interest.

[0029] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same, is included in a pharmaceutical composition further comprising at least one acceptable excipient.

[0030] In some embodiments, the cell population is a eukaryotic or prokaryotic cell population. In some embodiments, the cell population is a eukaryotic cell population. In some embodiments, the cell population is a prokaryotic cell population. In some embodiments, the method is performed in vitro. In some embodiments, the cell population is a population of cells cultured in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo.

[0031] In some embodiments, the cell population is included in a tissue or organ of a living body. In some embodiments, the organism is an animal. In some embodiments, the organism is a mammal. In some embodiments, the organism is a human.

[0032] In some embodiments, the method is contacting a cell population with a vector comprising a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof; culturing the cell population for a period ranging from 1 hour to 72 hours; contacting the cell population with a vector comprising a nucleic acid molecule encoding at least one target transgene and comprising.

[0033] In some embodiments, the method comprises contacting a cell population with a vector comprising a protein or polypeptide comprising a transposase or a fragment thereof; contacting the cell population with a vector comprising a nucleic acid molecule encoding at least one target transgene and comprising.

[0034] Definitions As used herein, the following terms have the following meanings.

[0035] "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease that includes a Cas9 protein or a fragment thereof (e.g., a protein containing an active or inactive DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 nuclease is also sometimes called casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, conjugative plasmids). A CRISPR cluster contains spacers, sequences complementary to a preceding mobile element, and a target for invading nucleic acids. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, accurate processing of pre-crRNA requires a small molecule RNA (tracrRNA) encoded in trans, an endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA functions as a guide for processing assisted by ribonuclease 3 of the pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA complex cleaves a linear or circular dsDNA target complementary to the spacer by an endonuclease. The target strand not complementary to the crRNA is first cleaved by an endonuclease and then trimmed by an exonuclease in a 3'-5' direction. Originally, DNA binding and cleavage typically require a protein and both RNAs. However, a single-guide RNA ("sgRNA" or simply "gRNA") can be engineered to incorporate both aspects of crRNA and tracrRNA into a single RNA species. Cas9 recognizes a short motif of the CRISPR repeat sequence (PAM or protospacer adjacent motif) and helps distinguish self from non-self. The sequence and structure of Cas9 nuclease are well known to those skilled in the art.Cas9 orthologs have been described in a variety of species including, but not limited to, Streptococcus pyogenes and Streptococcus thermophilus.

[0036] "Exogenous" refers to any molecule that is not naturally present in the cell or organism of interest but can be introduced into it by one or more genetic, biochemical, or other means. The natural presence of a molecule in a cell or organism can also be determined in the context of a particular developmental stage and its environmental conditions. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to adult muscle cells. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to cells that have not been heat shocked. Exogenous molecules can include, for example, a functional version of a malfunctioning endogenous molecule or a malfunctioning version of a normally functioning endogenous molecule. In contrast, the term "endogenous" means any molecule that is normally present in a cell or organism at a particular developmental stage under specific environmental conditions.

[0037] "Fusion protein" refers to a single-chain hybrid polypeptide that contains amino acid sequences from two or more (i.e., from two or more different proteins and / or peptides) that are fused together. The two or more amino acid sequences can be fused together directly via a peptide bond or indirectly via a peptide linker. A fusion protein may, in particular, be encoded entirely by a single nucleic acid sequence.

[0038] "Gene" typically refers to a DNA region that encodes a protein (i.e., the coding region). This term can also include DNA regions that do not themselves encode a protein (i.e., non-coding regions). The latter includes regions that are transcribed, for example, into functional non-coding RNA molecules (such as transfer RNA, ribosomal RNA, regulatory RNA, etc.). Other non-coding regions control the transcription and translation of coding regions (i.e., regulatory elements), or function as architectural elements (such as scaffold / matrix attachment regions), origins of DNA replication, centromeres, or telomeres. Regulatory elements include, but are not limited to, promoter sequences, terminators, translation control sequences (such as ribosome binding sites [RBS] and internal ribosome entry sites [IRES]), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0039] "Identity" or "identical," when used in reference to the relationship between two or more amino acid sequences or two or more nucleic acid sequences, refers to the degree of relatedness of the sequences between amino acid sequences or nucleic acid sequences, determined by the number of matches between chains of two or more amino acid residues or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences with (if present) gap alignment presented by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):1073 82. Preferred methods for determining identity are designed to provide the maximum match between the sequences being tested. Methods for determining identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two arrays include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387 95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403 10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0040] "Insertion" and "integration" refer to the addition of a nucleic acid sequence into a second nucleic acid sequence or its genome or part thereof. The insertion may be "specific", "site-specific", "targeted", or "on-target": these adjectives define the insertion of a nucleic acid into a specific site of the second nucleic acid or a specific site of its genome or part thereof (i.e., the site intentionally selected with respect to the insertion). Conversely, the adjectives "random", "untargeted", or "off-target" refer to the non-specific and / or unintended insertion of a nucleic acid into an undesirable site. The term "comprehensive" or "overall" refers to the total number of insertions.

[0041] "Linker" refers to a chemical group or molecule that links two adjacent molecules or parts.

[0042] "Modified" refers to a protein or nucleic acid sequence that is different from the corresponding unmodified protein or nucleic acid sequence.

[0043] "Mutated" in relation to a sequence (e.g., an amino acid sequence or a nucleic acid sequence) means that the sequence is different from a reference sequence such as a wild-type sequence. Typically, a mutated sequence contains at least one of substitutions, additions, or deletions of one or more residues as compared to a reference sequence such as the corresponding wild-type sequence.

[0044] "Mutation" refers to the substitution of a residue with another residue within a sequence, e.g., a nucleic acid or amino acid sequence, and / or the deletion or insertion of one or more residues within a nucleic acid or amino acid sequence. Mutations are typically described herein by identifying the original residue, then the position of the residue within the sequence, and then the identity of the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art and are described, for example, in Green & Sambrook, 2012 (Molecular cloning: a laboratory manual (4th Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0045] "Nuclease" refers to an enzyme that catalyzes the hydrolysis of nucleic acids within a nucleic acid sequence. Nuclease activity can result in the breakage of single-stranded or double-stranded nucleic acid molecules, which can be DNA or RNA.

[0046] "Nucleic acid (molecule / sequence)" and "nucleotide sequence" can be used interchangeably to refer to any molecule composed of or containing monomeric nucleotides. Nucleic acids may be oligonucleotides or polynucleotides; they may be DNA, RNA, or mixtures thereof. They may be chemically modified or artificial, for example, they may include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). These nucleic acids are each different from naturally occurring DNA or RNA due to changes in the molecular backbone. Phosphorothioate nucleotides may also be used. Other deoxynucleotide analogs include, but are not limited to, methylphosphonic acid, phosphoramidic acid, phosphorodithioate, N3’P5’ phosphoramidate and oligoribonucleotide phosphorothioate, and their 2’O-allyl analogs and 2’O-methyl ribonucleotide methylphosphonic acid used in the nucleic acids of the present disclosure.

[0047] "Polypeptide", "peptide", "protein", and "amino acid sequence" are used interchangeably to refer to a polymer of amino acid residues. Unless specified otherwise, the polymer of amino acid residues may be of any length. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

[0048] "Prevent" and any inflection thereof refer to prophylactic and preventive means, the purpose of which is to reduce the chance that a subject will develop a given disease state or disorder over a given period of time. Such reduction may be reflected, for example, by a delay in the onset of at least one symptom of the subject's disease state or disorder.

[0049] "Specificity" refers to the ability to selectively bind to a sequence that shares a degree of sequence identity to a selected sequence.

[0050] "Subject" refers to a mammal, preferably a human. The subject can be a "patient", i.e., a warm-blooded animal, preferably a human, who is waiting to receive medical treatment, or is receiving medical treatment, or has been / will be / may be the subject of medical treatment in the past / present / future, or is being monitored for the onset of a disease. The term "mammal" here refers to any mammal including humans, domesticated animals and livestock, as well as animals in zoos, for sports, or as pets, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

[0051] "Transduction" and any inflection thereof refer to the introduction of one or more nucleic acid molecules (DNA and / or RNA) into one or more cells using a viral vector carrier, such as, but not limited to, a retrovirus (including lentivirus), an adenovirus, an adeno-associated virus (AAV), and viruses, viral particles, or viral vectors derived therefrom.

[0052] "Transfection" and its inflections refer to the introduction of one or more nucleic acid molecules (DNA and / or RNA) into one or more cells by non-viral means, regardless of whether it is in vitro or in vivo. In other words, "transfection" refers to any method, technique, or vehicle known in the art that promotes or enhances the transduction of a nucleic acid molecule or cargo into the cytoplasm of a cell. Methods for transfection are well known in the art and include, for example, lipofection, PEI, and electroporation.

[0053] "Transgene" refers to an exogenous nucleic acid sequence encoding a gene product, particularly an exogenous DNA or cDNA. The gene product can be RNA, a peptide, or a protein. In addition to the coding region (CDS) of the gene product, the transgene may include or be associated with one or more operable sequences for promoting or enhancing expression, such as a promoter, enhancer, response element, reporter element, insulator element, polyadenylation signal, and / or other functional elements. Embodiments of the present disclosure may utilize any known suitable promoter, enhancer, response element, reporter element, insulator element, polyadenylation signal, and / or other functional elements, unless otherwise specified. Suitable elements and sequences are well known to those skilled in the art.

[0054] "Transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another location in the genome by a cut-and-paste mechanism or a replicative transposition mechanism. Within the scope of the present invention, the transposase can be a fragment of the transposase, such as an ITR-binding domain or a functional domain, preferably an ITR-binding domain.

[0055] "Treatment", "alleviation", "curative", and any inflection of these terms refer to therapeutic treatment excluding prophylactic or preventive measures, where the purpose is to delay, attenuate, arrest, or even (partially or completely) reverse the development of a target pathological condition or disorder. Those in need of treatment include those already having a disorder and those suspected of having a disorder. After a subject has received treatment for a target pathological condition or disorder, a "treatment" for that pathological condition or disorder is successful if it shows an observable and / or measurable reduction in one or more symptoms associated with the pathological condition or disorder; alleviation to some extent; a decrease in morbidity and / or mortality; and / or an improvement in quality of life. The above parameters for assessing treatment success and disease improvement are readily measurable by well-known and defined techniques by physicians.

[0056] "Vector", as used herein, refers to any polynucleotide that can, for example, have a second polynucleotide of interest and can, for example, transfer a gene sequence into a target cell. Thus, this term includes cloning, and expression vehicles, and integration vectors.

[0057] Detailed Description When attempting to express a transgene of interest in a population of cells, one of the main obstacles that can occur is an insufficient expression level that can be caused by the difficulty of getting a DNA molecule having the transgene of interest to reach the nucleus. The present invention addresses this problem.

[0058] Thus, the present invention relates to a method for increasing the expression of a nucleic acid molecule encoding at least one transgene of interest in a cell population.

[0059] The present invention also relates to a method for increasing the nuclear localization of a nucleic acid molecule encoding at least one transgene of interest in a cell population.

[0060] The present invention also relates to a method for editing the genome of a cell population by inserting a nucleic acid molecule encoding at least one transgene of interest into the genome of the cell population.

[0061] The present invention also relates to a method for transfecting a nucleic acid molecule encoding at least one transgene of interest in a cell population, preferably a non-dividing cell population.

[0062] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is transfected into the cell population by at least one technique selected from the group consisting of or comprising lipofection, electroporation, sonication, nanoparticles, microinjection, PEI, and viral vector infection including non-integrating viral vector infection and integrating viral vector infection.

[0063] The present invention is based on the finding by the inventors that cytoplasmic transposase can increase its nuclear localization by delivering nucleic acids to the nucleus of a cell. In particular, cytoplasmic transposase can transfer or facilitate the transfer of nucleic acids to the nucleus of cells including resting cells. Thus, the increased nuclear localization induces an increase in the expression level of the nucleic acid. Interestingly, the inventors observed that the increase in nuclear localization by transposase is independent of its enzymatic activity.

[0064] It is understood that a DNA molecule encoding at least one transgene of interest is detected by the immune system of the cell that induces a response (e.g., an inflammatory response) to the presence of foreign DNA when delivered to the cytoplasm of the cell. In addition, a DNA molecule encoding at least one transgene of interest also needs to cross the nuclear membrane to reach the nucleus. These problems are overcome when a DNA molecule encoding at least one transgene of interest is transferred by cytoplasmic transposase.

[0065] In the present invention, these methods involve a cell population A nucleic acid molecule encoding at least one target transgene, and A protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule Contacting.

[0066] In some embodiments, the nucleic acid molecule encoding at least one target transgene is delivered to the cell population by transfection. In some embodiments, the nucleic acid molecule encoding at least one target transgene is transfected into the cell population by at least one technique selected from the group consisting of lipofection, electroporation, sonication, nanoparticles, microinjection, PEI, and viral vector infection including non-integrating viral vector infection and integrating viral vector infection.

[0067] In some embodiments, the cell population may be contacted with a nucleic acid molecule encoding at least one target transgene and a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule, in any order.

[0068] In some embodiments, the cell population is contacted with a nucleic acid molecule encoding at least one target transgene simultaneously with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule.

[0069] In one embodiment, the cell population is contacted with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule, and then contacted with a nucleic acid molecule encoding at least one target transgene. In another embodiment, the cell population is contacted with a nucleic acid molecule encoding at least one target transgene and then contacted with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule.

[0070] The term "nucleic acid molecule encoding at least one transgene of interest" refers to a nucleic acid sequence that is inserted into the genome of a cell, preferably a eukaryotic cell, more preferably a mammalian cell (including human cells), and that encodes at least one product of interest. The product of interest may be a protein or a fragment thereof; in this case, the transgene of interest is referred to as the coding nucleic acid sequence. However, the term also encompasses non-coding nucleic acid sequences, i.e., nucleic acid sequences that do not encode a protein or a fragment thereof but that express "RNA genes" (or "non-coding RNAs") such as, for example, transfer RNA, ribosomal RNA, small molecule RNA, long non-coding RNA, etc.

[0071] In some embodiments, the transgene of interest is a nucleic acid sequence that encodes a peptide or a protein (including, but not limited to, an enzyme, a transcription factor, a growth factor, a trophic factor, a hormone, a cytokine, an antibody, an antigen, a receptor, an immunomodulatory factor, a differentiation factor, a suicide protein, a cell cycle modifying protein, an anti-proliferative protein, an angiogenesis factor, an anti-angiogenesis factor, a genome editor, a nuclease, a recombinase, a transposase, a neurotransmitter, and a reporter, including precursors and fusion proteins thereof). In this case, the sequence of interest can typically be (or be derived from) mRNA, cDNA, gDNA, synthetic nucleic acid, or any combination thereof.

[0072] In some embodiments, the transgene of interest is, alternatively, a nucleic acid sequence of a non-coding RNA.

[0073] Examples of non-coding RNAs include, but are not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), Y RNA, telomerase RNA component (TERC), splice leader RNA (SL RNA), catalytic RNA (i.e., ribozyme; e.g., ribonuclease P, ribonuclease MRP, etc.), antisense RNA (aRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding RNA (lncRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), enhancer RNA (eRNA), and RNA aptamer.

[0074] Examples of the gene of interest include any nucleic acid sequence encoding a molecule for therapeutic purposes, such as any nucleic acid sequence encoding a peptide or protein that is absent, defective, and / or non-functional in the subject, or includes non-coding RNA.

[0075] In some embodiments, the nucleic acid molecule encoding at least one gene of interest includes at least one gene and at least one control element. Examples of control elements include, but are not limited to, promoter, enhancer, silencer, insulator, etc. Preferably, at least one control element is located upstream, i.e., 5', of at least one gene of interest.

[0076] The nucleic acid molecule encoding at least one gene of interest can be double-stranded or single-stranded.

[0077] The nucleic acid molecule encoding at least one transgene of interest may be a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a mixture thereof, and preferably, the nucleic acid molecule encoding at least one transgene of interest is a DNA molecule.

[0078] The nucleic acid molecule encoding at least one transgene of interest typically contains natural nucleotides. However, it may also contain non-natural nucleotides. As used herein, "natural nucleotides" refers to adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term "non-natural nucleotides" refers to nucleotides of chemically modified A, T, U, C, or G.

[0079] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest has a length of at least 10 base pairs (bp), such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 bp, or more.

[0080] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is a DNA molecule selected from the group consisting of or comprising complementary DNA (cDNA) and genomic DNA (gDNA). In some embodiments, the DNA molecule is cDNA. In some embodiments, the DNA molecule is gDNA.

[0081] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest further comprises at least one inverted terminal repeat (ITR).

[0082] As used herein, the term "ITR sequence" refers to nucleic acid sequences that are naturally found at the ends of eukaryotic transposable elements (or transposons). These are referred to as "5' ITR" and "3' ITR". Typically, the 5' ITR and 3' ITR are complementary and can form hairpin structures. The ITR sequences are useful for the recognition of transposons by transposase enzymes. In some embodiments, the ITR sequence is a palindromic structure.

[0083] Some non-limiting examples of ITR sequences include SEQ ID NOs: 55-60, and their complementary sequences. SEQ ID NO: 55 ccctagaaagatagtctgcgtaaaattgacgcatg SEQ ID NO: 56 ccctagaaagatagtctgcgtaaaattgacgcatgagataatcaatattgtgacgtacgttaa SEQ ID NO: 57 ccctagaaagatagtctgcgtaaaattgacgcatgagataatcaatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatg SEQ ID NO: 58 gattatctttctaggg SEQ ID NO: 59 cacaatatgattatctttctaggg SEQ ID NO: 60 catgcgtcaattttacgcatgattatctttaacgtacgtacgtcacaatatgattatctttctaggg

[0084] In some embodiments, at least one ITR sequence interacts with or binds non-covalently to a protein or polypeptide comprising a transposase or a fragment thereof. In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof specifically recognizes or binds to at least one ITR sequence.

[0085] In some embodiments, at least one ITR sequence is adjacent to the transgene of interest. "Adjacent" means that there are no nucleotides between the at least one ITR sequence and the transgene of interest. Alternatively, the at least one ITR sequence can be separated from the transgene of interest by at least one nucleotide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or more nucleotides.

[0086] In some embodiments, a nucleic acid molecule encoding at least one transgene of interest comprises two ITR sequences. Preferably, the two ITR sequences are present at both ends of the transgene of interest. In some embodiments, the two ITR sequences are present immediately flanking the transgene of interest, i.e., there are no nucleotides between the two adjacent ITR sequences and the transgene of interest. Alternatively, the transgene of interest can be separated from one or both of the two ITR sequences by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or more nucleotides.

[0087] In some embodiments, a nucleic acid molecule encoding at least one transgene of interest is stably or transiently expressed by a cell population, preferably stably expressed.

[0088] In some embodiments, the nucleic acid molecule encoding the transgene for at least one purpose is transiently expressed. In a preferred embodiment, the nucleic acid molecule encoding the transgene for at least one purpose is transfected. In some embodiments, transfection involves the use of at least one vehicle and / or method that promotes or increases the cytoplasmic transduction of the nucleic acid molecule encoding the transgene for at least one purpose. Examples of transfection techniques include, but are not limited to, lipofection, electroporation, sonication, nanoparticles, microinjection, PEI, and viral vector infection including non-integrating viral vector infection and integrating viral vector infection.

[0089] In some embodiments, the nucleic acid molecule encoding the transgene for at least one purpose is a DNA molecule inserted into the genome of the cells of a cell population. The term "inserted into the genome" implies that the DNA molecule is replicated in parallel with the cell's genome during mitosis.

[0090] The transgene for the purpose can be an exogenous gene or an endogenous gene.

[0091] In some embodiments, the transgene is an exogenous gene. As used herein, "exogenous gene" refers to a gene that is not naturally present in a cell but can be introduced into the cell by one or more genetic, biochemical, or other methods. Whether a gene is naturally present in a cell can be determined with respect to a particular developmental stage and environmental conditions of the cell. For example, a molecule that is present only in cells during embryonic development is exogenous to adult cells. Similarly, a molecule induced by heat shocking a cell is exogenous to a cell that has not been heat shocked.

[0092] In some embodiments, the target transgene is an endogenous gene. As used herein, an "endogenous gene" refers to a gene in which at least one copy is naturally present in the genome of a population of cells. In some embodiments, at least one copy can be an allele, i.e., a version of the gene that contains at least one mutation. In certain embodiments, the expression of the transgenic endogenous gene increases the overall expression level (i.e., transcript) of the gene.

[0093] In some embodiments, the nucleic acid molecule encoding at least one target transgene is contained in a vector, such as, but not limited to, a plasmid, fosmid, cosmid, artificial chromosome, or viral vector.

[0094] In some embodiments, the nucleic acid molecule encoding at least one target transgene is contained in a plasmid. The plasmid may be circular or linear, preferably circular. In some embodiments, the nucleic acid molecule encoding at least one target transgene is contained in a fosmid. In some embodiments, the nucleic acid molecule encoding at least one target transgene is contained in a cosmid. In some embodiments, the nucleic acid molecule encoding at least one target transgene is contained in an artificial chromosome (e.g., a human artificial chromosome).

[0095] In some embodiments, the nucleic acid molecule encoding at least one target transgene is contained in a viral vector.

[0096] The viral vector can be a DNA virus-based vector. Some non-limiting examples of such DNA virus-based vectors include vectors derived from viruses in the Dupravirus realm, Monodnaviria realm, or Varidnaviria realm.

[0097] In some embodiments, the DNA virus-based vector is derived from a virus in the duplodnavirus realm. Viruses from the duplodnavirus realm include viruses of the order Herpesvirales. Thus, in some embodiments, the DNA virus-based vector is a vector derived from the Herpesviridae family, for example, a vector derived from Herpes simplex virus.

[0098] In some embodiments, the DNA virus-based vector is derived from a virus in the monodnavirus realm. Viruses from the monodnavirus realm include viruses of the families Papillomaviridae and Polyomaviridae. Thus, in some embodiments, the DNA virus-based vector is a vector derived from the Papillomaviridae family or the Polyomaviridae family.

[0099] In some embodiments, the DNA virus-based vector is derived from a virus in the varidnavirus realm. Viruses from the varidnavirus realm include viruses of the families Adenoviridae and Poxviridae. Thus, in some embodiments, the DNA virus-based vector is a vector derived from the Adenoviridae family, for example, a vector derived from Adenovirus, or a vector derived from the Poxviridae family, for example, a vector derived from Vaccinia virus.

[0100] In some embodiments, a nucleic acid molecule encoding at least one transgene of interest may be excised from the vector. In some embodiments, a nucleic acid molecule encoding at least one transgene of interest may be excised from the vector by at least one ITR sequence, typically by a transposase or a variant or fragment thereof.

[0101] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof increases the nuclear localization of a nucleic acid molecule encoding at least one transgene of interest.

[0102] As used herein, "increasing nuclear localization" means that the nuclear localization of a nucleic acid molecule encoding at least one target transgene in the presence of a protein or polypeptide comprising a transposase or a fragment thereof is at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more increased compared to the condition in the absence of the protein or polypeptide comprising a transposase or a fragment thereof.

[0103] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof transfers or facilitates the transfer of a nucleic acid molecule encoding at least one target transgene into the nucleus. In some embodiments, the transposase or a fragment thereof (i) interacts with and / or binds non-covalently to a nucleic acid molecule encoding at least one target transgene and (ii) transfers into the nucleus.

[0104] In some embodiments, the transposase or a fragment thereof does not insert and / or transfer a nucleic acid molecule encoding at least one target transgene into the genome.

[0105] In some embodiments, the transposase or a fragment thereof increases the passage of a nucleic acid molecule encoding at least one target transgene through the nuclear membrane by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more.

[0106] In some embodiments, the transposase or a fragment thereof protects a nucleic acid molecule encoding at least one transgene of interest from the cell's detection and / or defense mechanisms. In some embodiments, the transposase or a fragment thereof does not induce an inflammatory and / or inflammatory response.

[0107] In some embodiments, the transposase is selected from the group consisting of hyperactive PiggyBac transposase, PiggyBac transposase, Sleeping Beauty transposase, SB11 transposase, Tol2 transposase, Mos1 transposase, and Frog Prince transposase.

[0108] In some embodiments, the transposase is selected from the group consisting of hyperactive PiggyBac transposase and Sleeping Beauty transposase.

[0109] In some embodiments, the transposase is hyperactive PiggyBac transposase.

[0110] In some embodiments, the transposase is hyperactive PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1. [Table 1]

[0111] In some embodiments, the transposase is a modified hyperactive PiggyBac transposase. A "modified hyperactive PiggyBac transposase" is a transposase that contains one or more amino acid substitutions, typically 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less amino acid substitutions compared to the hyperactive PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1. More specifically, the modified hyperactive PiggyBac transposase may include (i) one or more amino acid substitutions for increasing excision activity compared to the hyperactive PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1, and / or (ii) one or more amino acid substitutions for decreasing DNA binding activity compared to the hyperactive PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1.

[0112] In some embodiments, the modified hyperactive PiggyBac transposase includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1.

[0113] In some embodiments, the modified hyperactive PiggyBac transposase includes one or more amino acid mutations for increasing excision activity.

[0114] In some embodiments, the modified hyperactive PiggyBac transposase includes one or more amino acid mutations for increasing excision activity selected from amino acid mutations within the region defined by amino acid position numbers [194 - 200], [214 - 222], [434 - 442], or [446 - 456]; for example, including amino acid substitutions at positions D198, D201, R202, M212, and / or S213 (the said position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1).

[0115] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations selected from amino acid mutations at positions 450, 560, 564, 573, 589, 592, and / or 594 (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1).

[0116] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations selected from amino acid mutations at positions M194 and / or D450 for increasing excision activity (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1, and preferably, the amino acid substitutions are M194V and / or D450N).

[0117] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations for decreasing DNA binding activity.

[0118] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations selected from amino acid mutations at positions 254, 275, 277, 347, 372, 375, and / or 465 for decreasing DNA binding activity (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1).

[0119] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations selected from R275, N347, R372, K375, R376, E377, and E380 for decreasing DNA binding activity (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1).

[0120] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations for reducing DNA binding activity selected from R372, K375, R376, E377, and E380 (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1, and preferably, the amino acid substitutions are R372A, K375A, R376A, E377A, and / or E380A).

[0121] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations for reducing DNA binding activity selected from N347, R372, and K375 (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1, and preferably, the amino acid substitutions are N347S, N347A, R372A, and / or K375A, and more preferably, the amino acid substitution is N347S or N347A).

[0122] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations for increasing excision activity as defined above and one or more amino acid mutations for reducing DNA binding activity as defined above.

[0123] In some embodiments, the modified hyperactive PiggyBac transposase comprises at least one amino acid substitution for increasing excision activity at position D245 and at least two amino acid substitutions for reducing DNA binding activity at positions N347, R372, and K375, and preferably, the transposase of the modified hyperactive PiggyBac comprises the double mutation N347S and D450N, or the triple mutation D450N, R372A, and K375A (the position numbers correspond to the amino acid numbers of hyperactive PiggyBac having SEQ ID NO: 1).

[0124] In some embodiments, the modified highly active PiggyBac transposase comprises double mutations N347S and D450N (the position numbers correspond to the amino acid numbers of the highly active PiggyBac having SEQ ID NO: 1).

[0125] In some embodiments, the modified highly active PiggyBac transposase disclosed in the above embodiments further comprises at least one mutation in the region defined by amino acid position numbers [158-169], such as A166S; and at least one mutation at positions Y527, R518, K525, N463.

[0126] In some embodiments, the modified highly active PiggyBac transposase further comprises one or more amino acid substitutions at positions 34, 43, 117, 202, 230, 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 388, 409, 411, 412, 432, 447, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592, and / or 594 (the position numbers correspond to the amino acid numbers of the highly active PiggyBac having SEQ ID NO: 1).

[0127] In some embodiments, the modified hyperactive PiggyBac transposase comprises one of the following amino acid substitutions or combinations of amino acid substitutions: V34M, T43I, Y177H, R202K, S230N, R245A, D268N, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R388A, K409A, A411T, K412A, K432A, D447A, D447N, D450N, R460A, K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, F594L, D450N / R372A / K375A, R275A / R277A, K409A / K412A, R460A / K461A, R275A / R277A / N347S / K375A / T560A / S573A / M589V / S592G, or R245A / R275A / R277A / R372A / W465A.

[0128] In some embodiments, the modified hyperactive PiggyBac transposase comprises one of the following amino acid substitutions or combinations of amino acid substitutions: -R372A / K375A / D450N, -R372A / K375A / R376A / D450N, -K375A / R376A / E377A / E380A / D450N, -R372A / K375A / R376A / E377A / E380A / D450N, -M194V, -M194V / R372A / K375A, -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, -R245A / R275A / R277A / R372A / W465A / M589V, -R275A / 325A / R372A / T560A, -N347A / D450N, -N347S / D450N / T560A / S573A / F594L, -R202K / R275A / N347S / R372A / D450N / T560A / F594L, -R275A / N347S / K375A / D450N / S592G, -R275A / N347S / R372A / D450N / T560A / F594L, -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, -V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, -G325A / N347S / K375A / D450N / S573A / M589V / S592G, -S230N / R277A / N347S / K375A / D450N, -T43I / R372A / K375A / A411T / D450N, -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G (The position numbers correspond to the amino acid numbers of highly active PiggyBac having SEQ ID NO: 1).

[0129] Some preferred modified highly active PiggyBac transposases include modified highly active PiggyBac containing one of the following combinations of amino acid substitutions: -R372A / K375A / D450N, -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, -R245A / R275A / R277A / R372A / W465A / M589V, -N347A / D450N, -N347S / D450N / T560A / S573A / F594L, -R202K / R275A / N347S / R372A / D450N / T560A / F594L, -R275A / N347S / K375A / D450N / S592G, -R275A / N347S / R372A / D450N / T560A / F594L, -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, -V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, -G325A / N347S / K375A / D450N / S573A / M589V / S592G, -S230N / R277A / N347S / K375A / D450N, -T43I / R372A / K375A / A411T / D450N, -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, or -R275A / 325A / R372A / T560A, (The above position numbers correspond to the amino acid numbers of highly active PiggyBac having SEQ ID NO: 1).

[0130] In some embodiments, the modified highly active PiggyBac transposase comprises one of the following combinations of amino acid substitutions: -R245A / R275A / R277A / R372A / W465A / M589V, -R275A / 325A / R372A / T560A, -N347A / D450N, -N347S / D450N / T560A / S573A / F594L, -R202K / R275A / N347S / R372A / D450N / T560A / F594L, -R275A / N347S / K375A / D450N / S592G, -R275A / N347S / R372A / D450N / T560A / F594L, -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, -G325A / N347S / K375A / D450N / S573A / M589V / S592G, -S230N / R277A / N347S / K375A / D450N, -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, (The above position numbers correspond to the amino acid numbers of highly active PiggyBac with SEQ ID NO: 1).

[0131] Some preferred modified highly active PiggyBac transposases contain one of the following combinations of amino acid substitutions: -N347A / D450N, -N347S / D450N / T560A / S573A / F594L, -R202K / R275A / N347S / R372A / D450N / T560A / F594L, -R275A / N347S / K375A / D450N / S592G, -R275A / N347S / R372A / D450N / T560A / F594L, -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, -G325A / N347S / K375A / D450N / S573A / M589V / S592G, -S230N / R277A / N347S / K375A / D450N, -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, (The position numbers correspond to the amino acid numbers of highly active PiggyBac having SEQ ID NO: 1).

[0132] In some embodiments, the modified highly active PiggyBac transposase comprises R372A / K375A / D450N substitutions, and the position numbers correspond to the amino acid numbers of highly active PiggyBac having SEQ ID NO: 1. The modified transposase has the amino acid sequence of SEQ ID NO: 4.

[0133] In some embodiments, the modified highly active PiggyBac transposase has an amino acid sequence selected from the group consisting of or comprising SEQ ID NOs: 5-26.

[0134] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group consisting of or comprising SEQ ID NOs: 5-13.

[0135] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group consisting of or comprising SEQ ID NOs: 14-26.

[0136] In some embodiments, the modified hyperactive PiggyBac transposase may include one or more mutations involved in the conserved catalytic triad at amino acids 268 and / or 346 (e.g., D268N and / or D346N) corresponding to the amino acid numbering of SEQ ID NO: 1, for example, compared to the hyperactive PiggyBac transposase.

[0137] In some embodiments, the modified hyperactive PiggyBac transposase may include one or more mutations important for excision at amino acids 287, 287 / 290, and / or 460 / 461 (e.g., K287A, K287A / K290A, and / or R460A / K461A) corresponding to the amino acid numbering of SEQ ID NO: 1, for example, compared to the hyperactive PiggyBac transposase.

[0138] In some embodiments, the modified hyperactive PiggyBac transposase may include one or more mutations involved in target binding at amino acids 351, 356, and / or 379 (e.g., S351E, S351P, S351A, and / or K356E) corresponding to the amino acid numbering of SEQ ID NO: 1, for example, compared to the hyperactive PiggyBac transposase.

[0139] In some embodiments, the modified hyperactive PiggyBac transposase may contain one or more mutations important for integration, for example, at amino acids 560, 564, 571, 573, 589, 592, and / or 594 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., T560A, S564P, S571N, S573A, M589V, S592G, and / or F594L), as compared to the hyperactive PiggyBac transposase.

[0140] In some embodiments, the modified hyperactive PiggyBac transposase may contain one or more mutations involved in alignment, for example, at amino acids 325, 347, 350, 357, and / or 465 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., G325A, N347A, N347S, T350A, and / or W465A), as compared to the hyperactive PiggyBac transposase.

[0141] In some embodiments, the modified hyperactive PiggyBac transposase may contain one or more mutations that are well conserved, for example, at amino acids 576 and / or 587 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., K576A and / or I587A), as compared to the hyperactive PiggyBac transposase.

[0142] In some embodiments, the modified hyperactive PiggyBac transposase may contain one or more mutations involved in the binding of Zn 2+ , for example, at amino acid 586 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., H586A), as compared to the hyperactive PiggyBac transposase.

[0143] In some embodiments, the modified hyperactive PiggyBac transposase may contain one or more mutations involved in integration, for example, at 315, 341, 372, and / or 375 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., R315A, R341A, R372A, and / or K375A), as compared to the hyperactive PiggyBac transposase.

[0144] In some embodiments, the modified hyperactive PiggyBac transposase is selected for its high specificity of DNA integration into the genome as compared to hyperactive PiggyBac.

[0145] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence having one or more of the modifications disclosed herein as compared to SEQ ID NO: 1, and retains at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with any of SEQ ID NOs: 5-26.

[0146] In some embodiments, the modified hyperactive PiggyBac transposase may include one or more mutations of amino acids selected from amino acids 245, 275, 277, 325, 347, 351, 372, 375, 388, 450, 465, 560, 564, 573, 589, 592, and 594 corresponding to the numbering of the amino acids of SEQ ID NO: 1.

[0147] In some embodiments, the modified hyperactive PiggyBac transposase may include one or more of the amino acid substitutions selected from R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G, and F594L corresponding to the numbering of the amino acids of SEQ ID NO: 1.

[0148] In some embodiments, the modified hyperactive PiggyBac transposase includes the amino acid substitution D450N corresponding to the numbering of the amino acids of SEQ ID NO: 1.

[0149] In some embodiments, the modified hyperactive PiggyBac transposase includes the amino acid substitutions R245A and D450 corresponding to the numbering of the amino acids of SEQ ID NO: 1.

[0150] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid substitutions R245A, G325A, and S573P corresponding to the amino acid numbering of SEQ ID NO: 1.

[0151] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid substitutions R245A, G325A, D450, and S573P corresponding to the amino acid numbering of SEQ ID NO: 1.

[0152] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid substitution N347S or N347A corresponding to the amino acid numbering of SEQ ID NO: 1.

[0153] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid substitutions N347S and D450N corresponding to the amino acid numbering of SEQ ID NO: 1.

[0154] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid substitutions N347A and D450N corresponding to the amino acid numbering of SEQ ID NO: 1. This modified hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 14.

[0155] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid sequence having SEQ ID NO: 1, the amino acid residue at position 34 is V or M, the amino acid residue at position 43 is T or I, the amino acid residue at position 177 is Y or H, the amino acid residue at position 202 is R or K, the amino acid residue at position 230 is S or N, the amino acid residue at position 245 is A, the amino acid residue at position 268 is D or N, The 277th amino acid residue is R or A, The 275th amino acid residue is R or A, The 277th amino acid residue is R or A, The 325th amino acid residue is A or G, The 347th amino acid residue is S or A, The 351st amino acid residue is E, P, or A, The 372nd amino acid residue is R or A, The 375th amino acid residue is K or A, The 388th amino acid residue is R or A, The 409th amino acid residue is K or A, The 411th amino acid residue is A or T, The 412th amino acid residue is K or A, The 450th amino acid residue is D or N, The 460th amino acid residue is R or A, The 465th amino acid residue is W or A, The 517th amino acid residue is S or A, The 560th amino acid residue is T or A, The 564th amino acid residue is P or S, The 571st amino acid residue is S or N, The 573rd amino acid residue is S or A, The 576th amino acid residue is K or A, The 586th amino acid residue is H or A, The 587th amino acid residue is I or A, The 589th amino acid residue is M or V, The 592nd amino acid residue is G or S, and / or The 594th amino acid residue is L or F.

[0156] In some embodiments, the modified hyperactive PiggyBac transposase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

[0157] In some embodiments, the modified hyperactive PiggyBac transposase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

[0158] In some embodiments, the modified hyperactive PiggyBac transposase is not a Himar1C9 variant.

[0159] In some embodiments, the transposase is a Sleeping Beauty transposase.

[0160] In some embodiments, the transposase is a hyperactive Sleeping Beauty SB100 transposase having the amino acid sequence of SEQ ID NO: 61. [Table 2]

[0161] In some embodiments, the transposase is a modified hyperactive Sleeping Beauty SB100 transposase that contains at least one amino acid substitution, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, compared to the amino acid sequence of the unmodified Sleeping Beauty transposase.

[0162] In some embodiments, the modified hyperactive Sleeping Beauty SB100 transposase contains one or more of the amino acid substitutions at positions selected from C176, H187, I212, P247, and K248 corresponding to the numbering of the amino acids of SEQ ID NO: 61.

[0163] In some embodiments, the modified hyperactive Sleeping Beauty SB100 transposase contains one or more of the amino acid substitutions selected from C176S, H187V / P, I212S, P247R / S, and K248A / C / I / L / M / N / R / S / T / V corresponding to the numbering of the amino acids of SEQ ID NO: 61.

[0164] In some embodiments, the transposase or a fragment thereof has a decreased catalytic activity. As used herein, "decreased catalytic activity" means a decrease of 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or more, compared to the wild-type transposase and / or the hyperactive transposase.

[0165] In some embodiments, the transposase or a fragment thereof has no catalytic activity. In some embodiments, the transposase or a fragment thereof exhibits no catalytic activity. In some embodiments, the transposase having no catalytic activity retains its ability to bind to other proteins, polypeptides, and / or nucleic acid molecules.

[0166] In some embodiments, a non-catalytic transposase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more sequence identity with a highly active non-catalytic PiggyBac transpose (dead hyPB) having SEQ ID NO: 3. In some embodiments, the non-catalytic hyPB comprises or consists of an amino acid sequence having SEQ ID NO: 3. [Table 3]

[0167] In some embodiments, the protein or polypeptide comprises a transposase fragment.

[0168] In some embodiments, the transposase fragment comprises or consists of at least one functional domain of a transposase.

[0169] In some embodiments, the transposase fragment comprises or consists of an ITR binding domain.

[0170] Examples of ITR binding domains include, but are not limited to, SEQ ID NOs: 62-64, where SEQ ID NOs: 62 and 63 are the ITR binding domains of highly active PiggyBac transposase, and SEQ ID NO: 64 is the ITR binding domain of Sleeping Beauty transposase (also referred to as the "N57 targeting domain"). SEQ ID NO: 62 ILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF SEQ ID NO: 63 MKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF SEQ ID NO: 64 MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYR

[0171] In some embodiments, the transposase fragment is a fragment of a hyperactive PiggyBac transposase or a Sleeping Beauty transposase.

[0172] In some embodiments, the transposase fragment is a fragment of a hyperactive PiggyBac transposase, preferably comprising an ITR binding domain.

[0173] In some embodiments, the transposase fragment is a fragment of a Sleeping Beauty transposase, preferably selected from the group consisting of the SB100 domain and the N57 domain of the Sleeping Beauty transposase.

[0174] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof is a fusion protein comprising the transposase or a fragment thereof and at least one additional polypeptide or protein.

[0175] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof is a fusion protein comprising the transposase or a fragment thereof and at least two additional polypeptides or proteins.

[0176] In some embodiments, the fusion protein comprises a single continuous polypeptide chain.

[0177] In some embodiments, the fusion protein further comprises at least one linker, particularly at least one peptide linker, between the two polypeptides or proteins of the fusion protein.

[0178] Exemplary linkers include, but are not limited to, (G) n , (GS) n , (GGS) n , (GGGS) n (with SEQ ID NO: 49), (GGGGS) n (with SEQ ID NO: 50), (EAAAK) n (with SEQ ID NO: 51), the XTEN linker, and (XP) n , and combinations thereof (where n is an integer from 1 to 50).

[0179] In some embodiments, the peptide linker is a glycine / serine-rich linker. In some embodiments, the peptide linker has an amino acid sequence selected from the group consisting of or including SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46. SEQ ID NO: 41: GGSGGGSGGG SEQ ID NO: 42: GGSGGSGGSGGS SEQ ID NO: 43: GGSGGSGGSGGSGGS SEQ ID NO: 44: GGSGGSGGSGGSGGSG SEQ ID NO: 45: GGSGGSGGSGGSGGSGGSGGS SEQ ID NO: 46: GGSGGSGGSGGSGGSGGSGGSGGS

[0180] In some embodiments, the peptide linker is an XTEN linker consisting of or including SEQ ID NO: 47. SEQ ID NO: 47: SGSETPGTSESATPES

[0181] In some embodiments, the peptide linker consists of or includes SEQ ID NO: 48. SEQ ID NO: 48: GSAGSAAGSGEF

[0182] In some embodiments, the peptide linker consists of or includes SEQ ID NO: 55. SEQ ID NO: 55: GIHGVPAA

[0183] Expression vectors containing the coding sequence of the fusion protein can be constructed using methods well known to those skilled in the art in conjunction with appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Sambrook et al., 2012 (Molecular cloning: A laboratory manual (4th ed.). Cold Spring Harbor Laboratory Press). The expression vector may be a plasmid, part of a virus, or a nucleic acid fragment. The expression vector contains an expression cassette in which a polynucleotide encoding the fusion protein (i.e., the coding region) is operably linked and cloned to a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, but this may be considered part of the coding region if present, although for example any adjacent sequences such as a promoter, ribosome binding site, transcription termination factor, intron, 5' and 3' untranslated regions, etc. are not part of the coding region. Two or more coding regions can be present in a single polypeptide construct, for example on a single vector, or in separate polynucleotide constructs, for example on separate (different) vectors. Furthermore, any vector may contain a single coding region or two or more coding regions, for example the vectors of the present invention may encode one or more polypeptides that are separated post-translationally or simultaneously with translation into the final product via proteolytic cleavage. Additionally, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions that are or are not fused to a polynucleotide encoding the fusion protein (fragment), or a variant or derivative thereof, of the present invention.The heterologous coding region includes, but is not limited to, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable linkage is a linkage in which a coding region for a gene product, such as a polypeptide, is joined to one or more regulatory sequences in such a way that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (e.g., a polypeptide coding region and a promoter joined thereto) are "operably linked" when induction of promoter function results in transcription of mRNA encoding the desired gene product and the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or with the ability of the DNA template to be transcribed. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide when the promoter is capable of effecting transcription of the nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of DNA in a given cell. Other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to a polynucleotide so as to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional control regions are known to those of skill in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments derived from cytomegalovirus (e.g., the immediate early promoter joined to intron A), promoter and enhancer segments derived from simian virus 40 (e.g., the early promoter), and promoter and enhancer segments derived from retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., the tetracycline-inducible promoter).Similarly, various translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation codons and translation termination codons, as well as elements derived from viral systems (in particular, internal ribosome entry sites or IRES, also known as CITE sequences). Expression cassettes may also include other characteristics, such as origins of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs), or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0184] Fusion proteins prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, molecular sieve chromatography, etc. The actual conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., which will be apparent to those of ordinary skill in the art. For purification by affinity chromatography, an antibody, ligand, receptor, or antigen to which the fusion protein binds can be used. The purity of the fusion protein can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high performance liquid chromatography, etc.

[0185] In some embodiments, the nucleic acid encoding the fusion protein can be expressed as a single nucleic acid molecule encoding the entire fusion protein or as multiple (e.g., two or more) nucleic acid molecules co-expressed. The polypeptides encoded by the co-expressed nucleic acid molecules may be joined, for example, via disulfide bonds or other means, to form a functional fusion protein.

[0186] In some embodiments, the fusion protein comprises or consists of a transposase or a fragment thereof fused directly or indirectly via a linker to the C-terminus of at least one protein or polypeptide. In other embodiments, the fusion protein comprises or consists of a transposase or a fragment thereof fused directly or indirectly via a linker to the N-terminus of at least one protein or polypeptide.

[0187] In some embodiments, the fusion protein is a triple fusion protein, i.e., the fusion protein comprises a transposase or a fragment thereof and at least two additional polypeptides or proteins.

[0188] In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). Generally, one or more NLSs have sufficient strength to drive the accumulation of the fusion protein in the cell nucleus. Generally, the strength of the nuclear localization activity is determined by the number and position of the NLSs and one or more specific NLSs used for the fusion protein.

[0189] In some embodiments, the NLS can be located at the N-terminus and / or C-terminus of the fusion protein. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the N-terminus. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the C-terminus. In some embodiments, the fusion protein comprises combinations thereof, e.g., one or more NLSs at the N-terminus and one or more NLSs at the C-terminus.

[0190] When more than one NLS is present, each NLS can be selected independently of the other NLSs. In some embodiments, the fusion protein comprises two NLSs, e.g., the two NLSs are located at the N-terminus and C-terminus, respectively.

[0191] Generally, an NLS consists of one or more short sequences of positively charged lysine or arginine residues exposed on the surface of a protein, although other types of NLSs are also known in the art. Non-limiting examples of NLSs include (M)KKRKV (with SEQ ID NO: 52), (M)PKKKRKV (with SEQ ID NO: 53), or (M)SGGSPKKKRKV (with SEQ ID NO: 54) (where (M) represents an initiator methionine that may be present if the NLS is located at the N-terminus or may be removed after translation and is not present if the NLS is located at the C-terminus).

[0192] Furthermore, the fusion protein may also include other localization sequences, such as a cytoplasmic localization sequence, a chloroplast localization sequence, a mitochondrial localization sequence, etc., depending on the desired localization of the fusion protein in the cell.

[0193] In some embodiments, at least one additional polypeptide or protein is a nuclease. In some embodiments, the nuclease is an endonuclease or an exonuclease. In some embodiments, the nuclease is a deoxyribonuclease or a ribonuclease.

[0194] In some embodiments, at least one additional polypeptide or protein is an RNA-guided nuclease.

[0195] In some embodiments, at least one additional polypeptide or protein is a Cas nuclease.

[0196] In some embodiments, the Cas nuclease is selected from the group consisting of or including Cas9, Cas12a (Cpf1), Cas12b, Cas12f, and CasX. It should be understood that variants and functional fragments thereof, such as nickase Cas (nCas) or dead Cas (dCas) variants, are also encompassed.

[0197] Examples of Cas9 nucleases include, but are not limited to, Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus haemolyticus (ShCas9), and Campylobacter jejuni Cas9 (CjCas9).

[0198] In some embodiments, the Cas nuclease has at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the sequence of SpCas9 having SEQ ID NO: 27, ShCas9 having SEQ ID NO: 28, Cpf1 having SEQ ID NO: 29, CjCas9 having SEQ ID NO: 30, nCas9 having SEQ ID NO: 31, and / or nCas9 having SEQ ID NO: 32. [Table 4] TIFF2025518638000005.tif242155TIFF2025518638000006.tif242159TIFF2025518638000007.tif242155TIFF2025518638000008.tif91159

[0199] In some embodiments, the Cas nuclease has at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of ShCas9 having SEQ ID NO: 28 or SpCas9 having SEQ ID NO: 27.

[0200] In some embodiments, the Cas nuclease is ShCas9 having SEQ ID NO: 28. In some embodiments, the Cas nuclease is SpCas9 having SEQ ID NO: 27.

[0201] In some embodiments, at least one additional polypeptide or protein is a variant or functional fragment of Cas9 nuclease. The Cas9 variant typically includes one or more amino acid substitutions compared to the wild-type amino acid sequence of said Cas9. In some embodiments, the Cas9 variant is a humanized Cas9 (hCas9) or a functional fragment thereof. As used herein, the term "humanized Cas9" or "hCas9" refers to a Cas9 protein sequence optimized for expression in human cells. In some embodiments, hCas9 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 33. In some embodiments, hCas9 has an amino acid sequence consisting of SEQ ID NO: 33.

Table 5

[0202] In some embodiments, at least one additional polypeptide or protein is CasX. In some embodiments, CasX has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 34. In some embodiments, CasX has an amino acid sequence consisting of SEQ ID NO: 34.

Table 6

[0203] In some embodiments, at least one additional polypeptide or protein is a dead Cas9 protein (dCas9). In some embodiments, dCas9 has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 35. In some embodiments, dCas9 has an amino acid sequence consisting of SEQ ID NO: 35.

Table 7

[0204] In some embodiments, at least one additional polypeptide or protein is TnpB (transposase B from transposon PsiTn554). In some embodiments, TnpB has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 36. In some embodiments, TnpB has an amino acid sequence consisting of SEQ ID NO: 36. [Table 8]

[0205] In some embodiments, at least one additional polypeptide or protein is Cas12f. In some embodiments, the Cas12f protein is derived from the bacterium Acidibacillus sulfuroxidans (AsCas12f). In some embodiments, Cas12f has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 37. In some embodiments, Cas12f has an amino acid sequence consisting of SEQ ID NO: 37. [Table 9]

[0206] In some embodiments, the protein or polypeptide comprising the transposase or a fragment thereof is a fusion protein comprising the transposase or a fragment thereof and the nuclease described above herein.

[0207] In some embodiments, the fusion protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity with SEQ ID NO: 2. In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 2.

Table 10

[0208] In one embodiment, at least one additional polypeptide or protein is an aptamer-binding protein.

[0209] In some embodiments, at least one additional polypeptide or protein is an aptamer-binding protein selected from the group consisting of or including the MS2 bacteriophage coat protein (MCP), the PP7 coat protein (PCP), the λN22 peptide, and COM.

[0210] In some embodiments, at least one additional polypeptide or protein is the MS2 bacteriophage coat protein (MCP). In some embodiments, the MCP has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 39.

Table 11

[0211] The MCP is capable of binding to the MS2 RNA tetraloop binding sequence (or “MS2 aptamer”). In some embodiments, the MS2 aptamer has an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 40.

Table 12

[0212] In some embodiments, the fusion protein can interact or be capable of interacting, covalently or non-covalently, via the MCP, with a guide RNA (gRNA) molecule comprising at least one MS2 aptamer.

[0213] Thus, the method of the invention may further comprise contacting a cell population with a guide RNA (gRNA) fused to at least one aptamer.

[0214] In some embodiments, the aptamer is an RNA sequence comprising a tetraloop. The term "tetraloop" refers to a 4-base hairpin loop motif. This term is used interchangeably herein with the terms "stem loop" or "hairpin loop".

[0215] In some embodiments, the aptamer is an MS2 RNA tetraloop sequence (or MS2 aptamer). In some embodiments, the MS2 aptamer has a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 99%, or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 40. In some embodiments, the aptamer comprises or consists of the nucleic acid sequence having SEQ ID NO: 40.

[0216] In some embodiments, the gRNA can form a complex with an RNA-guided nuclease described hereinabove, such as a Cas protein or a fusion protein comprising a Cas protein. In some embodiments, the gRNA molecule can interact or be capable of interacting, covalently or non-covalently, with an RNA-guided nuclease via at least one MS2 aptamer.

[0217] In some embodiments, the gRNA is capable of targeting an RNA-guided nuclease described herein to a specific sequence or region of the genome of a cell, preferably a mammalian cell, more preferably a human cell. In some embodiments, the specific sequence targeted by the gRNA is adjacent to a protospacer adjacent motif (PAM) specific to the Cas protein.

[0218] In some embodiments, the specific sequence targeted by the gRNA can be within a safe harbor locus in the genome of the cell. A "safe harbor locus" refers to a region of the genome of a cell that can appropriately express an integrated substance without disrupting the structure or function of an endogenous gene. Safe harbor loci include, but are not limited to, AAVS1 (intron 1 of PPP1R12C), HPRT, HI1, hRosa26, albumin, and the F region. A safe harbor locus can be an exon or intron of a gene with broad expression and / or a gene with tissue (e.g., muscle) - specific expression. A safe harbor locus can be selected from the group consisting of exon 1, intron 1, or exon 2 of PPP1R12C; exon 1, intron 1, or exon 2 of HPRT; exon 1, intron 1, or exon 2 of hRosa26; or intron 1 of the albumin gene. A safe harbor locus can also include a region of the genome with open chromatin that lacks an endogenous gene and allows for the expression of an introduced gene inserted without disrupting the structure or function of the genome.

[0219] In some embodiments, the gRNA comprises 20, 25, 30, 35, 40, 45, 50, or more nucleotides.

[0220] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell in protein form.

[0221] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell in the form of a nucleic acid encoding the transposase or a fragment thereof. The nucleic acid can be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0222] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is an RNA molecule. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is a messenger RNA (mRNA) molecule.

[0223] Alternatively, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof can be a DNA molecule. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is complementary DNA (cDNA). In some embodiments, the DNA molecule can be contained within a vector, such as a plasmid.

[0224] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is linear or circular. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is double-stranded or single-stranded.

[0225] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population by transfection or transformation.

[0226] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population by transfection. Examples of transfection techniques include, but are not limited to, lipofection, electroporation, sonication, nanoparticles, microinjection, and viral vector infection including non-integrating viral vector infection and integrating viral vector infection.

[0227] In some embodiments, transfection is performed by lipofection. Means of performing lipofection are known in the art and include, for example, using a lipofection reagent such as Lipofectamine™. In some embodiments, transfection is performed by electroporation. In some embodiments, transfection is performed by sonication. In some embodiments, transfection is performed by nanoparticles (e.g., polymeric nanoparticles, e.g., JetPEI). In some embodiments, transfection is performed by microinjection. In some embodiments, transfection is performed by viral vector infection. In some embodiments, the viral vector is integrating or non-integrating, preferably non-integrating. Non-limiting examples of non-integrating viral vectors include adenoviral vectors, adeno-associated virus (AAV) vectors, poxviral vectors, herpes simplex virus vectors, and the like.

[0228] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population by transformation. Examples of transformation techniques include, but are not limited to, the use of an integrating vector selected from the group consisting of or comprising an integrating viral vector, an integrating plasmid, an enzyme, or a genome editing method. In some embodiments, transformation comprises using an integrating viral vector or a modified integrating virus.

[0229] In some embodiments, the integrating viral vector or modified integrating virus is an integrating virus or a variant or mutant thereof selected from the group consisting of or including the viral families Retroviridae, Adenoviridae, Flaviviridae, Herpesviridae, Hepadnaviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, and Paramyxoviridae, preferably an integrating virus or a variant or mutant thereof selected from the group consisting of or including the viral families Retroviridae, Adenoviridae, and Flaviviridae. In some embodiments, the integrating viral vector belongs to the family Retroviridae. In some embodiments, the integrating viral vector is a lentivirus or a modified lentivirus.

[0230] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population together with a nucleic acid molecule encoding at least one transgene of interest. In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof and the nucleic acid molecule encoding at least one transgene of interest are delivered by the same vector. Alternatively, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof and the nucleic acid molecule encoding at least one transgene of interest are delivered simultaneously by separate vectors.

[0231] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population before the nucleic acid molecule encoding at least one transgene of interest, for example 1, 2, 3, 4, 5, 10, 30, 60 minutes, 2, 3, 4, 5, 6, 12, 24, 36, 48, 60, 72 hours, 4, 5, 6, 7 days, or more days before the nucleic acid molecule encoding at least one transgene of interest.

[0232] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population 1 to 72 hours before the nucleic acid molecule encoding at least one transgene of interest.

[0233] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population 2 to 72 hours, 3 to 72 hours, 4 to 72 hours, 5 to 72 hours, 6 to 72 hours, 7 to 72 hours, 8 to 72 hours, 9 to 72 hours, 10 to 72 hours, 11 to 72 hours, 12 to 72 hours, 24 to 72 hours, 36 to 72 hours, 48 to 72 hours before the nucleic acid molecule encoding at least one transgene of interest.

[0234] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population 1 to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 11 hours, 1 to 10 hours, 1 to 9 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours before the nucleic acid molecule encoding at least one transgene of interest.

[0235] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population 1 to 12 hours, 2 to 8 hours, 3 to 6 hours before the nucleic acid molecule encoding at least one transgene of interest.

[0236] In some embodiments, the nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population approximately 4 hours before the nucleic acid molecule encoding at least one transgene of interest.

[0237] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same, is included in a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

[0238] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that does not cause an adverse reaction, allergic reaction, or other undesirable reaction when administered to an animal, preferably a human, or a population of cells. This includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Acceptable excipients refer to any kind of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. For administration to humans, the preparation must meet the sterility, pyrogenicity, general safety, and purity requirements as required by the standards for biological products of the EMA or FDA.

[0239] In some embodiments, acceptable excipients are selected from the group consisting of or including solvents, diluents, carriers, dispersion media, coating agents, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and any combination thereof. The excipient must be "acceptable" in the sense that it is compatible with the protein, polypeptide, or nucleic acid of the method and must not be harmful after administration to an individual or a population of cells. Typically, an excipient does not cause an adverse reaction, allergic reaction, or other undesirable reaction when administered to an individual, preferably a human individual, or a population of cells.

[0240] Excipients acceptable for therapeutic use are well known in the art and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro ed. 1985). The selection of appropriate excipients can be made in accordance with the intended route of administration and standard practice.

[0241] In some embodiments, the cell population is a population of eukaryotic or prokaryotic cells. In some embodiments, the cell population is a population of eukaryotic cells. In some embodiments, the cell population is a population of prokaryotic cells.

[0242] In some embodiments, the cell population is isolated from a donor. As used herein, "donor" refers to an animal, preferably a mammal, more preferably a human. The donor may be alive or dead when the cell population is isolated, preferably alive. In some embodiments, the cell population is isolated from at least one organ or tissue of the donor, optionally from more than one organ or tissue of the donor. In some embodiments, the cell population is homogeneous (i.e., it contains a single type of cell) or heterogeneous (i.e., it contains more than one type of cell), preferably homogeneous.

[0243] In some embodiments, the cell population is cultured in vitro. In some embodiments, the cell population is contained in a primary cell culture.

[0244] In some embodiments, the cell population is derived from an immortalized cell line.

[0245] In some embodiments, the method is performed in vitro. In some embodiments, the cell population is a population of cells cultured in vitro.

[0246] Methods for culturing cells in vitro are well known in the art and are standard practice. Typically, cells are maintained in a controlled atmosphere (e.g., 37°C, 5% CO2) and in an appropriate culture medium (e.g., Dulbecco's Modified Eagle Medium).

[0247] In some embodiments, the cell population is cultured for 2 to 72 hours, 3 to 72 hours, 4 to 72 hours, 5 to 72 hours, 6 to 72 hours, 7 to 72 hours, 8 to 72 hours, 9 to 72 hours, 10 to 72 hours, 11 to 72 hours, 12 to 72 hours, 24 to 72 hours, 36 to 72 hours, 48 to 72 hours. In some embodiments, the cell population is cultured for 1 to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 11 hours, 1 to 10 hours, 1 to 9 hours, 1 to 8 hours, 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours.

[0248] In one embodiment, the cell population is non-dividing, non-replicating, terminally differentiated, or a quiescent cell population. The terms "non-dividing cells", "non-replicating cell population", "terminally differentiated", and "quiescent cell population" are used interchangeably herein.

[0249] As used herein, "non-dividing cells", "non-replicating cells", or "quiescent" cells mean that the cells remain outside the cell cycle but retain the ability to divide, in other words, the cells are in a state of reversible growth arrest. The quiescent state can be induced, alone or in combination, by contact inhibition, chemical or pharmacological agents, signaling proteins, hormones, inhibitors, etc., or by the absence of signals such as growth factors or contact with one or more cell types. In some embodiments, the cell population is in a quiescent state in the presence of one or more growth inhibitors in the culture medium. In some embodiments, the one or more growth inhibitors are selected from the group consisting of or including chemical agents, pharmacological agents, signaling proteins, hormones, and growth factors, and inhibitors. In some embodiments, the cell population is in a quiescent state in the absence of one or more agents that stimulate growth in the culture medium. In some embodiments, the one or more agents that stimulate growth are selected from the group consisting of chemical agents, pharmacological agents, signaling proteins, hormones, growth factors, amino acids, sugars, lipids, and fatty acids. As used herein, "terminally differentiated" cells mean that the cells remain outside the cell cycle and have lost the ability to divide, i.e., the cells are in a state of irreversible growth arrest.

[0250] In some embodiments, the non-dividing or quiescent cell population is a confluent cell culture (i.e., the quiescent state is induced by contact inhibition).

[0251] In some embodiments, the non-dividing or quiescent cell population consists of at least one cell type that does not replicate in culture.

[0252] In some embodiments, the non-dividing or quiescent cell population is senescent.

[0253] In some embodiments, the non-dividing or highly differentiated cell population consists of at least one cell type that has completely lost the ability to divide.

[0254] In some embodiments, the cell population is not actively dividing.

[0255] In another embodiment, the cell population is actively dividing, replicating, or proliferating. In some embodiments, the dividing cell population is a logarithmic-phase cell culture.

[0256] In some embodiments, the method is performed ex vivo.

[0257] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vivo in an animal subject, preferably a mammalian subject. In certain embodiments, the method is performed in vivo in a human subject. In some embodiments, the cell population is contained within a tissue or organ of a living body. In some embodiments, the living body is an animal. In some embodiments, the living body is a mammalian. In some embodiments, the living body is a human.

[0258] Furthermore, the present invention relates to a cell or cell population comprising at least one copy of a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule, and / or at least one copy of at least one transgene of interest. In some embodiments, the cell or cell population comprises at least one copy of both a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same molecule, and at least one copy of at least one transgene of interest.

[0259] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof, or the nucleic acid encoding the same substance, at least one transgene of interest, and the cell or cell population further comprise the characteristics disclosed above herein.

[0260] The present invention further provides a method for treating a genetic disease in a subject in need thereof, the method comprising administering to the subject A nucleic acid molecule encoding a transgene for at least one of the purposes described above in this specification, and A protein or polypeptide comprising a transposase or a fragment thereof described above in this specification, or a nucleic acid encoding the same molecule relates to a method comprising the step of administering.

[0261] The present invention also relates to a nucleic acid molecule encoding a transgene for at least one of the purposes described above in this specification for use in treating a genetic disease in a subject in need thereof, and a protein or polypeptide comprising a transposase or a fragment thereof described above in this specification, or a nucleic acid encoding the same molecule.

[0262] The present invention also relates to the use of a nucleic acid molecule encoding a transgene for at least one of the purposes described above in this specification, and a protein or polypeptide comprising a transposase or a fragment thereof described above in this specification, or a nucleic acid encoding the same molecule, for the manufacture of a medicament for treating a genetic disease in a subject in need thereof.

[0263] In some embodiments, the transgene of interest compensates for a gene defect that causes the genetic disease in the subject.

[0264] In some embodiments, the method further comprises the step of administering to the subject a guide RNA (gRNA) fused to at least one of the aptamers described above in this specification.

[0265] In some embodiments, the method may comprise the step of administering at least one additional therapeutic agent to a subject in need thereof. In some embodiments, the at least one therapeutic agent is for treating a genetic disease.

[0266] In some embodiments, a genetic disease is characterized in that at least one gene is mutated in the genome of a subject, such that the protein encoded by the at least one gene has a dysfunction or is non-functional, or is degraded by the cellular protein quality control (e.g., proteasome), or is not produced; in other words, the function of the at least one gene is at least partially lost, although not complete.

[0267] Non-limiting examples of genetic diseases include sickle cell anemia, cystic fibrosis, Huntington's disease, congenital muscular dystrophy, Duchenne muscular dystrophy, Fabry disease, Marfan syndrome, thalassemia, cystinosis, familial hypercholesterolemia, hemochromatosis, and the like.

[0268] In some embodiments, the transgene of interest partially or completely, preferably completely, restores the function of the at least one mutated gene. In some embodiments, the transgene of interest encodes the same protein as the at least one mutated gene. In some embodiments, the transgene of interest encodes a protein different from the protein encoded by the at least one mutated gene, but which has a similar or identical biological function. BRIEF DESCRIPTION OF THE DRAWINGS

[0269]

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[0270] Example The present invention is further illustrated by the following examples.

[0271] Example 1: Nuclear localization driven by transposase Materials and methods Electroporation of H9 stem cells Embryonic stem cells H9 were electroporated (CRG cell and tissue facility) either alone or with 1 μg of hyPB mRNA or 1.5 μg of FiCAT mRNA and 1.8 μg of a mini-circle GFP transposon combined with 1.75 μg of an sgRNA targeting the AAVS1 locus. Cells were imaged using bright field in a fluorescence microscope to estimate the survival rate and the % of GFP signal.

[0272] Lipofection of HepG2 cells Day 0: 400,000 cells / well were seeded in a p6 well plate.

[0273] Day 1: Either 1.25 μg of hyPB plasmid or puc19 mock plasmid was transfected with Lipofectamine 3000 according to the manufacturer's protocol. Cells were maintained with Optimem + transfection mix for 2 hours and then complete medium was added (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 2 mM glutamine, and 100 U of penicillin / 0.1 mg / ml of streptomycin).

[0274] Day 2: Transfected cells were harvested and seeded in a p12 well plate at 150,000 cells / well.

[0275] Day 3: Cells were transfected with 0.75 μg of RFP DNA transposon and 0.25 μg of puc19 filled with DNA using Lipofectamine 3000 according to the manufacturer's protocol. Cells were maintained with Optimem + transfection mix for 2 hours and then complete medium was added.

[0276] Day 4: Fluorescence was measured 24 hours after the transfection on day 4.

[0277] Alternatively, on day 1, cells were transfected with Cas9_SB100 plasmid and Cas9_N57 plasmid instead of hyPB. On day 3, cells were transfected with a GFP transposon containing SB100 ITR and a TCR1 gRNA plasmid.

[0278] Lipofection of Huh7 cells 400,000 cells were seeded 24 hours prior to transfection in a p6 well. Transfection was performed using Lipofectamine 3000 according to the manufacturer's instructions, using 1.8 μg of transposon DNA and 1.95 μg of hyPB or mock mRNA of a similar size. Cells were maintained with Optimem + transfection mix for 2 hours, and then complete medium was added. After transfection, cells were harvested every 3 hours or every 14 hours, and fluorescence was measured by flow cytometry.

[0279] Alternatively, on day 1, cells were transfected with Cas9_SB100 plasmid and Cas9_N57 plasmid instead of hyPB. On day 3, cells were transfected with a GFP transposon containing SB100 ITR and a TCR1 gRNA plasmid.

[0280] Hydrodynamic injection in vivo A total of 10 - 10.2 μg of nucleic acid was injected into 6 - 7 week - old mice (3.2 μg of MC - luciferase transposon, 1.42 μg of hyPB / hyPB_dead mRNA). The nucleic acid was diluted in PBS, and 7% (ml) of the animal's body weight was injected via retro - orbital systemic injection in less than 7 seconds.

[0281] In vivo transfection with JetPEI DNA payloads with different delivery methods and hyPb: 35.6 μg of MC-luciferase DNA transposon were intravenously injected into 5-week-old mice in vivo using JetPEI at an N / P ratio of 7 according to the manufacturer's instructions. Four hours later, LNP filled with 2.5 μg of hyPB mRNA was intravenously injected. The LNP was produced using Benchtop Nanossemblr (Precision Nanosystems).

[0282] DNA payloads with the same delivery method and hyPB: LNP filled with 15 μg of MC-luciferase DNA transposon was intravenously injected into 5-week-old mice with or without LNP filled with 2.5 μg of hyPB. The LNP was produced using Benchtop Nanossemblr (Precision Nanosystems).

[0283] The lipid ratio was 50% Dlin MC3, 10% DSPC helper lipid, 38.5% cholesterol, 1.5% PEG-2000, and the NP ratio was 6. The size of the nanoparticles was 100 - 130 nm, and the encapsulation efficiency was over 90%.

[0284] Whole-body imaging Whole-body imaging of luciferase expression was performed at different time points after administration of FiCAT gRNA transposon or transposon control using an IVIS spectrum imaging system (Caliper Life Sciences). Images were taken 5 minutes after intraperitoneal injection of D-luciferin potassium salt (Gold Biotechnology) according to the manufacturer's instructions. FiCAT:Cas9 hyPB fusion

[0285] Results Embryonic stem cells H9 electroporated with transposase (hyPB or FiCAT, where FiCAT is a Cas9 hyPB fusion protein) showed GFP signals (Figure 1), which means that the GFP transposon DNA entered the nucleus and was translated and transcribed. The episomal signal of GFP transposon DNA without hyPB or FiCAT was low (20% efficiency), and hyPB brought about a stronger GFP signal (70% transfection efficiency).

[0286] The HepG2 hepatocyte cell line already expressing hyPB showed better internalization of the RFP DNA transposon than cells not expressing hyPB (Figure 2). This result was also obtained with the Huh7 hepatocyte cell line (Figure 3). Furthermore, Huh7 cells transfected with hyPB mRNA showed the ability regarding higher DNA transposon uptake and expression compared to cells transfected with mock mRNA (Figures 4 and 5).

[0287] The Huh7 hepatocyte cell line transfected with mRNA having no catalytic action of hyPB showed the ability regarding higher DNA transposon uptake into the nucleus and expression compared to cells transfected with mock mRNA (Figure 6).

[0288] Interestingly, the above results showing that the ability of the transposon to be taken up into the nucleus by hyPB transposase is high compared to cells without hyPB can be generalized to other transposases such as SB100 and N57 (Figure 7).

[0289] Next, the requirements for ITRs related to the DNA payload were tested. The Huh hepatocyte cell line that already expresses hyPB showed better internalization of the GFP DNA transposon than cells that do not express hyPB (cells that had already been transfected with Puc19 plasmid DNA) (using GFP transposons with either one ITR or two ITRs in both cases) (Figures 8A - 8B). This data demonstrates that hyPB promotes the internalization of payload DNA into the nucleus even when only one ITR is present (thus the hyPB molecule can bind to its payload).

[0290] The ability of hyPB to improve transfection was further tested under in vivo conditions (Figure 9). The hyPB transposase shows an advantage in the internalization and expression of the DNA payload even when its catalytic activity is impaired (dead - hyPB). One out of three mice showed luciferase expression 24 hours after hydrodynamic injection of the single payload, which increased to 2 / 3 or 3 / 3 mice when co - injected with hyPB or hyPB without catalytic activity.

[0291] The hyPB transposase shows an advantage in the internalization and expression of the DNA payload when the payload DNA and hyPB mRNA are administered using different delivery methods (Figure 10). When DNA was injected, 50% of the mice showed a luciferase signal 24 hours later, while 100% of the mice showed a signal when hyPB was co - injected using LNP.

[0292] The hyPB transposase exhibits advantages in the internalization and expression of the DNA payload when the payload DNA and hyPB mRNA are administered using the same delivery method (Figure 11). When DNA alone is injected, mice do not show a luciferase signal, but in both cases, co-administering hyPB using LNP restores the signal. This result indicates that hyPB helps in the internalization of DNA into the nucleus and thus it can be expressed.

[0293] Example 2: Nuclear localization in non-dividing cells Materials and methods NIH / 3T3 culture NIH / 3T3 (ATCC) was cultured in DMEM supplemented with 10 or 1% heat-inactivated fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin in a humidified CO2 (5%) incubator at 37°C.

[0294] Assay of dose-dependent effects on quiescent NIH-3T3 cells 60,000 NIH-3T3 cells were seeded and senescence was induced by culturing them in DMEM supplemented with 1% FBS for 48 hours after reaching confluence. Next, the cells were transfected. Transfection was performed using 0.2 μg of transposon DNA alone (episomal) or in combination with a specified amount of hyPB-wt or catalytically inactive hyPB (dead-hyPB) with Lipofectamine 3000 according to the manufacturer's protocol. The cells were incubated overnight with Optimem containing the transfection mix, and then complete medium was added. One hour after adding DMEM, the cells were trypsinized and GFP expression was evaluated by flow cytometry. (Represented as mean ± SEM, n = 4).

[0295] Comparison of transfection in dividing NIH-3T3 cells and (ad) non-dividing NIH-3T3 cells In the cells during replication, 60,000 cells were seeded the day before transfection. In non-dividing 3T3, after reaching confluence, they were cultured in DMEM supplemented with 1% FBS for 48 hours to induce senescence. Next, the cells were transfected. Transfection was performed using Lipofectamine 3000 according to the manufacturer's protocol, alone (episomal) or in combination with 0.25 μg of transposon DNA with 0.25 μg of hyPB-wt or catalytically inactive hyPB (dead-hyPB), and trypsinized at 3-hour intervals for GFP assessment by flow cytometry.

[0296] Differences between groups were analyzed for statistical significance using a two-way analysis of variance (ANOVA) test.

[0297] Isolation of primary hepatocytes The liver was perfused with liver perfusion buffer (HBSS KCl 0.4 g.L -1 , glucose 1 g.L -1 , NaHCO3 2.1 g.L -1 , EDTA 0.2 g.L -1 ), and then digested with liver digestion buffer (DMEM-GlutaMAX 1 g.L -1 glucose, HEPES 15 mM pH 7.4, penicillin / streptomycin 1%, 5 mg / mouse of collagenase IV (C5138 Sigma)). After excision, the liver was placed on ice in plating medium (M199, fetal bovine serum 10%, penicillin / streptomycin 1%, sodium pyruvate 1%, L-glutamine 1%, 1 nM insulin, 1 mM dexamethasone, 2 mg.ml -1 of bovine serum albumin (BSA)). The tissue was homogenized using forceps and then filtered through seeding medium. Next, the cells were washed three times in seeding medium and then seeded onto plates (Thermo Fisher Scientific) coated with collagen in seeding medium. The medium was changed the next morning, and the cells were incubated for 48 hours before transfection.

[0298] Culture and transfection of primary hepatocytes Primary hepatocytes were isolated from wild-type 8-week-old mice and seeded at 75,000 cells per well. After culturing for 48 hours, the cells were transfected. Transfection was performed using 0.2 μg of transposon DNA alone (episomal) or in combination with 0.25 μg of hyPB-wt or catalytically inactive hyPB (dead-hyPB) with Lipofectamine 3000 according to the manufacturer's protocol. The cells were incubated with Optimem containing the transfection mix for 5 hours, and then complete medium was added. Twenty-four hours after transfection, the cells were washed once with PBS and lysed in the appropriate buffer. Luciferase activity was quantified 24 hours after transfection using the Luciferase Assay System (Promega, Madison, WI, USA), which was performed in duplicate. The data are shown as the fold change relative to the luciferase value of untransfected hepatocytes and are expressed in arbitrary units (AU).

[0299] Differences between groups were analyzed for statistical significance using one-way or two-way analysis of variance (ANOVA) tests. When applicable, pairwise comparisons were analyzed by two-sided Student's t-tests. The results are presented as mean ± s.e.m.

[0300] Results To further confirm the role of DNA transposases such as PiggyBac transposase in the nuclear import of DNA transposons, experiments were conducted in non-dividing cells.

[0301] Two different models were selected: in Figures 12 and 13A - 13B, the NIH / 3T3 fibroblast cell line is senescence-inducible, and in Figure 14, primary hepatocytes isolated from mice that do not replicate during culture were used to test the nuclear transfer activity of PiggyBac (hyPB) DNA in non-dividing (quiescent) cells.

[0302] The incremental PiggyBac transposase (hyPB) was tested to examine its dose-dependent effect; furthermore, a catalytically inactive version of the transposase (dead-hyPB) was added as a control for DNA transfer activity alone without transposase insertion activity. Figure 12 shows that the incorporation and expression (GFP) of the DNA transposon increased in cells expressing hyPB or dead-hyPB even at the lowest dose.

[0303] Furthermore, a control comparison of the transduction and expression efficiency of DNA payloads delivered into the cytoplasm of actively dividing NIH / 3T3 cells and non-dividing NIH / 3T3 cells was performed, which confirmed the role of the transposase protein in the nuclear localization of these payloads (Figures 13A - 13B). Figure 13B shows a significantly higher GFP payload expression efficiency in non-dividing NIH-3T3 cells expressing hyPB or dead-hyPB compared to the condition without using the transposase.

[0304] The results using primary cultured hepatocytes also supported this conclusion, indicating that the presence of PiggyBac transposase increased the expression of the transposon as a result of improved nuclear transport, regardless of whether it was catalytically active (hyPB) or inactive (dead-hyPB) (Figure 14).

[0305] In summary, these results demonstrate that DNA transposases increase the nuclear import of DNA transposons, particularly in non-dividing cells, independent of their insertion activity.

Claims

1. An in vitro method for increasing the nuclear localization of a nucleic acid molecule encoding at least one target transgene in a cell population, wherein the cell population is A protein or polypeptide containing a transposase or a fragment thereof, or a nucleic acid encoding such a transposase, and Nucleic acid molecule encoding at least one target transgene Step to bring into contact with Methods that include...

2. The in vitro method according to claim 1, wherein the nucleic acid molecule encoding the at least one target transgene further comprises at least one terminal inversion sequence (ITR).

3. The in vitro method according to claim 1, wherein the protein or polypeptide containing the transposase or fragment is a fusion protein comprising the transposase or fragment and at least one further polypeptide or protein.

4. The in vitro method according to claim 3, wherein the at least one further polypeptide or protein is a nuclease.

5. The in vitro method according to claim 3, wherein the fusion protein has at least 75% amino acid sequence identity with SEQ ID NO:

2.

6. The in vitro method according to claim 1, further comprising the step of bringing the cell population into contact with guide RNA (gRNA).

7. The in vitro method according to claim 3, wherein the at least one further polypeptide or protein is an aptamer-binding protein.

8. The in vitro method according to claim 7, wherein the aptamer-binding protein is MS2 bacteriophage coat protein (MCP).

9. The in vitro method according to claim 8, wherein the aptamer-binding protein is MS2 bacteriophage coat protein (MCP) having SEQ ID NO:

39.

10. The in vitro method according to claim 8, wherein the fusion protein interacts with or is capable of interacting with a gRNA molecule via MCP by covalent or non-covalent bonds, and the gRNA comprises at least one MS2 aptamer.

11. The in vitro method according to claim 9, wherein the fusion protein interacts with or is capable of interacting with a gRNA molecule via MCP by covalent or non-covalent bonds, and the gRNA comprises at least one MS2 aptamer having a ribonucleic acid sequence having Sequence ID No.

40.

12. The method according to claim 1, wherein the transposase is selected from the group consisting of highly active PiggyBac transposase, PiggyBac transposase, Sleeping Beauty transposase, SB11 transposase, Tol2 transposase, Mos1 transposase, and Frog Prince transposase.

13. The method according to claim 1, wherein the transposase is a modified highly active PiggyBac transposase comprising at least one amino acid mutation compared to the amino acid sequence of the highly active PiggyBac transposase having SEQ ID NO:

1.

14. The method according to claim 1, wherein the transposase or its fragment does not have catalytic activity.

15. The method according to claim 14, wherein the non-catalyzing transposase has at least 75% amino acid sequence identity with SEQ ID NO:

3.

16. A pharmaceutical composition comprising a protein or polypeptide containing a transposase or a fragment thereof, or a nucleic acid encoding such transposase, for use in the treatment of a genetic disorder requiring such treatment, The subject requiring the above is further administered a nucleic acid molecule encoding at least one target transgene, The expression of the target transgene in at least one cell of the subject requiring it compensates for the gene deficiency that causes the hereditary disease. Pharmaceutical composition for use.

17. A pharmaceutical composition for use according to claim 16, further comprising the properties described in any one or more of claims 2 to 15.

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