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    Enhanced system for construction of adenovirus vectors
    2.
    发明授权
    Enhanced system for construction of adenovirus vectors 失效
    增强腺病毒载体构建体系

    公开(公告)号：US06756226B2

    公开(公告)日：2004-06-29

    申请号：US09981648

    申请日：2001-10-16

    申请人： Frank L. Graham ,  Robin Parks ,  Philip Ng

    发明人： Frank L. Graham ,  Robin Parks ,  Philip Ng

    IPC分类号： C12N1564

    CPC分类号： C12N15/86 ,  A01K2217/05 ,  C12N2710/10343 ,  C12N2710/10351 ,  C12N2800/30

    摘要： In the present invention, viruses, plasmids or both are constructed which contain viral DNA, at least one head-to-head ITR junction, and optionally, recombinase recognition sites positioned such that site-specific recombination between recombinase recognition sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express a site-specific recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system, as well as compositions and methods for using such compositions as vaccines or in gene therapeutic applications. Enhancements in the efficiency of both site-specific and homologous recombination are provided by inclusion of at least one head-to-head ITR junction.

    摘要翻译： 在本发明中，构建了包含病毒DNA，至少一个头对头ITR连接和任选的重组酶识别位点的病毒，质粒或二者，使得分离质粒中重组酶识别位点之间的位点特异性重组导致 在已经被工程化以表达位点特异性重组酶的共转染的宿主细胞中高效率地产生感染性病毒DNA。 由于Cre酶的高效率和特异性，合适的工程改造质粒可以在共转染293细胞中高效率地重组，产生感染性病毒，同时产生野生型腺病毒，伴随着问题 用于去除。 除了lox位点之外的Cre和重组酶识别位点以及使用除293细胞之外的细胞的重组酶的使用也被公开和启用，以及包含位点特异性载体系统的试剂盒以及使用这些组合物作为疫苗的组合物和方法 或在基因治疗应用中。 通过包含至少一个头对头ITR连接来提供位点特异性和同源重组的效率的增强。

    Enhanced system for construction of adenovirus vectors
    3.
    发明授权
    Enhanced system for construction of adenovirus vectors 失效
    增强腺病毒载体构建体系

    公开(公告)号：US06855534B2

    公开(公告)日：2005-02-15

    申请号：US09978464

    申请日：2001-10-16

    申请人： Frank L. Graham ,  Robin Parks ,  Philip Ng

    发明人： Frank L. Graham ,  Robin Parks ,  Philip Ng

    IPC分类号： C12N15/09 ,  A61K35/76 ,  A61K48/00 ,  C12N5/10 ,  C12N7/00 ,  C12N15/861 ,  C12N1/861

    CPC分类号： C12N7/00 ,  C12N15/86 ,  C12N2710/10343 ,  C12N2710/10351 ,  C12N2800/30

    摘要： In the present invention, viruses, plasmids or both are constructed which contain viral DNA, at least one head-to-head ITR junction, and recombinase recognition sites positioned such that site-specific recombination between recombinase recognition sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express a site-specific recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system, as well as compositions and methods for using such compositions as vaccines or in gene therapeutic applications. Enhancement in the efficiency of site-specific recombination is provided by inclusion of a head-to-head ITR junction in each virus, plasmid, or other nucleic acid construct.

    摘要翻译： 在本发明中，构建了包含病毒DNA，至少一个头对头ITR连接和重组酶识别位点的病毒，质粒或二者，使得分离质粒中重组酶识别位点之间的位点特异性重组导致产生 感染性病毒DNA在共同转染的宿主细胞中以高效率工程化以表达位点特异性重组酶。 由于Cre酶的高效率和特异性，合适的工程改造质粒可以在共转染293细胞中高效率地重组，产生感染性病毒，同时产生野生型腺病毒，伴随着问题 用于去除。 除了lox位点之外的Cre和重组酶识别位点以及使用除293细胞之外的细胞的重组酶的使用也被公开和启用，以及包含位点特异性载体系统的试剂盒以及使用这些组合物作为疫苗的组合物和方法 或在基因治疗应用中。 通过在每个病毒，质粒或其他核酸构建体中包含头对头ITR连接来提供位点特异性重组的效率的提高。

    Adenovirus vectors generated from helper viruses and helper-dependent vectors
    4.
    发明授权
    Adenovirus vectors generated from helper viruses and helper-dependent vectors 失效
    由辅助病毒和辅助因子载体产生的腺病毒载体

    公开(公告)号：US06566128B1

    公开(公告)日：2003-05-20

    申请号：US09440809

    申请日：1999-11-15

    申请人： Frank L. Graham ,  Robin Parks ,  Liane Chen

    发明人： Frank L. Graham ,  Robin Parks ,  Liane Chen

    IPC分类号： A61K4800

    CPC分类号： C12N7/00 ,  A01K2217/05 ,  C12N15/86 ,  C12N2710/10343 ,  C12N2710/10351 ,  C12N2800/30 ,  C12N2830/002 ,  C12N2830/38

    摘要： The present invention provides an improved helper-dependent vector system for production of high capacity adenoviral cloning vectors. The invention makes use of the DNA size packaging constraints imposed on a pIX-defective Ad virion that prevent such virions from packaging DNA larger than approximately 35 kb. This constraint can be used to develop helper viruses that do not package their DNA. In one embodiment, the invention combines this methodology with the Cre-loxP helper-dependent system to decrease the quantity of contaminating helper virus in vector preparations. In another embodiment the invention is used for vector growth.

    摘要翻译： 本发明提供了用于产生高产量腺病毒克隆载体的改进的辅助依赖载体系统。 本发明利用施加在pIX缺陷型Ad病毒粒子上的DNA大小封装限制，其阻止这种病毒粒子包装大于约35kb的DNA。 该约束可用于开发不包装其DNA的辅助病毒。 在一个实施方案中，本发明将该方法与Cre-loxP辅助依赖性系统相结合，以减少载体制剂中污染性辅助病毒的量。 在另一个实施方案中，本发明用于载体生长。

    Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration
    5.
    发明授权
    Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration 失效
    替代血清型的辅助依赖性腺病毒载体的使用允许重复载体给药

    公开(公告)号：US06730507B1

    公开(公告)日：2004-05-04

    申请号：US09286874

    申请日：1999-04-06

    申请人： Frank L. Graham ,  Robin Parks

    发明人： Frank L. Graham ,  Robin Parks

    IPC分类号： C12N510

    CPC分类号： C12N7/00 ,  A01K2217/05 ,  A61K48/00 ,  C12N15/86 ,  C12N2710/10343 ,  C12N2710/10352 ,  C12N2800/30 ,  C12N2830/38

    摘要： This invention responds to a long felt need, by providing in one embodiment, a helper virus based on the Ad2 serotype for use in the Cre/loxP system for the generation of Ad vectors deleted of all Ad protein coding sequences. Using this and helper virus based on Ad5, genetically identical hdAd that differ only in the virion protein components, which are derived from the helper virus, were produced. The vectors have identical expression characteristics in vitro, regardless of the serotype, and the sequential use of hdAd of different serotypes allows for successful repeat vector administration in vivo.

    摘要翻译： 本发明通过在一个实施方案中提供基于用于Cre / loxP系统中的Ad2血清型的辅助病毒来响应长期的需要，用于产生缺失所有Ad蛋白编码序列的Ad载体。 使用基于Ad5的这种和辅助病毒，产生仅源自辅助病毒的病毒粒子蛋白质组分的遗传相同的hdAd。 载体在体外具有相同的表达特征，而不管血清型如何，并且不同血清型的hdAd的顺序使用允许体内成功重复载体给药。

    High-efficiency Cre/loxp based system for construction of adenovirus vectors
    7.
    发明授权
    High-efficiency Cre/loxp based system for construction of adenovirus vectors 有权
    用于构建腺病毒载体的高效Cre / loxp系统

    公开(公告)号：US06379943B1

    公开(公告)日：2002-04-30

    申请号：US09263650

    申请日：1999-03-05

    申请人： Frank L. Graham ,  Robin J. Parks ,  Philip Ng

    发明人： Frank L. Graham ,  Robin J. Parks ,  Philip Ng

    IPC分类号： C12N510

    CPC分类号： C12N7/00 ,  C12N15/86 ,  C12N15/907 ,  C12N2710/10343 ,  C12N2710/10351 ,  C12N2800/30

    摘要： In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox sites positioned such that site-specific recombination between lox sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system, as well as compositions and methods for using such compositions as vaccines or in gene therapeutic applications.

    摘要翻译： 在本发明中，构建了含有病毒DNA和lox位点的病毒，质粒或两者，其位置使得分离的质粒中的lox位点之间的位点特异性重组导致在共转染的宿主细胞中高效率地产生感染性病毒DNA 旨在表达Cre重组酶。 由于Cre酶的高效率和特异性，合适的工程改造质粒可以在共转染293细胞中高效率地重组，产生感染性病毒，同时产生野生型腺病毒，伴随着问题 用于去除。 除了lox位点之外的Cre和重组酶识别位点以及使用除293细胞之外的细胞的重组酶的使用也被公开和启用，以及包含位点特异性载体系统的试剂盒以及使用这些组合物作为疫苗的组合物和方法 或在基因治疗应用中。

    Recombinase-based system for combustion of adenovirus vectors
    8.
    发明授权
    Recombinase-based system for combustion of adenovirus vectors 有权
    用于腺病毒载体燃烧的基于重组酶的系统

    公开(公告)号：US07132290B2

    公开(公告)日：2006-11-07

    申请号：US09909414

    申请日：2001-07-19

    申请人： Frank L. Graham ,  Robin J. Parks ,  Philip Ng

    发明人： Frank L. Graham ,  Robin J. Parks ,  Philip Ng

    IPC分类号： C12N15/87 ,  C12N15/861

    CPC分类号： C12N7/00 ,  C12N15/86 ,  C12N15/907 ,  C12N2710/10343 ,  C12N2710/10351 ,  C12N2800/30

    摘要： In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox sites positioned such that site-specific recombination between lox sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system.

    摘要翻译： 在本发明中，构建了含有病毒DNA和lox位点的病毒，质粒或两者，其位置使得分离的质粒中的lox位点之间的位点特异性重组导致在共转染的宿主细胞中高效率地产生感染性病毒DNA 旨在表达Cre重组酶。 由于Cre酶的高效率和特异性，合适的工程改造质粒可以在共转染293细胞中高效率地重组，产生感染性病毒，同时产生野生型腺病毒，伴随着问题 用于去除。 除了含有lox位点之外的Cre和重组酶识别位点以及使用除293细胞之外的细胞的重组酶的使用也被公开和启用，以及包含位点特异性载体系统的试剂盒。

    Production of adenovirus vectors with reduced levels of replication competent adenovirus contamination
    9.
    发明授权
    Production of adenovirus vectors with reduced levels of replication competent adenovirus contamination 失效
    生产具有降低复制水平的腺病毒载体的腺病毒载体

    公开(公告)号：US07189561B2

    公开(公告)日：2007-03-13

    申请号：US10946484

    申请日：2004-09-21

    申请人： Frank L. Graham

    发明人： Frank L. Graham

    IPC分类号： C12N15/34 ,  C12N15/64 ,  C12N15/63 ,  C12N5/10 ,  C12N15/861 ,  C07H21/04

    CPC分类号： C12N15/86 ,  C12N2710/10343 ,  C12N2800/30 ,  C12N2830/42 ,  C12N2840/20

    摘要： Methods, cells and recombinant adenoviral vectors are disclosed that permit the production of recombinant adenoviral vector stocks with reduced levels of contamination by replication competent adenoviruses (RCA). In certain embodiments are disclosed early region 1 (E1) deficient recombinant adenoviral vectors and complementing E1 positive host cells whose sequences are designed to avoid formation of RCA by homologous recombination between sequences in the vector and E1 sequences in the cells. One aspect of the invention involves the inversion of the packaging signal in a recombinant adenoviral vector relative to an adjacent or nearby inverted terminal repeat (ITR). Methods include use of site-specific intregrase family recombinases such as Cre or FLP and recombinase recognition sites such as lox sites or frt sites.

    摘要翻译： 公开了方法，细胞和重组腺病毒载体，其允许通过复制性腺病毒（RCA）产生具有降低的污染水平的重组腺病毒载体原种。 在某些实施方案中公开了早期区域1（E1）缺陷型重组腺病毒载体和补体E1阳性宿主细胞，其序列被设计为避免通过载体中的序列和细胞中的E1序列之间的同源重组而形成RCA。 本发明的一个方面涉及重组腺病毒载体中相对于邻近或邻近倒置末端重复（ITR）的包装信号的反转。 方法包括使用位点特异性整合酶家族重组酶，如Cre或FLP以及重组酶识别位点，如lox位点或frt位点。