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1. 发明申请

US20060128020A1 Methods of unidirectional, site-specific integration into a genome, compositions and kits for practicing the same 有权
标题翻译：单向，位点特异性整合到基因组中的方法，用于实施它们的组合物和试剂盒
公开(公告)号：US20060128020A1
公开(公告)日：2006-06-15
申请号：US11198885
申请日：2005-08-04
申请人： Michele Calos
发明人： Michele Calos
IPC分类号： C12N15/86 , C12N15/74
CPC分类号： C12N15/90 , C12N15/8509
摘要： The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided system includes a site-specific integrating expression cassette (INTEC) vector, consisting of (a) a polynucleotide of interest operably linked to a promoter, (b) a single recombination site, and (c) a hybrid recombination site. In using the subject systems for site-specific integration, the INTEC vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific recombinase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.
摘要翻译：本发明提供了用于将核酸整合到靶细胞的基因组中的单向位点特异性整合系统。提供的系统包括位点特异性整合表达盒（INTEC）载体，其由（a）可操作地连接到启动子的目的多核苷酸，（b）单个重组位点和（c）杂交重组位点组成。在使用受试系统进行位点特异性整合时，将INTEC载体和整合酶引入靶细胞，并将细胞维持在足以通过重组事件提供核酸到靶细胞基因组的位点特异性整合的条件下由位点特异性重组酶介导。还提供了包括主题系统的套件。受试者系统，方法和试剂盒可用于各种不同的应用，其中还有几个代表性的这些应用也被详细描述。

2. 发明申请

US20050208021A1 Methods of unidirectional, site-specific integration into a genome, compositions and kits for practicing the same 有权
标题翻译：单向，位点特异性整合到基因组中的方法，用于实施它们的组合物和试剂盒
公开(公告)号：US20050208021A1
公开(公告)日：2005-09-22
申请号：US11003941
申请日：2004-12-03
申请人： Michele Calos
发明人： Michele Calos
IPC分类号： A61K48/00 , C07K14/505 , C12N9/00 , C12N9/22 , C12N15/00 , C12N15/52 , C12N15/82 , C12N15/85 , C12N15/86 , C12N15/87 , C12N15/90
CPC分类号： C12N15/85 , A61K48/005 , A61K48/0058 , A61K48/0066 , C07K14/505 , C12N9/22 , C12N15/8209 , C12N15/8213 , C12N15/8216 , C12N15/8217 , C12N15/8273 , C12N15/8286 , C12N15/90 , C12N2800/30 , C12N2800/90 , C12N2830/002 , C12N2830/38 , C12N2830/55 , C12N2830/85 , C12N2840/203 , Y02A40/162
摘要： The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided systems include a (1) a mutant, unidirectional site specific integrase, which can be provided by an integrase vector encoding the mutant integrase and (2) a targeting vector that includes: (a) a nucleic acid to be integrated; and (b) a vector attachment site, where the targeting vector attachment site serves as a substrate for the mutant, unidirectional site-specific integrase. In using the subject systems for site-specific integration, the targeting vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific integrase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.
摘要翻译：本发明提供了用于将核酸整合到靶细胞的基因组中的单向位点特异性整合系统。所提供的系统包括（1）突变单向位点特异性整合酶，其可以由编码突变型整合酶的整合酶载体提供，和（2）靶向载体，其包含：（a）待整合的核酸; 和（b）载体附着位点，其中靶向载体附着位点用作突变单向位点特异性整合酶的底物。在使用受试系统进行位点特异性整合时，靶向载体和整合酶被引入靶细胞，并且细胞维持在足以通过重组事件提供核酸到靶细胞基因组的位点特异性整合的条件由位点特异性整合酶介导。还提供了包括主题系统的套件。受试者系统，方法和试剂盒可用于各种不同的应用，其中还有几个代表性的这些应用也被详细描述。

3. 发明授权

US08420395B2 Methods of unidirectional, site-specific integration into a genome, compositions and kits for practicing the same 有权
标题翻译：单向，位点特异性整合到基因组中的方法，用于实施它们的组合物和试剂盒
公开(公告)号：US08420395B2
公开(公告)日：2013-04-16
申请号：US11003941
申请日：2004-12-03
申请人： Michele Calos
发明人： Michele Calos
IPC分类号： C12N15/63 , C12N15/11 , C12N15/52 , C12N9/00
CPC分类号： C12N15/85 , A61K48/005 , A61K48/0058 , A61K48/0066 , C07K14/505 , C12N9/22 , C12N15/8209 , C12N15/8213 , C12N15/8216 , C12N15/8217 , C12N15/8273 , C12N15/8286 , C12N15/90 , C12N2800/30 , C12N2800/90 , C12N2830/002 , C12N2830/38 , C12N2830/55 , C12N2830/85 , C12N2840/203 , Y02A40/162
摘要： The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided systems include a (1) a mutant, unidirectional site specific integrase, which can be provided by an integrase vector encoding the mutant integrase and (2) a targeting vector that includes: (a) a nucleic acid to be integrated; and (b) a vector attachment site, where the targeting vector attachment site serves as a substrate for the mutant, unidirectional site-specific integrase. In using the subject systems for site-specific integration, the targeting vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific integrase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.
摘要翻译：本发明提供了用于将核酸整合到靶细胞的基因组中的单向位点特异性整合系统。所提供的系统包括（1）突变单向位点特异性整合酶，其可以由编码突变型整合酶的整合酶载体提供，和（2）靶向载体，其包含：（a）待整合的核酸; 和（b）载体附着位点，其中靶向载体附着位点用作突变单向位点特异性整合酶的底物。在使用受试系统进行位点特异性整合时，将靶向载体和整合酶引入靶细胞，并且将细胞维持在足以通过重组事件提供核酸到靶细胞基因组的位点特异性整合的条件下由位点特异性整合酶介导。还提供了包括主题系统的套件。受试者系统，方法和试剂盒可用于各种不同的应用，其中还有几个代表性的这些应用也被详细描述。

4. 发明申请

US20070077589A1 Altered recombinases for genome modification 有权
标题翻译：改变基因组修饰的重组酶
公开(公告)号：US20070077589A1
公开(公告)日：2007-04-05
申请号：US11582836
申请日：2006-10-17
申请人： Michele Calos
发明人： Michele Calos
IPC分类号： C12Q1/68 , C12N9/22
CPC分类号： C12N9/00 , A61K48/00 , C12N9/22 , C12N15/90 , C12N15/902 , C12N2800/30
摘要： The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.
摘要翻译：本发明描述鉴定改变的重组酶及其组合物的方法，其中至少一个氨基酸不同于亲本野生型重组酶，并且改变的重组酶相对于野生型和/或伪att位点序列具有改善的重组效率亲本，野生型重组酶。本发明还包括使用改变的重组酶修饰细胞基因组的方法，包括将感兴趣的多核苷酸序列特异性整合到真核细胞的基因组中的方法。

5. 发明申请

US20070059835A1 Ocular gene therapy using avalanche-mediated transfection 有权
标题翻译：使用雪崩介导转染的眼基因治疗
公开(公告)号：US20070059835A1
公开(公告)日：2007-03-15
申请号：US11505249
申请日：2006-08-15
申请人： Thomas Chalberg , Mark Blumenkranz , Daniel Palanker , Alexander Vankov , Philip Hule , Michael Marmor , Michele Calos
发明人： Thomas Chalberg , Mark Blumenkranz , Daniel Palanker , Alexander Vankov , Philip Hule , Michael Marmor , Michele Calos
IPC分类号： C12N15/87
CPC分类号： C12N13/00 , A61K48/00 , C12N15/87
摘要： The present invention provides a method of treating an ocular disease in a subject. In a first step, a nucleic acid is introduced into cells or a tissue. The nucleic acid is introduced by electron avalanche transfection. With this technique, a high electric field induces a vapor bubble and plasma discharge between an electrode and the surrounding medium. The formation of a vapor bubble generates mechanical stress. Plasma discharge through the ionized vapor in the bubble enables connectivity between the electrode and the surrounding medium, so that mechanical stress and electric field are applied simultaneously, which results in permeabilization of the cells or tissue. This permeabilization in turn allows the nucleic acid to enter the cell or tissue. Cells or tissue containing the nucleic acid are then transplanted into an ocular region of the subject.
摘要翻译：本发明提供一种治疗受试者眼部疾病的方法。在第一步骤中，将核酸引入细胞或组织。通过电子雪崩转染引入核酸。利用这种技术，高电场在电极和周围介质之间引起蒸汽气泡和等离子体放电。气泡的形成产生机械应力。通过气泡中的电离蒸汽的等离子体放电使得电极和周围介质之间能够连接，使得同时施加机械应力和电场，这导致细胞或组织的透化。这种渗透反过来又允许核酸进入细胞或组织。然后将含有核酸的细胞或组织移植到受试者的眼部区域。

6. 发明申请

US20050003540A1 Altered recombinases for genome modification 有权
标题翻译：改变基因组修饰的重组酶
公开(公告)号：US20050003540A1
公开(公告)日：2005-01-06
申请号：US10836323
申请日：2004-04-29
申请人： Michele Calos
发明人： Michele Calos
IPC分类号： C12N15/09 , C12N9/00 , C12N9/90 , C12N15/90 , C12R1/01 , C12N15/63 , C12N15/85
CPC分类号： C12N9/00 , A61K48/00 , C12N9/22 , C12N15/90 , C12N15/902 , C12N2800/30
摘要： The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.
摘要翻译：本发明描述鉴定改变的重组酶及其组合物的方法，其中至少一个氨基酸不同于亲本野生型重组酶，并且改变的重组酶相对于野生型和/或伪att位点序列具有改善的重组效率亲本，野生型重组酶。本发明还包括使用改变的重组酶修饰细胞基因组的方法，包括将感兴趣的多核苷酸序列特异性整合到真核细胞的基因组中的方法。

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