Use of transposase for improving transgene expression and nuclear localization

Abstract

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

Description

Field of the Invention

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

[0003] Having an efficient method to introduce nucleic acids or proteins into cells and tissues is the key to genome engineering. For this purpose, different methods have been developed, such as viral infection, electroporation, polymer and nanoparticle transfection. Although these methods can greatly improve transfection efficiency, it is still a huge challenge to introduce the target DNA into the cell nucleus.

[0004] Indeed, after foreign DNA enters the cell, it will face various obstacles before reaching the nucleus. First, mammalian cells have defense mechanisms designed to sense and regulate foreign DNA, such as the interferon system. Semenova et al. (Nucleicacids research 2019, vol. 47, 19) reported that cytosolic DNA sensors bind to transfected DNA within hours after transfection, leading to a proinflammatory response. Secondly, foreign DNA molecules must pass through the barrier of the nuclear membrane. Vandenbroucke et al. (Nucleic acids research 2007, vol. 35, 12) proposed the use of amphiphilic molecules to regulate the activity of the nuclear pore complex (NPC) to increase nucleocytoplasmic transport; however, amphiphilic molecules are associated with a certain degree of toxicity, and they do not seem to be able to increase the nuclear uptake of certain DNA vectors.

[0005] Additional barriers to the translocation of foreign DNA into the nucleus may include transport, cell type specificity, and cell division (for details, see Bai et al. “Cytoplasmic transport and nuclear import of plasmid DNA.” Bioscience reports, 2017, vol. 37, 6).

[0006] Therefore, the difficulty of foreign DNA to reach the nucleus of eukaryotic cells (in vitro and in vivo) is a problem that many transfection methods try to overcome. The present invention provides a solution, including surrounding the target gene with ITRs and adding a transposase to the transfection mixture to help locate the foreign DNA into the nucleus.

[0007] Transposase is a naturally evolved enzyme that can "cut and paste" or "copy and paste" DNA fragments into the genomic DNA of prokaryotes (such as Tn3, Tn5 and Tn7) or eukaryotes (such as Sleeping Beauty and PiggyBac). These enzymes are found to have lower transposition efficiency in the genomes of some species to avoid genomic toxicity. Transposase binds to the payload DNA containing specific terminal inverted repeats (ITR) (called transposon), copies or cuts its sequence and pastes it into a random or semi-random genomic site. Like most DNA binding proteins, transposase is naturally located in the nucleus due to nuclear localization signals (for example, see Keith et al. "Analysis of the piggyBac transposase reveals afunctional nuclear targeting signal in the 94c-terminal residues." BMC Molecular Biology 2008, vol.9, 72).

[0008] The most advanced technologies include different transposases that have been engineered to increase their efficiency and create tools for mammalian genome editing, such as SB100 or highly active PiggyBac. Transposases have also been fused to DNA binding domains, such as Cas9 (see, for example, WO2022129438 or WO2020243085), dead-Cas9 (see, for example, Hew et al. "RNA-guided piggyBac transposition in human cells." Synthetic Biology 2019, vol. 4, 1), and ZNFs, in order to specifically target and edit a certain genomic DNA location.

[0009] However, although transposases have been described as tools for genome editing (e.g., see 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 insertional activity, either to insert genes into the DNA of host cells or to disrupt genes, which also raises the problem of insertional mutagenesis.

[0010] Furthermore, transfection is often performed on actively dividing (proliferating) cells because they internalize nucleic acids more easily than non-dividing quiescent cells (Bai et al. 2017), and it is particularly difficult to transfect confluent cells or certain cell types that do not divide in culture. Therefore, there is a need to improve transfection efficiency in non-dividing cells.

[0011] Thus, the present invention provides evidence that the use of a transposase and a vector that promotes cytoplasmic transduction of nucleic acids can increase nuclear localization or nuclear uptake of a DNA of interest, where it can then be expressed by the cell (i.e., transiently expressed, transfected) or inserted into genomic DNA. The methods of the present invention also enable efficient transfection of quiescent cells. Finally, the methods of the present invention also enable efficient transfection without insertion activity when using a catalytically inactive transposase. SUMMARY OF THE INVENTION

[0013] One 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, comprising contacting the cell population with:

[0014] - a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, and

[0015] - A nucleic acid molecule encoding at least one transgene of interest.

[0016] 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, comprising contacting the cell population with:

[0017] - a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, and

[0018] - A nucleic acid molecule encoding at least one transgene of interest.

[0019] Another object of the present invention is a method for editing the genome of a cell population, comprising contacting the cell population with:

[0020] - a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, and

[0021] - A nucleic acid molecule encoding at least one transgene of interest.

[0022] Another object of the present invention is a method of treating a genetic disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of:

[0023] - a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, and

[0024] - a nucleic acid molecule encoding at least one transgene of interest,

[0025] wherein expression of the transgene of interest in at least one cell of a subject in need thereof compensates for the genetic defect causing the genetic disease.

[0026] 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.

[0027] In some embodiments, the nucleic acid molecule encoding at least one transgenic target further comprises at least one inverted terminal repeat (ITR) sequence. In some embodiments, the nucleic acid molecule encoding at least one transgenic target further comprises two ITR sequences. In some embodiments, at least one ITR sequence is adjacent to the transgenic target. In some embodiments, the nucleic acid molecule encoding at least one transgenic target further comprises two ITR sequences flanking the transgenic target. In some embodiments, at least one ITR sequence interacts or non-covalently binds to a transposase.

[0028] 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.

[0029] In some embodiments, the transgene of interest is an exogenous gene. In some embodiments, the transgene of interest is an endogenous gene.

[0030] In some embodiments, the nucleic acid molecule encoding at least one transgenic target is contained in a plasmid, a fosmid, a cosmid, an artificial chromosome, or a viral vector. In some embodiments, the nucleic acid molecule encoding at least one transgenic target is contained in a plasmid. In some embodiments, the nucleic acid molecule encoding at least one transgenic target is contained in a viral vector. In some embodiments, the nucleic acid molecule encoding at least one transgenic target is contained in a DNA virus-based vector selected from the group consisting of viruses of the double-stranded DNA virus domain (Duplodnaviria), the single-stranded DNA virus domain (Monodnaviria), and the multi-stranded DNA virus domain (Varidnaviria).

[0031] 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 translocates a nucleic acid molecule encoding at least one transgene of interest to the nucleus, or promotes the translocation of the nucleic acid molecule to the nucleus.

[0032] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof is a fusion protein comprising a transposase or a fragment thereof and at least one additional polypeptide or protein. In some embodiments, at least one additional polypeptide or protein is a nuclease. In some embodiments, at least one additional polypeptide or protein is an RNA-guided nuclease. In some embodiments, at least one additional polypeptide or protein is a Cas nuclease. In some embodiments, at least one additional polypeptide or protein is a Cas9 nuclease.

[0033] In some embodiments, the fusion protein further comprises a linker.

[0034] In some embodiments, the fusion protein has at least 75% amino acid sequence identity to SEQ ID NO: 2. In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0035] In some embodiments, at least one additional polypeptide or protein is an aptamer binding protein. In some embodiments, at least one additional polypeptide or protein is an MS2 phage coat protein (MCP). In some embodiments, the fusion protein covalently or non-covalently interacts with a gRNA molecule comprising at least one MS2 aptamer through MCP, or is capable of covalently or non-covalently interacting. In some embodiments, the gRNA molecule covalently or non-covalently interacts with an RNA-guided nuclease through at least one MS2 aptamer, or is capable of covalently or non-covalently interacting.

[0036] In some embodiments, the transposase is selected from the group consisting of a highly active PiggyBac transposase, a PiggyBac transposase, a Sleeping Beauty transposase, an SB11 transposase, a Tol2 transposase, a Mos1 transposase, and a frog prince transposase. In some embodiments, the transposase is selected from the group consisting of a highly active PiggyBac transposase and a Sleeping Beauty transposase. In some embodiments, the transposase is a highly active PiggyBac transposase. In some embodiments, the transposase is a highly active PiggyBac transposase having an amino acid sequence of SEQ ID NO: 1. In some embodiments, 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 of SEQ ID NO: 1. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the transposase or a fragment thereof has reduced catalytic activity. In some embodiments, the transposase or a fragment thereof is catalytically inactive. In some embodiments, the catalytically inactive transposase has at least 75% amino acid sequence identity to SEQ ID NO: 3. In some embodiments, the catalytically inactive transposase comprises or consists of the amino acid sequence of SEQ ID NO:3.

[0037] In some embodiments, the protein or polypeptide comprises a transposase fragment. In some embodiments, the transposase fragment comprises at least one transposase functional domain or consists of at least one transposase functional domain. In some embodiments, the transposase fragment comprises an ITR binding domain or consists of an ITR binding domain. In some embodiments, the transposase fragment is a fragment of a highly active PiggyBac transposase or a Sleeping Beauty transposase. In some embodiments, the transposase fragment is a fragment of a highly active 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.

[0038] In some embodiments, the nucleic acid encoding the protein or polypeptide comprising a transposase or its fragment is a ribonucleic acid (RNA) molecule or a DNA molecule. In some embodiments, the nucleic acid encoding the protein or polypeptide comprising a transposase or its fragment is an RNA molecule. In some embodiments, the nucleic acid encoding the protein or polypeptide comprising a transposase or its fragment is a messenger RNA (mRNA) molecule. In some embodiments, the nucleic acid encoding the protein or polypeptide comprising a transposase or its fragment is a cDNA molecule or a plasmid. In some embodiments, the nucleic acid encoding the protein or polypeptide comprising a transposase or its fragment is delivered to a cell population by transfection or transformation. In some embodiments, transfection is selected from lipofection, electroporation, sonication, nanoparticles, microinjection and viral vector infection, including non-integrated and integrated viral vector infection. In some embodiments, transformation includes the use of an integrated viral vector or a modified integrated virus.

[0039] 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 transgenic target. In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population before a nucleic acid molecule encoding at least one transgenic target. In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population 1 hour to 72 hours before a nucleic acid molecule encoding at least one transgenic target. In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population about 4 hours before a nucleic acid molecule encoding at least one transgenic target.

[0040] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide is contained in a pharmaceutical composition, which further comprises at least one acceptable excipient.

[0041] In some embodiments, the cell population is a eukaryotic cell population or a 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 cell population cultured in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo.

[0042] In some embodiments, the cell population is contained in a tissue or organ of a living organism. In some embodiments, the organism is an animal. In some embodiments, the organism is a mammal. In some embodiments, the organism is a human.

[0043] In some embodiments, the method comprises the following steps:

[0044] - contacting the cell population with a vector comprising a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof,

[0045] - culturing the cell population for a period of time ranging from 1 hour to 72 hours, and

[0046] - contacting the cell population with a vector comprising a nucleic acid molecule encoding at least one transgene of interest.

[0047] In some embodiments, the method comprises the following steps:

[0048] - contacting the cell population with a vector comprising a protein or polypeptide comprising a transposase or a fragment thereof, and

[0049] - contacting the cell population with a vector comprising a nucleic acid molecule encoding at least one transgene of interest.

[0050] definition

[0051] In the present invention, the following terms have the following meanings:

[0052] "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease (e.g., a protein comprising an active or inactive DNA-cutting binding domain of Cas9 and / or a gRNA-binding domain of Cas9) comprising a Cas9 protein or a fragment thereof. Cas9 nucleases are sometimes also referred to as casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-related nucleases. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains a spacer (a sequence complementary to a previous mobile element) and targets invading nucleic acids. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In a type II CRISPR system, trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein are required for the correct processing of pre-crRNA. TracrRNA can be used as a guide sequence for ribonuclease 3 to assist in the processing of pre-crRNA. Subsequently, the Cas9 / crRNA / tracrRNA complex cuts a linear or circular dsDNA target complementary to the spacer region by endonucleolysis. The target chain that is not complementary to crRNA is first cut by endonucleolysis, and then 3'-5' is trimmed by exonucleolysis. In nature, DNA binding and cleavage generally require protein and two RNAs. However, single guide RNA ("sgRNA" or "gRNA" for short) can be engineered to integrate all aspects of crRNA and tracrRNA into a single RNA type. Cas9 recognizes short motifs (PAM or protospacer adjacent motifs) in CRISPR repeats to help distinguish between self and non-self. Cas9 nuclease sequences and structures 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 (S.pyogenes) and Streptococcus thermophilus (S.thermophilus).

[0053] "Exogenous" refers to any molecule that is not naturally present in the target cell or organism but can be introduced into it by one or more genetic, biochemical or other methods. The natural occurrence of a molecule in a cell or organism can also be determined based on its specific developmental stage and environmental conditions. Thus, for example, a molecule that is only present during muscle embryonic development is an exogenous molecule for an adult muscle cell. Similarly, for non-heat-shocked cells, heat-shock-induced molecules are exogenous molecules. Exogenous molecules can include, for example, functional versions of dysfunctional endogenous molecules or dysfunctional versions of functional endogenous molecules. In contrast, the term "endogenous" refers to any molecule that is normally present in a cell or organism at a specific developmental stage under specific environmental conditions.

[0054] "Fusion protein" refers to a single-chain hybrid polypeptide comprising two or more amino acid sequences fused together (i.e., from two or more different proteins and / or peptides). Two or more amino acid sequences can be fused together via a direct peptide bond or indirectly through a peptide linker. Fusion proteins can especially be completely encoded by a single nucleic acid sequence.

[0055] "Gene" generally refers to a DNA region (i.e., coding region) that encodes a protein. The term can also include DNA regions (i.e., non-coding regions) that do not encode proteins themselves. The latter include, for example, regions that are transcribed into functional non-coding RNA molecules (e.g., transfer RNA, ribosomal RNA, regulatory RNA, etc.). Other non-coding regions regulate the transcription and translation (i.e., regulatory elements) of the coding region, or are used as structural elements (e.g., scaffold / matrix attachment regions), origins of DNA replication, centromeres or telomeres, etc. Regulatory elements include, but are not limited to, promoter sequences, terminators, translation regulatory sequences (e.g., ribosome binding sites [RBS] and internal ribosome entry sites [IRES]), enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0056] "Identity" or "identity" when used in relation to two or more amino acid sequences or two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between two or more strings of amino acid residues or nucleic acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gapped alignments (if any) resolved 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 AM (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (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 MR & 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 give the largest match between the test sequences. Methods to determine identity are described in publicly available computer programs.Preferred computer program methods for determining the identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1Pt 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). Identity can also be determined using the well-known Smith Waterman algorithm.

[0057] "Insertion" and "integration" refer to the addition of one nucleic acid sequence into a second nucleic acid sequence or a genome or portion thereof. Insertion may be "specific," "site-specific," "targeted," or "on-targeted": these adjectives define the insertion of a nucleic acid into a specific site of a second nucleic acid or a specific site of a genome or portion thereof (i.e., a site specifically selected for insertion). In contrast, the adjectives "random," "non-targeted," or "off-targeted" refer to the non-specific and / or unintended insertion of a nucleic acid into an unwanted site. The terms "total" or "overall" refer to the total number of insertions.

[0058] "Linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties.

[0059] "Modified" refers to a protein or nucleic acid sequence that differs from the corresponding unmodified protein or nucleic acid sequence.

[0060] "Mutated" in relation to a sequence (e.g., an amino acid sequence or a nucleic acid sequence) refers to a sequence that is different from a reference sequence (e.g., a wild-type sequence). Typically, the mutant sequence comprises at least one of a substitution, addition, or deletion of one or more residues compared to a reference sequence (e.g., a corresponding wild-type sequence).

[0061] "Mutation" refers to the replacement of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) by another residue; and / or the deletion or insertion of one or more residues within a nucleic acid or amino acid sequence. Mutations are generally described herein by identifying the original residue, then identifying the position of the residue in the sequence, and then identifying the newly replaced residue. Various methods provided herein for performing amino acid substitutions (mutations) are well known in the art and are provided by, for example, Green & Sambrook, 2012 (Molecular cloning: a laboratory manual (4th Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0062] "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, wherein the nucleic acid molecule can be DNA or RNA.

[0063] "Nucleic acid (molecule / sequence)" and "nucleotide sequence" are used interchangeably and refer to any molecule consisting of or containing monomeric nucleotides. A nucleic acid can be an oligonucleotide or a polynucleotide; it can be DNA, RNA, or a mixture thereof. It can be chemically modified or artificial; for example, it includes peptide nucleic acids (PNA), morpholinos, and locked nucleic acids (LNA), as well as glycerol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these nucleic acids is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. In addition, thiophosphate nucleotides can also be used. Other deoxynucleotide analogs include, but are not limited to, methylphosphonates, phosphoamides, dithiophosphates, N3'P5'phosphoamides, and oligoribonucleotide thiophosphates and their 2'O-allyl analogs and 2'O-methylribonucleotide methylphosphonates, which can be used in the nucleic acids of the present disclosure.

[0064] "Polypeptide", "peptide", "protein" and "amino acid sequence" are used interchangeably to refer to a polymer of amino acid residues. Unless otherwise indicated, a 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 a corresponding naturally occurring amino acid.

[0065] "Prevention" and any of its conjugations refer to preventative and prophylactic measures, wherein the objective is to reduce the chance of a subject developing a given pathological condition or disorder within a given period of time. For example, such reduction may be reflected in a delayed onset of at least one symptom of the pathological condition or disorder in the subject.

[0066] "Specificity" refers to the ability to selectively bind to sequences that have a certain sequence identity with a selected sequence.

[0067] "Subject" refers to a mammal, preferably a human. The subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who is awaiting medical care or is receiving medical care or was / is / will be the subject of a medical procedure, or is being monitored for the development of a disease. The term "mammal" herein refers to any mammal, including humans, domestic and farm animals, and zoo, sport or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

[0068] "Transduction" and any variations 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 vehicle (e.g., a virus, viral particle, or viral vector, including but not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses (AAV) and vectors derived therefrom).

[0069] "Transfection" and any of its suffixes refer to the introduction of one or more nucleic acid molecules (DNA and / or RNA) into one or more cells by non-viral means, whether in vitro or in vivo. In other words, "transfection" refers to any method, technique or vehicle known in the art that promotes or increases cytoplasmic transduction of nucleic acid molecules or cargo. Transfection methods are well known in the art and include, for example, lipofection, PEI and electroporation.

[0070] "Transgenic" refers to an exogenous nucleic acid sequence, particularly an exogenous DNA or cDNA encoding a gene product. The gene product can be RNA, a peptide or a protein. In addition to the coding region (CDS) of the gene product, the transgenic can also include or be associated with one or more operating sequences to promote or enhance expression, such as promoters, enhancers, response elements, reporter elements, insulator elements, polyadenylation signals and / or other functional elements. Unless otherwise indicated, the embodiments of the present disclosure can utilize any known suitable promoters, enhancers, response elements, reporter elements, insulator elements, polyadenylation signals and / or other functional elements. Suitable elements and sequences are well known to those skilled in the art.

[0071] "Transposase" refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by a cut-and-paste mechanism or a copy-transposition mechanism. Within the scope of the present invention, the transposase may be a fragment of a transposase, for example, an ITR binding domain or a functional domain, preferably an ITR binding domain.

[0072] "Treatment," "relief," "cure," and any variations thereof refer to therapeutic treatment and do not include prophylactic or preventative measures; wherein the objective is to slow, alleviate, halt, or even reverse (partially or completely) the progression of the target pathological condition or disorder. Those in need of treatment include those already suffering from the disorder as well as those suspected of suffering from the disorder. A subject's target pathological condition or disorder is considered to be successfully "treated" if, after receiving treatment, the subject shows a significant and / or measurable reduction or disappearance of one or more symptoms associated with the pathological condition or disorder; is relieved to some extent; has reduced morbidity and / or mortality; and / or has improved quality of life issues. The above parameters for assessing successful treatment and disease improvement are readily measured by routine procedures with which physicians are familiar.

[0073] As used herein, "vector" refers to any polynucleotide that can carry, for example, a second polynucleotide of interest and, for example, can transfer a gene sequence to a target cell. Thus, the term includes cloning and expression vectors as well as integration vectors. DETAILED DESCRIPTION OF THE INVENTION

[0075] One of the major obstacles that may arise when attempting to express a transgene of interest in a cell population is poor expression levels, which may be caused by the difficulty of the DNA molecule carrying the transgene of interest to reach the cell nucleus. The present invention solves this problem.

[0076] 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.

[0077] 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.

[0078] The present invention also relates to a method for editing the genome of a cell population, in particular by inserting a nucleic acid molecule encoding at least one transgene of interest into the genome of the cell population.

[0079] The present invention also relates to a method for transfecting a cell population, preferably a non-dividing cell population, with a nucleic acid molecule encoding at least one transgene of interest.

[0080] In some embodiments, a nucleic acid molecule encoding at least one transgene of interest is transfected into a 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 and integrating viral vector infection.

[0081] The present invention is based on the inventor's observation that cytoplasmic transposases can deliver nucleic acids to the nucleus, thereby increasing their nuclear localization. Specifically, cytoplasmic transposases can translocate or promote translocation of nucleic acids to the nucleus of cells (including resting cells). Therefore, an increase in nuclear localization can induce an increase in the level of nucleic acid expression. Interestingly, the inventors observed that the increased nuclear localization of the transposase was not dependent on its enzymatic activity.

[0082] It will be appreciated that the DNA molecule encoding at least one transgene of interest, when delivered to the cytoplasm of a cell, will be sensed by the cell's immune system, thereby initiating a response (e.g., an inflammatory response) to the presence of the foreign DNA. In addition, the DNA molecule encoding at least one transgene of interest must also pass through the nuclear membrane to reach the nucleus. These problems are overcome when the DNA molecule encoding at least one transgene of interest is translocated by a cytoplasmic transposase.

[0083] According to the invention, these methods comprise contacting a cell population with:

[0084] - a nucleic acid molecule encoding at least one transgene of interest, and

[0085] - A protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide.

[0086] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is delivered to the cell population by transfection. 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 comprising or consisting of: lipofection, electroporation, sonication, nanoparticles, microinjection, PEI, and viral vector infection, including non-integrating and integrating viral vector infection.

[0087] In some embodiments, the cell population can be contacted with a nucleic acid molecule encoding at least one transgene of interest and a protein or polypeptide comprising or a nucleic acid encoding a transposase or fragment thereof in any order.

[0088] In some embodiments, a population of cells is contacted with a nucleic acid molecule encoding at least one transgene of interest and simultaneously contacted with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the same.

[0089] 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 protein or polypeptide, prior to contacting the cell population with a nucleic acid molecule encoding at least one transgene of interest. In another embodiment, the cell population is contacted with a nucleic acid molecule encoding at least one transgene of interest prior to contacting the cell population with a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide.

[0090] The term "nucleic acid molecule encoding at least one target transgene" refers to a nucleic acid sequence to be inserted into the genome of a cell (preferably a eukaryotic cell, more preferably a mammalian cell (including a human cell)), which encodes at least one target product. The target product can be a protein or a fragment thereof; in this case, the target transgene is referred to as a coding nucleic acid sequence. However, the term also encompasses non-coding nucleic acid sequences, such as nucleic acid sequences that do not encode proteins or fragments thereof but express "RNA genes" (or "non-coding RNA") (e.g., transfer RNA, ribosomal RNA, small RNA, long non-coding RNA, etc.).

[0091] In some embodiments, the target transgene is a nucleic acid sequence encoding a peptide or protein (such as, 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 immunomodulator, a differentiation factor, a suicide protein, a cell cycle modification protein, an antiproliferative protein, an angiogenic factor, an anti-angiogenic factor, a genome editor, a nuclease, a recombinase, a transposase, a neurotransmitter, and a reporter factor, including any precursor thereof, and a fusion protein). In this case, the target sequence can generally be (or is derived from) mRNA, cDNA, gDNA, a synthetic nucleic acid, or any combination thereof.

[0092] In some embodiments, the transgene of interest is optionally a nucleic acid sequence of a non-coding RNA.

[0093] Examples of non-coding RNA 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), splicing leader RNA (SL RNA), catalytic RNA (i.e., ribozymes; such as ribonuclease P, ribonuclease MRP, etc.), antisense RNA (aRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 7SK RNA (7SK), enhancer RNA (eRNA), and RNA aptamers.

[0094] Examples of transgenes of interest include any nucleic acid sequence encoding a molecule of therapeutic interest, such as any nucleic acid sequence encoding a peptide or protein, or a non-coding RNA, that is absent, deficient and / or non-functional in a subject.

[0095] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest comprises at least one transgene and at least one regulatory element. Examples of regulatory elements include, but are not limited to, promoters, enhancers, silencers, insulators, etc. Preferably, at least one regulatory element is located upstream, i.e., 5', of at least one transgene of interest.

[0096] The nucleic acid molecule encoding at least one transgene of interest may be double-stranded or single-stranded.

[0097] 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; preferably, the nucleic acid molecule encoding at least one transgene of interest is a DNA molecule.

[0098] The nucleic acid molecule encoding at least one transgene of interest typically comprises natural nucleotides. However, it may also comprise non-natural nucleotides. As used herein, "natural nucleotides" refer to adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). As used herein, the term "non-natural nucleotides" refers to chemically modified A, T, U, C or G nucleotides.

[0099] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is at least 10 base pairs (bp) in length, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 bp or more.

[0100] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is a DNA molecule selected from the group comprising or consisting of complementary DNA (cDNA) and genomic DNA (gDNA). In some embodiments, the DNA molecule is cDNA. In some embodiments, the DNA molecule is gDNA.

[0101] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest further comprises at least one inverted terminal repeat (ITR) sequence.

[0102] As used herein, "ITR sequence" refers to a nucleic acid sequence naturally present at the end of a eukaryotic transposable element (or transposon). They are referred to as "5'ITR" and "3'ITR". Typically, 5'ITR and 3'ITR are complementary and can form a hairpin structure. ITR sequences can be used for transposase recognition of transposons. In some embodiments, the ITR sequence is palindromic.

[0103] Some non-limiting examples of ITR sequences include SEQ ID NOs: 55 to 60, and complementary sequences thereof.

[0104] SEQ ID NO:55ccctagaaagatagtctgcgtaaaattgacgcatg

[0105] SEQ ID NO:56

[0106] ccctagaaagatagtctgcgtaaaattgacgcatgagataatcaatattgtgacgtacgttaa

[0107] SEQ ID NO:57

[0108] ccctagaaagatagtctgcgtaaaattgacgcatgagataatcaatattgtgacgtacgttaaagataatcat gcgtaaaattgacgcatg

[0109] SEQ ID NO:58gattatctttctaggg

[0110] SEQ ID NO:59cacaatatgattatctttctaggg

[0111] SEQ ID NO:60

[0112] catgcgtcaattttacgcatgattatctttaacgtacgtacgtcacaatatgattatctttctaggg

[0113] In some embodiments, at least one ITR sequence interacts or non-covalently binds to a protein or polypeptide comprising a transposase or a fragment thereof. In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof specifically recognizes and binds to at least one ITR sequence.

[0114] In some embodiments, at least one ITR sequence is adjacent to the transgene of interest. "Adjacent" means that no nucleotides separate the transgene of interest from the at least one ITR sequence. Alternatively, at least one ITR sequence can be separated from the transgene of interest by at least one nucleotide, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides.

[0115] In some embodiments, the nucleic acid molecule encoding at least one transgenic gene of interest comprises two ITR sequences. Preferably, the two ITR sequences are located on either side of the transgenic gene of interest. In some embodiments, the two ITR sequences are directly located on either side of the transgenic gene of interest, i.e., no nucleotides separate the transgenic gene of interest from the two flanking ITR sequences. Alternatively, the transgenic gene of interest may be separated from at least one or both of the two ITR sequences by at least one nucleotide, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides.

[0116] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is stably or transiently expressed by the cell population, preferably stably expressed.

[0117] In some embodiments, the nucleic acid molecule encoding at least one transgenic target is transiently expressed. In a preferred embodiment, the nucleic acid molecule encoding at least one transgenic target is transfected. In some embodiments, transfection includes using at least one vector and / or method to promote or increase cytoplasmic transduction of the nucleic acid molecule encoding at least one transgenic target. Examples of transfection techniques include, but are not limited to, lipofection, electroporation, sonication, nanoparticles, microinjection, PEI, and viral vector infection, including non-integrating and integrating viral vector infection.

[0118] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is a DNA molecule that is inserted into the genome of a cell of the cell population. It should be understood that the term "inserted into the genome" means that the DNA molecule is replicated with the cell genome during mitosis.

[0119] The transgene of interest can be an exogenous gene or an endogenous gene.

[0120] In some embodiments, the transgene of interest 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 a cell by one or more genetic, biochemical or other methods. The natural presence in a cell can be determined based on the specific developmental stage and environmental conditions of the cell. For example, a molecule that is only present in a cell during embryonic development is exogenous to a cell in the adult stage. Similarly, a molecule induced by heat shock cells is exogenous to a non-heat shock cell.

[0121] In some embodiments, the transgenic gene of interest is an endogenous gene. As used herein, "endogenous gene" refers to a gene that naturally exists in at least one copy in the genome of a cell population. In some embodiments, at least one copy can be an allele, i.e., a gene version that contains at least one mutation. In certain embodiments, the expression of a transgenic endogenous gene increases the overall expression level (i.e., transcript) of the gene.

[0122] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is contained in a vector, such as, but not limited to, a plasmid, a fosmid, a cosmid, an artificial chromosome, or a viral vector.

[0123] In some embodiments, the nucleic acid molecule encoding at least one transgenic target gene is contained in a plasmid. The plasmid can be circular or linear, preferably circular. In some embodiments, the nucleic acid molecule encoding at least one transgenic target gene is contained in a fosmid. In some embodiments, the nucleic acid molecule encoding at least one transgenic target gene is contained in a cosmid. In some embodiments, the nucleic acid molecule encoding at least one transgenic target gene is contained in an artificial chromosome (e.g., a human artificial chromosome).

[0124] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest is contained in a viral vector.

[0125] 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 of the double-stranded DNA virus domain, the single-stranded DNA virus domain, or the multi-stranded DNA virus domain.

[0126] In some embodiments, the vector based on DNA virus is derived from a virus of the double-stranded DNA virus domain. The virus of the double-stranded DNA virus domain includes a Herpesvirales virus. Therefore, in some embodiments, the vector based on DNA virus is a vector derived from the Herpesviridae family, for example, from herpes simplex virus.

[0127] In some embodiments, the vector based on DNA virus is derived from a virus of the single-stranded DNA virus domain. The viruses of the single-stranded DNA virus domain include viruses of the papillomaviridae family and the polyomaviridae family. Therefore, in some embodiments, the vector based on DNA virus is a vector derived from the papillomaviridae family or the polyomaviridae family.

[0128] In some embodiments, the vector based on a DNA virus is derived from a virus of the multi-stranded DNA virus domain. The viruses of the multi-stranded DNA virus domain include viruses of the Adenoviridae and Poxviridae families. Therefore, in some embodiments, the vector based on a DNA virus is a vector derived from the Adenoviridae family, such as from an adenovirus; or a vector derived from the Poxviridae family, such as from a vaccinia virus.

[0129] In some embodiments, the nucleic acid molecule encoding at least one transgene of interest can be excised from the vector. In some embodiments, the nucleic acid molecule encoding at least one transgene of interest can be excised from the vector via at least one ITR sequence (usually by a transposase or a variant or fragment thereof).

[0130] 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.

[0131] As used herein, "increased nuclear localization" means that nuclear localization of a nucleic acid molecule encoding at least one transgene of interest is increased 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 in the presence of a protein or polypeptide comprising a transposase or a fragment thereof as compared to the absence of a protein or polypeptide comprising a transposase or a fragment thereof.

[0132] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof translocates or facilitates translocation of a nucleic acid molecule encoding at least one transgene of interest to a cell nucleus. In some embodiments, a transposase or a fragment thereof (i) interacts and / or non-covalently binds to a nucleic acid molecule encoding at least one transgene of interest, and (ii) translocates to a cell nucleus.

[0133] In some embodiments, the transposase or fragment thereof does not insert and / or translocate a nucleic acid molecule encoding at least one transgene of interest into the genome.

[0134] In some embodiments, the transposase or fragment thereof increases the movement of a nucleic acid molecule encoding at least one transgene of interest across 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.

[0135] In some embodiments, the transposase or fragment thereof protects the nucleic acid molecule encoding at least one transgene of interest from the sensing and / or defense mechanisms of the cell. In some embodiments, the transposase or fragment thereof does not induce a pro-inflammatory and / or inflammatory response.

[0136] In some embodiments, the transposase is selected from the group consisting of a hyperactive PiggyBac transposase, a PiggyBac transposase, a Sleeping Beauty transposase, an SB11 transposase, a Tol2 transposase, a Mosl transposase, and a Frog Prince transposase.

[0137] In some embodiments, the transposase is selected from the group consisting of a hyperactive PiggyBac transposase and a Sleeping Beauty transposase.

[0138] In some embodiments, the transposase is a hyperactive PiggyBac transposase.

[0139] In some embodiments, the transposase is a hyperactive PiggyBac transposase having the amino acid sequence of SEQ ID NO:1.

[0140]

[0141]

[0142] In some embodiments, the transposase is a modified high-activity PiggyBac transposase. "Modified high-activity PiggyBac transposase" refers to a transposase comprising one or more amino acid substitutions, typically no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, compared to the high-activity PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1. More specifically, the modified high-activity PiggyBac transposase may comprise (i) one or more amino acid substitutions to increase excision activity compared to the high-activity PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1, and / or (ii) one or more amino acid substitutions to reduce DNA binding activity compared to the high-activity PiggyBac transposase having the amino acid sequence of SEQ ID NO: 1.

[0143] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:1.

[0144] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity.

[0145] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity, wherein the amino acid mutations are 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, amino acid substitutions at positions D198, D201, R202, M212 and / or S213; the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0146] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity, wherein the amino acid mutations are 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 the hyperactive PiggyBac having SEQ ID NO: 1.

[0147] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity, wherein the amino acid mutations are selected from amino acid mutations at positions M194 and / or D450, wherein the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1; preferably, the amino acid substitutions are M194V and / or D450N.

[0148] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to reduce DNA binding activity.

[0149] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to reduce DNA binding activity, wherein the amino acid mutations are selected from amino acid mutations at positions 254, 275, 277, 347, 372, 375 and / or 465; the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0150] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to reduce DNA binding activity, wherein the mutations are selected from R275, N347, R372, K375, R376, E377 and E380, and the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac of SEQ ID NO: 1.

[0151] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to reduce DNA binding activity, wherein the mutations are selected from R372, K375, R376, E377 and E380, and the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1; preferably, the amino acid substitutions are R372A, K375A, R376A, E377A and / or E380A.

[0152] In some embodiments, the modified high-activity PiggyBac transposase comprises one or more amino acid mutations to reduce DNA binding activity, wherein the mutations are selected from N347, R372 and K375, and the position numbers correspond to the amino acid numbers of the high-activity PiggyBac with SEQ ID NO: 1; preferably, the amino acid substitution is N347S, N347A, R372A and / or K375A; more preferably, the amino acid substitution is N347S or N347A.

[0153] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations as defined above to increase excision activity; and one or more amino acid mutations as defined above to reduce DNA binding activity.

[0154] In some embodiments, the modified high-activity PiggyBac transposase includes at least one amino acid substitution to increase the excision activity at position D450, and at least two amino acid substitutions to reduce the DNA binding activity at positions N347, R372 and K375; preferably, the modified high-activity PiggyBac transposase includes double mutations N347S and D450N or triple mutations D450N, R372A and K375A, and the position numbers correspond to the amino acid numbers of the high-activity PiggyBac having SEQ ID NO:1.

[0155] In some embodiments, the modified hyperactive PiggyBac transposase comprises double mutations N347S and D450N, wherein the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0156] In some embodiments, the modified hyperactive PiggyBac transposase as disclosed in the previous embodiments further comprises at least one mutation in the region defined by amino acid position numbers [158-169], e.g., A166S; and / or at least one mutation at positions Y527, R518, K525, N463.

[0157] In some embodiments, the modified hyperactive 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, where the position numbers correspond to the amino acid numbering of the hyperactive PiggyBac having SEQ ID NO: 1.

[0158] In some embodiments, the modified hyperactive piggyBac transposase comprises one or a combination of the following 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, N 357A, 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.

[0159] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or a combination of the following amino acid substitutions:

[0160] -R372A / K375A / D450N, <h2 style=";text-align:left;direction:ltr">

[0161] <h2 style=";text-align:left;direction:ltr"> -R372A / K375A / R376A / D450N,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0162] <h2 style=";text-align:left;direction:ltr"> -K375A / R376A / E377A / E380A / D450N,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0163] <h2 style=";text-align:left;direction:ltr"> -R372A / K375A / R376A / E377A / E380A / D450N,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0164] <h2 style=";text-align:left;direction:ltr"> -M194V,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0165] <h2 style=";text-align:left;direction:ltr"> -M194V / R372A / K375A,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0166] <h2 style=";text-align:left;direction:ltr"> -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0167] <h2 style=";text-align:left;direction:ltr"> -R245A / R275A / R277A / R372A / W465A / M589V,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0168] <h2 style=";text-align:left;direction:ltr"> -R275A / 325A / R372A / T560A,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0169] <h2 style=";text-align:left;direction:ltr"> -N347A / D450N,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0170] <h2 style=";text-align:left;direction:ltr"> -N347S / D450N / T560A / S573A / F594L,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0171] <h2 style=";text-align:left;direction:ltr"> -R202K / R275A / N347S / R372A / D450N / T560A / F594L,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0172] <h2 style=";text-align:left;direction:ltr"> -R275A / N347S / K375A / D450N / S592G,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0173] <h2 style=";text-align:left;direction:ltr"> -R275A / N347S / R372A / D450N / T560A / F594L,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0174] <h2 style=";text-align:left;direction:ltr"> -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0175] <h2 style=";text-align:left;direction:ltr"> -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0176] <h2 style=";text-align:left;direction:ltr"> -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0177] -V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P,

[0178] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0179] -S230N / R277A / N347S / K375A / D450N,

[0180] -T43I / R372A / K375A / A411T / D450N,

[0181] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or

[0182] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,

[0183] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0184] Some preferred modified hyperactive PiggyBac transposases include modified hyperactive PiggyBac comprising one of the following combinations of amino acid substitutions:

[0185] -R372A / K375A / D450N,

[0186] -S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L,

[0187] -R245A / R275A / R277A / R372A / W465A / M589V,

[0188] -N347A / D450N,

[0189] -N347S / D450N / T560A / S573A / F594L,

[0190] -R202K / R275A / N347S / R372A / D450N / T560A / F594L,

[0191] -R275A / N347S / K375A / D450N / S592G,

[0192] -R275A / N347S / R372A / D450N / T560A / F594L,

[0193] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0194] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0195] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0196] -V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P,

[0197] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0198] -S230N / R277A / N347S / K375A / D450N,

[0199] -T43I / R372A / K375A / A411T / D450N,

[0200] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G,

[0201] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, or

[0202] -R275A / 325A / R372A / T560A,

[0203] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0204] In some embodiments, the modified hyperactive PiggyBac transposase comprises one of the following combinations of amino acid substitutions:

[0205] -R245A / R275A / R277A / R372A / W465A / M589V,

[0206] -R275A / 325A / R372A / T560A,

[0207] -N347A / D450N,

[0208] -N347S / D450N / T560A / S573A / F594L,

[0209] -R202K / R275A / N347S / R372A / D450N / T560A / F594L,

[0210] -R275A / N347S / K375A / D450N / S592G,

[0211] -R275A / N347S / R372A / D450N / T560A / F594L,

[0212] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0213] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0214] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0215] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0216] -S230N / R277A / N347S / K375A / D450N,

[0217] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or

[0218] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,

[0219] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0220] In some preferred embodiments, the modified hyperactive PiggyBac transposase comprises one of the following combinations of amino acid substitutions:

[0221] -N347A / D450N,

[0222] -N347S / D450N / T560A / S573A / F594L,

[0223] -R202K / R275A / N347S / R372A / D450N / T560A / F594L,

[0224] -R275A / N347S / K375A / D450N / S592G,

[0225] -R275A / N347S / R372A / D450N / T560A / F594L,

[0226] -R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L,

[0227] -R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G,

[0228] -R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L,

[0229] -G325A / N347S / K375A / D450N / S573A / M589V / S592G,

[0230] -S230N / R277A / N347S / K375A / D450N,

[0231] -G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, or

[0232] -Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,

[0233] The position numbers correspond to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1.

[0234] In some embodiments, the modified hyperactive PiggyBac transposase comprises R372A / K375A / D450N substitutions, the position numbers corresponding to the amino acid numbers of the hyperactive PiggyBac having SEQ ID NO: 1. The modified transposase has the amino acid sequence of SEQ ID NO:4.

[0235] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs: 5-26.

[0236] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs: 5-13.

[0237] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NOs: 14-26.

[0238] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that participate in the conserved catalytic triad, such as at amino acids 268 and / or 346 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., D268N and / or D346N).

[0239] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that are critical for excision, such as at amino acids 287, 287 / 290, and / or 460 / 461 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., K287A, K287A / K290A, and / or R460A / K461A).

[0240] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that are involved in target ligation, such as at amino acids 351, 356, and / or 379 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., S351E, S351P, S351A, and / or K356E).

[0241] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that are essential for integration, such as 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).

[0242] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that participate in the alignment, such as 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).

[0243] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that are highly conserved, such as at amino acids 576 and / or 587 corresponding to the amino acid numbering of SEQ ID NO: 1 (e.g., K576A and / or I587A).

[0244] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that are involved in Zn 2+ Binds, for example, at position 586 corresponding to the amino acid number of SEQ ID NO: 1 (eg, H586A).

[0245] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to the hyperactive PiggyBac transposase that are involved in integration, e.g., at 315, 341, 372, and / or 375 corresponding to amino acid numbering of SEQ ID NO: 1 (e.g., R315A, R341A, R372A, and / or K375A).

[0246] In some embodiments, the modified hyperactive PiggyBac is selected because it has a higher specificity for integrating DNA into the genome than the hyperactive PiggyBac.

[0247] In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence having one or more modifications disclosed herein relative to SEQ ID NO: 1, and maintains at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO: 5-26.

[0248] In some embodiments, the modified hyperactive PiggyBac transposase may comprise a mutation in one or more 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 amino acid numbering of SEQ ID NO: 1.

[0249] In some embodiments, the modified hyperactive PiggyBac transposase mutation may comprise one or more amino acid substitutions corresponding to the amino acid numbers of SEQ ID NO: 1 selected from the group consisting of R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G, and F594L.

[0250] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid substitution D450N corresponding to the amino acid numbering of SEQ ID NO:1.

[0251] In some embodiments, the modified hyperactive PiggyBac transposase comprises amino acid substitutions R245A and D450 corresponding to the amino acid numbering of SEQ ID NO:1.

[0252] In some embodiments, the modified hyperactive PiggyBac transposase comprises amino acid substitutions R245A, G325A, and S573P corresponding to the amino acid numbering of SEQ ID NO:1.

[0253] In some embodiments, the modified hyperactive PiggyBac transposase comprises amino acid substitutions R245A, G325A, D450, and S573P corresponding to the amino acid numbering of SEQ ID NO:1.

[0254] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid substitution N347S or N347A corresponding to the amino acid numbering of SEQ ID NO:1.

[0255] In some embodiments, the modified hyperactive PiggyBac transposase comprises amino acid substitutions N347S and D450N corresponding to the amino acid numbering of SEQ ID NO:1.

[0256] In some embodiments, the modified hyperactive PiggyBac transposase comprises amino acid substitutions N347A and D450N corresponding to the amino acid numbering of SEQ ID NO: 1. The modified hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO:14.

[0257] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence having SEQ ID NO: 1, wherein:

[0258] - the amino acid residue at position 34 is V or M,

[0259] - the amino acid residue at position 43 is T or I,

[0260] - the amino acid residue at position 177 is Y or H,

[0261] - the amino acid residue at position 202 is R or K,

[0262] - the amino acid residue at position 230 is S or N,

[0263] - the amino acid residue at position 245 is A,

[0264] - the amino acid residue at position 268 is D or N,

[0265] - the amino acid residue at position 277 is R or A,

[0266] - the amino acid residue at position 275 is R or A,

[0267] - the amino acid residue at position 277 is R or A,

[0268] - the amino acid residue at position 325 is A or G,

[0269] - the amino acid residue at position 347 is S or A,

[0270] - the amino acid residue at position 351 is E, P or A, - the amino acid residue at position 372 is R or A,

[0271] - the amino acid residue at position 375 is K or A,

[0272] - the amino acid residue at position 388 is R or A,

[0273] - the amino acid residue at position 409 is K or A,

[0274] - the amino acid residue at position 411 is A or T,

[0275] - the amino acid residue at position 412 is K or A,

[0276] - the amino acid residue at position 450 is D or N,

[0277] - the amino acid residue at position 460 is R or A,

[0278] - the amino acid residue at position 465 is W or A,

[0279] - the amino acid residue at position 517 is S or A,

[0280] - the amino acid residue at position 560 is T or A,

[0281] - the amino acid residue at position 564 is P or S,

[0282] - the amino acid residue at position 571 is S or N,

[0283] - the amino acid residue at position 573 is S or A,

[0284] - the amino acid residue at position 576 is K or A,

[0285] - the amino acid residue at position 586 is H or A, - the amino acid residue at position 587 is I or A,

[0286] - the amino acid residue at position 589 is M or V,

[0287] - the amino acid residue at position 592 is G or S, and / or,

[0288] - The amino acid residue at position 594 is L or F.

[0289] 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 to an amino acid sequence selected from the group comprising or 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.

[0290] 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 to an amino acid sequence selected from the group comprising or 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.

[0291] In some embodiments, the modified transposase is not a HimarlC9 mutant.

[0292] In some embodiments, the transposase is a Sleeping Beauty transposase.

[0293] In some embodiments, the transposase is the highly active Sleeping Beauty SB100 transposase having the amino acid sequence of SEQ ID NO:61.

[0294]

[0295]

[0296] In some embodiments, the transposase is a modified hyperactive Sleeping Beauty SB100 transposase comprising at least one amino acid substitution, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions compared to the amino acid sequence of an unmodified Sleeping Beauty transposase.

[0297] In some embodiments, the modified hyperactive Sleeping Beauty SB100 transposase comprises one or more amino acid substitutions at positions selected from C176, H187, I212, P247, and K248 corresponding to the amino acid numbering of SEQ ID NO:61.

[0298] In some embodiments, the modified hyperactive Sleeping Beauty SB100 transposase comprises one or more 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 amino acid numbering of SEQ ID NO:61.

[0299] In some embodiments, the transposase or fragment thereof has reduced catalytic activity. As used herein, "reduced 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 a wild-type transposase and / or a hyperactive transposase.

[0300] In some embodiments, the transposase or its fragment is catalytically inactive. In some embodiments, the transposase or its fragment does not exert any catalytic activity. In some embodiments, the catalytically inactive transposase retains its ability to bind to other proteins, polypeptides and / or nucleic acid molecules.

[0301] In some embodiments, the catalytically inactive transposase has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more sequence identity to a catalytically inactive hyperactive PiggyBac transposase (inactive hyPB) having SEQ ID NO: 3. In some embodiments, the inactive hyPB comprises or consists of an amino acid sequence having SEQ ID NO: 3.

[0302]

[0303]

[0304] In some embodiments, the protein or polypeptide comprises a transposase fragment.

[0305] In some embodiments, the transposase fragment comprises or consists of at least one transposase functional domain.

[0306] In some embodiments, the transposase fragment comprises or consists of an ITR binding domain.

[0307] Examples of ITR binding domains include, but are not limited to, SEQ ID NOs: 62 to 64, wherein SEQ ID NOs: 62 and 63 are ITR binding domains of the hyperactive PiggyBac transposase, and SEQ ID NO: 64 is the ITR binding domain of the Sleeping Beauty transposase (also referred to as the "N57 targeting domain").

[0308] SEQ ID NO:62

[0309] ILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVI CREHNIDMCQSCF

[0310] SEQ ID NO:63

[0311] MKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF

[0312] SEQ ID NO:64

[0313] MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYK HHGTTQPSYR

[0314] In some embodiments, the transposase fragment is a fragment of the hyperactive PiggyBac transposase or Sleeping Beauty transposase.

[0315] In some embodiments, the transposase fragment is a fragment of a hyperactive PiggyBac transposase, preferably comprising an ITR binding domain.

[0316] In some embodiments, the transposase fragment is a fragment of Sleeping Beauty transposase, preferably selected from the group consisting of the SB100 domain and the N57 domain of Sleeping Beauty transposase.

[0317] 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 at least one additional polypeptide or protein.

[0318] 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 at least two additional polypeptides or proteins.

[0319] In some embodiments, the fusion protein comprises a single contiguous polypeptide chain.

[0320] In some embodiments, the fusion protein further comprises at least one linker, in particular at least one peptide linker, between the two polypeptides or proteins of the fusion protein.

[0321] Exemplary linkers include, but are not limited to (G) n , (GS) n 、(GGS) n 、(GGGS) n (having SEQ ID NO:49), (GGGGS)n (having SEQ ID NO:50), (EAAAK) n (having SEQ ID NO: 51), an XTEN linker and (XP) n linker, and any combination thereof, wherein n is an integer between 1 and 50.

[0322] 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: 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.

[0323] SEQ ID NO:41:GGSGGGSGGG

[0324] SEQ ID NO:42:GGSGGSGGSGGS

[0325] SEQ ID NO:43:GGSGGSGGSGGSGGS

[0326] SEQ ID NO:44:GGSGGSGGSGGSGGSG

[0327] SEQ ID NO:45:GGGSGGSGGSGGSGGSGGSGGS

[0328] SEQ ID NO:46:GGSGGSGGSGGSGGSGGSGGSGGS

[0329] In some embodiments, the peptide linker is an XTEN linker comprising or consisting of SEQ ID NO:47.

[0330] SEQ ID NO:47:SGSETPGTSESATPES

[0331] In some embodiments, the peptide linker comprises or consists of SEQ ID NO:48.

[0332] SEQ ID NO:48:GSAGSAAGSGEF

[0333] In some embodiments, the peptide linker comprises or consists of SEQ ID NO:55.

[0334] SEQ ID NO:55:GIHGVPAA

[0335] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the fusion protein and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination. For example, refer to Sambrook et al., 2012 (Molecular cloning: A laboratory manual (4th edition). Cold Spring Harbor Laboratory Press) described in the technology. The expression vector can be a part of a plasmid, a virus, or it can be a nucleic acid fragment. The expression vector includes an expression cassette, and the polynucleotide encoding the fusion protein (i.e., the coding region) is connected and cloned into the expression cassette in an operable manner with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a nucleic acid portion composed of codons translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated into amino acids, if present, it can be regarded as a part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5'- and 3'-untranslated regions, etc., are not a part of the coding region. Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may contain two or more coding regions, for example, the vector of the present invention may encode one or more polypeptides, which are separated into final proteins via proteolytic cleavage after translation or during co-translation. In addition, the vector, polynucleotide or nucleic acid of the present invention may encode heterologous coding regions, which may or may not be fused to a polynucleotide encoding a fusion protein (fragment) of the present invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable connection refers to the connection of a coding region of a gene product (e.g., a polypeptide) to one or more regulatory sequences, so that the expression of the gene product is affected or controlled by the regulatory sequences. If the induction of promoter function results in the transcription of mRNA encoding the desired gene product, and the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed, then the two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably connected". Thus, a promoter region will be operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of the nucleic acid. The promoter may be a cell-specific promoter that directs bulk transcription of the DNA only in a predetermined cell. Other transcription control elements other than promoters, such as enhancers, operators, repressors, and transcription termination signals, may also be operably linked to a polynucleotide to direct cell-specific transcription.Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer fragments from cytomegalovirus (e.g., immediate early promoters bound to intron-A), simian virus 40 (e.g., early promoters), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include transcription control regions derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit α-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline inducible promoters). Similarly, a variety of 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 and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites or IRES, also referred to as CITE sequences). The expression cassette may also include other features, such as an origin of replication and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0336] 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, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens to which the fusion protein is bound 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 pressure liquid chromatography, etc.

[0337] 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 a plurality of (e.g., two or more) nucleic acid molecules of co-expression. The polypeptides encoded by the co-expressed nucleic acid molecules can be associated, for example, by disulfide bonds or other means, to form a functional fusion protein.

[0338] In some embodiments, the fusion protein comprises or consists of a transposase or 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 fragment thereof fused directly or indirectly via a linker to the N-terminus of at least one protein or polypeptide.

[0339] In some embodiments, the fusion protein is a triple fusion protein, ie, the fusion protein comprises a transposase or a fragment thereof, and at least two additional polypeptides or proteins.

[0340] In some embodiments, the fusion protein also includes a nuclear localization sequence (NLS). In general, one or more NLS should have enough strength to drive the fusion protein to accumulate in the nucleus of the cell. In general, the intensity of nuclear localization activity depends on the number and position of the NLS, and the one or more specific NLS used in the fusion protein.

[0341] In some embodiments, 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 NLS. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS near the N-terminus or the N-terminus. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS near the C-terminus or the C-terminus. In some embodiments, the fusion protein comprises a combination of these, such as one or more NLSs located at the N-terminus and one or more NLSs located at the C-terminus.

[0342] When more than one NLS is present, each NLS can be selected independently of the other NLS. In some embodiments, the fusion protein comprises two NLSs, for example, the two NLSs are located at the N-terminus and the C-terminus, respectively.

[0343] In general, NLS consists of a short sequence of one or more positively charged lysine or arginine amino acids exposed on the surface of the protein, but other types of NLS are also known in the art. Non-limiting examples of NLS include (M) KKRKV (having SEQ ID NO: 52), (M) PKKKRKV (having SEQ ID NO: 53) or (M) SGGSPKKKRKV (having SEQ ID NO: 54), wherein (M) represents a promoter methionine, which may be present when the NLS is located at the N-terminus or removed after translation, and is absent when the NLS is located at the C-terminus.

[0344] In addition, the fusion protein may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, etc., depending on the desired localization of the fusion protein in the cell.

[0345] 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.

[0346] In some embodiments, at least one additional polypeptide or protein is an RNA-guided nuclease.

[0347] In some embodiments, at least one additional polypeptide or protein is a Cas nuclease.

[0348] In some embodiments, the Cas nuclease is selected from the group comprising or consisting of Cas9, Cas12a (Cpf1), Cas12b, Cas12f and CasX. It should be understood that variants and functional fragments thereof are also included, such as nickase Cas (nCas) or inactivated Cas (dCas) variants.

[0349] Examples of Cas9 nucleases include, but are not limited to, Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus haemolyticus Cas9 (ShCas9), and Campylobacter jejuni Cas9 (CjCas9).

[0350] In some embodiments, the Cas nuclease has at least 80%, 85%, 90%, 95%, 99% or more amino acid sequence identity to a 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.

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357] In some embodiments, the Cas nuclease has at least 80%, 85%, 90%, 95%, 99% or more amino acid sequence identity to the amino acid sequence of ShCas9 having SEQ ID NO:28 or SpCas9 having SEQ ID NO:27.

[0358] 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.

[0359] In some embodiments, at least one additional polypeptide or protein is a variant or functional fragment of the Cas9 nuclease. Compared with the wild-type amino acid sequence of the Cas9, the Cas9 variant generally comprises one or more amino acid substitutions. 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 an optimized Cas9 protein sequence 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.

[0360]

[0361]

[0362] In some embodiments, the 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 to the amino acid sequence of SEQ ID NO: 34. In some embodiments, CasX has an amino acid sequence consisting of SEQ ID NO: 34.

[0363]

[0364]

[0365] In some embodiments, at least one additional polypeptide or protein is a deactivated 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 to the amino acid sequence of SEQ ID NO: 35. In some embodiments, dCas9 has an amino acid sequence consisting of SEQ ID NO: 35.

[0366]

[0367]

[0368] In some embodiments, the at least one additional polypeptide or protein is TnpB (transposase B from transposon PsiTn554). In some embodiments, TnpB has an amino acid sequence that has at least 80%, 85%, 90%, 95%, 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, TnpB has an amino acid sequence consisting of SEQ ID NO: 36.

[0369]

[0370] In some embodiments, at least one additional polypeptide or protein is Cas12f. In some embodiments, the Cas12f protein is 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.

[0371] In some embodiments, Cas12f has an amino acid sequence consisting of SEQ ID NO:37.

[0372]

[0373] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof is a fusion protein comprising a transposase or a fragment thereof and a nuclease as described above.

[0374] In some embodiments, the fusion protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 2. In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0375]

[0376]

[0377] In some embodiments, at least one additional polypeptide or protein is an aptamer binding protein.

[0378] In some embodiments, the at least one additional polypeptide or protein is an aptamer binding protein selected from the group comprising or consisting of MS2 bacteriophage coat protein (MCP), PP7 coat protein (PCP), λN22 peptide, and COM.

[0379] In some embodiments, at least one additional polypeptide or protein is a MS2 bacteriophage coat protein (MCP). In some embodiments, the MCP has an amino acid sequence that has at least 80%, 85%, 90%, 95%, 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 39.

[0380]

[0381] MCP is capable of binding to MS2 RNA tetraloop binding sequences (or "MS2 aptamers"). In some embodiments, the MS2 aptamer has a nucleic acid sequence that has at least 80%, 85%, 90%, 95%, 99% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO:40.

[0382]

[0383] In some embodiments, the fusion protein covalently or non-covalently interacts or is capable of covalently or non-covalently interacting with a guide RNA (gRNA) molecule comprising at least one MS2 aptamer through the MCP.

[0384] Thus, the methods of the invention may further comprise contacting the cell population with a guide RNA (gRNA) fused to at least one aptamer.

[0385] In some embodiments, the aptamer is an RNA sequence comprising a tetraloop. The term "tetraloop" refers to a four-base hairpin loop motif. The term is used interchangeably with the term "stem loop" or "hairpin loop" herein.

[0386] In some embodiments, the aptamer is a 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 a nucleic acid sequence having SEQ ID NO: 40.

[0387] In some embodiments, the gRNA is capable of forming a complex with a nuclease guided by RNA as described above, such as a Cas protein or a fusion protein comprising a Cas protein. In some embodiments, the gRNA molecule covalently or non-covalently interacts with the nuclease guided by RNA through at least one MS2 aptamer, or is capable of covalently or non-covalently interacting.

[0388] In some embodiments, gRNA is capable of targeting the nuclease guided by RNA as described above 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 gRNA is adjacent to a protospacer adjacent motif (PAM) specific to a Cas protein.

[0389] In some embodiments, the specific sequence targeted by the gRNA may be located within a safe harbor locus in the cell genome. "Safe harbor locus" refers to a region of the cell genome in which the integrated material can be fully expressed without disrupting the structure or function of the endogenous gene. Safe harbor loci include, but are not limited to, AAVS1 (intron 1 of PPP1R12C), HPRT, HI 1, hRosa26, albumin, and FA regions. The safe harbor locus can be an exon or intron of a gene that is ubiquitously expressed and / or has tissue-specific expression (e.g., muscle). The 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. The safe harbor locus can also include a genomic region that lacks endogenous genes and has open chromatin, which allows the expression of inserted transgenes without disrupting the structure or function of the genome.

[0390] In some embodiments, the gRNA comprises 20, 25, 30, 35, 40, 45, 50, or more nucleotides.

[0391] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell in the form of a protein.

[0392] In some embodiments, the protein or polypeptide comprising the transposase or a fragment thereof is delivered to the cell in the form of a nucleic acid encoding the transposase or a fragment thereof. The nucleic acid may be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0393] 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.

[0394] 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 a complementary DNA (cDNA). In some embodiments, the DNA molecule can be contained in a vector, such as a plasmid.

[0395] 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.

[0396] 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 or transformation.

[0397] In some embodiments, nucleic acids encoding proteins or polypeptides comprising transposases or fragments thereof are 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 and integrating viral vector infection.

[0398] In some embodiments, transfection is performed by lipofection. Methods for performing lipofection are known in the art and include using a lipofection reagent, such as lipofectamine TM . In some embodiments, transfection is performed by electroporation. In some embodiments, transfection is performed by ultrasonic treatment. In some embodiments, transfection is performed by nanoparticles (e.g., polymer nanoparticles, such as 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 an integrating or non-integrating type, preferably a non-integrating type. Non-limiting examples of non-integrating viral vectors include adenoviral vectors, adeno-associated virus (AAV) vectors, poxvirus vectors, herpes simplex virus vectors, etc.

[0399] In some embodiments, nucleic acids encoding proteins or polypeptides comprising transposases or fragments thereof are delivered to a cell population by transformation. Examples of transformation techniques include, but are not limited to, the use of an integrative vector selected from the group consisting of: an integrative viral vector, an integrative plasmid, an enzyme, or a genome editing method. In some embodiments, transformation comprises the use of an integrative viral vector or a modified integrative virus.

[0400] 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 comprising or consisting of Retroviridae, Adenoviridae, Flaviviridae, Herpesviridae, Hepadnaviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae and Paramyxoviridae, preferably selected from the group comprising or consisting of Retroviridae, Adenoviridae and Flaviviridae. In some embodiments, the integrating viral vector belongs to the Retroviridae family. In some embodiments, the integrating viral vector is a lentivirus or a modified lentivirus.

[0401] 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 and a nucleic acid molecule encoding at least one transgene of interest are delivered in the same vector. Alternatively, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof and a nucleic acid molecule encoding at least one transgene of interest are delivered simultaneously in different vectors.

[0402] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to a cell population prior to a nucleic acid molecule encoding at least one transgene of interest, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or more prior to a nucleic acid molecule encoding at least one transgene of interest.

[0403] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population between 1 hour and 72 hours prior to a nucleic acid molecule encoding at least one transgene of interest.

[0404] 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 hours to 72 hours, 3 hours to 72 hours, 4 hours to 72 hours, 5 hours to 72 hours, 6 hours to 72 hours, 7 hours to 72 hours, 8 hours to 72 hours, 9 hours to 72 hours, 10 hours to 72 hours, 11 hours to 72 hours, 12 hours to 72 hours, 24 hours to 72 hours, 36 hours to 72 hours, or 48 hours to 72 hours prior to the nucleic acid molecule encoding at least one transgene of interest.

[0405] 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 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 11 hours, 1 hour to 10 hours, 1 hour to 9 hours, 1 hour to 8 hours, 1 hour to 7 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 4 hours, 1 hour to 3 hours, or 1 hour to 2 hours prior to the nucleic acid molecule encoding at least one transgene of interest.

[0406] 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 12 hours, 2 hours to 8 hours, 3 hours to 6 hours prior to a nucleic acid molecule encoding at least one transgene of interest.

[0407] In some embodiments, a nucleic acid encoding a protein or polypeptide comprising a transposase or a fragment thereof is delivered to the cell population about 4 hours prior to a nucleic acid molecule encoding at least one transgene of interest.

[0408] In some embodiments, a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide is contained in a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable excipient.

[0409] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that does not produce adverse, allergic or other undesirable reactions when applied to an animal (preferably a human) or a cell population. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. An acceptable excipient refers to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation excipient. For human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards of the EMA or FDA Office of Biological Products Standards.

[0410] In some embodiments, acceptable excipients are selected from the group comprising or consisting of: solvents, diluents, carriers, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delay agents, and any combination thereof. Excipients must be "acceptable" in the sense that they are compatible with the protein, polypeptide or nucleic acid molecule of the method and are not harmful after being applied to an individual or cell group. Typically, excipients do not produce adverse, allergic or other unwanted reactions when applied to an individual (preferably a human individual) or a cell group.

[0411] Acceptable excipients for therapeutic use are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985). Suitable excipients may be selected with regard to the intended route of administration and standard practice.

[0412] In some embodiments, the cell population is a eukaryotic cell population or a 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.

[0413] In some embodiments, the cell population is separated from a donor. As used herein, "donor" refers to an animal, preferably a mammal, more preferably a human. When the cell population is separated, the donor can be alive or dead, preferably alive. In some embodiments, the cell population is separated 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.

[0414] In some embodiments, the cell population is cultured in vitro. In some embodiments, the cell population is contained in a primary cell culture.

[0415] In some embodiments, the cell population is from an immortalized cell line.

[0416] In some embodiments, the method is performed in vitro. In some embodiments, the cell population is a cell population cultured in vitro.

[0417] Methods of culturing cells in vitro are well known in the art and are routine practice. Typically, cells are maintained in a controlled atmosphere (eg, 37° C., 5% CO 2 ) and in a suitable culture medium (eg, Dulbecco's modified Eagle's medium).

[0418] In some embodiments, the cell population is cultured for 2 hours to 72 hours, 3 hours to 72 hours, 4 hours to 72 hours, 5 hours to 72 hours, 6 hours to 72 hours, 7 hours to 72 hours, 8 hours to 72 hours, 9 hours to 72 hours, 10 hours to 72 hours, 11 hours to 72 hours, 12 hours to 72 hours, 24 hours to 72 hours, 36 hours to 72 hours, 48 ​​hours to 72 hours. In some embodiments, the cell population is cultured for 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 11 hours, 1 hour to 10 hours, 1 hour to 9 hours, 1 hour to 8 hours, 1 hour to 7 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours.

[0419] In one embodiment, the cell population is a non-dividing, non-replicating, terminally differentiated or quiescent cell population.The terms "non-dividing cell population", "non-replicating cell population", "terminally differentiated" and "quiescent cell population" are used interchangeably herein.

[0420] As used herein, "non-dividing" cells, "non-replicating" cells or "quiescent" cells mean that the cells are still outside the cell cycle, but retain the ability to divide, in other words, the cells are in a state of reversible growth arrest. Quiescence can be induced by non-mimetic contact inhibition, chemical or pharmacological agents, signaling proteins, hormones, inhibitors, etc., or by lack of signals (such as growth factors) or contact with one or more cell types. In some embodiments, in the presence of one or more growth inhibitors in the culture medium, the cell group is static. In some embodiments, one or more growth inhibitors are selected from the group comprising or consisting of: chemicals, pharmacological agents, signaling proteins, hormones, growth factors and inhibitors. In some embodiments, in the absence of one or more growth stimulants in the culture medium, the cell group is static. In some embodiments, one or more growth stimulants are selected from the group comprising or consisting of: chemicals, pharmacological agents, signaling proteins, hormones, growth factors, amino acids, sugars, lipids and fatty acids. As used herein, "terminal differentiation" cells refer to cells that are out of the cell cycle and lose the ability to divide. In other words, the cells are in an irreversible state of growth arrest.

[0421] In some embodiments, the population of non-dividing or quiescent cells is a confluent cell culture (ie, quiescence is induced by contact inhibition).

[0422] In some embodiments, the population of non-dividing or quiescent cells consists of at least one cell type that does not replicate in culture.

[0423] In some embodiments, the population of non-dividing or quiescent cells is senescent.

[0424] In some embodiments, a population of non-dividing or terminally differentiated cells consists of at least one cell type that has completely lost the ability to undergo cell division.

[0425] In some embodiments, the population of cells is not actively dividing.

[0426] In another embodiment, the cell population is actively dividing, replicating or proliferating. In some embodiments, the dividing cell population is in the exponential phase of cell culture.

[0427] In some embodiments, the method is performed ex vivo.

[0428] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in an animal subject (preferably a mammalian subject). In some embodiments, the method is performed in a human subject. In some embodiments, the cell population is contained in a tissue or organ of a living organism. In some embodiments, the organism is an animal. In some embodiments, the organism is a mammal. In some embodiments, the organism is a human.

[0429] The present invention also relates to a cell or cell population comprising at least one copy of a protein or polypeptide containing a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, 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 a protein or polypeptide containing a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, and at least one copy of at least one transgene of interest.

[0430] In some embodiments, the protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, at least one transgene of interest, and the cell or cell population further comprise the features as disclosed above.

[0431] The present invention also relates to a method of treating a genetic disease in a subject in need thereof, comprising administering to the subject:

[0432] - a nucleic acid molecule encoding at least one transgene of interest as described above, and

[0433] - A protein or polypeptide comprising a transposase or a fragment thereof as described above, or a nucleic acid encoding the protein or polypeptide.

[0434] The present invention also relates to a nucleic acid molecule encoding at least one desired transgene as described above, and a protein or polypeptide comprising a transposase or a fragment thereof as described above, or a nucleic acid encoding the protein or polypeptide, for use in treating a genetic disease in a subject in need thereof.

[0435] The present invention also relates to the use of a nucleic acid molecule encoding at least one target transgene as described above, and a protein or polypeptide comprising a transposase or a fragment thereof as described above, or a nucleic acid encoding the protein or polypeptide in the manufacture of a medicament for treating a genetic disease in a subject in need thereof.

[0436] In some embodiments, the transgene of interest complements a gene detected to cause a genetic disease in the subject.

[0437] In some embodiments, the method further comprises administering to the subject a guide RNA (gRNA) fused to at least one aptamer as described above.

[0438] In some embodiments, the method may include administering to a subject in need thereof at least one additional therapeutic agent. In some embodiments, the at least one therapeutic agent is used to treat a genetic disease.

[0439] In some embodiments, the genetic disease is characterized by a mutation in at least one gene in the subject's genome such that the protein encoded by the at least one gene is impaired or non-functional, or is degraded by cellular protein quality control (e.g., the proteasome), or is not produced; in other words, the function of the at least one gene is at least partially (if not completely) lost.

[0440] Non-limiting examples of genetic diseases include sickle cell anemia, cystic fibrosis, Huntington disease, congenital muscular dystrophy, Duchenne muscular dystrophy, Fabry disease, Marfan syndrome, thalassemia, cystinosis, familial hypercholesterolemia, hemochromatosis, etc.

[0441] In some embodiments, the transgenic gene of interest partially or completely (preferably completely) restores the function of the at least one gene that has been mutated. In some embodiments, the transgenic gene of interest encodes the same protein as the at least one gene that has been mutated. In some embodiments, the protein encoded by the transgenic gene of interest is different from the protein encoded by the at least one gene that has been mutated, but has similar or identical biological functions.

[0442] BRIEF DESCRIPTION OF THE DRAWINGS

[0443] Figure 1 is a set of photographs illustrating the viability and transfection efficiency of GFP-expressing transposons and transposases in embryonic stem cells H9 electroporated with transposases (hyPB alone or hyPB fused to Cas9 nuclease). BF: bright field; GFP: green fluorescent protein.

[0444] Figure 2 is a histogram showing the transcription efficiency of the red fluorescent protein (RFP) reporter gene in HepG2 cells expressing or not expressing hyPB.

[0445] Figure 3 is a histogram showing the transcription efficiency of the RFP reporter gene in Huh7 cells expressing or not expressing hyPB.

[0446] Figure 4 and Figure 5 Figure 2 shows the expression of GFP reporter gene in Huh7 cells expressing or not expressing hyPB over time ( Figure 4 The middle ones are 3 hours, 6 hours, 9 hours and 12 hours; Figure 5 Figure 3 is a graph of transcription efficiency at 8 hours, 10 hours, 12 hours, 14 hours and 16 hours.

[0447] Figure 6 is a histogram showing the transcription efficiency of the GFP reporter gene in Huh7 cells expressing or not expressing catalytically inactive hyPB.

[0448] Figure 7 is a histogram showing the transcription efficiency of the GFP reporter gene in Huh7 cells expressing a fusion protein comprising Cas9 nuclease and SB100 transposase or N57 transposase.

[0449] Figures 8A-8B is a set of histograms comparing the transcription efficiency of the GFP reporter gene in Huh7 cells expressing or not expressing hyPB, where the GFP DNA transposon contains an inverted terminal repeat (ITR) ( Fig. 8A ) or two ITRs( Figure 8B ).

[0450] Fig. 9 is a set of photographs showing the transcription efficiency of the luciferase reporter gene in mice one day or four weeks after injection with hyPB mRNA or catalytically inactive hyPB mRNA.

[0451] Fig.10 is a set of photographs showing the transcription efficiency of the luciferase reporter gene in vivo using a polymer nanoparticle carrier (jetPEI) one day or one week after injection in mice transfected or not transfected with hyPB DNA.

[0452] Fig.11 is a set of photographs showing the transcription efficiency of the luciferase reporter gene in vivo one day after injection in mice transfected with or without hyPB, where the DNA payload (luciferase reporter gene) and hyPB mRNA were co-transfected using the same vector (lipid nanoparticles).

[0453] Fig.12 is a histogram showing that non-dividing NIH-3T3 cells transfected with hyPB have higher DNA transposon uptake and expression capacity compared to cells not expressing hyPB. Results are expressed as mean ± sem, n = 4.

[0454] Figures 13A-13B is a panel showing the number of replicates measured at 20-hour intervals after transfection ( Fig.13A ) and non-dividing ( Fig. 13B ) Graph of nuclear DNA translocation in NIH / 3T3 cells. Results are presented as mean ± sem, n = 2. Statistical significance is presented as *** (p value < 0.001) and ** (p value < 0.005).

[0455] Fig.14 is a histogram showing that non-dividing primary mouse hepatocytes in culture transfected with hyPB have higher uptake and expression capacity of DNA luciferase encoding transposon compared to cells not expressing hyPB. Data are presented as mean ± SEM, n=3. Example

[0456] The present invention is further illustrated by the following examples.

[0457] Example 1: Transposase-driven nuclear localization

[0458] Materials and methods

[0459] Electroporation of H9 stem cells

[0460] Embryonic stem cells H9 were electroporated with 1.8 μg of minicircle GFP transposon alone or with 1 μg of hyPB mRNA or 1.5 μg of FiCAT mRNA and 1.75 μg of sgRNA targeting the AAVS1 locus (CRG Cell and Tissue Facility). Cells were imaged using a fluorescent microscope and bright field, and the percentage of viability and GFP signal was estimated.

[0461] Lipofection of HepG2 cells

[0462] Day 0: 400k cells / well were seeded in P6 well plates.

[0463] Day 1: Transfect 1.25 μg of hyPB plasmid or pucl9 mock plasmid using lipofectamine 3000 according to the manufacturer's protocol. Keep cells with Optimem+ transfection mix for 2 hours and then add complete medium (Dule's modified Eagle's medium 10% fetal bovine serum, 2 mM glutamine and 100 U penicillin / 0.1 mg / ml streptomycin)

[0464] Day 2: The transfected cells were removed and seeded into a P12 well plate at a density of 150k cells / well

[0465] Day 3: Transfect cells following the manufacturer's protocol with 0.75 μg RFP DNA transposon and 0.25 μg puCl9 filler DNA and Lipofectamine 3000. Keep cells with Optimem+ transfection mix for 2 h before adding complete medium.

[0466] Day 4: Measure fluorescence 24 h after transfection on day 4

[0467] Alternatively, transfect cells with Cas9_SB100 plasmid and Cas9_N57 plasmid instead of hyPB on day 1. On day 3, transfect cells with GFP transposon containing SB100 ITR and TCR1 gRNA plasmid.

[0468] Lipofection of Huh7 cells

[0469] 400,000 cells were seeded in p6 wells 24 hours before transfection. Transfection was performed with lipofectamine 3000 using 1.8 μg of transposon DNA and 1.95 μg of hyPB or mock mRNA of similar size according to the manufacturer's protocol. Cells were kept with Optimem+ transfection mix for 2 hours before complete medium was added. Cells were extracted every 3 hours or every 14 hours after transfection and fluorescence was measured by flow cytometry.

[0470] Alternatively, cells were transfected with Cas9_SB100 plasmid and Cas9_N57 plasmid instead of hyPB on day 1. On day 3, cells were transfected with a GFP transposon containing the SB100 ITR as well as with a TCR1 gRNA plasmid.

[0471] In vivo hydrodynamic injection

[0472] 6-7 week old mice were injected with a total of 10 to 10.2 μg of nucleic acid (3.2 μg MC-luciferase transposon, 1.42 μg hyPB / hyPB_dead mRNA). Nucleic acid was diluted in PBS and 7% of the animal's body weight (in ml) was injected via retro-orbital systemic injection in less than 7 seconds.

[0473] JetPEI in vivo transfection

[0474] DNA payload and hyPB were used with different delivery methods: 35.6 μg MC-luciferase DNA transposon was injected intravenously into 5-week-old mice at a N / P ratio of 7 using in vivo JetPEI according to the manufacturer's instructions. LNPs loaded with 2.5 μg hyPB mRNA were injected 4 hours after the intravenous injection. LNP manufacturing was performed using a Benchtop Nanossemblr (PrecisionNanosystems).

[0475] The same delivery method was used with DNA payload and hyPB: 15 μg of LNPs loaded with MC-luciferase DNA transposon were injected intravenously into 5-week-old mice with or without 2.5 μg of LNPs loaded with hyPB. LNP manufacturing was performed using a Benchtop Nanossemblr (Precision Nanosystems).

[0476] The lipid ratio was 50% Dlin-MC3, 10% DSPC helper lipid, 38.5% cholesterol, 1.5% PEG-2000 and N / P ratio was 6. The size of the nanoparticles was 100-130 nm and the encapsulation efficiency was higher than 90%.

[0477] Whole body imaging

[0478] Whole-body imaging of luciferase expression at different time points after administration of FiCAT-gRNA transposon or transposon control was performed 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.

[0479] result

[0480] Embryonic stem cells H9 electroporated with transposase (hyPB or FiCAT, FiCAT is a Cas9-hyPB fusion protein) show GFP signal ( Figure 1), which means that the GFP transposon DNA has entered the nucleus and has been translated and transcribed. Although the episomal signal of the GFP transposon without hyPB or FiCAT was low (efficiency of 20%), hyPB produced a stronger GFP signal (transfection efficiency of 70%).

[0481] It was previously shown that the HepG2 hepatocyte cell line expressing hyPB exhibited better internalization of the RFP DNA transposon than cells not expressing hyPB ( Figure 2 This result was also obtained using the Huh7 liver cell line ( Figure 3 ). In addition, Huh7 cells transfected with hyPB mRNA showed a higher capacity to take up and express DNA transposons compared to cells transfected with mock mRNA ( Figure 4 and 5 ).

[0482] Huh7 hepatocytes transfected with hyPB catalytically inactive mRNA showed increased nuclear uptake and expression of DNA transposons compared to cells transfected with mock mRNA ( Figure 6 ).

[0483] Interestingly, the above results showing that the hyPB transposase has a higher nuclear uptake capacity for transposons compared to cells without hyPB can be extended to other transposases, such as SB100 and N57 ( Figure 7 ).

[0484] The ITR requirement of the DNA payload was then tested. Huh7 hepatocytes previously transfected with hyPB showed better internalization of the GFP DNA transposon than cells not expressing hyPB (previously transfected with Pucl9 plasmid DNA), both with GFP transposons carrying either 1 ITR or 2 ITRs ( Figures 8A-8B ). These data suggest that hyPB can promote nuclear internalization of payload DNA even when only 1 ITR is present (so that the hyPB molecule can bind to its payload).

[0485] The ability of hyPB to improve transfection was further tested under in vivo conditions ( Fig. 9 ). The hyPB transposase showed superiority in DNA payload internalization and expression even when its catalytic activity was compromised (inactive-hyPB). 24 hours after hydrodynamic injection of the payload alone, 1 in 3 mice showed luciferase expression, whereas this increased to 2 / 3 or 3 / 3 mice when co-injected with hyPB or catalytically inactive hyPB.

[0486] When payload DNA and hyPB mRNA were administered using different delivery methods, hyPB transposase showed advantages in DNA payload internalization and expression ( Fig.10 ). When DNA was injected, 50% of the mice showed luciferase signal after 24 hours, while 100% of the mice showed signal when hyPB was co-administered with LNP.

[0487] When payload DNA and hyPB mRNA were administered using the same delivery method, the hyPB transposase showed superiority in DNA payload internalization and expression ( Fig.11 ). When injected with DNAsolo, the mice showed no luciferase signal, whereas in both cases the signal was restored when hyPB was co-administered with LNPs. This result suggests that hyPB facilitates the internalization of DNA into the nucleus so it can be expressed.

[0488] Example 2: Nuclear localization in non-dividing cells

[0489] Materials and methods

[0490] NIH / 3T3 culture

[0491] NIH / 3T3 (ATCC) cells were cultured in DMEM containing 10% or 1% heat-inactivated fetal bovine serum, 100 U / ml penicillin and 0.1 mg / ml streptomycin at 37°C in a humidified CO2 (5%) incubator.

[0492] Determination of dose-dependent effects on quiescent NIH-3T3 cells

[0493] 60,000 NIH-3T3 cells were seeded and cultured in DMEM supplemented with 1% FBS for 48 hours after reaching confluence to induce senescence. Then, cells were transfected. Transfection was performed with lipofectamine 3000 using 0.2ug transposon DNA alone (episomes) or in combination with the specified amount of hyPB-wt or catalytically inactive hyPB (inactive-hyPB) according to the manufacturer's protocol. Cells were incubated with the transfection mixture in Optimem overnight and complete medium was then added. One hour after DMEM addition, cells were trypsinized and GFP expression was assessed by flow cytometry. (Expressed as mean ± SEM, n = 4)

[0494] Comparison of transfection in dividing and non-dividing NIH-3T3 cells

[0495] In replicating cells, 60,000 cells were seeded the day before transfection. For non-dividing 3T3, once they reached confluence, they were cultured in DMEM supplemented with 1% FBS for 48 hours to induce senescence. Then, cells were transfected. Transfection was performed with lipofectamine 3000 according to the manufacturer's protocol using 0.25ug transposon DNA alone (episomes) or in combination with 0.25ug hyPB-wt or catalytically inactive hyPB (inactive-hyPB) and trypsinized at 3 hour intervals for GFP evaluation by flow cytometry.

[0496] Two-way analysis of variance (ANOVA) test was used to analyze the statistical significance of differences among the groups.

[0497] Primary hepatocyte isolation

[0498] Use liver perfusion buffer (HBSS KCl 0.4 g / L -1 , glucose 1g.L -1 , NaHCO3 2.1gL -1 , EDTA 0.2 g.L -1 ) was perfused and then the liver was digested with liver digestion buffer (DMEM-GlutaMAX 1 g.L -1 Glucose, HEPES 15mM pH 7.4, penicillin / streptomycin 1%, 5mg collagenase IV (C5138 Sigma) per mouse) for digestion. 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%, 1nm insulin, 1mM dexamethasone, 2mg.mL -1 Bovine serum albumin (BSA). Tissues were homogenized using forceps and then filtered in plating medium. Cells were then washed three times in plating medium and then plated onto collagen-coated plates (Thermo Fisher Scientific) in plating medium. The medium was changed the next morning and cells were incubated for 48 hours before transfection.

[0499] Culture and transfection of primary hepatocytes

[0500] Primary hepatocytes were isolated from 8-week-old wild-type mice and 75,000 cells were inoculated per well. After 48 hours of culture, cells were transfected. According to the manufacturer's protocol, 0.2ug transposon DNA (episome) alone or in combination with 0.25ug hyPB-wt or catalytically inactivated hyPB (inactivated-hyPB) was transfected with lipofectamine 3000. Cells were incubated with the transfection mixture in Optimem for 5 hours, and then complete medium was added. 24 hours after transfection, cells were washed once with PBS and lysed with appropriate buffer. 24 hours after transfection, luciferase activity was quantified using a luciferase detection system (Promega, Madison, WI, USA), performed in duplicate. Data are shown as the multiple change relative to the luciferase value of untransfected hepatocytes, and expressed in arbitrary units (AU).

[0501] One-way or two-way analysis of variance (ANOVA) tests were used to analyze the statistical significance of differences between groups. Where applicable, pairwise comparisons were analyzed using a two-sided Student's t-test. Results are expressed as mean ± sem.

[0502] result

[0503] To further support the role of DNA transposases such as PiggyBac transposase in the nuclear import of DNA transposons, experiments were performed in non-dividing cells.

[0504] Two different models were chosen: Fig.12 and Figures 13A-13B In the NIH / 3T3 fibroblast cell line, senescence was induced. Fig.14 In this study, primary hepatocytes isolated from mice (which do not replicate in culture) were used to test the nuclear translocation activity of PiggyBac (hyPB) DNA in non-dividing (quiescent) cells.

[0505] Increasing amounts of PiggyBac transposase (hyPB) were tested to explore its dose-dependent effect; in addition, a catalytically inactive version of the transposase (inactive-hyPB) was added as a control for DNA translocation activity alone without the insertion activity of the transposase. Fig.12 showed that even with the lowest dose, uptake and expression of the DNA transposon (GFP) was increased in cells expressing hyPB or inactive-hyPB.

[0506] In addition, a side-by-side comparison of transduction and expression efficiencies of DNA payloads delivered into the cytoplasm of actively dividing and non-dividing NIH / 3T3 cells was performed to support a role for transposase proteins in the nuclear localization of these payloads. Figures 13A-13B ). Fig. 13Bshowed that the expression efficiency of the GFP payload was significantly increased in non-dividing NIH-3T3 cells expressing hyPB or inactive-hyPB compared to the absence of the transposase.

[0507] Results from primary hepatocytes also support this conclusion and show that the presence of the PiggyBac transposase, either catalytically active (hyPB) or inactive (inactive-hyPB), increases transposon expression ( Fig.14 ), which is a result of its improved nuclear transport.

[0508] Overall, these results demonstrate that DNA transposases increase nuclear import of DNA transposons independently of their insertional activity, particularly in non-dividing cells.

Claims

1. An in vitro method for increasing the nuclear localization of a nucleic acid molecule encoding at least one transgene of interest in a cell population, comprising contacting the cell population with: - a protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, and - a nucleic acid molecule encoding said at least one transgene of interest.

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

3. The in vitro method according to claim 1 or 2, wherein the protein or polypeptide comprising the transposase or a fragment thereof is a fusion protein comprising the transposase or a fragment thereof and at least one additional polypeptide or protein.

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

5. The in vitro method according to claim 3 or 4, wherein the fusion protein has at least 75% amino acid sequence identity with SEQ ID NO:

2.

6. The in vitro method according to any one of claims 1 to 5, further comprising contacting the cell population with a guide RNA (gRNA).

7. The in vitro method according to any one of claims 3 to 6, wherein the at least one additional polypeptide or protein is an aptamer binding protein.

8. The in vitro method according to claim 7, wherein the aptamer binding protein is the MS2 bacteriophage coat protein (MCP), preferably having SEQ ID NO:

39.

9. The in vitro method according to claim 8, wherein the fusion protein covalently or non-covalently interacts with the gRNA molecule through MCP, or is capable of covalently or non-covalently interacting with the gRNA through MCP, wherein the gRNA comprises at least one MS2 aptamer, preferably, the MS2 aptamer has a ribonucleic acid sequence of SEQ ID NO:

40.

10. The method according to any one of claims 1 to 9, wherein the transposase is selected from the group consisting of a hyperactive PiggyBac transposase, a PiggyBac transposase, a Sleeping Beauty transposase, a SB11 transposase, a Tol2 transposase, a Mos1 transposase and a Frog Prince transposase.

11. The method according to any one of claims 1 to 10, wherein the transposase is a modified hyperactive PiggyBac transposase comprising at least one amino acid mutation compared to the amino acid sequence of the hyperactive PiggyBac transposase having SEQ ID NO:

1.

12. The method according to any one of claims 1 to 11, wherein the transposase or fragment thereof is catalytically inactive.

13. The method of claim 12, wherein the catalytically inactive transposase has at least 75% amino acid sequence identity to SEQ ID NO:

3.

14. A protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, for use in treating a genetic disease in a subject in need thereof, wherein a nucleic acid molecule encoding at least one transgene of interest is further administered to a subject in need thereof, wherein expression of the transgene of interest in at least one cell of a subject in need thereof compensates for the genetic defect causing the genetic disease.

15. A protein or polypeptide comprising a transposase or a fragment thereof, or a nucleic acid encoding the protein or polypeptide, for use according to claim 14, wherein the use further comprises the features according to any one or more of claims 2 to 13.

Images