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。

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。

公开(公告)号：US07135187B2

公开(公告)日：2006-11-14

申请号：US10355330

申请日：2003-01-31

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

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

IPC分类号： A61K39/23 ,  C07H21/04 ,  C12N7/00 ,  C12N15/00

CPC分类号： C12N15/86 ,  A01K2217/05 ,  A61K48/00 ,  C12N15/85 ,  C12N2710/10343 ,  C12N2710/10351 ,  C12N2800/30 ,  C12N2800/80 ,  C12N2830/38 ,  C12N2830/42 ,  C12N2840/203

摘要： The present invention relates to methods for efficient and reliable construction of adenovirus vectors which contain and express foreign DNA and are useful for gene transfer into mammalian cells, for vaccines and for gene therapy. The invention provides for the growth and purification of adenovirus vectors (helper dependent vectors or HDVs) from which all or most of the viral genes have been removed. The vector system described herein is a new method designed to eliminate helper viruses from the final HDV preparation by cleavage of the helper virus DNA with an endonuclease, alone or in combination with other methods known to limit the level of helper virus contamination of helper dependent vector preparations. The disclosed methods and compositions also provide for regulated control of gene expression.

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。

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。

公开(公告)号：US6080569A

公开(公告)日：2000-06-27

申请号：US719217

申请日：1996-09-25

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

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

IPC分类号： C12N15/09 ,  A61K48/00 ,  C12N5/10 ,  C12N7/00 ,  C12N15/83 ,  C12N15/861 ,  C12Q1/70 ,  C12N15/63

CPC分类号： C12N7/00 ,  C12N15/86 ,  A01K2217/05 ,  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.

公开(公告)号：US20070014769A1

公开(公告)日：2007-01-18

申请号：US11526429

申请日：2006-09-25

申请人： Frank Graham ,  Silvia Bacchetti ,  Philip Ng ,  Robin Parks ,  Mauro Anglana

发明人： Frank Graham ,  Silvia Bacchetti ,  Philip Ng ,  Robin Parks ,  Mauro Anglana

IPC分类号： A61K48/00 ,  C12N15/861

CPC分类号： C12N15/86 ,  A61K48/00 ,  A61K2039/5256 ,  C12N7/00 ,  C12N9/22 ,  C12N15/85 ,  C12N2710/10343 ,  C12N2800/30 ,  C12N2800/80 ,  C12N2830/38 ,  C12N2830/42 ,  C12N2840/203

摘要： The present invention relates to methods for efficient and reliable construction of adenovirus vectors which contain and express foreign DNA and are useful for gene transfer into mammalian cells, for vaccines and for gene therapy. The invention provides for the growth and purification of adenovirus vectors (helper dependent vectors or HDVs) from which all or most of the viral genes have been removed. The vector system described herein is a new method designed to eliminate helper viruses from the final HDV preparation by cleavage of the helper virus DNA with an endonuclease, alone or in combination with other methods known to limit the level of helper virus contamination of helper dependent vector preparations. The disclosed methods and compositions also provide for regulated control of gene expression.

摘要翻译： 本发明涉及用于有效和可靠地构建含有和表达外源DNA的腺病毒载体的方法，并且可用于将基因转移到哺乳动物细胞中，用于疫苗和基因治疗。 本发明提供了从其中除去所有或大部分病毒基因的腺病毒载体（辅助因子依赖载体或HDV）的生长和纯化。 本文描述的载体系统是设计用于通过用内切核酸酶单独或与已知限制辅助病毒污染辅助性依赖载体水平的其它方法结合的内切核酸酶切割辅助病毒DNA从最终HDV制备中消除辅助病毒的新方法 准备工作 所公开的方法和组合物还提供基因表达的调控。

公开(公告)号：US20070128165A1

公开(公告)日：2007-06-07

申请号：US11556936

申请日：2006-11-06

申请人： Frank Graham ,  Robin Parks ,  Philip Ng

发明人： Frank Graham ,  Robin Parks ,  Philip Ng

IPC分类号： A61K48/00 ,  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。