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1. 发明申请

US20110061118A1 VECTORS AND METHODS FOR GENERATING VECTOR-FREE INDUCED PLURIPOTENT STEM (IPS) CELLS USING SITE-SPECIFIC RECOMBINATION 审中-公开
公开(公告)号：US20110061118A1
公开(公告)日：2011-03-10
申请号：US12933362
申请日：2009-03-17
申请人： Ralf Kühn , Wolfgang Wurst
发明人： Ralf Kühn , Wolfgang Wurst
IPC分类号： A01K67/02 , C07H21/04 , C12N15/63 , C12N5/10 , C12N15/79 , C12Q1/68 , C12Q1/02 , C12N5/071
CPC分类号： C07K14/4702 , C12N5/0696 , C12N15/85 , C12N2501/602 , C12N2501/603 , C12N2501/604 , C12N2501/606 , C12N2510/00 , C12N2800/30 , C12N2830/003 , C12N2840/206
摘要： The present invention relates to a DNA molecule comprising: (a) a first DNA sequence comprising: (aa) a coding sequence giving rise upon transcription to a factor that contributes to the reprogramming of a somatic cell into an induced pluripotent stem (iPS) cell; (ab) a promoter mediating the transcription of said coding sequence; and (ac) two sequence motifs that mediate excision of (aa) and/or (ab) from the DNA molecule, wherein one sequence motif is positioned 5′ and the other sequence motif is positioned 3′ of the sequence to be excised; (b) a second DNA sequence comprising a sequence motif that mediates site-specific integration of (a) into another DNA molecule. Further, the invention relates to DNA molecule comprising: (a) a first DNA sequence comprising: (aa) a coding sequence giving rise upon transcription to a factor that contributes to the reprogramming of a somatic cell into an induced pluripotent stem cell; and (ab) a promoter mediating the transcription of said coding sequence; (b) a second DNA sequence comprising: (ba) a sequence motif that mediates extrachromosomal self-replication of the DNA-molecule; and (bb) two sequence motifs that mediate excision of at least said sequence motif of (ba) from the second DNA sequence (b), wherein one sequence motif is located 5′ of (ba) and the other sequence motif 3′ of (ba). Also, the invention relates to a vector comprising the DNA molecule of the invention, a method for assembly of said vector and a somatic cell comprising said DNA molecule or said vector of the invention. Furthermore, the invention relates to methods to generate an induced pluripotent stem (iPS) cell, an induced pluripotent stem cell obtainable by said methods, to a kit comprising the DNA molecule of the invention, to a cell line or cell culture collection comprising the induced pluripotent stem cell of the invention, to the use of said cell or cell line as a research tool, to a method to generate a transgenic non-human animal and to a non-human animal generated by said method. Finally, the invention relates to a composition for gene therapy, regenerative medicine, cell therapy or drug screening.

2. 发明申请

US20130212725A1 FUSION PROTEINS COMPRISING A DNA-BINDING DOMAIN OF A TAL EFFECTOR PROTEIN AND A NON-SPECIFIC CLEAVAGE DOMAIN OF A RESTRICTION NUCLEASE AND THEIR USE 审中-公开
标题翻译：包含T细胞效应蛋白的DNA结合域和限制性核酸的非特异性切割域及其使用的融合蛋白
公开(公告)号：US20130212725A1
公开(公告)日：2013-08-15
申请号：US13702231
申请日：2011-06-07
申请人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
发明人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
IPC分类号： C12N15/62
CPC分类号： C12N15/62 , A01K67/0276 , A01K67/0278 , A01K2207/05 , A01K2217/07 , A01K2217/072 , A01K2227/105 , A01K2267/03 , A01K2267/0393 , C07K2319/80 , C12N9/22 , C12N15/907
摘要： The present invention relates to a method of modifying a target sequence in the genome of a eukaryotic cell, the method comprising the step: (a) introducing into the cell a fusion protein comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease or a nucleic acid molecule encoding the fusion protein in expressible form, wherein the fusion protein specifically binds within the target sequence and introduces a double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the modification of the target sequence is by homologous recombination with a donor nucleic acid sequence further comprising the step: (b) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal or vertebrate carrying a modified target sequence in its genome. Furthermore, the present invention relates to a fusion protein comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease.
摘要翻译：本发明涉及一种修饰真核细胞基因组中的靶序列的方法，所述方法包括以下步骤：（a）将包含Tal效应蛋白的DNA结合结构域的融合蛋白引入细胞，限制性核酸酶或编码可表达形式的融合蛋白的核酸分子的特异性切割结构域，其中所述融合蛋白在靶序列内特异性结合并在靶序列内引入双链断裂。本发明还涉及本发明的方法，其中靶序列的修饰是通过与供体核酸序列的同源重组而进一步包括步骤：（b）将核酸分子引入细胞，其中核酸分子包含供体核酸序列和与靶序列同源的区域。本发明还涉及在其基因组中产生携带修饰的靶序列的非人哺乳动物或脊椎动物的方法。此外，本发明涉及包含Tal效应蛋白的DNA结合结构域和限制性核酸酶的非特异性切割结构域的融合蛋白。

3. 发明申请

US20140304847A1 RECOMBINATION EFFICIENCY BY INHIBITION OF NHEJ DNA REPAIR 审中-公开
标题翻译：通过抑制NHEJ DNA修复的重组效率
公开(公告)号：US20140304847A1
公开(公告)日：2014-10-09
申请号：US14124106
申请日：2012-06-06
申请人： Ralf Kühn , Wolfgang Wurst
发明人： Ralf Kühn , Wolfgang Wurst
IPC分类号： C12N15/85
CPC分类号： C12N15/85 , C12N15/1024 , C12N15/8509 , C12N15/907
摘要： The present invention relates to a method for modifying a target sequence in the genome of a mammalian cell, the method comprising the step of introducing into a mammalian cell: a. one or more compounds that introduce double-strand breaks in said target sequence; b. one or more DNA molecules comprising a donor DNA sequence to be incorporated by homologous recombination into the genomic DNA of said mammalian cell within said target sequence, wherein said donor DNA sequence is flanked upstream by a first flanking element and downstream by a second flanking element, wherein said first and second flanking element are different and wherein each of said first and second flanking sequence are homologous to a continuous DNA sequence on either side of the double-strand break introduced by said one or more compounds of a. within said target sequence in the genome of said mammalian cell; and c. one or more compounds that decrease the activity of the non-homologous end joining (NHEJ) DNA repair complex in said mammalian cell. Further, the invention relates to a method of producing a non-human mammal carrying a modified target sequence in its genome.
摘要翻译：本发明涉及一种用于修饰哺乳动物细胞基因组中靶序列的方法，所述方法包括引入哺乳动物细胞的步骤：a。一种或多种在所述靶序列中引入双链断裂的化合物; b。一个或多个DNA分子，其包含通过同源重组并入所述靶序列内的所述哺乳动物细胞的基因组DNA中的供体DNA序列，其中所述供体DNA序列在第一侧翼元件的上游侧和第二侧翼元件的下游，其中所述第一和第二侧翼元件是不同的，并且其中所述第一和第二侧翼序列中的每一个与由所述一种或多种a的化合物引入的双链断裂的任一侧上的连续DNA序列同源。在所述哺乳动物细胞的基因组中的所述靶序列内; 和c。一种或多种降低所述哺乳动物细胞中非同源末端连接（NHEJ）DNA修复复合物的活性的化合物。此外，本发明涉及在其基因组中生产携带修饰的靶序列的非人哺乳动物的方法。

4. 发明申请

US20120276537A1 HOMOLOGOUS RECOMBINATION IN THE OOCYTE 审中-公开
标题翻译：同卵生物的重组
公开(公告)号：US20120276537A1
公开(公告)日：2012-11-01
申请号：US13504587
申请日：2010-10-28
申请人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
发明人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
IPC分类号： C12N15/90 , C12Q1/68 , C12N15/89
CPC分类号： A01K67/0278 , A01K2217/072 , A01K2227/105 , A01K2267/01 , C12N9/22 , C12N15/8509 , C12N15/907
摘要： The present invention relates to a method of modifying a target sequence in the genome of a mammalian or avian oocyte by homologous recombination with a donor nucleic acid sequence, the method comprising the steps (a) introducing into the oocyte a zinc finger nuclease or a nucleic acid molecule encoding the zinc finger nuclease in expressible form, wherein the zinc finger nuclease specifically binds within the target sequence and introduces a double strand break within the target sequence; and (b) introducing a nucleic acid molecule into the oocyte, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention further relates to a method of producing a non-human mammal or an avian carrying a modified target sequence in its genome.
摘要翻译：本发明涉及通过与供体核酸序列同源重组来修饰哺乳动物或禽类卵母细胞基因组中的靶序列的方法，所述方法包括以下步骤：（a）向卵母细胞中引入锌指核酸酶或核酸编码锌指核酸酶的可分解形式的酸分子，其中所述锌指核酸酶在靶序列内特异性结合并在靶序列内引入双链断裂; 和（b）将核酸分子引入所述卵母细胞中，其中所述核酸分子包含供体核酸序列和与靶序列同源的区域。本发明还涉及在其基因组中产生携带修饰的靶序列的非人哺乳动物或禽类的方法。

5. 发明申请

US20100299771A1 MEANS AND METHODS FOR shRNA MEDIATED CONDITIONAL KNOCKDOWN OF GENES 审中-公开
公开(公告)号：US20100299771A1
公开(公告)日：2010-11-25
申请号：US12678645
申请日：2008-09-17
申请人： Ralf Kühn , Wolfgang Wurst , Patricia Steuber-Buchberger
发明人： Ralf Kühn , Wolfgang Wurst , Patricia Steuber-Buchberger
IPC分类号： A01K67/027 , C12N15/63 , A01K67/00 , C12N5/10 , C12N15/09 , C12Q1/68
CPC分类号： C12N15/111 , C12N2310/111 , C12N2310/14 , C12N2320/12 , C12N2320/50
摘要： The present invention relates to a combination of DNA segments comprising: (a) a first segment comprising in 5′ to 3′ or 3′ to 5′ order: (aa) a promoter; (ab) a first DNA sequence comprising: (i) a DNA sequence giving rise upon transcription to the sense strand of an shRNA molecule; (ii) a transcriptional stop element which is flanked by a first type of recombinase recognition sequences; and (iii) a DNA sequence giving rise upon transcription to the antisense strand of an shRNA molecule; (b) a second segment comprising in 5′ to 3′ or 3′ to 5′ order: (ba) a promoter; (bb) a second DNA sequence comprising: (i) a DNA sequence giving rise upon transcription to the sense strand of an shRNA molecule; (ii) a transcriptional stop element which is flanked by a second type of recombinase recognition sequences; and (iii) a DNA sequence giving rise upon transcription to the antisense strand of an shRNA molecule; wherein (i) said first type of recombinase recognition sequences are recognized and recombined by a recombinase but not recombined with said second type of recombinase recognition sequences; (ii) said second type of recombinase recognition sequences are recognized and recombined by the recombinase of (i) but not recombined with said first type of recombinase recognition sequences; and (iii) said DNA sequence of (ab) and (bb) is expressed under the control of said promoters of (aa) and (ba) upon removal of said transcriptional stop elements of (ab) and (bb) by the activity of a recombinase, resulting in transcription of said shRNA molecule in a cell. Further, the invention relates to a genetically engineered non-human animal and a method to produce said transgenic non-human animal. Also, the invention relates to a cell genetically engineered with the DNA molecule of the invention and a method of simultaneously knocking down two genes in a cell. Furthermore, envisaged is a method of identifying a combination of two target genes as a potential drug target and the use of the DNA molecule of the invention for the preparation of a composition for gene therapy.

6. 发明申请

US20100242127A1 METHODS TO IDENTIFY MODULATORS OF B-RAF PROTEIN KINASE AND THEIR USE FOR THE TREATMENT OF ANXIETY AND DEPRESSION 审中-公开
标题翻译：鉴定B-RAF蛋白激酶调节剂的方法及其用于治疗放射性疾病和抑郁症的用途
公开(公告)号：US20100242127A1
公开(公告)日：2010-09-23
申请号：US12602753
申请日：2008-06-03
申请人： Christiane Hitz , Sabine Hölter , Ralf Kühn , Wolfgang Wurst , Benedikt Wefers
发明人： Christiane Hitz , Sabine Hölter , Ralf Kühn , Wolfgang Wurst , Benedikt Wefers
IPC分类号： A01K67/00 , C12Q1/68 , C12Q1/48 , G01N33/53 , A61K31/44 , A61P25/00 , C07D213/62
CPC分类号： C12Q1/485 , C12Q1/6883 , C12Q2600/136 , C12Q2600/158 , G01N33/6893 , G01N2500/02 , G01N2500/10 , G01N2800/301 , G01N2800/304
摘要： The present invention relates to a method for identifying a compound capable of modulating an anxiety or depression disorder comprising the steps of: (a) contacting a composition comprising a B-Raf protein or a B-Raf gene in expressible form or a transcript thereof with a compound under conditions that allow for an interaction of the B-Raf protein or the B-Raf gene or a transcript thereof and the compound; and (b) measuring whether said interaction, if any, results in (i) a change of B-Raf kinase activity compared to B-Raf kinase activity in the absence of said compound; (ii) a modulation of the expression of the B-Raf gene compared to B-Raf gene expression in the absence of said compound; or (iii) the formation of a complex between the compound and the B-Raf protein, wherein such a change in activity, modulation of expression or the formation of a complex is indicative of the compound being a modulator of an anxiety or depression disorder. Further, the invention relates to a method for treating an anxiety or depression disorder in an individual comprising administering to the individual an effective amount of a compound inhibiting B-Raf kinase activity or gene expression and to a use of a compound that inhibits B-Raf kinase activity or gene expression in the manufacture of a pharmaceutical composition for treating an anxiety or depression disorder. Moreover, the invention relates to a method of diagnosing a B-Raf associated anxiety or depression disorder and to a genetically engineered mouse. Finally, the invention also relates to a method of identifying another gene contributing to the pathophysiology of an anxiety or depression disorder apart from B-Raf.
摘要翻译：本发明涉及一种鉴定能够调节焦虑或抑郁症的化合物的方法，包括以下步骤：（a）将包含可表达形式的B-Raf蛋白或B-Raf基因或其转录物的组合物与在允许B-Raf蛋白或B-Raf基因或其转录物与所述化合物相互作用的条件下的化合物; 和（b）测量所述相互作用（如果有的话）导致（i）与不存在所述化合物的B-Raf激酶活性相比，B-Raf激酶活性的变化; （ii）与不存在所述化合物的B-Raf基因表达相比，B-Raf基因表达的调节; 或（iii）化合物与B-Raf蛋白质之间的复合物的形成，其中这种活性变化，表达调节或复合物的形成指示该化合物为焦虑或抑郁症的调节剂。此外，本发明涉及一种治疗个体中的焦虑或抑郁症的方法，包括向个体施用有效量的抑制B-Raf激酶活性或基因表达的化合物以及使用抑制B-Raf的化合物在制备用于治疗焦虑或抑郁症的药物组合物中的激酶活性或基因表达。此外，本发明涉及一种诊断B-Raf相关焦虑症或抑郁症病症和遗传工程化小鼠的方法。最后，本发明还涉及一种识别除了B-Raf之外有助于焦虑或抑郁症病理生理学的另一种基因的方法。

7. 发明申请

US20140298505A1 PROTEIN HAVING NUCLEASE ACTIVITY, FUSION PROTEINS AND USES THEREOF 有权
标题翻译：蛋白具有核酸活性，融合蛋白及其用途
公开(公告)号：US20140298505A1
公开(公告)日：2014-10-02
申请号：US14124117
申请日：2012-06-06
申请人： Ralf Kühn
发明人： Ralf Kühn
IPC分类号： C12N9/16 , A01K67/027
CPC分类号： C12N15/907 , A01K67/0275 , A01K2207/05 , A01K2217/07 , A01K2217/075 , A01K2227/105 , A01K2267/03 , C07K14/195 , C07K2319/80 , C12N9/16 , C12N9/22 , C12N15/62 , C12N15/8509 , C12N2800/80 , C12N2999/007 , C12Y301/21004
摘要： The present invention relates to a nucleic acid molecule encoding (I) a polypeptide having the activity of an endonuclease, which is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2; (c) a nucleic acid molecule encoding an endonuclease, the amino acid sequence of which is at least 70% identical to the amino acid sequence of SEQ ID NO: 1; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 50% identical to the nucleotide sequence of SEQ ID NO: 2; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (e) wherein T is replaced by U; (II) a fragment of the polypeptide of (I) having the activity of an endonuclease. Also, the present invention relates to a vector comprising the nucleic acid molecule and a protein encoded by said nucleic acid molecule. Further, the invention relates to a method of modifying the genome of a eukaryotic cell and a method of producing a non-human vertebrate or mammal.
摘要翻译：本发明涉及编码（I）具有内切核酸酶活性的多肽的核酸分子，其是（a）编码包含或由SEQ ID NO：1的氨基酸序列组成的多肽的核酸分子; （b）包含SEQ ID NO：2的核苷酸序列或由其组成的核酸分子; （c）编码内切核酸酶的核酸分子，其氨基酸序列与SEQ ID NO：1的氨基酸序列至少70％相同; （d）包含与SEQ ID NO：2的核苷酸序列至少50％相同的核苷酸序列或由其组成的核酸分子; （e）相对于（d）的核酸分子简并的核酸分子; 或（f）与（a）〜（e）中任一项所述的核酸分子对应的核酸分子，其中，T由U代替; （II）具有内切核酸酶活性的（I）多肽的片段。此外，本发明涉及包含核酸分子和由所述核酸分子编码的蛋白质的载体。此外，本发明涉及一种修饰真核细胞的基因组的方法和一种生产非人脊椎动物或哺乳动物的方法。

8. 发明授权

US09410134B2 Protein having nuclease activity, fusion proteins and uses thereof 有权
标题翻译：具有核酸酶活性的蛋白质，融合蛋白质及其用途
公开(公告)号：US09410134B2
公开(公告)日：2016-08-09
申请号：US14124117
申请日：2012-06-06
申请人： Ralf Kühn
发明人： Ralf Kühn
IPC分类号： C07H21/04 , C12N9/16 , A01K67/027 , C12N15/62 , C12N15/85 , C12N15/90 , C12N9/22
CPC分类号： C12N15/907 , A01K67/0275 , A01K2207/05 , A01K2217/07 , A01K2217/075 , A01K2227/105 , A01K2267/03 , C07K14/195 , C07K2319/80 , C12N9/16 , C12N9/22 , C12N15/62 , C12N15/8509 , C12N2800/80 , C12N2999/007 , C12Y301/21004
摘要： The present invention relates to a nucleic acid molecule encoding (I) a polypeptide having the activity of an endonuclease, which is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2; (c) a nucleic acid molecule encoding an endonuclease, the amino acid sequence of which is at least 70% identical to the amino acid sequence of SEQ ID NO: 1; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 50% identical to the nucleotide sequence of SEQ ID NO: 2; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (e) wherein T is replaced by U; (II) a fragment of the polypeptide of (I) having the activity of an endonuclease. Also, the present invention relates to a vector comprising the nucleic acid molecule and a protein encoded by said nucleic acid molecule. Further, the invention relates to a method of modifying the genome of a eukaryotic cell and a method of producing a non-human vertebrate or mammal.
摘要翻译：本发明涉及编码（I）具有内切核酸酶活性的多肽的核酸分子，其是（a）编码包含或由SEQ ID NO：1的氨基酸序列组成的多肽的核酸分子; （b）包含SEQ ID NO：2的核苷酸序列或由其组成的核酸分子; （c）编码内切核酸酶的核酸分子，其氨基酸序列与SEQ ID NO：1的氨基酸序列至少70％相同; （d）包含与SEQ ID NO：2的核苷酸序列至少50％相同的核苷酸序列或由其组成的核酸分子; （e）相对于（d）的核酸分子简并的核酸分子; 或（f）与（a）〜（e）中任一项所述的核酸分子对应的核酸分子，其中，T由U代替; （II）具有内切核酸酶活性的（I）多肽的片段。此外，本发明涉及包含核酸分子和由所述核酸分子编码的蛋白质的载体。此外，本发明涉及一种修饰真核细胞的基因组的方法和一种生产非人脊椎动物或哺乳动物的方法。

9. 发明申请

US20060218654A1 Sirna mediated gene silencing in transgenic animals 审中-公开
标题翻译： Sirna介导转基因动物的基因沉默
公开(公告)号：US20060218654A1
公开(公告)日：2006-09-28
申请号：US10531347
申请日：2003-10-10
申请人： Jost Seibler , Frieder Schwenk , Ralf Kühn , Birgit Küter-Luks
发明人： Jost Seibler , Frieder Schwenk , Ralf Kühn , Birgit Küter-Luks
IPC分类号： A01K67/027
CPC分类号： C12N15/111 , A01K67/0275 , A01K2217/05 , A01K2217/058 , A01K2217/20 , A01K2227/105 , A01K2227/40 , A01K2267/03 , A61K48/00 , C12N15/63 , C12N15/8509 , C12N2310/111 , C12N2310/14 , C12N2330/30 , C12N2800/30
摘要： The present invention relates to a process that enables constitutive and inducible gene knock down in living organisms using a short hairpin RNA expression vector integrated into the genome of the organism.
摘要翻译：本发明涉及使用整合到生物体的基因组中的短发夹RNA表达载体来使活生物体中的组成型和诱导型基因敲低的方法。

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