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71 条记录 (耗时 0.047 秒)

    SUPERCHARGED PROTEINS FOR CELL PENETRATION
    41.
    发明公开
    SUPERCHARGED PROTEINS FOR CELL PENETRATION 审中-公开
    SUPRA电荷的蛋白质用于细胞渗透

    公开(公告)号：EP2424877A2

    公开(公告)日：2012-03-07

    申请号：EP10772365.2

    申请日：2010-04-28

    申请人： President and Fellows of Harvard College

    发明人： LIU, David, R. ,  MCNAUGHTON, Brian, R. ,  CRONICAN, James, Joseph ,  THOMPSON, David, B.

    IPC分类号： C07K14/00

    CPC分类号： C07K14/43595 ,  C07K7/06 ,  C07K7/08 ,  C07K14/001 ,  C07K2319/01 ,  C12N5/0602 ,  C12N2501/40 ,  C12N2501/60 ,  C12N2740/13043

    摘要： Compositions, preparations, systems, and related methods for delivering a supercharged protein, or a complex of a supercharged protein and an agent (e.g., nucleic acids, peptides, proteins, small molecules) to cells are provided. Such systems and methods include the use of supercharged proteins. For example, superpositively charged proteins may be associated with nucleic acids (which typically have a net negative charge) via electrostatic interactions. In some embodiments, such systems and methods involve altering the primary sequence of a protein in order to "supercharge" the protein (e.g., to generate a superpositively-charged protein). In some embodiments, complexes comprising supercharged proteins and one or more agents to be delivered are useful as therapeutic agents. In some embodiments, complexes and/or pharmaceutical compositions thereof are administered to a subject in need thereof. The inventive complexes or pharmaceutical compositions thereof may be used to treat proliferative diseases, infectious diseases, cardiovascular diseases, inborn errors in metabolism, genetic diseases, etc.

    DELIVERY OF NEGATIVELY CHARGED PROTEINS USING CATIONIC LIPIDS
    43.
    发明公开
    DELIVERY OF NEGATIVELY CHARGED PROTEINS USING CATIONIC LIPIDS 审中-公开
    用阳离子脂质递送带负电的蛋白质

    公开(公告)号：EP3212165A1

    公开(公告)日：2017-09-06

    申请号：EP15797506.1

    申请日：2015-10-30

    申请人： President and Fellows of Harvard College

    发明人： LIU, David, R. ,  THOMPSON, David, B. ,  ZURIS, John, Anthony

    IPC分类号： A61K9/00 ,  C07K14/435

    CPC分类号： C07K14/43595 ,  A61K9/1272 ,  A61K47/557

    摘要： Compositions, methods, strategies, kits, and systems for the delivery of negatively charged proteins, protein complexes, and fusion proteins, using cationic polymers or lipids are provided. Delivery of proteins into cells can be effected
    in vivo ,
    ex vivo , or
    in vitro . Proteins that can be delivered using the compositions, methods, strategies, kits, and systems provided herein include, without limitation, enzymes, transcription factors, genome editing proteins, Cas9 proteins, TALEs, TALENs, nucleases, binding proteins (
    e.g. , ligands, receptors, antibodies, antibody fragments; nucleic acid binding proteins,
    etc .), structural proteins, and therapeutic proteins (
    e.g. , tumor suppressor proteins, therapeutic enzymes, growth factors, growth factor receptors, transcription factors, proteases,
    etc. ), as well as variants and fusions of such proteins.

    摘要翻译： 提供了使用阳离子聚合物或脂质递送带负电的蛋白质，蛋白质复合物和融合蛋白的组合物，方法，策略，试剂盒和系统。 蛋白质进入细胞可以在体内，离体或体外进行。 可以使用本文提供的组合物，方法，策略，试剂盒和系统递送的蛋白包括但不限于酶，转录因子，基因组编辑蛋白，Cas9蛋白，TALE，TALEN，核酸酶，结合蛋白（例如配体，受体 ，抗体，抗体片段，核酸结合蛋白等），结构蛋白和治疗性蛋白（例如肿瘤抑制蛋白，治疗性酶，生长因子，生长因子受体，转录因子，蛋白酶等），以及 这些蛋白质的变体和融合体。

    CAS9 PROTEINS INCLUDING LIGAND-DEPENDENT INTEINS
    44.
    发明公开
    CAS9 PROTEINS INCLUDING LIGAND-DEPENDENT INTEINS 审中-公开
    CAS9蛋白，包括配体依赖性蛋白

    公开(公告)号：EP3177718A2

    公开(公告)日：2017-06-14

    申请号：EP15830407.1

    申请日：2015-07-30

    申请人： President and Fellows of Harvard College

    发明人： LIU, David, R. ,  DAVIS, Kevin

    IPC分类号： C12N9/22 ,  C07K14/315 ,  C07K14/195

    CPC分类号： C12N15/907 ,  C07K14/35 ,  C07K14/721 ,  C07K2319/92 ,  C12N9/0071 ,  C12N9/22 ,  C12N9/78 ,  C12N15/63 ,  C12P19/34 ,  C12Y114/11 ,  C12Y305/04

    摘要： Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

    摘要翻译： 本公开的一些方面提供了用于控制RNA可编程核酸内切酶例如Cas9的活性或用于控制包含与功能性效应子结构域融合的Cas9变体的蛋白质的活性的组合物，方法，系统和试剂盒，所述功能性效应器域例如核酸酶 ，切口酶，重组酶，脱氨酶，转录激活物，转录阻遏物或表观遗传修饰结构域。 例如，提供的本发明蛋白质包含配体依赖性内含肽，其存在抑制蛋白质的一种或多种活性（例如，gRNA结合，酶促活性，靶DNA结合）。 配体与内含肽的结合导致内含肽的自我切除，恢复蛋白质的活性。

    NUCLEASE PROFILING SYSTEM
    45.
    发明公开
    NUCLEASE PROFILING SYSTEM 审中-公开
    核苷酸分析系统

    公开(公告)号：EP3030650A1

    公开(公告)日：2016-06-15

    申请号：EP14755947.0

    申请日：2014-08-08

    申请人： President and Fellows of Harvard College

    发明人： LIU, David, R. ,  PATTANAYAK, Vikram

    IPC分类号： C12N9/22

    CPC分类号： C12Q1/44 ,  A61K38/465 ,  C12N9/22 ,  C12Q1/6816 ,  C12Q1/6869 ,  C12Q1/6874 ,  C12Q1/6883 ,  C12Q2600/156 ,  C12Y301/00 ,  C12Q2521/301 ,  C12Q2522/101 ,  C12Q2535/122 ,  C12Q2563/131 ,  C12Q2563/149

    摘要： Some aspects of this disclosure provide strategies, methods, and reagents for determining nuclease target site preferences and specificity of site-specific endonucleases. Some methods provided herein utilize a novel “one-cut” strategy for screening a library of concatemers comprising repeat units of candidate nuclease target sites and constant insert regions to identify library members that can been cut by a nuclease of interest via sequencing of an intact target site adjacent and identical to a cut target site.

    摘要翻译： 本公开的一些方面提供了用于确定位点特异性内切核酸酶的核酸酶靶位点偏好和特异性的策略，方法和试剂。 本文提供的一些方法利用新颖的“单切”策略来筛选包含候选核酸酶靶位点和恒定插入区的重复单元的串联体文库，以鉴定可通过测序完整靶标而被目的核酸酶切割的文库成员 与切割目标站点相邻并相同。 本公开的一些方面提供了用于基于确定其靶位点偏好和特异性来选择位点特异性核酸内切酶的策略，方法和试剂。 还提供了用于确定目标位点偏好和特异性的方法和试剂。

    EVALUATION AND IMPROVEMENT OF NUCLEASE CLEAVAGE SPECIFICITY
    47.
    发明公开
    EVALUATION AND IMPROVEMENT OF NUCLEASE CLEAVAGE SPECIFICITY 审中-公开
    核酸酶切割特异性的评估和改进

    公开(公告)号：EP2734621A2

    公开(公告)日：2014-05-28

    申请号：EP12845790.0

    申请日：2012-07-22

    申请人： President and Fellows of Harvard College ,  Liu, David R. ,  Guilinger, John Paul ,  Pattanayak, Vikram

    发明人： LIU, David, R. ,  GUILINGER, John, Paul ,  PATTANAYAK, Vikram

    IPC分类号： C12N9/16 ,  C12N15/55 ,  C12Q1/44 ,  C12Q1/68 ,  C40B40/06 ,  A61K38/43

    CPC分类号： C12N9/22 ,  C12Q1/44 ,  C12Q1/68 ,  C12Q1/6874 ,  C12Q1/6802 ,  C12Q2521/30

    摘要： Engineered nucleases (e.g., zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and others) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for their ability to be cleaved by active, dimeric nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future nuclease design. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

    摘要翻译： 工程核酸酶（例如锌指核酸酶（ZFN），转录激活物样效应物核酸酶（TALEN）等）是用于基因组操作的有希望的工具并且确定这些酶的脱靶切割位点是非常有意义的。 我们开发了一种体外筛选方法，其询问10 11个DNA序列被活性二聚体抑制肽（例如ZFN和TALEN）裂解的能力。 该方法揭示了成千上万的DNA序列，一些存在于人类基因组中，可以通过两种分别靶向内源性人CCR5和VEGF-A基因的ZFNs，CCR5-224和VF2468在体外切割。 在培养的人类细胞中鉴定的位点的分析揭示了在9个脱靶位点处的CCR5-224诱导的诱变。 同样，我们在培养的人类细胞中观察到了VF2468切割的31个脱靶位点。 我们的研究结果建立了ZFN特异性的能量补偿模型，其中过量结合能量有助于脱靶ZFN切割并提出改进未来核酸酶设计的策略。 还观察到TALEN可以达到类似于或高于在ZFN中观察到的切割特异性。