Abstract: The present invention relates to enhanced Sleeping Beauty-type transposons and methods of transposition. In particular the invention relates to a polynucleotide comprising a cargo nucleic acid flanked by a left and a right inverted repeat/direct repeat (IR/DR), wherein IR/DRs, having specific sequences, are recognized by a Sleeping Beauty transposase protein and the polynucleotide is capable of integrating into the DNA of a cell.

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transpsoson, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: An in vitro method for identifying, isolating and/or enriching primate naive pluripotent stem cells, the method including analyzing transcription of a type 7 long terminal repeat (LTR7) nucleic acid sequence of a type H human endogenous retrovirus (HERVH) (LTR7/HERVH-associated transcription), and identifying, isolating and/or enriching primate naive pluripotent stem cells based on LTR7/HERVH-associated transcription, wherein LTR7/HERVH-associated transcription is a marker for primate naive pluripotent stem cells. An isolated in vitro population of primate naive pluripotent stem cells is obtained by the method, wherein in the cells LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are primed pluripotent stem cells or differentiated cells.

Abstract: The present invention relates to the field of genetic engineering, in particular, to a transposon-based transfection kit suitable for transfection of primary cells, such as T cells, comprising mRNA encoding a transposase, or reagents for generating mRNA encoding said transposase, as well as minicircle DNA comprising the transposon. The invention also relates to a nucleic acid, preferably, a DNA minicircle, comprising a transposon, wherein the transposon encodes a protein and at least one miRNA, wherein the sequences encoding the miRNA are located in an intron and expression of the protein and the miRNA is regulated by the same promoter. The invention also provides a population of cells obtainable with the method of the invention. Methods of transfection are also provided, as well as medical use, e.g. in immunotherapy, in particular, in adoptive T cell therapy or T cell receptor (TCR) or chimeric antigen receptor (CAR) gene therapy.

Abstract: The present invention relates to the field of genetic engineering, in particular, to a transposon-based transfection kit suitable for transfection of primary cells, such as T cells, comprising mRNA encoding a transposase, or reagents for generating mRNA encoding said transposase, as well as minicircle DNA comprising the transposon. The invention also relates to a nucleic acid, preferably, a DNA minicircle, comprising a transposon, wherein the transposon encodes a protein and at least one miRNA, wherein the sequences encoding the miRNA are located in an intron and expression of the protein and the miRNA is regulated by the same promoter. The invention also provides a population of cells obtainable with the method of the invention. Methods of transfection are also provided, as well as medical use, e.g. in immunotherapy, in particular, in adoptive T cell therapy or T cell receptor (TCR) or chimeric antigen receptor (CAR) gene therapy.

Abstract: An in vitro method for identifying, isolating and/or enriching primate naive pluripotent stem cells, the method including analyzing transcription of a type 7 long terminal repeat (LTR7) nucleic acid sequence of a type H human endogenous retrovirus (HERVH) (LTR7/HERVH-associated transcription), and identifying, isolating and/or enriching primate naive pluripotent stem cells based on LTR7/HERVH-associated transcription, wherein LTR7/HERVH-associated transcription is a marker for primate naive pluripotent stem cells. An isolated in vitro population of primate naive pluripotent stem cells is obtained by the method, wherein in the cells LTR7/HERVH-associated transcription is elevated in comparison to control cells, wherein control cells are primed pluripotent stem cells or differentiated cells.

Abstract: The present invention relates to enhanced Sleeping Beauty-type transposons and methods of transposition. In particular the invention relates to a polynucleotide comprising a cargo nucleic acid flanked by a left and a right inverted repeat/direct repeat (IR/DR), wherein IR/DRs, having specific sequences, are recognized by a Sleeping Beauty transposase protein and the polynucleotide is capable of integrating into the DNA of a cell.

Abstract: The present invention relates to the field of genetic engineering, in particular, to a transposon-based transfection kit suitable for transfection of primary cells, such as T cells, comprising mRNA encoding a transposase, or reagents for generating mRNA encoding said transposase, as well as minicircle DNA comprising the transposon. The invention also relates to a nucleic acid, preferably, a DNA minicircle, comprising a transposon, wherein the transposon encodes a protein and at least one miRNA, wherein the sequences encoding the miRNA are located in an intron and expression of the protein and the miRNA is regulated by the same promoter. The invention also provides a population of cells obtainable with the method of the invention. Methods of transfection are also provided, as well as medical use, e.g. in immunotherapy, in particular, in adoptive T cell therapy or T cell receptor (TCR) or chimeric antigen receptor (CAR) gene therapy.

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: One or more type 7 long terminal repeat (LTR7) nucleic acid sequences of type H human endogenous retroviruses (HERVH) (“LTR7/HERVH nucleic acid sequences”) can be used for identifying primate naïve pluripotent stem cells. LTR7/HERVH-associated transcription can be used as a marker for primate naive pluripotent stem cells. A reporter construct that includes LTR7/HERVH nucleic acid sequences can be used for optimizing culture conditions for naïve primate pluripotent stem cells. A cell growth medium can be used for cultivation of primate naive pluripotent stem cells, which can exhibit elevated levels of LTR7/HERVH-associated transcription in comparison to control cells.

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transpsoson, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: The present invention relates to a targeting system comprising, preferably as distinct components (a) a transposon which is devoid of polynucleotide encoding a functional transposase comprising (aa) a polynucleotide of interest and; (ab) a DNA sequence specifically recognized by a DNA binding domain; and (ba) a fusion protein comprising (i) said DNA binding domain; or (ii) a (poly)peptide binding domain binding to a (poly)peptide comprising said DNA binding domain; and (iii) a DNA targeting domain; or (iv) a (poly)peptide binding domain that binds to a cellular or engineered (poly)peptide that comprises a DNA targeting domain; or (bb) a polynucleotide encoding the fusion protein of (ba); and (ca) a transposase or a fragment or derivative thereof having transposase function; or (cb) a polynucleotide encoding the transposase or fragment or derivative thereof having transposase function of (ca).

Abstract: The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transpsoson, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Abstract: The present invention relates to a targeting system comprising, preferably as distinct components, (a) a transposon which is devoid of a polynucleotide encoding a functional transposase comprising a polynucleotide of interest; and (b) a fusion protein comprising (ba) a transposase or a fragment or derivative thereof having transposase function; and (bb) a DNA targeting domain; or (bc) a (poly)peptide binding domain that binds to a cellular or engineered (poly)peptide comprising a DNA targeting domain; or (bd) a (poly)peptide comprising the DNA targeting domain of (bb) or the (poly)peptide binding domain of (bc), wherein the transposase or a fragment or derivative thereof having transposase function of (a) is joined by a linker to the domain of (bb) or to the domain of (bc) or to the (poly)peptide of (bd); or (c) a polynucleotide encoding the fusion protein of (b).

Abstract: The present invention provides for transposon vectors encoding expression control region-traps and gene-traps. Also provided are dicistronic vectors. Certain embodiments of the invention contain internal ribosome entry sites.

Abstract: This invention relates to a system for introducing nucleic acid into the DNA of a cell. The system includes the use of a member of the SB family of transposases (SB) or nucleic acid encoding the transposase and a nucleic acid fragment that includes a nucleic acid sequence with flanking inverted repeats. The transposase recognizes at least a portion of an inverted repeats and incorporates the nucleic acid sequence into the DNA. Methods for use of this system are discussed.

Abstract: The present invention relates to a targeting system comprising, preferably as distinct components (a) a transposon which is devoid of polynucleotide encoding a functional transposase comprising (aa) a polynucleotide of interest and; (ab) a DNA sequence specifically recognized by a DNA binding domain; and (ba) a fusion protein comprising (i) said DNA binding domain; or (ii) a (poly)peptide binding domain binding to a (poly)peptide comprising said DNA binding domain; and (iii) a DNA targeting domain; or (iv) a (poly)peptide binding domain that binds to a cellular or engineered (poly)peptide that comprises a DNA targeting domain; or (bb) a polynucleotide encoding the fusion protein of (ba); and (ca) a transposase or a fragment or derivative thereof having transposase function; or (cb) a polynucleotide encoding the transposase or fragment or derivative thereof having transposase function of (ca).